key: cord-0041651-hpzj1qz7
authors: nan
title: Poster Presentations
date: 2008-06-28
journal: FEBS J
DOI: 10.1111/j.1742-4658.2008.06448.x
sha: eb19e1f53e8f7cad7eb5d29c888d1a9ae16fe62e
doc_id: 41651
cord_uid: hpzj1qz7

nan

PP1A-3 A rare form of chromosomal instability in neoplasia leads to spontaneous emergence of human minute chromosomes In the alternative pathway of telomere lengthening (ALT), endogenous telomere dysfunction is accompanied by frequent telomeric chromosomal rearrangements. We demonstrate that ALT is unexpectedly characterized by increased pericentromeric instability leading to spontaneous emergence of mitotically functional human neo-acrocentric chromosomes. Molecular and conventional cytogenetics in a panel of 10 telomerase positive and 5 ALT human cell lines showed that ALT recombination break-points were clustered at pericentromeric regions. Chromatin immunoprecipitation, centromere specific and M-FISH, indicated that ALT neo-acrocentric chromosomes were mainly produced by true centromeric fissions. We focus on an interesting subgroup of ALT neo-acrocentric chromosomes, composed of DNA entities that are 2-5 times smaller in size than HSA21. These ALT minute chromosomes exhibit a two-chromatid structure, carry non-heterochromatic genomic DNA, centromeric and telomeric repeats, they associate with CENP-A, and display regular mitotic functionality. The frequencies of ALT minute chromosomes were elevated by clastogenic c-irradiation, and suppressed by reconstitution of telomerase activity or inhibition of the RAD50 and MRE11. Continuous growth after telomerase inhibition/depletion in two telomerase positive human cancer cell lines was followed by increased rates of telomeric sister chromatid recombination and the emergence of minute chromosomes. Together our data suggest that in the ALT pathway, pericentromeric instability, chromosomal Breakage/Fusion/Bridge cycles and stochastic recombinatorial telomere capping, are capable to generate in vitro mitotically functional human minute chromosomes.

Human PIF1 suppresses genomic instability through its evolutionary conserved role in telomere maintenance PP1A-5 High osmolality activates the G1 and G2 cell cycle checkpoints and affects the DNA integrity of nucleus pulposus intervertebral disc cells triggering an enhanced DNA repair response E. Mavrogonatou and D. Kletsas Laboratory of Cell Proliferation and Ageing, Institute of Biology, National Centre For Scientific Research 'Demokritos', Athens, GREECE Nucleus pulposus intervertebral disc cells experience a broad range of physicochemical stimuli in their native environment including osmotic fluctuations. Here we show that hyperosmotic treatment reduced nucleus pulposus cells' proliferation by activating the G2 and G1 cell cycle checkpoints. p38 MAPK was found to participate in the manifestation of the G2 arrest under conditions of increased osmolality, since inhibition of its activity released the cells from G2 phase into mitosis. High osmolality resulted in the ATM-mediated phosphorylation of p53 on Ser15, the up-regulation of p21 WAF1 and the hypophosphorylation of the retinoblastoma protein in accordance to the observed G1 arrest. Furthermore, comet assay revealed the presence of DNA damage after hyperosmotic treatment, possibly attributed to the abrupt alterations in chromatin configuration observed early after exposure of the cells to this stress. Under these conditions, the MRN complex remained in the nucleus and H2A.X was phosphorylated on Ser139. In addition, nucleus pulposus cells residing within a hyperosmotic environment clearly retained their ability to sense newly introduced DNA damage, as confirmed by the re-activation of p53 by ionizing radiation. Even more, when the DNA repair efficiency of the cells was directly measured by a host cell reactivation of luciferase activity assay, it was found to be significantly increased under hyperosmotic pressure. Overall, our data provide the first evidence that high osmolality delays nucleus pulposus cells' proliferation and affects their DNA integrity triggering an enhanced DNA repair response.

Introduction: Male infertility is a common health problem. Microdeletions of the human Y chromosome (AZF region) are associated with spermatogenic failure. CFTR gene (chromosome 7) also is related to male infertility. In males without cystic fibrosis, mutations in CFTR gene phenotypicaly look like obstructive azoospermy, with results CBAVD -lack of vas deferens. The aims of our research were: to detect Y chromosomal microdeletions in AZF region, and to detect poly-T, poly-GT polymorphisms and delF508 mutation of CFRT gene in infertile male. Material and Methods: Objects for analysis were 79 individuals (DNA isolated from the blood cells) with different spermatogenic arrest and normal kariotype. Microdeletions in AZF region were determined by multiplex STS based PCR reactions. Mutation delF508 in CFTR gene was determined by appropriate PCR reaction. Polymorphisms poly-T and poly-GT were detected by sequencing. Results: Out of 79 analysed samples microdeletions were found in five cases (6%). Three of five patients had deletions of AZFc region and two had deletion of all three AZF regions. Mutation delF508 was not found in analysed 79 samples. Analysis of poly-T and poly GT polymorphisms in CFTR gene showed interesting results. In infertile males more common variant was 11GT/12GT heterozygous form. However, it does not give statistically valid interpretation. Conclusion: The frequency of Y-chromosomal microdeletions is low (6%) in Latvian infertile male population. Analysis of mutation delF508 and polymorphisms poly-T and poly-GT does not show correlation with infertility. However, Y chromosome microdeletion screening and CFTR gene analysis is important in case of genetic counselling. (1) . The function of human Par14 is still elusive. In consequence hereof, the theme of this work is devoted to unravel its sub-nuclear localisation and function. Methods and Results: On the basis of sequence similarity of Par14 to the well known mitotic regulator Pin1 and Par14's enrichment in the cellular nucleus with a dsDNA binding ability, Par14 has been posited to play a role in cell cycle regulation and chromatin remodelling. Different biochemical sub-nuclear fractionation methods were applied to study the localisation of the endogenous Par14 protein within the cellular nucleus. In an orthogonal approach, indirect immunofluorescence was used to establish the localisation of Par14 in polythene chromosomes. Immunoblots for the biochemical fractions revealed that Par14 was associated with both the nuclear matrix and chromatin with a higher affinity to the latter. In a similar light, chromosome bands were observed in polythene chromosomes. Conclusion: Taken together, these experiments show the sub-nuclear localisation of Par14. Proteins which are chromatin and/or nuclear matrix bound have been shown to be involved in a variety of DNAdependent activities such as transcription, replication, recombination, DNA-repair and genomic stability. The attachment between the two large architectural units of bacterial cell, the membrane and the nucleoid, is presumably brought about through RNA-protein bridges resulting from concurrent transcription/ translation and insertion of integral proteins into the membrane, the 'transertion' process. The transertion model proposes an evolutionary reasonable mechanism for DNA-membrane interaction and consequent membrane domain formation. The hyperfunction of this mechanism is its participation in spatio-temporal organization of the bacterial cell. Coupling between the first two points of the transertion was directly demonstrated by visualizing acting polysomes. The aim of this project is to test the transertion model regarding the nature of the connection between the nucleoid and the membrane in the third point. According to the model, membrane domains should be enriched with Sec translocation machinery proteins and their preferred phospholipids. Plasmids encoding fusions of green fluorescent protein (eGFP) with different components of the translocation machinery (SecA, SecY and SecE) were constructed. We observed a heterogenic localization of SecA-eGFP and eGFP-SecE fusion proteins in E. coli membrane. Examination of nucleoids released from such cells revealed regions of eGFP florescence attached to the nucleoid body. These regions presumably represent membrane patches enriched with SecA-eGFP, eGFP-SecE and eGFP-SecY. Our results confirm the existence of a continuous connection between the nucleoid and translocation complexes in the bacterial membrane through nascent mRNA and peptides.

1B. Imprinting and Epigenetic Regulation PP1B-1 E6 human papillomavirus type16 methylation in clinical specimens In vivo effects of histone-deacetylase inhibitortrichostatin A (TSA) on chromatin dynamics during murine spermatogenesis L. Burlibasa, O. Zarnescu and L. Gavrila University of Bucharest, Faculty of Biology, Bucharest, ROMANIA Spermatogenesis is an extreme case of chromatin reorganisation in somatic cells with a view to differentiating the respective germ line in highly differentiating gamete cells like spermatozoa. This structural dynamics is controlled both at the level of genetic information and at the one of epigenetic information (chemical modifications of chromatin components-DNA and histones). Our studies were focused on molecular architecture of chromatin during mouse spermatogenesis. The dynamics of histone H4 hypercetylation during spermatogenesis has been investigated. The transmission electron microscopy analysis, immunohistochemistry technique and specific molecular investigations were employed for deciphering some peculiar aspects of chromatin organization and evolution in spermatogenesis of Mus musculus . An increasing chromatin condensation in final stages of spermatogenesis has been encountered. Spermatogonia, preleptotene spermatocytes and spermatids contained hyperacetylated H4, whereas no acetylated H4 was noticed in leptotene or pachytene spermatocytes. In order to gain an insight into mechanism cotrolling histone hyperacetylation during spermatogenesis we treated the mouse with Trichostatin A a histone deacetylase inhibitor. Ultrastructural and molecular analysis of the mouse testicular tissue after TSA treatment indicated a major chromatin remodelling in sperm nuclei, evidenced by chromatin decompactation and the absence of protamines and an obvious increase of histone acetylation in round spermatid. Thus, histone acetylation could be interpreted as an early signal of histone replacement by highly basic sperm-specific proteins a process tightly linked to nuclear condensation. Our investigations suggest that the regulation of histone deacetylase activity appears to play a major role in modulating histone hyperacetylation and perhaps histone replacement during spermiogenesis. Institute of Virology, 'Stefan S. Nicolau', Bucharest, ROMANIA, 2 'Coltea' Hospital, Bucharest, ROMANIA Introduction: Epstein-Barr virus (EBV) infects nearly all humans and then persists for the life of the host. EBV has been associated with various types of cancer, including non-Hodgkin and Hodgkin lymphoma (NHL and HL) and nasopharyngeal carcinoma. In the current study, we analyzed EBNA1 (EB viral nuclear antigen-1) gene expression and Cp promoter in different subtypes of NHL. Methods: Peripheral blood samples from 50 patients diagnosed with different types of NHL in 'Coltea' Hospital were collected. DNA was extracted and then EBV presence was investigated by EBNA1 gene expression using TaqMan real time PCR. We also studied the methylation status in the Cp region of EBNA1 gene using methylation specific PCR analysis in the same DNA samples. Results: Methylation of CpG is an important mechanism of promoter regulation and EBV latent gene expression. EBNA gene expression has been shown to be inversely correlated with the methylation status of the promoter Cp in 15 patients (30%) of the total NHL cases. Methylation may inhibit transcription by a number of mechanisms, either by direct blocking of the binding of transcription factors to DNA or through mediation of methyl-CpG-binding proteins, which recruit histone deacetylases to the DNA, leading to the remodeling of chromatin into an inactive configuration. Conclusions: Hypermethylation in the Cp region occurs in various degrees regardless of the phenotypic difference in NHL. So far, no differences are discernible between lymphoma types and further studies on a larger cohort of patients are required.

Set9-mediated methylation of E2F-1 H. Kontaki and I. Talianidis Biomedical Sciences Research Center Al. Fleming, Athens, GREECE Set9 (KMT7) is a member of the SET domain-containing methyltransferases, that can specifically methylate lysine 4 residue of histone 3. Recent studies suggested that histones are not the sole substrates for Set9, implying that Set9-mediated transcriptional activation involves methylation of other proteins as well. The non-histone substrates for Set9 identified so far are TAF10 and p53. Set9-mediated methylation of TAF10 enhances its interaction with RNA pol-II at specific promoters, while methylation of p53 leads to an increased stability of the protein, which contributes to p53-mediated apoptosis. In this study we show that E2F-1, a key factor regulating S-phase specific as well as several apoptotic genes, is a bona fide substrate for Set9. Set9 monomethylates E2F1 at lysine 185 residue, located in the DNA-binding/DP heterodimerization domain, both in vitro and in vivo. We provide evidence that Set9 prevents DNA damage-induced accumulation of E2F-1. The results point to a novel role of Set9 in regulating E2F-1-induced p53-independent apoptosis. PP1B-7 Genetic instability, splicing and epigenetic modification of p53 G. Tzimagiorgis 1 , A. Fragou 1 , A. Malousi 1 , E. Georgiou 1 , N. Barbetakis 2 , C. Tsilikas 2 and S. Kouidou 1 1 Aristotle University of Thessaloniki, School of Medicine, Thessaloniki, GREECE, 2 Theageneion Anticancer Hospital, Thessaloniki, GREECE Introduction: Analysis of silent mutations in p53 sporadic mutation databases reveals that genetic instability in p53 is frequently related to the loss of methylation and exonic splicing-regulatory sequences. Furthermore, the presence of asymmetric non-CpG cytosine methylation in an oligoC sequence (codon 177) is also commonly detected in lung carcinomas. Methods and Results: We presently report that direct methylation sequencing analysis of 20 lung carcinomas shows frequent non-CpG methylation in CCs, CAs and CGCs in most lung carcinoma specimens but not in adjacent non-cancerous tissue; methylation-specific PCR (MSP) analysis of the same specimens also shows frequent presence of several asymmetric methylation sites in the vicinity of codon 133 (start codon of D133p53 isoform), (sequences 131-137, and 141-147) in addition to methylation in codon 177 (sequences 176-178). RT-PCR analysis reveals correlation of methylation in codons 177 and 133 with truncated p53 transcripts missing exon 5 and carrying frequent C > T transcriptional single base aberrations at CCs and CAs as well as A > T transversions at CAs in all carcinomas tested. Analysis of the p53 sporadic mutation databases reveals that the C > T mutation distribution in sporadic cancers is related to the loss of SC35 splicing protein binding sites (p = 0.194), while computational evaluation of germline mutations shows that the same aberrations (i.e. splice-site introducing mutations in codon 257) are frequent in the Li Fraumeni, cancer-related syndrome. Conclusion: Non-CpG epigenetic modifications of p53 in lung carcinomas is probably associated with the frequently observed C > T transcriptional aberrations resulting in splicing anomalies.

Plant specific phi class GST, OSGSTF3: herbicide specificity and catalytic roles X chromosome inactivation in female mammals is initiated by accumulation on the future inactive X of a 17-to 19-kb long non coding RNA, Xist. The Xist RNA is thought to recruit specific protein partners which are responsible for the transcriptional silencing of the genes on the inactive X chromosome. Genetic evidence has established the absolute requirement for the silencing process, of an approximately 500-nt long conserved region in the 5'-terminal region of the Xist RNA called the 'A-repeat'. This region composed of 7 or 8 repetitions of a 24 nt long GC-rich element separated by U-rich spacers. By using chemical and enzymatic probes of the RNA structure, we found that the A-repeat region of both mouse and human Xist RNAs can be folded into two long irregular stem-loop structures. The repeats are involved in base-pair interactions either with another repeat or with nucleotides of the spacer sequences. However, most of the second element of each repeat is in a single stranded conformation. Bulges and loops in each stem loop mostly correspond to U-rich linker sequences and the 3' termini of the repeated sequences. By using an affinity chromatography method, we identified several nuclear proteins from human and mice that can interact with this region. We show that the Polypyrimidine tract binding protein (PTB) has a strong affinity for this region and may participate to the establishment of its peculiar structure.

Oncogenic RAS protein drives covalent modifications in histone H3 that contribute to colorectal tumor progression I. Mazon Pelaez and A. Pintzas Laboratory of Signal Mediated Gene Expression, Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, Athens, GREECE Introduction: Cancer develops through a collaboration of genetic and epigenetic alterations. Epigenetic abnormalities are manifested in global changes in chromatin packing and in localized gene promoter changes that regulate gene transcription. Covalent modifications in histones can be considered as active or repressive markers of gene expression. Activation of ras signaling pathways through oncogenic Ki-ras or BRAF mutations is a hallmark of colorectal cancer. Phosphorylation of H3 at Ser10 is involved in gene transcription and it is elevated in ras transformed cells (1) . We focused on the role of histone H3 epigenetic modifications in ras and raf signal transduction pathway and oncogenic transformation. Methods: Two systems have been used to assess H3 covalent modifications, a cell model created by selecting commercially available cell lines that beard different combination of genetic mutations and0 reflect different tumor stages and a system based in stable transfected cell lines with oncogenic Ki-, Ha-ras (2) and BRAF proteins. Western Blot, chromatin immunoprecipitation, siRNA technology and inhibitors for ras oncogenic activated pathways were used to study the role of histone modifications in ras mediated gene expression. Results: Ras oncogenic affects, through ERK/MAPK pathway, covalent modifications in H3 that lead to gene expression changes. Further, acetylation and methylation patterns of lysine in H3 are altered from early adenomacarcinoma cell lines to late colorectal tumor stages cells lines. Conclusion: Our results show that some histone covalent modifications play a role in progression of colorectal cancer in a ras dependent manner and may be useful as molecular markers. Introduction: The histone H3 demethylase Ndy1/KDM2B is a physiological inhibitor of replicative senescence and proposed to have protective effects from DNA damage. Based on these data we investigated whether Ndy1 protects from DNA damage by regulating redox homeostasis. Methods: Primary MEFs were infected with retrovirus constructs of Ndy1, and deletion mutants or transfected with Ndy1 siRNA. Cell survival, apoptosis and cell cycle distribution were studied using the MTT assay, TUNEL and Et.Br nuclear staining, respectively. DNA oxidation and damage were analyzed by immunofluorescence and the Comet assay. Reactive Oxygen Species accumulation, antioxidant activity and the expression of redox regulatory genes were assessed by FACs, the antioxidant assay kit and RT 2 -PCR array, respectively. Ndy1 binding to the DNA and histone methylation were studied by chromatin immunoprecipitation. Results: Ndy1 overexpression protects cells from H 2 O 2 -induced apoptosis and G2/M arrest and inhibits ROS-mediated signaling and DNA damage, whereas knockdown of Ndy1 has the opposite effects. Ndy1 promotes H 2 O 2 detoxification through the up-regulation of the antioxidant genes Aass, Nqo1, Prdx4 and Serpinb1b, whereas Ndy1 knockdown has the opposite effects. The induction of at least Nqo1 and Prdx4 depends on the binding of Ndy1 to specific sites within their promoters and the demethylation of promoter-associated histone H3 dimethylated at K36. Knockdown of Aass, Nqo1, Prdx4 and Ser-pinb1b in Ndy1-expressing cells to levels equivalent to those in control cells was sufficient to suppress the Ndy1 redox phenotype. Conclusion: Ndy1 epigenetically regulates several redox genes, thus modulating H 2 O 2 levels and the resistance of Ndy1-expressing cells to oxidative stress.

Transcriptional regulation of gas1 in Hepa 1-6 cells N. Sacilotto and G. Lopes-Rodas Chromatin Laboratory, University of Valencia, Valencia, SPAIN

The tumor suppressor gene gas1 (Growth Arrest Specific gene 1) plays an important role in the mammalian cell cycle control, blocking cellular proliferation at the G 1 /S transition or inducing apoptosis, depending on the cellular context. After analyzing gas1 expression in the hepatocarcinoma cell line Hepa 1-6 we found a tight regulation, appearing two peaks of expression around 3 and 12 hours after the administration of serum into a synchronized cell culture, and a minimum around 24 hours. To better comprehend the molecular mechanisms of gas1 transcriptional regulation at chromatin level, we studied gas1 promoter performing Chromatin Immunoprecipitation assays (ChIPs). This gave us information about the recruitment of transcriptional factors, chromatin modifying and remodeling complexes implicated in such regulation. Our results suggest the presence of the transcriptional factors C/EBPb, NF-jB, pELK1 and SRF, as well as GCN5 and PCAF containing histone acetyltransferases complexes in the transcriptionally active state. On the other hand, the remodeling complex NuRD, generally implicated in generating chromatin suppressive structures, is present when the gene is silenced and, concomitantly with the activation of the gene, it is released from the promoter while the remodeling complex SWI/SNF is recruited to it. These results suggest that structural changes of the chromatin at the promoter of the gene are also relevant in controlling gas1 transcriptional state. Therefore, special attention has to be paid on how epigenetic regulation influences the control of gene expression, because is in the context of chromatin where different signals must integrate to modulate cell proliferation.

Age-related changes at the histone H10 gene region P. Salpea 1 , V. R. Russanova 2 , K. Sekeri-Pataryas 1 Introduction: Epigenetic changes, such as posttranslational histone modifications, affect gene expression during ageing. Acetylation is the most studied histone modification and is associated with transcriptionally competent chromatin. The H1 histone subtype, H1 0 , is known to accumulate during terminal differentiation and has also been shown to accumulate during cellular senescence. Moreover, the acetylation of the promoter of this gene has been shown to be regulated during development and differentiation. In light of the above, the focus of this study is to investigate changes in histone acetylation levels that may occur in the H1 0 gene region during cellular ageing. Methods: The systems used were isolated human peripheral blood lymphocytes in the quiescent state or activated in vitro with phytohemagglutinin and skin fibroblasts from young and elderly donors. Polymerase chain reaction (PCR) was carried out with primers designed for the H1 0 gene region and the PCR products were analyzed using denaturing high-performance liquid chromatography. Results: Histone acetylation levels of the H1 0 gene region up to 11 kb upstream were found to be higher in both activated and non activated lymphocytes from elderly donors as compared to those from young donors. The same age-related changes in acetylation levels were also detected in skin fibroblasts. Conclusions: Acetylation loosens the interaction between core histones and DNA and as such is a prerequisite for transcriptional activation. The elevated acetylation levels in the lymphocytes and skin fibroblasts from the elderly donors reveals that epigenetic changes may play a role in H1 0 accumulation previously found to occur during cellular ageing. Methods: Here, we induced neural differentiation of an embryonal carcinoma stem cell line (N-Tera 2) by retinoic acid (RA) treatment and analyzed histone variants and modifications. Results: We observed significant changes in histone variants as well as histone modification patterns of the differentiated cells, during differentiation. Western blot analysis of histone extracts has shown that beside a global change in acetylation/methylation pattern of histones especially histone H3, the expression of some histone variants such as H3.3, H2A.Z and H1.0 has changed after RA treatment of the cells. The histone variants were further identified by mass spectrometry. Using chromatin immunoprecipitation (ChIP) technique coupled with real-time PCR analysis, we have also shown that these epigenetic changes occur on the regulatory regions of the genes involved in stemness (Oct4, Nanog) and neural differentiation (Pax6, Nestin). Conclusions: This report demonstrates the dynamic interplay of histone variants in regulating gene expression during stem cell differentiation. To study the in vivo role of TFIID in the transcriptional regulation of hepatic genes we generated mice with liver-specific disruption of TAF10 gene. Inactivation of TAF10 in hepatocytes resulted in the dissociation of TFIID into individual components. This correlated with the down-regulation of most hepatocyte-specific genes during embryonic life and a dramatic defect in liver organogenesis. Unexpectedly however, the transcription of less than 5% of active genes was affected by TAF10 inactivation and TFIID disassembly in adult liver. The extent of changes in transcription of the affected genes was dependent on the timing of their activation during liver development, relative to that of TAF10 inactivation. Interestingly, TFIID dissociation from promoters correlated with the re-expression of several postnatally silenced hepatic genes. The results point to the functional plasticity of TFIID and suggest that TFIID is required for the initial activation of genes, but is dispensable for maintaining ongoing transcription.

The role of dADA2b proteins in dSAGA In Drosophila melanogaster two ADA2 proteins (dADA2a and dADA2b) have been described in two GCN5-containing histone acetyltransferase complexes, ATAC and dSAGA. The dADA2b-containing dSAGA complex acetylates histone H3 tails and in the lack of dADA2b H3 K9 and K14 acetylation is seriously reduced. However, there are two isoforms of dADA2b (dADA2bS and dADA2bL) that are differentially expressed during development. To characterize dADA2b function in dSAGA complexes we generated dAda2b mutations which eliminate the production of either or both dADA2b isoforms. Genetic studies showed that neither dAda2b isoform alone could provide a complete restoration of dAda2b function, suggesting that both are required for development. On the other hand, either dADA2b isoform can productively participate in dSAGA com-plexes and render those at least partially active in histone H3 acetylation. To study the molecular consequences of the loss of H3 K9 and K14 acetylation we compared the mRNA profiles of wild-type and dAda2b mutant animals by cDNA microarray. Global gene expression profiling indicates that the expression of less than 5% of the genes is significantly changed in dAda2b null mutants. More than half of the affected genes are up-regulated, of which the high number of defenserelated genes is particularly striking. Our data support the notion that different dADA2 proteins contribute to the functional versatility of multiprotein complexes and demonstrate that the loss of dSAGA specific H3 K9 and K14 acetylation affects (up-or down-regulates) the expression of a rather small subset of genes but does not cause a general transcription deregulation.

Histone deacetylase inhibition regulates the immune response 1C. Nuclear Extrachromosomal Structures PP1C-1 H2AX and lamin A/C interact dynamically in apoptosis indicating the existence of H2AX-organized machinery centers 1D. Evolving Genomes and Synthetic Biology PP1D-1 Genetic chracterisation of various cat breeds by using microsatellites in Turkey Methods: Ninety six of animals of cat populations, Turkish Van, Turkish Angora, Turkish Tekir, Siamese and Persian were genetically characterized at the DNA level by using 10 microsatellite loci including FCA069, FCA075, FCA105, FCA149, FCA220, FCA229, FCA240, FCA310, FCA441 and FCA678. The amplified polymerase chain reaction (PCR) products were separated by capillary electrophoresis using a CEQ-8000 Beckman Coulter Genetic Analysis System and alleles were determined. Total allel numbers, observed heterozygosity (H O ), expected heterozygosity (H E ) and deviation from Hardy-Weinberg Equilibrium were calculated. Results: A total of 274 different alleles were observed ranging from 3.7 (Siamese) to 8.3 (Turkish Van). The observed heterozygosity (H O ) and expected heterozygosity (H E ) were ranged from 1.0000 to 0.1667 and from 0.8939 to 0.871, respectively. Neighbour-Joining Phylogenetic trees based on D A distances and Factorial Correspondence Analysis (FCA) suggested the fact that genetic relationships of present-day cat Turkish breeds are consisted with their historical origins. Conclusion: Lower genetic diversities were observed in these breed. It is well known that increased numbers of deleterious mutations are observed in pure cat breeds, it is therefore suggested that attention should be paid on cat breed management plans.

The determination and comparative study of the complete rDNA sequences of G. pallida, G. rostochiensis and G. artemisiae K. Nowaczyk 1 , A. Obrepalska-Steplowska 1 and X. Dobosz 2 1 Interdepartmental Laboratory of Molecular Biology, Institute of Plant Protection, Poznan, POLAND, 2 Department of Zoology, Institute of Plant Protection, Poznan, POLAND Introduction: Nematodes from genus Globodera belong to potato cyst nematodes (PCN) and are responsible for severe damage in cultivated Solanaceous plants.

Our attempt was to analyse the genetic diversity between two quarantine species: G. pallida and G. rostochensis and G. artemisiae -parasite of Artemisia regarding the differences in development, host preference and adaptation requirements. Methods: Total DNA was isolated from intact cysts of Polish populations of G. rostochiensis, G. pallida and G. artemisiae. On the basis of sequences of rDNA from Globodera ssp available in the GenBank database specific primers were designed to amplify rDNA sequence in PCR reaction followed by sequencing. Obtained sequences were put together and compared with available sequences of other populations from genus Globodera using MEGA 3.1 program. Results: For three species of Globodera complete rDNAs have been sequenced and aligned. Similarity between Polish populations of G. pallida and G. rostochiensis is 97%, between G. pallida and G. artemisiae 95% and between G. rostochiensis and G. artemisiae -95%. The ITS fragments of rDNA of G. pallida and G. rostochiensis show high variability level in G. rostochiensis and G. pallida, whereas these regions are identical between available in the GenBank sequences of three populations of G. artemisiae and the Polish one. Conclusions: Analysis of rDNA sequence confirms close relationship betweeen G. rostochiensis and G. pallida and more distant relationship between these two species and G. artemisiae. Higher variability level of ITS fragments among populations of G. rostochiensis and G. pallida could be the effect of parallel evolution of a parasite in adaptation to various species of hosts.

PP1E-1 Vitamin D receptor gene polymorphisms and calcipotriol response in Turkish patients with psoriasis PP1E-5 BCL-2, BAX and BCL2L12 are modulated at the mRNA level in response of leukemic cells to the ABL protein tyrosine kinase inhibitor, Gleevec Cytochrome P450 (CYP) isoenzymes are one major kind of phase I enzymes and play an important role in the oxidation of xenobiotics, often resulting in the formation of highly reactive compounds that are the ultimate carcinogens. CYP1A1 and CYP2A6 play important key role in phase I metabolism and metabolize several important toxins and procarcinogens. Microsomal epoxide hydrolase (mEPH) is mainly involved in the detoxification of wide variety of endogenous or exogenous carcinogens. Genetic differences in CYP1A1, CYP2A6 and mEPH genes have been reported to be associated with susceptibility to various cancers. In the present study, we determined the genotype frequencies of the main CYP1A1*2, CYP2A6*1 and *3, mEPH*3 and *4 polymorphisms in a group of 114 healthy volunteers representing Turkish population. For this purpose, whole blood samples were collected from and genomic DNA was isolated and was used to determine the frequency of CYP1A1*2, CYP2A6*1 and *3, mEPH*3 and *4 allelic variants by the PCR-RFLP. The genotype frequency, for CYP1A1*2 was determined as 0.95 for homo-and hemi-zygous wild type and 0.05 for homozygous mutants, respectively which was statistically different (p < 0.05). The CYP2A6*1 and *3 frequencies were not found in the population studied. Regarding the mEPH*3 and mEPH*4 genotypes, we found frequencies of 0.94 and 0.96 for homo-and hemi-zygous wild type (p < 0.05) and 0.06 and 0.04 for homozygous mutants, respectively (p < 0.05). This preliminary study presents CYP1A1, CYP2A6 and mEPH mutant allele distribution in the Turkish population and will provide understanding of the etiology of several cancers in Turkish patients. Down syndrome (DS) is a chromosomal abnormality (trisomy 21) associated with mental retardation and Alzheimer-like dementia, characteristic change of the individual's phenotype and premature aging. Oxidative stress is known to play a major role in this pathology since a gene dose effect leads to elevated ratio of superoxide dismutase to catalase/glutathione peroxidase. Accumulation of uric acid, the end product of purine metabolism, is another feature of DS and this might also be associated with genomic instability via overexpression of GARS-AIRS-GART, the gene coding for the protein complex that catalyzes different steps in de novo purine synthesis. Hyperuricemia has an interesting relationship with oxidative stress since it represents an important free radical scavenger and ROS themselves could influence its increase since, under oxidative stress conditions, xantine-dehydrogenase (XDH) is converted in xanthine oxidase (XO) which leads to concomitant production of free radicals. ROS might finally represent an important modulator of gene expression of non trisomic genes affecting penetration and expressivity of the pathology. Oxidative stress could be therefore an important pharmacological target to be addressed in this important genomic abnormality syndrome. Here we report that plasma sample from DS patients in pediatric age despite an increased total antioxidant capacity, largely due to elevated uric acid content, do not differ from healthy control plasma samples in terms of susceptibility to peroxidative stimuli. Instead, they present significantly elevated markers of oxidative stress such as increased allantoin levels. Moreover by treating the patients with CoQ10 (4 mg/kg/die) for two moths we were able to decrease markers of oxidative stress such as uric acid conversion to allantoin and oxidative DNA damage in peripheral blood lymphocytes.

The effect of endothelial nitric oxide synthase gene polymorphisms in patients with erectile dysfunction in Turkish population Introduction: Penile erection depends on the balanced action between vasoactive molecules such as nitric oxide (NO). By the catalytic function of endothelial NO synthase (eNOS), NO is synthesized from L-arginine, which is a key mediator of endothelial function. Endothelial dysfunction is the common denominator in erectile dysfunction (ED). To date very scarce data exits about the relationship between the eNOS polymorphisms and ED. Our aim is whether the three potential polymorphisms in the eNOS gene (T786C in the promoter region, 27 bp repeat in intron 4) were associated with an increased risk of ED in Turkish population. Methods: Seventy-three patients with ED and 41 healthy controls were analyzed. To assess erectile status penile, color doppler USG and International Index of erectile dysfunction (IIEF) questionnare was used. ED group consists of only patients with proved vascular pathology. Patients who has an IIEF score greater than 26 consisted the control group. Genotypes were determined through polymerase chain reaction with or without restriction endonuclease digestions. Multilocus haplotypes tested to overcome the problems caused by using single polymorphisms in genetic association studies. Results: Although genotype distribution was not significantly different between patients with ED and controls statistically (p = 0.106), for 27 bp repeat in intron 4 the presence of a deletion seems to have 2.0 times (95% CI for OR:0.9-4.9) protective effect. Polymorphism of the promoter region is not significantly different between cases and controls. Conclusion: The study is recruting more controls and 27 bp deletion genotype in intron 4 of eNOS gene seems to be protective for ED in Turkish population.

Correlation of serotonin transporter gene polymorphism with psychological characteristics S. Veletza, M. Samakouri, A. Mamatsiou, G. Tripsianis and M. Livaditis Division of Medicine, Department of Biology, University of Thrace, Alexandroupolis, GREECE Introduction: A 44 bp indel at the promoter region of the gene encoding serotonin transporter has been the focus of several association studies of mood disorders, showing a modest association with bipolar depression and suicidal behaviour. Allele S has been previously shown to result in lower levels of mRNA and protein as well as slower reuptake of serotonin from the synapse. Moreover, patients with the S/ S genotype respond worse to SSRIs. We attempted to study the relation of the serotonin transporting gene L/S polymorphism with depression-related trait scores in a group of University students. Methods: One hundred eighty one volunteer students were genotyped for the L/S polymorphism and were evaluated with a battery of psychological tests (Zung Depression Rating Scale, Symptoms Check-List-90-R, Eysenck Personality Inventory). They also answered questionnaires regarding adverse experiences. The group of carriers of the S allele were compared to non-carriers. We used MANOVA to evaluate the effect of the genotype and its interaction with both adverse life events and scores in the psychological tests. Results: We did not detect any significant association of genotype with the measured psychological scores. However, an interaction of adversities and genotype was detected: Among carriers of the S allele, students having faced adverse life events showed higher depressionrelated trait scores compared to those with no adversities. Among stu-dents without adversities those with no S allele scored higher than the S allele carriers. Conclusions: The S and L alleles may contribute differently to geneenvironment interaction that leads to psychological vulnerability.

Introduction: The CYP19 gene encodes the enzyme aromatase, which catalyzed the conversion of androgens into estrogens. To investigate whether polymorphisms of CYP19 gene are associated with the risk of breast cancer, we analysed the frequency and distribution of single nucleotide polymorphisms a tetranucleotide (TTTA) tandem repeat polymorphism and a 3 bp insertion (I)/deletion (D) polymorphism in intron 4 of CYP19 gene. Methods: We studied 70 patients with breast cancer and 102 healthy control women. Results: The distribution of the genotypes of the (TTTA)n polymorphism of CYP19 was not significantly different groups, but high copy number of repeat (over 10) was increased in ER negative patient and low copy number of repeat (under 10) was increased in ER positive patient (p = 0.019) . On the other hand an increased frequency of the D allele was observed in the breast cancer group as compared with the control group (p = 0.001). When the control and patient group compared for there insertion/deletion (I/D) genotype, the frequency of patient with ID was significantly higher than the frequency of the healthy group with the same genotypes (p = 0.005, OR = 2.62) and DD was significantly higher, too (p = 0.004, OR = 12.81). Conclusions: These results suggest that the 3 bp I/D polymorphism of the CYP19 gene would be useful for selecting Korean women at a high risk of breast cancer. And the repeat number of (TTTA) polymorphism of CYP19 is associated with ER-positive and negative patient, it would be useful in the selection of candidate for chemoprevention with tamoxifen.

Idarubicin (4-demethoxy-analogue of daunomycin) is an anthracycline DNA-binding drug that is being used for treatment of various cancers. The main target of this drug is DNA in which inhibits DNA replication and transcription. We have previously shown that, in chromatin, apart from DNA, chromatin proteins, specially histones, could be considered as a target for idarubicin. In the present study, we have investigated the interaction of different concentrations of idarubicin with histone H1 in solution, using ultraviolet/visible and fluorescence spectroscopy. The results show that in the presence of idarubicin the absorbance of histone H1 at 210 nm is decreased and the binding induces hypochromicity in the absorption spectrum of the molecule. Fluorescence emission data shows that the interaction of idarubicin with histone H1 decreases the emission intensity at 308 nm which corresponds to the fluorescence emission intensity of tyrosine in the histone H1. The results suggest that idarubicin binds to histone H1 and the position of its binding is probably located in the globular domain.

1. Not allocated to any session PP1-1 PKU-beta/TLK1 plays a dual role in both centrosome separation and chromosome segregation PP1-4 RNA is not cleaved by the major apoptotic nuclease DFF40/CAD in cells undergoing programmed cell death J. Hanus, M. Kalinowska-Herok and P. Widlak MSC Cancer Center and Institute of Oncology, Gliwice, POLAND DFF40/CAD endonuclease is primarily responsible for internucleosomal DNA cleavage during the terminal stages of apoptosis and introduces double strand breaks into chromatin. However, potential mechanism of RNA cleavage by this nuclease has not been explored so far. Here, we performed a detailed study on the specificity of DFF40/CAD nuclease using synthetic single-stranded and double-stranded ribo-and deoxyribo-oligonucleotides as substrates in vitro. In addition, correlation between DFF40/CAD activation and integrity of RNA was analyzed in cells undergoing apoptosis in vivo. We have found that neither singlestranded RNA, single-stranded DNA, double-stranded RNA nor RNA-DNA heteroduplexes are cleaved by DFF40/CAD. Noteworthy, all types of oligonucleotides that are not cleaved by the nuclease inhibit cleavage of double-stranded DNA. We have observed that neither the activation of DFF40/CAD nor oligonucleosomal chromatin fragmentation was temporally correlated with total cellular or nuclear RNA degradation, indicating lack of a relationship between integrity of RNA and activity of the nuclease in vivo. We conclude that DFF40/CAD is exclusively specific for double-stranded DNA. In addition, although the nuclease is inhibited by RNA in vitro, the activity of DFF40/CAD is unlikely affected by the integrity of cellular RNA in vivo in cells undergoing apoptosis.

Analyses of the polymorphic Dmc-1-M200V variant In many cancers, different types of gross chromosomal rearrangements (GCRs) are observed including translocations, interstitial deletions, terminal deletions with de novo telomere additions, and chromosome fusions. In order to study the roles of essential genes for GCR suppression, we chose Smc5-Smc6 complex, which is involved in structural maintenance of chromosomes and required for interchromosomal and sister chromatid recombination. The Smc6-Smc9 temperature-sensitive mutation increased the GCR rates up to 76-fold compared to wild type. The inactivation of Smc6 with mutations in different GCR mutator genes synergistically increased the GCR rates. The breakpoint analysis of individual GCRs showed that loss of Smc6 resulted in mainly the translocation types of GCRs. These results point towards a role for the Smc5-Smc6 complex in suppressing gross chromosomal rearrangements (GCR formation) and ensuring the proper chromosome structures. DNA double-strand breaks (DSB) can arise during DNA replication or after exposure to DNA-damaging agents, and their correct repair is fundamental for cell survival and genomic stability. Here, we show that the Smc5-Smc6 complex is recruited to DSBs de novo to support their repair by homologous recombination between sister chromatids. Our findings show that the Smc5-Smc6 complex is essential for genome stability as it promotes repair of DSBs by error-free sisterchromatid recombination (SCR), thereby suppressing inappropriate non-sister recombination events. The eukaryotic Rad51 protein, a homologue of the bacterial RecA protein, plays a central role in mitotic recombinational repair of double strand DNA breaks and meiotic homologous recombination. Rad51 is known to catalyze homologous pairing and strand exchange during the recombination process. In human, the conventional Rad51 has a Lys residue at position 313(Rad51-K313). But the Rad51-Q313 that has Glu at position 313 was reported as an isofrom. In this study, we purified the Rad51-K313 and the Rad51-Q313 isoform as recombinant proteins, and performed their biochemical analyses in vitoro. Interestingly, we found that the strandexchange activity of Rad51-Q313 was significantly higher than that of Rad51-K313 under conditions with Ca 2+ , although the difference was not observed under conditions without Ca 2+ . To know their filament-formation ability, we performed a double-stranded DNA unwinding assay and that result showed the filament-formation ability of Rad51-Q313 was also enhanced. From mutational analyses, the positively charged residues (Lys and Arg), but not negatively charged, polar, and hydrophobic residues (Glu, Gln, and Met, respectively), at position 313 reduced the strand-exchange and DNA unwinding abilities of the HsRad51 protein. These findings suggest that the recombinase activity of HsRad51 is regulated by the electrostatic environment around position 313. The major apoptotic nuclease DFF40/CAD is primarily responsible for internucleosomal DNA cleavage during the terminal stages of programmed cell death. It has been reported that several proteins, including chromatin proteins histone H1, HMGB1/2 and topoisomerase II, can stimulate activity of this nuclease. Here, we investigated the mechanism of stimulation of DFF40/CAD by major non-histone chromatin protein HMGB1. Naked DNA was used as a substrate for recombinant DFF40/CAD nuclease in the presence of HMGB1 and other HMG-box containing proteins, including truncated forms of HMGB1. Efficiency of DNA cleavage, protein-protein and protein-DNA interactions were then analyzed in such in vitro experimental model. We have observed that HMGB1 significantly stimulates naked DNA cleavage by DFF40/CAD nuclease in a dose-dependent manner. However, we show that HMGB1 neither binds to DFF40/CAD nor enhances its ability for stable binding to DNA. Comparison of the stimulatory activities of different HMG-box-containing proteins indicates that a structural array of two HMG-boxes is required for such stimulation. HMG-boxes are known to confer specific local distortions of DNA structure upon binding. Interestingly, the presence of DNA strand cross-links formed by cisplatin or transplatin, which may somehow mimic distortions induced by HMG-boxes, also affects DNA cleavage by the nuclease. The data presented suggest that changes induced in DNA conformation upon HMG-box binding makes the substrate more accessible to cleavage by DFF40/CAD nuclease and thus may contribute to preferential linker DNA cleavage during apoptosis.

Human DNA ligase IV and Artemis independently contribute to cytotoxicity in the presence of replication-associated DNA damage: implications for double-strand break repair A. Kurosawa and N. Adachi Yokohama City University, Yokohama, JAPAN Nonhomologous end-joining (NHEJ) and homologous recombination (HR) are two major pathways for repairing DNA double-strand breaks (DSBs); however, their respective roles in human somatic cells remain obscure. Here, we generate human knockout cell mutants genetically deleted for the HR protein Rad54 and/or the critical NHEJ factor DNA ligase IV. We show that although NHEJ and HR are both important for repairing ionizing radiation-induced DSBs, NHEJ repairs nearly all of the topoisomerase-II as well as low-dose irradiationinduced DNA damage. Intriguingly, we find that DNA ligase IV negatively affects survival of cells treated with genotoxic agents capable of causing replication-associated DSBs (i.e. the topoisomerase I poison camptothecin and the PARP inhibitor NU1025). These results indicate that replication-associated DSBs are deleterious to cells when dealt with by the NHEJ pathway. Loss of Artemis, an NHEJ factor with endonuclease activity, also conferred resistance to camptothecin and NU1025. However, this effect was independent of DNA ligase IV deficiency, as the LIG4/ARTEMIS double-mutant was even more resistant to these agents than either single mutant. We also show that loss of Artemis significantly alleviates hypersensitivity of LIG4 null cells to radiation-and topoisomerase II-induced DNA damage. Based on our data, we propose a novel model for DSB repair, in which NHEJ tries to repair virtually all DSBs, with HR only active when NHEJ is missing or abortive, and Artemis is involved in pathway switching from incomplete NHEJ to HR.

In vitro recombinase activities of the rice Dmc1A and Dmc1B proteins H. Kurumizaka, I. Sakane, C. Kamataki and Y. Takizawa Graduate School of Advanced Science & Engineering, Waseda University, Tokyo, JAPAN The Dmc1 protein is an eukaryotic homologue of the bacterial RecA protein, which catalyzes homologous pairing and strand exchange during homologous recombination processes. The Dmc1 protein shares significant homology with the Rad51 protein. The Dmc1 protein is only present in meiotic cells, but the Rad51 protein is expressed in both meiotic and mitotic cells in yeast and vertebrates. Therefore, the Dmc1 protein functions as a specific factor for meiotic homologous recombination. In rice, two Dmc1 genes, Dmc1A and Dmc1B, have been reported, but their biochemical properties have not been defined. Therefore, in the present study, we purified the Oryza sativa Dmc1A and Dmc1B (OsDmc1A and OsDmc1B, respectively) proteins. Electron microscopic analyses indicated that the OsDmc1A and OsDmc1B proteins formed helical filaments along single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). Biochemical analyses revealed that the OsDmc1 proteins promoted robust strand exchange between ssDNA and dsDNA over five thousand base pairs, even in the absence of RPA, which is an essential factor for strand exchange by the human and yeast Dmc1/Rad51 proteins. The strand-exchange activity of the OsDmc1A protein was obviously higher than that of the OsDmc1B protein. This work represents the thorough biochemical analyses of the rice OsDmc1A and OsDmc1B proteins, and provides important insight into their functions in rice meiosis.

The human PSF protein is a novel Rad51-interacting protein Y. Morozumi, Y. Takizawa and H. Kurumizaka Graduate School of Advanced Science and Engineering, Waseda University, Tokyo, JAPAN Homologous recombination is important for the repair of DNA double-strand breaks caused by DNA-damaging agents, such as ionizing radiation and DNA cross-linking reagents. The human Rad51 protein is a homolog of the Escherichia coli RecA protein, and plays a key role in Homologous recombination. However, previous biochemical studies indicated that the homologous DNA pairing activity of the Rad51 protein is extremely lower than that of the RecA protein in vitro. To find novel Rad51-interacting factors, in this study, we performed the yeast two-hybrid screening with human cDNA pools, and the human polypyrimidine tract-binding protein associated splicing factor (PSF) protein was identified as a candidate for the novel Rad51-interacting protein. The PSF protein was originally identified as a component of spliceosomes, suggesting a role in pre-mRNA splicing. In addition, previous studies have shown that the PSF protein promotes homologous DNA paring by itself. We purified the human Rad51 and PSF proteins as recombinant, and found that the PSF protein directly interacts with the Rad51 protein. The PSF protein enhanced the Rad51-mediated double-stranded DNA unwinding, which may depends on the Rad51-filament formation. These results suggested that the PSF protein may function in the Rad51-filament formation step. PP1-13 BRCA1-mediated stabilization of MLH1 DNA mismatch repair protein in response to adryamicin-induced DNA damage PP1-15 The replication licensing regulator CDT1 is overexpressed in breast cancer nin. The balance between Cdt1 and Geminin is important for maintaining genomic stability: loss of Geminin or overexpression of Cdt1 drive rereplication while Cdt1 functions as an oncogene and promotes aneuploidy. Despite the documented oncogenic activity of Cdt1, few data are available concerning its expression in tumors and its potential use as a biomarker. We wished to investigate the mRNA and protein expression levels of Cdt1 and Geminin in cancer. Quantitative analysis of Geminin and Cdt1 mRNA levels was performed in human tumour cell lines and primary fibroblasts by Real-Time PCR. Cdt1 mRNA showed a 3-4 fold increase in breast cancer derived MCF7 cells and cervical cancer derived HeLa cells compared to primary cells, while 3-4 fold increase in Geminin mRNA levels was observed in HeLa cells compared to MCF7 and primary human fibroblasts, indicating that different tumour-derived cell lines exhibit different ratios of Cdt1/ Geminin levels. In order to determine Cdt1 and Geminin protein expression in breast cancer, immunohistochemistry was performed in a panel of breast cancer specimens. Both proteins exhibit higher expression levels in tumour cells in comparison to adjacent normal tissue. Cdt1 expression levels were evaluated at different stages of tumor progression, and correlated with HER2/neu, cyclin-A, ki67 and p53 expression.

Expression correlations between clusters of genes distantly positioned in human genome T. Szczepinska and K. Pawlowski Nencki Institute of Experimental Biology, Warsaw, POLAND Introduction: There is increasing interest in how a genome is spatially and temporally organized within the cell nucleus. Evidence supports the idea that basic nuclear functions, such as transcription, are structurally integrated. Several studies demonstrated non-random, gene-density related positioning of chromosomes. We have used the large sets of gene expression data in public repositories to explore relations between functionality and gene localization. Methods: We have used a graph-based data-mining approach [1] to efficiently identify frequent co-expression clusters in 105 assembled human microarray datasets. The potential transcription modules have been integrated with gene position and density information. Literature relationship based tools have been used for functional analysis. Results: We identified and functionally annotated distant genomic clusters within co-expression clusters. The number of such groups of genomic clusters is statistically higher than random. The genes in expression clusters show correlation with overall chromosome genedensity.

Conclusions: This large-scale microarray data-mining analysis has shown the occurrence of functionally related co-expressed genes from regions distant in genome. Findings enrich our knowledge of the overall organization of human genome and allow interpretation in the nuclear architecture context. 

Identification of a novel Rad51B-interacting protein M. Takaku, Y. Takizawa, S. Nakayama, S. Machida and H. Kurumizaka Graduate School of Advanced Science and Engineering, Waseda University, Tokyo, JAPAN Homologous recombinational repair (HRR) is an important pathway for the repair of double-strand breaks. The eukaryotic Rad51 protein plays a central role in the HRR pathway, but many other proteins are required for this pathway. In vertebrates, five proteins (the Rad51 paralogs; Rad51B, Rad51C, Rad51D, Xrcc2, Xrcc3) that share homology with the Rad51 protein are found as factors involved in the HRR pathway. The Rad51B protein reportedly interacts with the Rad51C protein, and the Rad51B-Rad51C complex functions in the Rad51 assembly onto the double strand break (DSB) sites during an early stage of the HRR pathway. On the other hand, we previously showed that the Rad51B protein preferentially binds to the Holliday junction DNA substrate in the presence of authentic double-stranded DNA and the Y-form DNA, suggesting that the Rad51B protein may also function in the late stage of the HRR pathway. Therefore, these facts suggest that the Rad51B protein have multiple functions in the early to late stages of the HRR pathway. To identified novel Rad51Binteracting proteins, we performed the yeast two-hybrid screening with human cDNA pools, and obtained seventy-three positive clones. Thirty clones contained DNA fragments from the Evl gene. We found that the purified Evl protein directly bound to the Rad51B protein in vitro. Therefore, the Evl protein may be a novel Rad51B-interacting protein. Results: In the absence or overexpression of Rad9, the transcripts of 1-2% of the yeast genes was found to change significantly and at least twofold compared to the corresponding wild type strain. In the former case, among the significantly up-regulated genes were a group with role in telomere maintenance via recombination and a group with helicase activity and among the down-regulated were genes with role in gene regulation such as RPB1 and CLN3. In the latter case, genes implicated in metabolism and carbohydrate transport along with HSP30 were found significantly up-regulated, while genes concerning nucleolus, ribosome biogenesis and assembly and cohesin complex were down-regulated. Conclusions: The genome-wide expression analysis showed for the first time, under physiological conditions implication of Rad9 in the expression of an appreciable number of genes belonging to particular functional categories. Under these unchallenged conditions, Rad9 seems to have an effect in pathways distinct from the known DNA damage check point. This role of Rad9 in gene regulation may reveal new aspects of signalling pathways cross-talking with cell cycle regulation. Whether Rad9 effect is direct or indirect is being examined by chromatin immunoprecipitation experiments and remains to be elucidated.

The new criteria to investigate internal ribosome entry initiation mechanism for cellular mRNAs The two major and principally different mechanisms of translation initiation in eukaryotes are cap-dependent scanning and internal ribosome entry. The latter one requires specific structures known as internal ribosome entry sites (IRES). IRESes were initially found in the 5' untranslated regions (5' UTR) of some cytoplasmic viral mRNAs. Later, the presence of IRES elements were also suggested for some cellular mRNAs. However, the existence of 'cellular IRESes' is still questionable. The conventional approach to identify IRES-elements is the method of di-cisrtonic constructs. The only criterion used so far is the comparison of various di-cistronic constructs, i.e. second cistron translation driven by the sequence tested with one driven by known (previously characterized) IRES element. This criterion is rather subjective. Moreover, in natural conditions all cellular mRNAs are monocistronic and possess m7G-cap, and theoretically are able to use the conventional cap-dependent way. Here we suggest new criteria to evaluate an actual mechanism of mRNA translation: 1) comparison of translation driven by the 5' UTRs in monocistronic context versus dicistronic one; 2) comparison of capped mono-cistronic RNA translation versus uncapped transcript. We applied these criteria to several human mRNAs with complex 5'UTRs previously reported to contain IRES elements: c-myc, Apaf-1, L1 and Hsp70. For all of them the translation was much more efficient for mono-cistronic mRNAs. Moreover, it was strongly depen-dent on the presence of the 5'-cap. These data lead us to conclude that mRNAs with long structured 5'UTRs are able to translate efficiently by the cap-dependent mechanism, at least under normal conditions. Poly(A)-specific ribonuclease (PARN) is a poly(A)-specific and capinteracting 3'-exoribonuclease that efficiently degrades mRNA poly(A) tails. Although many strategies have been proposed for the regulation of PARN activity, very few is known about the modulation of PARN activity by small molecule effectors, such as nucleotides. Based on the enzyme's preference for its natural substrates and regulators, we examined the role of purine nucleotides as potent effectors of human PARN activity. Detailed kinetic analysis revealed that RTP nucleotides tested behave as non-competitive inhibitors, while RDP and RMP exhibit competitive inhibition. Mg 2+ can release inhibition of RTP and RDP but not that of RMP. The same inhibitory effect was observed when the same analysis was performed for cytosine nucleotides. We further synthesized and used modified purine and pyrimidine nucleoside analogues with known anti-tumor growth effects. These compounds bear a six-membered ring carbohydrate moiety and purine or pyrimidine modified on the amine with a benzoyl-group. Two of the modified nucleotides tested, one with modified adenine and one with modified cytosine could effectively inhibit PARN activity. The six-membered sugar ring compound alone, or the nucleosides lacking the benzoyl-modified purine or pyrimidine had weaker or no effect on the enzyme. Our data imply that PARN activity can be modulated by natural nucleotides in vitro, thus providing a simple regulatory mechanism. Moreover, the analogues used in this work can serve as leading compounds for development of novel inhibitors of PARN. The Negative Regulator of Splicing (NRS) is a 230 nt, cis-acting RNA sequence contained within the gag gene of Rous sarcoma virus (RSV). This intronic element inhibits splicing between an uptream 5¢ splice site and the 3¢ splice site. It appears to function as a non-productive 5¢splice site that forms abortive splicing complexes with the 3¢-splice site. NRS contains at least two functional regions: overlapping U1 and U11 binding sites at the 3¢ end and an SR protein binding region in the 5¢ part. By using chemical and enzymatic probes for RNA secondary structure we investigated the 2D structure of the NRS element. We provide evidence that NRS adopts a cruciform 2D structure where the SR (SLS2) and U1/U11 (SLS6) binding sites form individual stem loops. Gel shift and footprint analyses showed that hnRNPA1 binds the SLS2, SLS6 and H III structures, therefore the hnRNPA1 sites overlap the SR protein binding sites. By using an affinity purification technique and mass spectrometry we identified HeLa nuclear proteins that bind the NRS element. We confirmed that the SR proteins ASF/ SF2, 9G8 and SC35 bind this element. By using deletion mutants we showed that elimination of SLS2 and SLS6 strongly influences the binding of the SR proteins but not the binding of hnRNPA1 and hnRNP H. Thus, we propose that binding of these antagonist splicing factors on NRS strongly modulates its inhibition action on RSV RNA.

Interactive partners and molecular characteristics of the mammalian hnRNP A3 protein G. Boukakis, M. Patrinou-Georgoula, C. Papadopoulou and A. Guialis National Hellenic Research Foundation, Institute of Biological Research and Biotechnology, Athens, GREECE hnRNP proteins are considered important participants in almost every step of mRNA maturation processes. In particular, the group of hnRNP A/B comprises abundant nuclear proteins with a major role in basic as well as alternative splicing and ability for nuclear/cytoplasmic shuttling. Compared to the best known members (hnRNP A1 and A2/ B1), hnRNP A3 is a relatively new protein, for which we have an interest in ascribing biochemical and molecular properties. To this extent, the auxiliary domain of hnRNP A3 was expressed as a GST-fused product and applied, in parallel with a GST-fused hnRNP A2, in pulldown assays using nuclear and cytoplasmic extracts from human (HeLa, A549) cell lines. These studies revealed the unique ability of this domain of hnRNP A3 to form an extensive network of protein-protein interactions, not only with the other A/B, but also with hnRNP M and L proteins, in both cellular compartments. All interactions, with the exception of A1, were highly enhanced by previous RNase digestion of the extract. Furthermore, immunochemical studies applying a commercial and a home made rabbit polyclonal antibody provided clear evidence for the existence of two discrete hnRNP A3 isoform types, one common to human and rodent and the second comprising a major isoform unique to rodent cells. Evidence is also provided for an increased expression of hnRNP A3 in human lung cancer, in common to other A/B type proteins. Our findings provide evidence for novel characteristics related to hnRNP A3 and give further support to its anticipated involvement, together with the other hnRNP A/B proteins, in mRNA metabolism.

Regulation of brain fatty acid-binding protein and glial fibrillary acidic protein in malignant glioma by nuclear factor I PP2A-13 Searching for downstream genes of Hoxc8 by chip-cloning H. J. Chung, M. Kang and M. Hee. Kim Department of Anatomy, Embryology Lab Brain Korea 21, Project for Medical Science, Yonsei University, College of Medicine, Seoul, KOREA Introduction: Hox genes encode homeodomain-containing transcription factors that pattern an anterior-posterior body axis and regulate gene expression during vertebrate embryogenesis. Homeodomain proteins regulate specific genes either activator or repressor when they bind DNA binding motif (TAAT/ATTA, TTAT/ATAA) in target genes. However the specific DNA-Hox protein interaction has not been well defined. The present study we used chromatin immunoprecipitation (ChIP) system to identify responsible transcriptional targets of Hoxc8, a member of Hox genes. Methods: In order to screen for downstream target genes of Hoxc8, we performed ChIP cloning. First, we prepared the murine E11.5 embryo because Hoxc8 expression is somewhat high. The embryo's chromatin DNAs were cross-linked with 1% formaldehyde and then sonicated, immunoprecipitated with Hoxc8 monoclonal antibody. Secondary ChIP DNA fragments were cloned into pBluescript vector and then sequenced to analyze cloned DNA. NCBI blast search were performed to collect gene information. For functional study luciferase reporter assay was performed. Results: We found 12 putative downstream genes of Hoxc8. Cloned genomic fragments are located in intron of seven genes individually. Four cloned genomic fragments are situated in intergenic regions as non-coding regions. One fragment is spread from intron 5 to exon 6. Among them Msh5 and Ttbk2 genes were found five or four times repeatedly. Luciferase reporter assay showed that Msh5 and Ttbk2 were up-regulated by overexpressed Hoxc8 in F9 cells. Conclusions: ChIP cloning method using Hoxc8 specific antibody suggested new 12 putative downstream target genes of Hoxc8. Among them Msh5 and Ttbk2 might be responsible for Hoxc8 directly. The chemical compound of early earth atmosphere such as oxygen species and nitric oxide play role of signaling molecules in biological processes. Methanol of modern earth atmosphere is generated by ubiquitous pectin methylesterase (PME) from plant cell wall pectin. PME gene transcription is increased by stress impact. We suggest that PME and methanol may take part in regulation of plant mRNA stability. To check this hypothesis we isolated and sequenced the full-size tobacco proPME cDNA. Intracellular PME targeting was studied in transient expression experiments by agroinjection of Nicotiana benthamiana leaves. Our results showed that PME activity depends on proPME leader part providing mature PME delivery to cell wall pectin. Co-agroinjection of N. benthamiana leaves with proPME gene and crTMV: GFP vector resulted in stimulation of virus-induced RNA silencing. Conversely, co-expression of crTMV: GFP with either antisense PME construct or with enzymatically inactive proPME restored synthesis of viral RNA. Furthermore, expression of proPME enhanced GFP transgene-induced gene silencing accompanied by relocation of the DCL1 protein from nucleus to the cytoplasm, activation of siRNAs and miRNAs production. Then it has been shown that mechanical stress, transgene expression and even spontaneous transcription resulted in activation of PME gene expression. We showed on plants stored in hermetically isolated boxes that (i) methanol evaporation stimulated gene silencing and (ii) methanol donor plants agroinjected with proPME had inhibitory effect on crTMV: GFP reproduction in methanol accepting plants. We concluded that methanol is likely to be silencing signal molecule.

Ets-2 is required for telomerase activity and cancer cell survival J. Dwyer, H. Li and J-P. Liu Molecular Signalling Laboratory, Department of Immunology, Monash University, Prahran, VIC, AUSTRALIA Introduction: Telomerase maintains telomeres and underlies continuous cell proliferation in up to 90% of cancers. However, the mechanisms of telomerase activation remain to be elucidated. The regulation of telomerase activity takes place at multiple levels, most pre-dominantly transcriptional regulation of the telomerase catalytic subunit (human telomerase reverse transcriptase, or hTERT). The family of Ets transcription factors has been implicated in regulating hTERT gene expression. Hitherto, the roles and underlying mechanisms of specific Ets members in regulating hTERT gene remain to be investigated. Method: RNA interference technology was exploited to silence the Ets transcription factors, Ets-1 and Ets-2, in various types of cancer cells. Gene expression of hTERT and telomerase activity was determined in the cells in parallel with cell proliferation and apoptotic analyses. Results: Silencing Ets-2, but not Ets-1, resulted in significant repression of the hTERT gene and inhibitions of telomerase activity, in association with increased apoptosis in multiple cancer cell lines. The apoptosis induced by silencing Ets-2 was inhibited by either transducing Ets-2 protein or gene expression of hTERT, suggesting that Ets-2 gene silencing-induced cancer cell death was telomerase inhibitiondependent. Furthermore, Ets-2 bound the hTERT gene. Mutagenesis identified two non-canonical Ets binding sites in the hTERT promoter; mutation of each Ets-2 binding site resulted in depressed hTERT gene promoter activity. Conclusion: The transcription factor Ets-2 is required for hTERT gene expression, thereby telomerase activity and cancer cell proliferation. Ets-2 may be a potential target for inducing telomerase inhibition and cancer cell death.

Ochratoxin A interferes with the transcriptional process of heat shock and apoptotic proteins in cultured human hepatoma cells Introduction: The mycotoxin ochratoxin A (OTA) is well known to have diverse toxicological effects on animals. However, the aspect of current concern is the validity for human risk assessment. The present investigation was conducted to better explain the molecular aspects of its toxicity. Methods: Using human hepatoma cells (HepG2), we tried to elucidate the regulation process involved in some proteins and genes expression by OTA using western blotting and RT-PCR analysis. The oxidative status was analysed by measuring the reactive oxygen species (ROS) using DCFH-DA as a fluorescent probe. Results: Our study demonstrated that, there is a down-regulation for Hsp 70, Hsp 27 mRNAs, and proteins levels. We showed a slight timedependent increase of ROS, which was sufficient to provoke Heat shock proteins induction. To verify this apparent transcriptional inhibitor potential of Heat shock protein genes expression by OTA, we have studied the regulation of some targets of the transcriptional factor p53: p21, mdm2, Bax and PUMA. We have demonstrated that OTA increased p53 stabilization and phosphorylation, in these conditions p21 and mdm2 mRNAs levels decreased remarkably. Similar results were observed with p21, Bax and PUMA protein levels. This suggested that OTA may acts as an inhibitor of genes transcription. This hypothesis was confirmed by expression of a metabolic enzyme, gapdh and we found that gapdh mRNA level decreased significantly in presence of OTA. Conclusion: These data might suggest that this inhibition of transcriptional process in response to OTA treatment seems to be a general mechanism.

PP2A-17 Characterization of a downstream YY1 regulatory site as a translation initiator in short 5' UTR mRNAs The stimulation-specific regulation by negative transcription elongation factors of immediate early gene expression controls programmed hormone production in neuroendocrine cells T . Fujita, I. Piuz and W. Schlegel Fondation Pour Recherches Medicales, University of Geneva, Geneva, SWITZERLAND Introduction: The transcription rate of immediate early genes (IEGs) is controlled strictly by positive transcription elongation factor b (P-TEFb) and negative transcription elongation factors, negative elongation factor (NELF) and 5,6-dichloro-1-b-D-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor (DSIF), at critical steps of transcription elongation. We herein addressed whether NELF and DSIF functions for IEG transcription are stimulation-dependent. In addition, we examined whether IEG transcription controlled by NELF and DSIF affects hormone production in neuroendocrine cells. Methods: The expression of NELF (NELF-E) and DSIF (Spt5) was knocked down using RNA interference (RNAi) in neuroendocrine GH4C1 cells. Effects of the knock-downs on expression of IEGs (c-fos, MKP-1, junB) and hormone genes (prolactin and growth hormone) were evaluated by using real-time PCR, Western blotting and chromatin immunoprecipitation assay. Results: In cellular resting condition, either knock-down increased basal levels of IEGs transcription. NELF-E knock-down reduced thyrotropin-releasing hormone (TRH)-induced IEGs transcription. In surprising contrast, epidermal growth factor (EGF)-induced IEGs transcription was unaltered or even slightly increased by NELF-E knock-down. Conversely, Spt5 knock-down reduced both TRH-and EGF-induced IEGs transcription. Dynamic analysis of pol II distribution revealed that NELF-E knock-down reduced TRH-induced IEGs transcription by slowing down pol II elongation rate. As for hormone production, knockdown of NELF-E strongly induced prolactin production while that of Spt5 strongly suppressed growth hormone production. Conclusions: NELF regulates IEG transcription in a stimulation-specific manner by modulating pol II elongation rates. NELF-E and Spt5 knock-down alters hormonal transcription pattern possibly as a result of indirect regulation via transcription factors encoded by IEGs.

Transcriptional co-regulator homolog Prp45 is required for splicing of non-consensual substrates in yeast Homolog of the human transcriptional co-regulator and spliceosomal component SNW/SKIP in budding yeast, Prp45, was implicated in pre-mRNA splicing. Its participation in the splicing process, however, was not studied in detail. We identified a conditionally lethal allele, prp45 , which exhibits temperature sensitivity and cell wall-and cell cyclerelated phenotypes at permissive temperatures. Comprehensive synthetic lethal analysis of this mutation revealed genetic interactions with the alleles of the Nineteen Complex (NTC) members and the factors acting in the 3' splice site recognition and during the second catalytic step. Furthermore, tandem affinity purified Cwc2-associated spliceosomal complexes from the prp45 (1-169) strain contained significantly less Prp22 than the wild-type complexes. Prp22 is an ATP dependent DEAHbox RNA helicase involved in mRNA release, which was recently shown to affect the fidelity of 3' splice site usage. Based on these observations, we tested the efficiencies of both steps of splicing in vivo using various ACT1-CUP1 fusion constructs mutated in the consensus nucleotides of 5' and 3' splice sites or branch-point sequences. We found that while prp45 (1-169) did not affect the splicing of canonical ACT1-CUP1 template it lowered the splicing efficiency of various first and second step limiting reporters. The data suggest that Prp45 contributes to the interactions between the substrate and the spliceosomal active site during both steps of splicing. These interactions may affect the splicing efficiency of pre-mRNAs nonconforming to the consensus. The role of Prp45 in modulating splicing efficiency is intriguing because of the involvement of human SNW/SKIP in the regulation of elongating RNA Pol II and in the expression of alternately spliced genes. In Staphylococcus aureus at least four isoacceptor tRNA Gly molecules have been identified that, besides their prominent role in protein synthesis, they participate also in cell wall synthesis. All tRNA Gly molecules are aminoacylated by a single glycyl-tRNA synthetase (GlyRS), but they follow different pathways depending mainly on idiosyncratic structural features. The S. aureus cell wall consists of more than 20 layers of linear glycan chains that are cross-linked via characteristic pentaglycine bridges. Glycine is incorporated by the non-ribosomal peptidyltransferases FemX, FemA and FemB and glycyl-tRNA Gly molecules serve as glycine donors. In the present study, we identified through genomic survey a pseudogene encoding an atypical tRNA Gly , apart from the four tRNA Gly coding genes that have been reported that exist in S. aureus. Initial screening of S. aureus cultures with RT-PCR showed that this pseudogene is expressed and subsequent cloning and in vitro transcription showed that the pseudo-tRNA Gly is suitable substrate for aminoacylation by GlyRS. Moreover, bioinformatic and structural analysis strongly suggests that this pseudo-tRNA Gly carries specific identity elements that do not favour the interaction with EfTu and protein synthesis machinery. In contrast, this tRNA molecule more likely serves solely the incorporation of glycine residues in the cell wall of S. aureus. The identification of a pseudogene that encodes a functional tRNA Gly is important towards the elucidation of the essential biochemical events that take place during the tRNA-dependent cell wall synthesis pathway in S. aureus, which represents a novel molecular target for inactivation.

Reconstitution of archaeal C/D box sRNPs: towards synthetically programmable sequencespecific labelling of RNA PP2A-27 Corepressor Tup1, histone deacetilase Hda1 and repressor Nrg1 direct chromatin remodeling at GDH3 promoter and specific repression during Saccharomyces cerevisiae fermentative growth J. Herrera, C. E. Flores and A. Gonzalez Instituto de Fisiologia Celular, Universidad Naciona Autonoma de Mexico, Mexico City, MEXICO Introduction: Chromatin organization at promoters determines the dynamics of transcriptional state of eukaryotic genes. In the course of transcriptional repression, promoter-resident nucleosomes serve as substrates for a variety of modifying and/or re-modeling activities that have their reorganization as a result. This process is often facilitated by the presence of specific proteins such as repressors, co-repressors, histone modifiers and chromatin remodelers. The expression of Saccharomyces cerevisiae GDH3 gene is strongly repressed in the presence of glucose and is enhanced in ethanol. However, the mechanism does not involve the classic glucose repression mediated by Mig1p. Methods: We tested the involvement of histone deacetylase Hda1p, corepressor Tup1/Cyc8 and several putative repressors in the repression of GDH3 promoter using reporter (-gal and RT-PCR assays. Further, we analyzed the chromatin state by chromosomal MNase assays. Results: In the presence of glucose, expression of b-gal under GDH3 promoter was incremented about eightfold in hda1D, tup1D, cyc8D, and hda1/tup1D strains, and about threefold in each nrg1D and nrg2D strains, as compared to WT. The result was consistent with RT-PCR for the endogenous GDH3 expression. A particular cleavage pattern by MNase was observed for GDH3 promoter under glucose, and particularly changed under ethanol at the TATA box. S change was also observed under glucose for the above deletion strains. Conclusions: The particular chromatin organization of GDH3 promoter under repressive conditions is dependent on Hda1p. Hda1p is recruited by the co-repressor Tup1p-Cyc8p complex which in turn is recruited to the GDH3 promoter via Nrg1p or Nrg2p.

Analyses on regulatory mechanism of nuclear and organelle ribosomal DNA transcription in a unicellular red alga Cyanidioschyzon merolae S. Imamura 1 , K. Tokunaga 2 , A. Iseki 2 , H. Nanamiya 2 , F. Kawamura 2 and K. Tanaka 1   1 The University of Tokyo, IMCB, Tokyo, JAPAN, 2 Rikkyo University, College of Science, Tokyo, JAPAN Although ribosomal DNA (rDNA) transcription is the integral step for ribosome biogenesis, the underlying regulatory mechanism in plant cell remains unclear. As a first step to understand the rRNA synthesis in plant cell, we investigated transcription factors that are involved in nuclear and plastid rDNA transcription in a unicellular red alga Cyanidioschyzon merolae. Chromatin immunoprecipitation (ChIP) analysis with a polyclonal antibody against CmRRN3, of which homologues in yeast and mammals is key transcription factors to assemble RNA polymerase I (Pol I) pre-initiation complex, showed that CmRRN3 specifically occupied the 18S rDNA promoter region. Pol I occupation was also observed at the same region, suggesting that RRN3 function is conserved in plant cell as well as in other eukaryotes. To identify an RNA polymerase sigma factor for rDNA transcription in the plastid, we prepared polyclonal antibodies against each four RNA polymerase sigma factors, SIG1-SIG4, and subjected to ChIP analyses. Results indicated that SIG1 was specifically co-immunoprecipitated with the rrn16 promoter region, and the level was increased upon light exposure of the dark-adapted cells. Those occupancies were proportional to de novo rrn16 synthesis rate estimated by run-on transcription assay. These results suggested that SIG1 is positively involved in the rRNA synthesis in the plastid. We also present data obtained by run-on transcription analyses suggesting how the cell coordinates transcription of three kinds of rDNA in nucleus, plastid, and mitochondria.

Auto-amplification of dmpR transcription through non-overlapping divergent transcription The dmp-system of P. putida CF600 confers the ability to utilize (methyl) phenols as carbon sources. This catabolic system is composed of the dmp-operon encoding the pathway enzymes and the divergently transcribed regulatory dmpR gene product. Transcription of the dmpoperon from the Po promoter is dependent on the activity of dmpR, which binds pathway substrates to take up its transcriptional promoting form. Because the Po promoter is dependent on r 54 while the Pr promoter of dmpR is dependent on r 70 , this regulatory circuit is controlled by two different forms of RNA polymerase. Utilization of phenolics via the dmp-system is dominated by global regulation that leads to inactivity of the Po promoter even in the presence of pathway substrates under high energy conditions such as rapid growth on rich medium. However, robust transcription occurs under conditions that result in synthesis of the alarmone ppGpp, which has been proposed to ultimately control the cellular r 54 -RNA polymerase levels and thereby Po promoter occupancy. Using specific mutations and in vitro and in vivo transcription assays, we present evidence that the activity of the r 70 -Pr promoter is regulated by a novel mechanism in which r 54 -RNA polymerase occupancy and activity of the Po promoter stimulate Pr output. Thus, dmpR auto-stimulates Pr activity through its action on the nonoverlapping and divergently transcribed Po promoter. We propose a model in which this positive feed-forward loop reinforces both silencing in the presence of alternative carbon sources with robust transcription when the dmp-encoded catabolic enzymes are needed.

Transcriptional regulation of the COUP-TF gene in the sea urchin L. Kalampoki, C. Petropoulou and C. Flytzanis Department of Biology, University of Patras, GREECE COUP-TFs are transcription factors, members of the steroid-thyroidretinoic acid receptor super family. Studies in a plethora of species have shown that COUP-TFs are extremely conserved genes preferentially expressed in the developing nervous system. They also play an important role in organogenesis, homeostasis and cellular differentiation during embryonic development. The sea urchin COUP-TF is expressed in oogenesis and the stored maternal mRNA is spatially localized in the egg. The early embryonic COUP-TF protein represses transcription of the CyIIIb actin gene in the aboral ectoderm and relocates from the nuclear periphery to the condensed chromatin in a cell cycle depended manner. At the final stages of embryogenesis (pluteus), COUP-TF transcripts accumulate in the neurogenic cell layer of the oral ectoderm. To study the transcriptional regulation of the sea urchin COUP-TF gene we cloned and sequenced a 2000 bp up-stream region and determined the transcription initiation site. This up-stream region was inserted in a vector containing the green fluorescence protein gene (GFP) and the basal promoter of the sea urchin gene Endo16. The Endo16 promoter is unable to drive expression of the GFP gene without the presence of additional regulatory elements. In addition to this vector, a series of up-stream deletions were constructed, as well as insertions of isolated small fragments from the COUP-TF up-stream region in front of the Endo16/GFP cassette. All constructs were microinjected in fertilized Paracentrotus lividus eggs and resulting transgenic embryos were let developed to gastrula and pluteus embryonic stages. Analysis of GFP fluorescence in series of transgenic embryos, indicate the presence of a regulatory module that is sufficient to drive expression of the GFP cassette in the oral ectoderm, where the endogenous gene is also expressed. A number of putative response elements were recognized within this module and using electrophoretic mobility shift assays (EMSA), the corresponding binding factors were detected in nuclear protein extracts from developing embryos. Methods: Interactions between AP-1 proteins and TAFs were studied through immunoprecipitations in nuclear extracts of different colon adenoma cell lines. Small interfering RNAs (siRNA), migration assays, Chromatin Immunoprecipitation and Confocal microscopy were performed. Results: An interaction between c-Jun and TAF4b was identified, while both proteins co-localize in the nucleus. An increased cell migration and a change in cell morphology after the use of TAF4b siRNA, indicate an involvement of TAF4b in cell transformation and EMT. Furthermore, c-Jun and TAF4b bind on the AP-1 binding sites of EMT markers, while the expression of these genes is up-regulated when c-Jun or TAF4b are down-regulated through siRNA. Conclusion: Cells undergo the final steps of tumor progression leading to metastasis via a complex and not yet fully characterized transcriptional network. We suggest that TAFs together with AP-1 members are implicated in cancer and further on in metastasis through a synergistic action.

Oligonucleotide recognition by the RNA-binding protein TIAR in post-transcriptional regulation of gene expression PP2A-37 Prognostic significance of L-dopa decarboxylase (DDC) expression in colon cancer Apolipoprotein B mRNA editing enzyme catalytic polypeptides (Apobec) is a family of cytidine deaminase showing cytidine to uridine RNA editing characteristics. Modification of mRNA sequence through mRNA editing is crucial in gene expression modulation such as antibody diversification. However, the biological function of the most ancient family member, Apobec2 is unknown. Apobec2 is specifically expressed in skeletal and cardiac muscles that it may posses a distinct role. In the present study, characterization of intracellular localization and biological significance of apobec2 in mouse myoblasts C2C12 is studied. Apobec2 was found to be localized in the nucleus of myoblasts in the undifferentiated state. However, cytoplasmic accumulation of apobec2 in myotubes was observed upon differentiation. A nuclear export signal sequence is predicted near the C-terminal of the Apobec2 sequence and away from the conserved catalytic motif. Previous studies have demonstrated that nucleocytoplasmic shuttling of apobec1 is mediated by exportin 1 pathway. Mutagenesis study is being carried out to identify protein segment essential to apobec2 translocation and whether exportin 1 is involved. Furthermore, we have initiated siRNA transfection experiment to investigate the biological significance of apo-bec2 in myogenesis. Knowledge obtained will grant us key information to understand the molecular basis of apobec2 in muscle differentiation.

Combined effects of Elk-1/NRSF and TGIF/E2F regulate junctional adhesion molecule-B gene expression in mouse testicular cells PP2A-43 Changes of active octamer and inactive dimer of human geranylgeranyl diphosphate synthase are caused by redox C205 and C247

The Institute for Multidisciplinary Research of Advanced Materials, Tohoku University, Sendai, JAPAN Introduction: Geranylgeranyl diphosphate (GGPP) is an important intermediate in mevalonate pathway, serving as a precursor for geranylgeranylation of Rho, Rac and Rab proteins and acts as a negative regulator for DNA binding of liver X receptor. It is synthesized from farnesyl diphosphate by the action of GGPP synthase. We demonstrated interconversion between the active octamer and the inactive dimer of human GGPP synthase through redox of cysteine residues. In order to clarify which cysteine residues are involved in the interconversion, we analyzed to relationship between oligomeric structure and activity of wildtype and cysteine mutants.

Methods: Single, double and triple mutants with respect to three cysteine residues (C138, C205 and C247) per one subunit were prepared by using the QuikChange site-directed mutagenesis kit (Stratagene). Oligomeric structure of cysteine mutants were analyzed by gel filtration (Superdex 200 column) and blue Native-PAGE (Invitrogen). Enzymatic activities were assayed with a combination of farnesyl diphosphate and [1-14 C]isopentenyl diphosphate.

Results: The mutants were each recovered in a soluble form and active.

C138S, C205S and C138S/C205S mutants were kept in octamer similar to the case of the wildtype enzyme. However, C247S and C138S/C247S were analyzed to be in hexamer and both C205S/C247S and C138S/C205S/C247S were to be in dimer, suggesting that the C247 is critical for the octameric form. On the other hand, in similar analysis of mutants treated with copper sulfate, the dimer form observed in the case of wildtype treated with the oxidizing reagent was observed only for the C138S mutants, suggesting that the disulfide formation between C205 and C247 is requisite for the dimeric form.

Conclusions: Geranylgeranyl diphosphate synthase active octamer is converted to inactive dimers through the formation of C205-C247 disulfide bond upon oxidation.

Results: Our studies revealed the presence of the following putative elements in the jhbp promoter: )29TATAAA-24, +14TCAGTA+19, +38AGGTG+42, corresponding to TATAbox, Inr, DPE, respectively. We have found that the region )39/+51 is responsible for basic transcriptional activity. Furthermore, the luciferase assay has shown, that promoter with deleted +37/+51 fragment exhibited only slight reduction of transcriptional level, whereas deletion in +28/+51 and +19/+51 region caused a 2,5 and 3,5-fold increase of transcriptional activity, respectively. Further deletion +13/+51 encompassing putative Inr element resulted in promoter activity reduction by about 2/3.

Conclusions: Functional analysis of jhbp core promoter has shown that it has composite structure with a suppressive region. Moreover the data has shown, that Inr plays a key role in a regulation of basic transcription level. We have found that this region is responsible for high transcriptional activity of the jhbp gene. Mutational tests in vivo have shown that TATA box and Inr elements are necessary for maintaining of basal transcription level. Our observation that does not influence a basal transcription level suggests, that this region is no functional core element. The data has shown that the jhbp gene. For characterization of jhbp promoter we cloned the 90 bp located at position -39/+51 relative to transcription start site (tsp) into the promoterless pGL3-Basic luciferase reporter plasmid and reporter constructs containing wild promoter fragment or deletion mutants were generated. Up until now, the jhbp gene structure has only been described for M. sexta and G. mellonella. (In order to show which of the potential regulatory elements are functional) DPE is not required for the core promoter activity of jhbp. Our earlier sequence analysis and EMSA studies revealed the presence of the following putative elements in jhbp promoter: +29TATAAA-24, +14TCAGTA+19 and +38AGGTG+42, corresponding to TATA box, Inr, and DPE, respectively. Moreover, TATA box play a key role in a regulation of basic transcription level. Inr element seems to cooperate with TATA box increasing the basic transcriptional activity. Introduction: Clinical data suggest that iron is a negative factor in chronic hepatitis C; however, the molecular mechanisms by which iron modulates the life cycle of hepatitis C virus (HCV) remain elusive. Likewise, little is known on how HCV affects cellular iron homeostasis. Methods and Results: We utilized Huh7 hepatocytes expressing a HCV subgenomic replicon as a well-established model for viral replication. We demonstrate that iron administration to replicon cells dramatically inhibits the expression of viral RNA, while iron chelation elicits opposite responses. Pulse-chase experiments show that this is not due to alterations in viral RNA stability. Rather, tryptophan fluorescence emission spectroscopy with purified recombinant HCV RNA polymerase (NS5B) reveals that iron binds specifically and with high affinity (apparent K d : 6 and 60 lM for Fe 2+ and Fe 3+ , respectively) to the protein's Mg 2+ -binding pocket, thereby inhibiting its enzymatic activity. Analysis of expression profiles of iron metabolism genes suggests that replicon cells undergo a genetic reprogramming to support subgenomic viral replication, resulting in reduction of intracellular iron levels.

Conclusions: We propose that iron impairs HCV replication by inactivating NS5B, and that its negative effects in chronic hepatitis C may be primarily due to attenuation of antiviral immune responses. Our data provide a direct molecular link between iron and HCV replication, and warrant consideration in the design of antiviral therapies.

2A. Transcription, RNA Processing and Export

The novel member of the kallikrein gene family, KLK4, is overexpressed in breast tumours and significantly correlated with the apoptosis related genes BCL2 and BCL2L12

Introduction: The steroid-regulated KLK4 gene is a recently identified member of the human kallikrein gene family. BCL2L12 is a novel gene of the BCL2 family of the apoptosis related genes. The aim of this study is to analyze the KLK4 expression in relation to BCL2 and BCL2L12 in breast tumours. Methods: Total RNA was isolated from 100 breast tissue specimens.

After testing RNA quality, cDNA was prepared by reverse transcription. A highly sensitive quantitative real-time PCR (qRT-PCR) method for KLK4 mRNA quantification was developed using the SYBR Green chemistry. GAPDH was used as endogenous control gene. Relative quantification analysis was performed using the comparative C T method 2 -DDC T . Results: A statistically significant elevation of KLK4 mRNA expression was observed in the malignant (mean ± SE: 73.6 ± 18.1 mRNA copies of KLK4/mRNA copies of GAPDH, c/Kc), compared to benign tumour specimens (mean ± SE: 2.8 ± 1.3 c/Kc). KLK4 mRNA expression analysis of malignant tumours revealed a significant increase in advanced grade III as compared to grade II and even more so in low grade I patients (p = 0.034). Moreover, a statistical significant correlation between KLK4 expression status and the expression of the apoptosis related genes BCL2, BCL2L12 was observed (p < 0.01). Conclusions: Our results recommend that KLK4, BCL2 and BCL2L12 may be used as a new potential panel of prognostic biomarkers in breast cancer. Acknowledgements: This work was supported by a research grant from Empirikion Foundation, Athens, Greece, as well as through the grant 'KAPODISTRIAS' of the University of Athens. Introduction: The Elavl/Hu family of RNA-binding proteins is composed of the ubiquitously expressed HuA/HuR and the neuronrestricted HuB, HuC and HuD. All members share similarities towards the post-transcriptional regulation of their target mRNAs. Thus it is currently unclear whether they share functional properties in neurons or they have distinctive functions. In this report we aim to identify the potential role of HuR in CNS neurons and correlate it with expression of the neuronal Hu proteins. Methods: We utilize immunohistochemical and immunoblotting methodologies to identify the expression of HuR in different CNS compartments. We further aim to identify the functional role of HuR in the CNS using a transgenic system of HuR-ablation in murine neurons in the hipoccampus and frontal lobe. Results: We found that HuR is expressed in the nucleus of many neuronal and glial CNS compartments, albeit at lower levels than the neuronal Hu's that appear to have a cytoplasmic expression pattern. Mice with a HuR-deficient neurons develop with no gross defects in the CNS. In a model of kainate-induced excitotoxicity a partial protection was observed in stages proximal to kainate administration; however at later stages neuronal death was much more severe in the absence of HuR. In addition, HuR-deficient neurons exhibited increases in the expression of neuronal Hu's suggesting potential compensatory effects. Conclusions: Our results demonstrate a differential role for HuR in kainate-receptor instigated signals versus apoptotic neuronal damage. They further demonstrate a potential interplay between HuR neuronal Hu's towards the modulation of neuronal gene expression. Post-transcriptional processes that include splicing, polyadenylation, transport, stability and translation of mRNA, are important events in regulating gene expression in higher eukaryotes. These are brought about by an extensive network of interactions of a large number of RNA-binding proteins (RBPs), operating within diverse multifactorial RNP complexes. We focus here on interactions between hnRNP proteins, having a major role in RNA splicing, and the HuR and TIA-1 proteins with an established role in stability/translation of target mRNAs. Both groups of proteins have a major nucleoplasmic localization and ability to shuttle between the nucleus and cytoplasm, as well as an apparent increased expression in cancer (mainly, in colon and lung). Currently, a likely functional interaction between HuR and AUF-1/hnRNP D is most considered. We report here our findings on additional interactions between hnRNPs and HuR that were obtained by an extensive application of immunoprecipitation and pull-down assays on nuclear and cytoplasmic extracts prepared from human and mouse cell lines. We have identified specific associations between hnRNP A1, A3 and C1/C2 with HuR (but not TIA-1) and have ascribed discrete biochemical properties with respect to nuclear/cytoplasmic presence, stability to RNase and salt. We presently apply fractionation studies to look at their presence in specific RNP complexes. The biological significance, if any, of these interactions will be tested under conditions related to stress or to cellular transformation. These initial findings on the association of specific hnRNPs with HuR implicate a discrete role in RNA processing events, operating in the nucleus and/or the cytoplasm that remains to be established.

Resetting of the mammalian clock by TSA has phase-dependent effects on the expression levels of c-myc and wee1 genes in N2A murine cells A. Repouskou 1 , K. E. Sekeri-Pataryas 2 , T. G. Sourlingas 2 and A. Prombona 1 1 Laboratory of Chronobiology, Institute of Biology, National Centre For Scientific Research 'DEMOKRITOS', Athens, GREECE, 2 Laboratory of Histone Biochemistry And Ageing, Institute of Biology, National Centre For Scientific Research 'DEMOKRITOS', Athens, GREECE Introduction: Trichostatin A (TSA), a histone deacetylase inhibitor, causes changes in the acetylation status of histones and other cellular non-histone proteins. Especially in malignant cells, this has dramatic effects on the expression levels of cell cycle genes, on cell cycle progression and/or the induction of apoptosis. Recent reports have shown that circadian clock component proteins regulate the expression levels of cell cycle genes, like cyclin D1, wee-1 and c-myc. In this investigation, we apply TSA in N2A murine neuroblastoma cells at peaks and troughs of per1 rhythm, in order to study how circadian phasing influences the expression levels of the cell cycle-related genes, wee1 and c-myc. Methods: The circadian clock in N2A cell cultures is synchronized by 100 nM dexamethasone and TSA (100 ng/ml) is added to the medium for 2 hours at various circadian times. Expression levels of per1, c-myc and wee1 are monitored by reverse transcription and qPCR. Results: Our results show that TSA causes an acute response in per1 transcription in all cases examined. Moreover, the circadian phase of per1 is critical in modulating the effect of TSA on the expression levels of the cell cycle genes c-myc and wee1. In particular, wee1 expression is suppressed when cells are treated with TSA at per1 peak levels only. Conclusions: We conclude that circadian phasing is important for TSA-induced changes in the expression levels of cell cycle genes. Therefore, circadian timing may modulate the effect of drugs (e.g. TSA) on cell cycle progression in cancer cell lines. NPAS is a transcription factor that regulates the mammalian circadian rhythm and contains a heme-binding site in each of two PAS domains (A and B). The heme domain has been suggested to act as a sensor for gaseous molecules such as carbon monoxide (CO). To characterize the role of the heme domain in this function, we overexpressed mouse NPAS2 and mouse BMAL1 with a reporter gene in NIH3T3 cells, and investigated the effects of PASA domain mutants, in the context of full-length NPAS2, on the transcriptional activity of the mouse Period 1 (mPer1) gene. H138A, H148A, and C170A mutants with 6-coordinated low-spin ferric heme acted as a transactivator formPer1 like the wild type. In contrast, H119A and H171A mutants at the endogenous ligands for heme showed remarkably reduced transcriptional activity. In gel-shift assays, H119A or H171A mutations in the isolated basic helix-loop-helix (bHLH)-PASA domain impaired heterodimer formation with BMAL1, resulting in loss of DNA binding to the canonical E-box (CACGTT). The other mutants such as H138A, H148A and C170A had significant binding activities similar to that of wild type. These results indicate that the transcriptional activities of the mutants correlated well with their DNA binding activities, suggesting that local conformational changes near the axial ligands of the PASA domain are responsible for its regulation of transcription. The similar results were also observed in the ferrous forms, suggesting that the DNA-binding activities of the mutants are independent of heme redox states. Placental type glutathione transferase (GST-P) is not expressed in normal liver but is highly induced during rat haptocarcinogenesis, and has been used as a reliable tumor marker for experimental hepatocarcinogenesis in the rat. The molecular mechanism of any tumor marker, once elucidated, may shed a light on the mechanism of the particular tumorigenesis. This idea in mind, we have been pursuing the mechanism of specific induction of the GST-P gene during hepatocarcinogenesis. Making use of advanced technologies of molecular and cellular biology, including proteomic analysis, gene cloning, analyses of specific interaction between enhancer element and regulatory proteins in vitro and in vivo, production of specific transgenic rats etc. We were able to identify the enhancer element (GPE1). We also identified the activator and repressor proteins that interact with GPE1 and responsible for this tumor marker expression. In particular, C/EBP was required for the suppression of GST-P gene in normal liver, whereas the Nrf2/MafK heterodimer was required for the activation of this gene during hepatocarcinogenesis. The DNA damage response is a complex signaling network that is induced by DNA lesions and vigorously activated by double strand breaks (DSBs). The DSB response is mobilized by the nuclear protein kinase ATM that phosphorylates key players in the damage response pathway. ATM deficiency leads to the genome instability syndrome ataxia-telangiectasia (A-T). Here, we show that Matrin 3 (Matr3) is an ATM substrate. Matr3's function is unknown, but its functional link with the proteins p54 nrb and PSF implies a role in RNA processing. We found that Matr3 phosphorylation is important for proper activation of the DSB-induced cell cycle checkpoints. Using proteomic analysis we demonstrated that the RNA helicase DHX9 and the p53 co-activators hnRNP K and DDX5 interact with Matr3. Importantly, hnRNP K is known to be induced following DNA damage in an ATM-dependent manner and both hnRNP K and DDX5 are involved in activating the cell cycle checkpoints. DHX9 plays an active role in gene expression by bridging CREB-binding protein (CBP) and BRCA1 with RNA polymerase II. CBP and BRCA1 are known ATM substrates and we found that DHX9 was an ATM target as well. The emerging picture is that of a protein complex whose members are ATM targets in the cell cycle checkpoint pathway. Further investigation of the role of Matr3 and other members of this complex in the checkpoint pathway is underway.

Deciphering the complex in vivo role of AUF1/ hnRNPD N. Sgantzis 1 , M. Dimitriou 1 , A. Paterou 1 , D. Graf 1 , A. Economides 2 and D. L. Kontoyiannis 1 1 Institute of Immunology, B.S.R.C. 'Alexander FLEMING', Vari, GREECE, 2 Genomic Engineering Technologies, Regeneron Pharmaceuticals, Inc., Tarrytown, NY, USA Introduction: AUF1 (hnRNPD) is a member of the hnRNP family and exists as four protein isoforms (p37, p40, p42, p45) produced as a result of alternative splicing. The molecular effects of AUF1 are deciphered by its primary involvement in posttranscriptional regulation of several mRNAs (maturation, N/C shuttling, stability/degradation, translation) relating to a pleiotropy of cellular responses from survival to senescence. However the in vivo role of AUF1 remains unclear, although indications for its involvement in inflammation and neoplasia have been provided. Methods: We created a 'functional knock-out' for AUF1 using gene targeting and application of the Flp/FRT and Cre/loxP systems of recombination. The induced mutation abolishes the functions of the central RNA-recognition motifs (AUF1 DRRM ) in all AUF1 isoforms. Results: Obligatory homozygous AUF1 DRRM showed multiple phenotypes. Specifically, 50% of the mutant embrya die past midgestation relating to defects in fetal growth. Similarly, the mutant mice born are growth retarded and more than 60% of those succumb at the age of 4 weeks from unknown aetiology. The remaining mice survive but both male and female carriers appear sterile. With respect to inflammation, we also generated an innate immune-restricted system for AUF-1 ablation which demonstrated a pro-inflammatory role for AUF1, which did not relate to any effects on the biosynthesis of key cytokines. Conclusion: Our studies demonstrate a pleiotropic role for AUF1 in a number of cellular responses supporting embryonic growth, fertility and inflammation.

The zinc finger protein Cas5 is required for morphogenesis of Candida albicans W. Song, H. Wang and J. Chen State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, SIBS, Chinese Academy of Sciences, Shanghai, CHINA Candida albicans is a commensal fungus of mucosal surfaces that can cause disease in susceptible hosts. Its ability of morphological transition is associated with the virulence of this human pathogen. Many transcription factors have been proved to regulate morphogenesis of C. albicans. Cas5 is a putative zinc finger transcription factor which was reported to have a role in the C. albicans cell wall damage response, but its role in morphogenesis of C. albicans remains unknown. To determine its transcriptional activity, we fused different Cas5 domains to the lexA DNA binding domain and measured the transcriptional activities of the fusion proteins in a lacZ reporter system. Beta-galactosidase activity assay showed that the N-terminal polyQ domain is necessary and sufficient for the transcriptional activity of Cas5. Through homologous recombination using a hisG-URA3-hisG cassette, we sequentially knocked out both alleles of CAS5 gene and examined the phenotype of the cas5/cas5 mutant under different growth conditions. The cas5/cas5 cells showed severe growth defect and formed wrinkled colonies on several solid media. In yeast growth conditions, the cas5/cas5 mutant cells enhanced filaments formation. Our results suggest that Cas5 functions as a transcription factor both in cell growth and morphogenesis of C. albicans.

PP2A-59 CIITA isoforms display a different preference in the MHC class II genes they regulate P. Stavride 1,2 and J. Papamatheakis 1,2 1 IMBB-FORTH, Heraklion, GREECE, 2 Biology Department, University of Crete, Heraklion, GREECE Introduction: Expression of the major MHC class II transactivator CIITA is under developmental and IIFN c control. In addition, utilisation of alternative cell type promoters generates CIITA isoforms that are distinguished by different N-terminal regions: Types I and III are constitutively expressed in dendritic and B-cells respectively and Type IV is IIFN inducible in various cell types. We investigated the ability of those variants to regulate different MHC class II isotypes. Methods: Rj2.2.5 (CIITA-deficient B-cells) and HeLa cells (in which CIITA is not normally expressed), were used. Different CIITA isoforms were transfected in each cell line, and total RNA isolated was analysed by real-time PCR for MHCII transcripts. The CIITA isoforms were also co-transfected with different MHCII gene promoters fused to the luciferase gene. Results: We found that each CIITA isoform has a different preference in the MHCII genes that it activates, resulting in different ratios of these genes in each cell line. As expected, the observed total levels of transcription vary significantly, in the order Type IV<III<I. Conclusion: MHC class II genes show a differential response to the CIITA isoforms. This may be the basis of differential MHC isotype expression patterns, as well as quantitative and/or qualitative cell type preferences in antigen presentation function in various cell types.

The role of the RPD3 and ROM2 genes in regulation of multidrug resistance in yeast S. Borecka, Z. Kozovska, I. Hikkel, V. Dzugasova and J. Subik Department of Microbiology and Virology, Faculty of Natural Sciences, Comenius University, Bratislava, SLOVAKIA Introduction: In the multidrug resistant Saccharomyces cerevisiae cells, the hyperactive mutant forms of the Pdr1p or Pdr3p transcriptional activators are responsible for the upregulation of PDR5 and other genes encoding multidrug resistance efflux transporters. Methods: Transposon mutagenesis, DNA sequencing, drug susceptibility assay, Northern blot analysis. Results: In this report we show that the RPD3 and ROM2 genes participate in modulation of multidrug resistance in yeast. Transposon insertion mutations in ROM2, RPD3 and some other genes encoding specific subunits of the large Rpd3L chromatin remodeling protein complex resulted in enhanced susceptibility of mutant cells to antifungals. In the rpd3D and rom2D mutants both the PDR5 mRNA level determined either in the absence or in the presence of cycloheximide and the rate of rhodamine 6G efflux were reduced. Unlike to rpd3D, in rom2D mutant cells the drug hypersensitivity and the defect in PDR5 expression were suppressed by the hyperactive pdr3-9 mutant allele and by PDR1 or PDR3 overexpressed from heterologous promoters.

The results indicate that Rpd3p histone deacetylase participating in chromatin remodeling and Rom2p representing the GDP/ GTP exchange protein for Rho1p and Rho2p participating in several signal transduction pathways are involved in the transcriptional activation of the PDR5 gene and regulation of multidrug resistance in yeast.

Osteoprotegerin (OPG) expression in synovial tissues from rheumatoid arthritis patients -an immunohistochemical and RT-PCR assay D. Teletin, C. Iosif, M. Ceausu, F. Andrei, M. Neagu, A. Georgescu, G. Butur and C. Ardeleanu 'Victor Babes' National Institute of Pathology, Bucharest, ROMANIA Introduction: Rheumatoid arthritis (RA) is a systemic autoimmune inflammatory disease involving the breakdown of cartilage and juxtaarticular bone, which is often accompanied by decreased bone mineral density (BMD), deformity of synovial joints, and increased risk of fracture. Several lines of evidence indicate that an imbalance in the OPG/ RANK/RANKL system that controls osteoclast activity contributes significantly to the skeletal erosions that are characteristic of RA. OPG is a soluble osteoblast-derived protein that influences bone resorption by inhibiting osteoclast differentiation and activation. Methods: Formalin fixed paraffin embedded synovial tissues, surgically removed from knee joints of patients with RA were analysed using standard HE and the indirect tristadial ABC-peroxidase immunohistochemical technique for OPG. mRNA for OPG was detected by reverse-transcription polymerase chain reaction (RT-PCR) from fresh tissues. Results: Strong immunohistochemical staining for OPG in synovial tissue from patients with RA was detected in samples with mild inflammatory infiltrate and patients with mild osteolysis as evaluated by DEXA. Levels of OPG mRNA detected by RT-PCR correlates significantly with protein expression and severity of bone loss. Conclusions: In this study, high levels of OPG reflect a protective mechanism of the skeleton, preventing most of the bone loss that develops in immune-mediated arthritis. These results suggest that RANKL-RANK-OPG system is an excellent therapeutic target, and OPG could represent a potent new agent for treating human patients with RA. Introduction: Earlier studies from our laboratory have shown that DNA/RNA methylation inhibitors block murine erythroleukemia (MEL) cell differentiation and promote cytoplasmic accumulation of discrete non-poly(A) RNA transcripts. These RNAs were hybridized with DNA fragments derived from 3¢-flanking sequences of b major globin gene locus in which a 235bp silent region was found to contain a B1 repetitive element and structural elements with potential capacity to regulate gene transcription. In this study, we have attempted to characterize the region of 235bp for its structural elements, ability to recruit transcriptional factors and encode RNA transcripts. Methods: Mobility shift banding, reporter gene assays to assess the promoter-like activity, RT-PCR and Northern blot hybridization analysis were employed. Results: The data obtained thus far has shown that the examined region of 235bp at the 3¢-flanking b major globin gene sequence: (i) recruits the binding of several proteins; (ii) possesses promoter-like activity that is developmentally regulated; and (iii) encodes at least one RNA molecule encoded at mouse chromosome 3 that contains short direct repeats (SDR) at its 5¢-and 3¢-ends. Moreover, this RNA transcript shares the same identical 108bp sequence previously identified at the 5¢-end of a B1 element located within the 235bp region. Conclusions: Overall, the results presented indicate that the 235bp sequence contains structural elements with potential to drive gene transcription, whereas treatment of MEL cells with methylation inhibitors activates the transcription of a region at chromosome 3. The role of these events in the blockade of commitment to erythroid differentiation of MEL cells is discussed.

Stimulation of T7 RNA polymerase in Xenopus oocyte and egg extracts A. Tokmakov, T. Iwasaki and Y. Fukami Kobe University, Kobe, JAPAN Introductions: Bacteriophage T7 RNA polymerase (T7RNAP) has found the widest applications in molecular biology and biotechnology. It is universally used for in vivo and in vitro transcription of genes put under the control of T7RNAP promoter. Recently, T7RNAP is increasingly employed for high-throughput protein expression aimed at establishing protein structure and structure-based drug development. Methods: To facilitate quantification of T7RNAP activity, we developed a highly sensitive, luciferase-based coupled-enzyme assay. Chemiluminescence in this system was dependent on the amount of T7 RNAP in a range extending over three orders of concentration magnitude (0.05-50 ng/assay). Results: Using this assay, the studies have been performed to evaluate the stability of T7RNAP in Xenopus oocytes and Xenopus cell-free system. It has been demonstrated that not only T7RNAP is remarkably stable in the oocyte cytoplasm, but also it can be stimulated, under certain conditions, in the cell-free oocyte and egg extracts. We found that the T7RNAP stimulator is a low molecular weight, thermo-stable substance, which increases the affinity of T7RNAP for DNA template. It is inactivated by proteinase treatment of the extracts, suggesting the presence of a peptide moiety in the stimulator molecule. MALDI TOF mass-spectrometry of the His-tagged T7RNAP incubated in Xenopus egg extracts revealed the presence of several specific peaks in the region of 900-1,700 Da. Currently, their molecular identification is being carried out. Conclusions: Identification of T7RNAP stimulator(s) will help to develop the novel experimental conditions favorable for enhanced transcription with T7RNAP, resulting in more efficient protein production. Methods: Cells were grown in glucose M9 minimal media at 37°C. CspE levels were estimated by western blot analysis. Results: The CspE expression was found to be considerably lower during stationary phase than in the exponential phase. A drastic reduction in cellular CspE levels was observed when Dibutyryl-cAMP (Db-cAMP) was added to the growth medium during the early exponential phase. Conclusions: cspE is regulated in a growth-phase-dependent manner with the cellular levels of the protein being considerably lower during stationary phase. This down regulation could be due to the increased cAMP levels caused by glucose depletion during stationary phase. Further, the presence of a putative CRP (cAMP receptor protein) binding site in the upstream region of cspE, supports this possibility. This is the first report demonstrating the role of cyclic AMP in the regulation of a cold-shock protein.

The taxon-level variability of the 2-5A synthetases expressed in sponges K. Vallmann, N. Aas, A. Kuusksalu, A. Lopp, T. Reintamm and M. Kelve Tallinn University of Technology, Department of Gene Technology, Tallinn, ESTONIA Introduction: Like mammals, the evolutionarily lowest metazoan, sponges, are capable of synthesizing 2',5'-oligoadenylates (2-5A). The activation mechanism of 2-5A synthetases in sponges seems to be different from that known for mammals; the known sponge 2-5A synthetase primary structures are highly variable. In this study 2-5A synthetase primary structure of the marine sponge Geodia barretti was determined. Methods: Primers designed against 2-5A synthetase cDNA from the marine sponge Geodia cydonium were used in suboptimal conditions to amplify 2-5A synthetase fragments from G. barretti. The resulting PCR products were sequenced and used to design primers specific to G. barretti 2-5A synthetase. The full-length sequence of G. barretti 2-5A synthetase was elucidated using the RLM RACE method. Multiple sequence alignment with sponge 2-5A synthetases was performed by using ClustalW program. Results: It was possible to obtain the sequence covering the full functional cDNA of the 2-5A synthetase from G. barretti by starting from the primers specific to that from another member of the genus Geodia. Compared to the corresponding sequence from G. cydonium, the coding sequence had approximately 75% identity on the amino acid level, the nucleotide substitutions being randomly distributed. Conclusions: The comparison of the coding sequences from two species of Geodia revealed their degree of similarity, close to that between higher vertebrates, e.g. human and pig. The further studies of gene regulation and activation mechanisms of sponge 2-5A synthetases will lead us to understanding the role(s) of these enzymes in sponges which may be totally different from that in higher vertebrates.

Molecular and functional changes associated with APE1/Ref-1 loss of expression reveal its multifunctional nature in mammalian cells APE1/Ref-1 is essential for cell protection toward oxidative stress by acting as a transcriptional regulator of antioxidant genes and as a central enzyme in base excision repair pathways of DNA lesions. While it is known that loss of APE1 expression is associated to significant biological effects, the molecular mechanisms involved in these processes are still not completely known. By using an unbiased approach, we combined both mRNA expression profiling and proteomics to define the molecular changes associated with APE1 loss-of-expression by siRNA technology. This approach identified the influence of APE1 on apoptosis, cell growth, intracellular redox state, mitochondrial function and cytoskeletal structure. To dissect both direct and indirect effects, we performed knock-in experiments with wild type and different APE1 mutants. Two of them (i.e. C65S and C310S) were able to rescue mitochondrial depolarisation and cytoskeleton perturbation but not cell cycle arrest and apoptosis. These findings indicate that the wild-type APE1 protein has structural components that function in several distinct central cellular roles and strongly support the notion that, besides its DNA-repair activity, additional functions of APE1 are important for cell viability. Our data highlight the molecular reasons for the multifunctional nature of APE1 protein, providing the basis for future studies on the comprehension of the causal role played by APE1 in regulating different biological mechanisms through the control of signal transduction and gene expression.

Involvement of ADA2b, a member of the histone acetyltransferase SAGA complex, in the induction of COR genes upon salt stress in Arabidopsis thaliana Introduction: Plants respond to environmental stresses by inducing gene expression. In response to cold stress, Arabidopsis thaliana induce the expression of COR genes by the action of the CBF transcription factors. CBFs might require the function of co-activators ADA2b and GCN5, members of the histone acetyltransferase SAGA complex (Vlachonasios et al., Plant Cell 2003; 15: 626-638). Here, we investigated the role of ADA2b in the induction of COR genes upon salt stress. Methods: RT-PCR analysis used to determine the expression levels of several COR genes in ada2b-1 mutants in response to salt stress. Moreover, we performed ChIP assays, using H3Ac antibodies, at the promoters of COR genes to examine histone acetylation patterns in ada2b-1 mutants upon salt stress. Results: COR genes were induced at a high level 3 hours after onset of the stress in wild type plants. In ada2b-1, RD29b, RAB18 and COR6.6 were induced at a lower level. At the promoters of RD29b, RAB18 and COR6.6 genes, H3 acetylation levels remained constant or slightly increased upon salt stress in wild type, while in ada2b-1 were generally lower, suggesting that ADA2b is required for the H3 acetylation in salt-induced gene expression. Conclusions: For some of the COR genes studied, there was a positive correlation between H3 acetylation and expression levels upon salt stress. ADA2b could act as a positive regulator of H3 acetylation in response to salt stress.

Introduction: In eukaryotes, TAF5 is a 90kDa subunit of TFIID and SAGA complexes involving in RNA polymerase II transcription initiation, chromatin modification and histone acetylation. Arabidopsis genome encodes one TAF5 (At5g25150) of approximately 744 kDa, with highly identiy to yeast (34%) and human TAF5 (37%) particularly in WD40 domain. To study the biological role of TAF5 in Arabidopsis we screened SALK T-DNA mutant collection. Methods: We use PCR analysis to genotype the putative taf5 mutants and RT-PCR analysis to characterize the mutations. Results: Three mutants bearing a T-DNA insertion were identified in fourth intron (taf5-1), fifth exon (taf5-2) and in fifteenth intron (taf5-3). Although in yeast TAF5 is an essential gene, in Arabidopsis, homozygous taf5-1 mutants are viable and exhibit developmental phenotypes. All three heterozygous mutants showed folded cauline leaves and reduced length of inflorescence compared to wildtype plants. Moreover, taf5-1 homozygous plants dipslayed a terminal flower phenotype; characterized by a shoot meristem convertion to a terminal flower in long and short days unlike the wild type plants, where they grow indefinitely, never differentiating and only finally senescing. Conclusions: TAF5 in Arabidopsis thaliana is not essential for viability but is important for shoot meristem identity.

The role of mesenchymal cell supernatant in the apoptosis of primary hepatocyte cultures after liver injury A. Xagorari 1 , E. Siotou 1 , M. Yiangou 2 , E. Yannaki 1 , G. Karponi 1 , N. Psatha 1 , K. Koukoulias 1 , A. Anagnostopoulos 1 and A. Fassas 1 1 Gene and Cell Therapy Center, Hematology-BMT Unit, G.Papanicolaou Hospital, Thessaloniki, GREECE, 2 Department of Gen., Dev. & Mol. Biol., Sch. of Biology, Aristotle University of Thessaloniki, Thessaloniki, GREECE Introduction: Carbon tetrachloride (CCL4) intoxification in mice is a widely used model for acute liver injury. Mesenchymal stem cells from bone marrow (MSCs) might have the potential to ameliorate CCL4 effect into hepatocytes. This study aimed to investigate the effect of MSC supernatant on primary cultures of mouse hepatocytes after CCL4-induced acute liver injury. Methods: Mice were treated intraperitoneally with 2 ml/kg CCL4 and after 24 hour intact viable hepatocytes were isolated by liver perfusion and were cultured for 6 days in defined medium. MSC from bone marrow were cultured to passage 4 and their supernatant was collected and was supplemented to the hepatocyte primary cultures. RT-PCR of albumin, HNF4 and GDF15 were performed. Apoptosis was detected by FCM analysis of annexinV-FITC staining of hepatocytes. Results: Expression of the hepatic markers albumin and HNF4 was observed in the hepatocytes cultures. Flow cytometric analysis of hepatocytes stained with annexin-V demonstrated that hepatocytes from CCL4-treated mice supplemented with MSC supernatant exhibited lower levels of apoptosis at day 3 of culture as compared to non-supplemented (0.14% vs. 0.99%, p = 0.037). Furthermore the expression of growth/differentiation factor 15 (GDF15), a member of the TGF-b family, which is upregulated in liver injury and its role has not yet been elucidated, was detected in culture day 1 in CCl4-treated hepatocytes independently of the presence of MSC supernatant. Introduction: Vascular endothelial growth factor (VEGF) acts as the central angiogenic factor in both physiological and pathological conditions through binding to its cell surface receptors. Soluble VEGF receptors, soluble Flt-1 (sFlt-1) and soluble neuropilin-1 (sNRP-1), are potent angiostatic factors, which trap extracellular VEGF. We previously found that human microvascular endothelial cells (HMVEC) express sFlt-1 and sNRP-1 and they have crucial roles in the autocrine regulation of angiogenesis [1] . sFlt-1 and sNRP-1 mRNAs are produced by alternative 3'-end processing from the same genes that produce the cell surface receptors. However, the mechanisms underlying the regulation of alternative 3'-end processing of VEGF receptor pre-mRNAs are poorly understood. Methods: The cleavage/polyadenylation sites of sFlt-1 mRNA were determined by 3'-RACE method using HMVEC RNA. The Flt-1 minigene was constructed by inserting a DNA fragment containing exon 12-exon 14 of human Flt-1 gene into a pCIneo expression vector. Results: We determined the alternative 3'-end processing sites in the intron 13 sequence of sFlt-1 pre-mRNA and established an assay system for sFlt-1 pre-mRNA alternative 3'-end processing with the human Flt-1 mini-gene. Using this system with site-directed mutant minigenes, we identified cis-acting elements on sFlt-1 pre-mRNA, which can be involved in the regulation of alternative 3'-end processing. We also identified RNA binding proteins that can affect the generation of the soluble-type RNA. Conclusions: The present study unveiled a molecular mechanism for the regulation of alternative 3'-end processing of VEGF receptor pre-mRNAs in vascular endothelial cells. Introduction: The mammalian complement system is a phylogenetically ancient cascade system that has a major role in innate and adaptive immunity. Activation of component C3 is central to the three complement pathways, leading to the terminal Membrane Attack Complex (MAC) and results in inflammation and elimination of self and non-self targets. Recent evidences have shown that C3 is also implicated in tissue regeneration and cell differentiation. Methods and Results: In order to study the spatial and temporal mRNA expression profile of MAC components (C6, C7, C8a, C8b, and C8c) and MAC regulatory genes (CD59, clusterin and vitronectin) as well as the potential developmental regulation of C3 gene, chicken (Galus gallus) was used as a model organism. Concerning the MAC components, our results show a full expression of MAC genes in the heart, brain and liver, while their developmental expression patterns show that all of them are expressed in both 12 and 17 day embryo, while no expression was observed in early embryo (day 6) and the neonatal individual (day 2). On the contrary, both MAC regulatory genes and C3 are expressed in whole embryo (day 4 and 6) and in the liver of a 12 and 17 day embryo, as well as in postnatal individual (day 2 and 5) and adult organism. Further studies by in situ hybridization show a highly differential distribution of C3 mRNA in chick embryo. Conclusion: This is the first report on the spatial and temporal expression of complement genes (C3, C6-C8, CD59, clusterin and vitronectin) in chicken. Introduction: Survivin belongs to the family of the inhibitors of apoptosis and has a role in cell division and proliferation. There are at least five isoforms of survivin which seem to have opposing roles regarding apoptosis and may be necessary for the regulation of the action of survivin. In the present study, the relationship between the expression of four of the isoforms of survivin and the promoter polymorphism -31G/ C was assessed in colon cancer. Methods: Blood and colonic tissue (neoplastic and normal) was obtained from 46 patients with primary colon cancer. Survivin, survivin-2B, survivin-dEx3 and Survivin-2a mRNA levels in tissue samples were assessed with quantitative RT-PCR. The promoter polymorphism -31G/C was detected in DNA from the blood samples with real time PCR. Results: All isoforms were expressed in higher levels in neoplastic tissue compared to normal (P < 0.001). Regarding the -31G/C polymorphism, 52.2% (24/46) of the patients were heterozygotes and 43.4% (20/46) homozygotes GG, while CC were only 4.4% (2/46). In neoplastic tissues, the presence of the C allele correlated with higher levels of expression of the isoforms studied (not statistically significant for survivin) although there was no difference in the levels in the normal tissue. Conclusions: All isoforms are over-expressed in neoplastic tissue. The G allele was more frequently detected than the C and it correlates with lower expression levels of survivin-2B, -dEx3 and -2a in neoplastic tissue. Introduction: The transcription factors peroxisome-proliferator activated receptors (PPARs) were recently identified in fish and, like mammals, may to be involved in lipid homeostasis and also in the innate immune response. Herein, we investigate the regulation of the expression of PPARs in immunological and metabolic challenges in fish. Methods: PPAR expression was studied after an in vivo 72 hours lipopolysaccharide (LPS) injection (6 mg/kg body weight) in sea bream (Sparus aurata). Moreover, sea bream PPAR expression was studied after a 24 hours insulin (17 U/kg body weight) administration and after a fasting/refeeding experiment. The expression of PPARs was studied by Real-Time PCR. Results: In most tissues, LPS administration resulted in an overall decrease in the expression of PPARs. Most prominently, LPS decreased the expression of PPARa, b, and c in liver and gills and of PPARa and PPARb in red and white muscle. In contrast, the expression of all three PPARs was up-regulated in adipose tissue. Insulin increased the hepatic expression of PPAR a and diminished PPARc, while in anterior intestine PPARa expression was decreased. Fasting decreased the expression of PPARc and refeeding reversed this effect in liver, red and white muscle. However, fasting increased the expression of PPARb in white and red skeletal muscle but decreased after refeeding. Conclusions: The expression of PPAR isoforms in fish is regulated by immune, nutritional, and hormonal factors, indicating that PPARs may play a role in the innate immune response and metabolic regulation in fish. Acknowledgements: This work was partly supported by GSRT of the Ministry of Development under the Research and Technology Cooperation scheme between Greece and Spain.

Involvement of the CTNS gene in thiol/disulfide systems regulation F. Bellomo, A. Taranta, S. Corallini, E. Giorda and F. Emma Department of Nephrology and Urology, Nephrology Division, 'Bambino Gesu' Children's Hospital, Rome, ITALY Introduction: The human CTNS gene encodes for the cystinosin protein that mediates the efflux of cystine from lysosomes and cause, when mutated, nephropathic cystinosis. In this work, we have studied the role of the CTNS gene in the redox pathway using the HK2 Human Kidney cell line. Methods: GSH/GSSG and Cys/CySS ratios were measured by HPLC, ROS production by FACS/Flow cytometry and CTNS gene expression by RT-PCR. Results: Cystine and methionine deprivation resulted in decreased GSH/GSSG (from 64 ± 5.7 to 13.5 ± 0.7) and Cys/CySS (from 35.6 ± 5 to 9.9 ± 3.1) ratios, in increased ROS production (from 33.7 ± 0.5 to 57 ± 5) and in increased CTNS gene expression (from 0.41 ± 0.07 to 0.91 ± 0.14), at 24 and 48 hours. The same effects were obtained with tert-butyl hydroperoxide and buthionine sulfoximine, which oxidate glutathione or inhibit its synthesis, respectively. CTNS over-expression and RNA silencing did not influenced cell redox state. Conclusions: These results show that changes of thiol/disulfide couples induce the transcription of the CTNS gene. We speculate that CTNS plays a crucial role in supplying cells with cystine, directly for the Cys/CySS redox couple, or indirectly for glutathione synthesis. This is also supported by the presence of ARE sequences (Antioxidant Responsive Element) in the CTNS gene.

Regulation of the leukemia associated oncogene and developmental regulator EVI1 by all-trans retinoic acid S. Bingemann and R. Wieser Department for Medical Genetics, Medical University Vienna, Vienna, AUSTRIA The EVI1 gene codes for a zinc finger protein with important roles in embryonic development and in myeloid leukemogenesis. It is transcribed into several mRNA species with variable 5¢-ends. One of these mRNA variants, MDS1/EVI1, gives rise to an EVI1 protein with an extended N-terminus and with functions partially different from those of the shorter EVI1 protein type. The other EVI1 5¢-end variants are most likely translated into the same protein, i.e. the short EVI1 protein variant, but their variable 5¢-UTRs can be expected to affect the regulation of protein expression. So far all-trans retinoic acid (ATRA) is the only known physiological regulator of EVI1 in mammalian cells. Using the teratocarcinoma cell line NT-2, we have investigated the induction of EVI1 by ATRA. Time course analyses showed that ATRA rapidly induces the EVI1 mRNA in NT-2 cells. A maximum is reached after approximately 48 hours, except for the MDS1/EVI1 mRNA, which was noticeably induced only after 48 hours. This response was already detectable with as little as 10 nM ATRA and affected all EVI1 mRNA variants, albeit to variable degrees. Using reporter gene assays, an ATRA responsive region of 200 bp was identified within exon 1a of EVI1. Chromatin immunoprecipitation (ChIP) experiments confirmed the binding of retinoic acid receptors RAR and RXR to a retinoic acid response element (RARE) in this region. We conclude that the EVI1 gene is a direct target of retinoic acid signaling and that its regulation via this pathway is mediated through an intragenic RARE. Chitosanases catalyze the hydrolysis of chitosan, a polymer of beta-1,4 D-glucosamine (GlcN). Chitosanases from Streptomyces sp. N174 (CsnN174) and Kitasatospora sp. N106 (CsnN106) were extensively studied. They are efficiently produced in the industrial heterologous host Streptomyces lividans. However, few works were dedicated to the regulation of csn gene expression. A palindromic sequence is observed up-stream the coding sequences of CsnN174 and CsnN106. Similar boxes are present in the genomes of several actinomycetes, mostly upstream genes encoding other chitosanases, such as sco0677 in S. coelicolor A3(2) and its ortholog in S. lividans but also up-stream the gene sco2657, encoding a transcription regulator belonging to the ROK family. sco2657 is the first gene of a six-gene operon probably involved in the transport of GlcN oligosaccharides. To find if the ROK protein was involved in csn gene regulation, we performed gene disruption of the sco2657 homolog (W2657h) in S. lividans. An electrophoretic shift of the palindromic box was observed with crude extracts from GlcNinduced cultures of S. lividans while it was absent using extracts from disruption mutants. An N-terminal His 6 -tagged version of the ROK protein was expressed in E. coli and purified. The recombinant ROK exhibited binding to the palindromic box as tested by EMSA. Binding of the ROK protein to the palidromic box in vitro is affected by the presence of GlcN or chitosan pentamer indicating that ROK is probably a repressor. Binding of ROK is also observed to the palindromic boxes of the heterologous genes encoding CsnN106 and CsnN174.

Searching for a functional relationship between breast cancer gene 1 (BRCA1) and the progesterone receptor in breast cancer cells V. Calvo Vidal and M. Beato del Rosal Fundacio Centre de Regulacio Genomica-PRBB, Barcelona, SPAIN Germ-line mutations in the BRCA1 gene strongly increase the risk of developing breast and ovary cancer. BRCA1 protein interacts with and inhibits estrogen receptor activity, considered to be the main sex-steroid responsible for breast tumorigenesis. However, much data have been published recently pointing to a key role of the progesterone receptor (PR) in normal and breast cancer formation. This study aimed to search for a functional relationship between BRCA1 and PR in breast cancer cells. We have detected a ligand-independent interaction between BRCA1 and PR by co-immunoprecipitation. BRCA1 also inhibits the transcriptional activity of endogenous PR. Using RNAi against BRCA1 we enhanced the transcription of several PR-target genes and the over-expression caused their down-regulation. BRCA1 was found present at the hormone responsive region of some of these genes, by chromatin immunoprecipitation. Its presence is dependent on the recruitment of the receptor to those sites and the amount of BRCA1 being expressed affects the amount of receptor recruited.

Another key observation is that the BRCA1/BARD1 heterodimer is able to ubiquitinate the receptor in vivo and in vitro, and it regulates the stability of the receptor protein. We are now further studying the connection between the control on PR stability and ubiquitination, regulation of transcription and other events BRCA1 might drive on these promoters. These findings support a connection between BRCA1, the main tumour suppressor responsible for familial breast cancer, and the progesterone receptor, of grand importance for the maintenance of breast homeostasis. Introduction: Breast cancer is one of the leading causes of death among women in the western world. Plants are important sources of active natural products which differ widely in terms of structure and biological properties. They have a remarkable role in the traditional medicine in different countries. Pistacia atlantica wih local name of Baneh (Annacardiaceae) is growing in parts of Iran, which has been used traditionally in the treatment of peptic ulcer and various diseases. In this study we evaluated the effects of extract on gene expression of p53 and Men1 as tumor suppressor genes and CCND1 as an oncogenen in T-47D cell line. Methods: Cytotoxicity of extract on T-47D cells was determined by MTT assay in comparison to RPMI and Doxorubicin (DOX). In addition, total cellular RNA was isolated from T-47D treated cells with Baneh 1 mg/ml (EC 50 ), DOX (250 nM) and RPMI using Trizol. RNA samples were reverse transcribed to cDNA and PCR amplified using thermal cycler.Results were then analyzed by agarose gel electrophoresis. Results: Baneh extract at 1 mg/ml showed significant growth inhibitory effects on proliferation of T-47D cells. Extract also showed significant decrease in mRNA levels of CCND1, Men1 and p53 in comparison to RPMI and DOX when data normalized to b-actin. Conclusions: The extract of Baneh has significant anti-proliferative effect (time and concentration dependent) against T-47D cells. Growth rate of cells significantly reduced following treatment with Baneh extract similar to DOX in comparison to RPMI. The level of gene expression (CCND1, Men1 and p53) showed significant decrease.

Interaction between endothelin-1 and cyclic AMP signaling pathways in the regulation of Glut1 transcription in 3T3-L1 adipocytes J. Fong and Y.-S. Kao National Yang-Ming University, Taipei, TAIWAN

We have shown previously that chronic exposure to both endothelin-1 (ET-1) and cAMP results in a synergistic increase in Glut1 transcription in 3T3-L1 adipocytes. In the present study, we further examined the molecular mechanism involved. Using various deletion mutants of a luciferase reporter driven by Glut1 promoter and enhancer 1 and 2, we identified a specific region on enhancer 2 as essential for the synergistic effect of ET-1 and cAMP on Glut1 transcription. By employing inhibitory agents to suppress the activities of corresponding putative transcription factors in this region, mutation of the putative responsive sites, chromatin immunoprecipitation and co-immunoprecipitation experiments, we were able to demonstrate that a ternary complex containing Sp1, pCREB and AP-1 was mediating the synergistic effect of ET-1 and cAMP on Glut1 transcription. The results also suggest that while an increase in nuclear Sp1 is induced by ET-1, the accumulation of nuclear pCREB is only found in the presence of cAMP. An increase in nuclear AP-1, on the other hand, can be achieved by both ET-1 and cAMP. Of interest is the finding that ET-1 alone may induce the formation of NF-jB/Sp1/AP-1 complex; however, in the simultaneous presence of cAMP, NF-jB in the complex is replaced by pCREB. In concord, ET-1 activation of NF-jB was suppressed by cAMP. Taken together, our results indicate that ET-1 and cAMP together provide a circumstance favoring the formation of nuclear Sp1/AP-1/pCREB complex that may facilitate the transcription of Glut1 gene.

The identification of new signaling pathways involved in estrogen receptor-alpha (ERa) transcriptional activity G. Giamas, S. Ali, C. Apostolopoulos, L. Castellano, C. Coombes and J. Stebbing Department of Medical Oncology, Imperial College, Hammersmith Campus, London, UK Introduction: Approximately half of estrogen receptor (ER) positive breast cancers develop resistance to tamoxifen, usually caused by its phosphorylation. Transcriptional activation occurs through at least two distinct transactivation domains located in the amino-terminal A/B region (AF-1) and the carboxy terminal E region of the receptor (AF-2). The AF-1 domain is hormone-independent, whereas the AF-2 domain is ligand-dependent. Both AF domains are required for maximal ERa transcriptional activity. It has been shown that ERa can be phosphorylated at serine residues (Ser104, Ser106, Ser118, Ser167, Ser236) by protein kinases, (including MAPK, Rsk, Cdk2, Akt, PKA) which influence the recruitment of co-activators resulting in ERmediated transactivation. Methods: Post-transcriptional gene silencing (PTGS), using RNA interference (RNAi), has become a useful technique for targeted gene knockdown by introduction of siRNA into cells. A systematic mediumthroughput RNAi screening, has been performed in order to search for prospective protein kinases that are able to phosphorylate and/or modulate ERa. Results: We have identified a novel kinase that is able to phosphorylate ERa in vitro and up-regulate its transcriptional activity in a direct and/or indirect way. Interestingly, inhibition of this kinase resulted in stabilisation of ERa protein levels and decrease of proliferation as demonstrated in MCF7 cells. Conclusions: Since phosphorylation of ERa has been shown to be important for the stimulation of its transcriptional activity, focusing on the role of kinases and the influence of site specific phosphorylation events in modulating ERa's activity is considered essential.

Histone deacetylases modulating nuclear receptors regulate bile acid synthesis and lipid metabolism Transcriptional control of metabolic homeostasis requires specific signals to be transduced to the cell nucleus, where defined sets of genes are targeted. In this study we explore the role of some NRs and associated co-regulators in the regulation of cholesterol 7a-hydroxylase (CYP7A1) gene which encodes the rate-limiting enzyme in the conversion of cholesterol to bile acids. Chromatin immunoprecipitations show that bile acids promote the recruitment on CYP7A1 promoter of a repressive complex containing the histone deacetylases (HDAC) 7, 1, 3 and the corepressor SMRT, that affects the transcriptional activity of HNF-4, a strong activator of CYP7A1. By laser confocal microscopy we found that bile acids increase the nuclear localization of HDAC7. By siRNA assays we found that HDAC7 is the key factor required for the repression of CYP7A1 transcription. In vivo administration of the HDAC inhibitors, valproic acid (VPA) or trichostatin A (TSA), to hypercholesterolemic Lld-r KO mice derepresses Cyp7a1 transcription and increases cholesterol conversion to bile acids consequently reducing total plasma and LDL cholesterol. HDACs inhibitors also display effects in extrahepatic tissues: VPA and TSA increase the number and the oxidative activity of mitochondria in skeletal muscle cells, in adipocytes and in hepatocytes, thus favoring the catabolism of fatty acids in these cells. In conclusion our study highlights the importance of HDACs in the regulation of CYP7A1 gene transcription and identifies HDACs as potential targets to modulate bile acid synthesis and lipid homeostasis. HER2/ErbB2 over-expression is associated with tamoxifen resistance in estrogen receptor a (ERa)-positive breast tumors. This is thought to be due to modifications of ERa activity leading to alteration of estrogen/ antiestrogen-triggered changes in the expression of estrogen target genes. However, since the link between estrogen effects on cell proliferation and on gene expression is still a matter of debate, we evaluated in detail the effects of HER2 over-expression on the growth of MCF-7 breast cancer cells and correlated these effects with changes in the expression of three estrogen-target genes regulated by ER through different mechanisms: pS2, involving direct binding of ER to Estrogen Responsive Element (ERE), cyclin D1 (CCND1), involving Activator Protein-1 (AP-1) and IEX-1 (linked to apoptosis), involving Nuclear Factor Kappa B (NFjB). HER2-over-expressing cells displayed higher proliferation rate, estrogen-independent growth and reduced sensitivity to estradiol and tamoxifen. Furthermore, while the regulation of the CCND1 and IEX-1 genes in HER2 over-expressing cells exhibited consistency with the growth characteristics of these cells, this was not the case for EREdependent gene regulation. Our results point to the IEX-1 gene as a potentially interesting marker of tamoxifen resistance in breast cancer cells. They also suggest that HER2-associated tamoxifen resistance is not likely to be only due to alterations of classical ERE-dependent gene transcription, but also of the activities of other transcription factors co-operating with ERa, such as AP-1 and NFjB, which might thus be considered as potential new targets for breast cancer treatment.

Introduction: Although juvenile hormone (JH) is an important regulator of insect development and reproductive maturation, neither the molecular mechanism of JH action nor JH receptor is known. Met is one of proposed and very probable candidate for this function. It has been shown that Met belongs to the family of bHLH-PAS transcription factors. Their functional activity often is correlated with shuttling between nucleus and cytoplasm according to the masking and unmasking, by interacting partners, signals directing this proteins to nuclear or cytoplasmic compartment of the cell. The first report concerning Drosophila melanogaster Met has informed about localization of this protein in cytoplasm. Later on it has been shown as nuclear protein by in vivo immunostaining of Drosophila tissues and by using chimeric proteins with GFP in S2 cells. Till now, no NLS and NES motifs has never been elucidated for Met. Methods: In order to determine the sequences of nuclear localization signals (NLSs) and nuclear export signals (NESs), series of deletion and point mutants tagged by yellow fluorescence protein (YFP) were prepared by cloning in to EYFP-C1 vector and expressed in mammalian cells. Mithramycin is one of the oldest chemotherapy drugs used for decades. It is a DNA-binding agent and has been found to play a role in the increase of foetal haemoglobin (HbF) in erythroid Burst Forming Units (BFUe) in normal individuals. Specificity protein 1 (Sp1) is a transcription factor involved in gene expression in the early development of an organism. It is generally known as a ubiquitous activator; however increased binding of Sp1 to the c-globin gene promoter upon site-specific cytosine methylation indicates the possible functional role of Sp1 as a repressor of c-globin synthesis. Some correlation has been found between Mithramycin and Sp1 since both seem to bind at GC rich sites on the genome. Activation of the c-globin gene by Mithramycin and repression by Sp1 has lead to the theory that these two may act as antagonists. With the goal to dissect the molecular mechanisms of action of Mithramycin, in the current study we used chromatin immunoprecipitations to identify whether a direct effect was involved in activating the c-globin gene, or if activation was induced through suppression of Sp1. This was assessed by exploring the binding activities of Sp1 in BFUe cells that were not treated and BFUe cells that were treated with Mithramycin. Our results indicate that treatment of cells with Mithramycin reduces or even suppresses the binding of Sp1 on the c-promoter, thus revealing a new mechanism of c-globin gene activation and subsequent production of foetal haemoglobin which may lead to an improvement in the treatment of b-thalassaemia and other haemoglobinopathies.

Design of gene-switches in mammalian cells: can insect ecdysteroid hormones be useful? 3 Intrexon Corp, PA, USA Introduction: The ecdysteroid receptor (EcR) is a ligand-inducible transcriptor factor activated by the insect moulting hormone, ecdysteroids. This nuclear receptor can be exploited for the design of ligandinducible gene regulatory systems (gene-switches) in mammalian cells, which find a variety of biomedical applications, including gene therapy. Methods: To investigate the nature of the ecdysteroid ligand-receptor interactions, we performed multi-dimensional quantitative structureactivity relationship (QSAR) studies, including Comparative Molecular Field Analysis (CoMFA) and four-dimensional (4D)-QSAR analysis, after collecting an extensive set of natural and semi-synthetic compounds and screening their activities using the Drosophila melanogaster BII bioassay.

Results: Significant 3D-and 4D-QSAR models could be generated from the assembled ecdysteroid library. These models were examined in relation to the EcR ligand-binding domain protein structure from the recently solved ecdysteroid-bound EcR crystal structure (1) . Conclusions: While the effectiveness of EcR-based ligand-inducible gene expression systems have been demonstrated in mouse models (2) , further studies are needed to improve the potency and efficacy of ecdysteroids in mammals. Focussing on elucidation of the ecdysteroid structure-activity relationships, this work contributes to improve our understanding of the structural features determining the molecular recognition between the EcR and ecdysteroid ligands, and therefore may be useful for a rational design of novel EcR-based gene-switch actuators. GILZ is an ubiquitous protein whose expression is induced by glucocorticoids in thymocytes and peripheral T-lymphocytes. We showed that GILZ expression is rapidly and transiently induced upon IL-2deprivation of the T-cell line CTLL-2, protecting these cells from the onset of apoptosis. This protection is conferred through inhibition of FoxO3 transcriptional activity, resulting in down-regulation of proapoptotic Bim expression. Nuclear import of FoxO factors follows stress signals such as oxidative stress or growth factor-deprivation, leading to up-regulation of target genes (bim, fasl), whereas nuclear export mainly results from Protein Kinase B phosphorylation. The aim of this work was to determine the mechanism of FoxO factors inhibition by GILZ. First, we showed that GILZ inhibits FoxO1, FoxO3 and FoxO4 transcriptional activities in the acute promyelocytic leukaemia cells HL60 independently of PKB phosphorylation, suggesting that GILZ is a general inhibitor of FoxO factors with an original mechanism of action. We demonstrated by fluorescence microscopy that GILZ is mainly cytoplasmic in HL60 cells and identified a Nuclear Export signal which appears to be functional and Crm-1-dependent. Experiments using cytoplasmic and nuclear GILZ mutants suggested that GILZ cytoplasmic localization is a pre-requisite for FoxO3 inhibition.

Interestingly, we showed that GILZ expression favors a cytoplasmic locolization of FoxO3, avoiding the transcription of its targets genes within the nucleus. GILZ could affect FoxO3 import or export. All together, our findings raise the possibility that deregulated expression of GILZ could lead to a general inhibition of FoxO signalling pathways.

Amorphastilbol enhances adipocyte differentiation in 3T3-L1 preadipocytes through PPAR-gamma transactivation KIST Gangneung Institute, Gangwon-do, KOREA, 2 Kwandong University, Gangwon-do, KOREA, 3 Korea University, Seoul, KOREA Introduction: Peroxisome proliferator activated receptor gamma (PPAR-gamma) is a ligand-dependent transcriptional factor that belongs to the nuclear hormone receptor superfamily and forms a heterodimer with a retinoid X receptor. PPAR-gamma plays a key role in adipocyte differentiation and insulin sensitivity. In the present study, we show that amorphastilbol leads to adipocyte differentiation through the trans-activation of PPAR-gamma in 3T3-L1 cells. Methods: An established luciferase reporter gene assay system was used to test trans-activation of amorphastilbol to human PPARgamma. Fluorescence polarization (FP) ligand binding assay was based on purified recombinant human PPAR-gamma ligand-binding domain and a selective fluorescent PPAR-gamma ligand. EC 50 values (concentrations at which 50% of the fluorescent ligand is displaced) was determined for amorphastilbol by measuring millipolarization values for a series of concentrations. To further characterized the effects of amorphastilbol on differentiation, cellular triglyceride content and GPDH enzyme activity were measured. And we observed the changes of C/ EBP alpha, PPAR-gamma, aP2, adiponectin and resistin mRNA by using RT-PCR and Real-time PCR. Results: Amorphastilbol was determined to be a novel PPAR-gamma agonist because it strongly induced luciferase activity on human PPARgamma and directly bind to PPAR-gamma LBD (EC 50 = 1.26 uM). Amorphastilbol enhances adipocyte differentiation as evidenced by increased triglyceride accumulation, glycerol-3-phosphate dehydrogenase (GPDH) activity. Using RT-PCR and quantitative RT-PCR analyses, we also confirmed that amorphastilbol increased the expression of genes critical for acquisition of adipocyte phenotype. Conclusions: We suggested that amorphastilbol is a novel agonist of PPAR-gamma and may be beneficial for reducing insulin resistance through its potency to regulate adipocyte differentiation.

Abstract Withdrawn PP2B-18 Co-ordinate induction of PPARa and SREBP2 in multifunctional protein 2 deficient mice Introduction: Mice with inactivation of the D-specific multifunctional protein 2 (MFP2), a crucial enzyme of peroxisomal b-oxidation, develop multiple pathologies in diverse tissues already starting in the post-natal period and causing death before the age of 6 months. Methods and results: Gene expression profiling performed on liver of 2-day-old pups revealed up-regulation of PPARa responsive genes in knockout mice. Surprisingly, also genes involved in cholesterol biosynthesis were markedly induced. Real-time PCR confirmed the induction of PPARa target genes and of HMGCR and SREBP2, both involved in cholesterol synthesis, as well in 2-day-old pups as in adults. In accordance, the rate of cholesterol biosynthesis was markedly increased in liver of knockout mice but the hepatic cholesterol concentration was unaltered. In PPARa/MFP2 double knockout mice, upregulations of SREBP2 and HMGCR were less pronounced suggesting a link between PPARa induction and cholesterol synthesis. Conclusion: These data indicate that impaired peroxisomal b-oxidation causes an accumulation of PPARa ligands in the intact animal and a concomitant up-regulation of genes of cholesterol biosynthesis through increased expression of SREBP2. Since the hepatic cholesterol concentration was not different between the genotypes, it appears that the up-regulation was not triggered by a reduced level of cholesterol, neither resulted in increased cholesterol levels.

Dominant negative mutant of CREB inhibits TrkCinduced activation of the nur77 promoter J. Matuszyk and D. Klopotowska L.Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, POLAND nur77 (also termed NGFI-B, TR3, NR4A1) and its family members Nurr1 and Nor-1 are orphan nuclear receptors that play important roles in neuronal differentiation, memory consolidation, stress response, and apoptosis. The promoter region of the nur77 gene contains four near AP-1 (NAP) sites 5'-TGCGTCA. Results of Yoon & Lau [1] supported the hypothesis that JunD, but not CREB, binding to two proximal NAP elements is responsible for induction of the transcription of nur77 in response to nerve growth factor. However, the results of the present study indicate that A-CREB (the dominant negative mutant of CREB) inhibits the activation of the nur77 promoter in response to the activation of TrkC. In conclusion it is suggested that activation of the nur77 promoter in response to neurotrophins requires the co-operation of both CREB and JunD. PP2B-20 Genome-wide location of glucocorticoid receptor binding sites Introduction: Glucocorticoids (GCs) are essential steroid hormones that regulate a variety of physiological processes including growth, differentiation, programmed cell death, glucose homeostasis and protein, lipid and carbohydrate metabolism. GCs exert their functions through the glucocorticoid receptor (GR), a ligand inducible transcription factor that binds to a variety of promoter elements, including glucocorticoid response elements (GREs), and regulates gene transcription in a cell-specific manner. The mechanisms by which GR selectively regulates cell-specific transcription and the global networks regulated by GR are not well established. In the present study we mapped the human GR binding sites on a genome-wide scale. Methods: To identify the global profile of GR binding sites we used chromatin immunoprecipitation (ChIP), with a monoclonal antibody against GR, combined with either hybridization on genomic microarrays covering the entire genome (ChIP-on-chip) or massively parallel sequencing (ChIP-seq). Results: We mapped the genome-wide GR binding sites in immortalized human hepatocytes (IHH) and HeLa cells and identified known and novel cis regulatory elements and target genes in cell-specific contexts. This study revealed the presence of GR binding sites in previously un-explored regions of the genome as well as networks of transcription factors underlying glucocorticoid signalling. Our data demonstrated distinct mechanisms of glucocorticoid-mediated gene regulation and shed light on the global networks modulated by GR. Conclusions: Analysis of GR chromatin occupancy in human genome substantially contributes to our understanding of the mechanisms and molecular networks underlying the diverse biological effects of glucocorticoids.

PP2B-21 CD40 ligation induces immunoproteasome gene expression via the co-ordinated action of NF-jB and of NF-jB mediated de novo synthesis of IRF1 Interferon regulatory factors (IRFs) comprise a family of pleiotropic transcription factors which influence development, immune homeostasis and disease pathogenesis. In this study, we demonstrate that stimulation of CD40, a TNF receptor family member with a pivotal role in adaptive and innate immunity, rapidly induces the expression of IRF-1 in carcinoma cells. Using a combination of small molecule kinase inhibitors, RNA interference and CD40 mutagenesis approaches, we show that p65 (RelA) NF-jB but not MAPK signaling is required for the CD40 ligand-induced up-regulation of IRF-1. Chromatin immunoprecipitation and gel-shift assays demonstrated recruitment of p65 NF-jB to the IRF-1 promoter. When fused to a luciferase reporter gene, the IRF-1 promoter responds to CD40 stimulation whereas mutations which abolish NF-jB binding render it unresponsive. Evaluation of NF-jB pathway components suggests the involvement of TAK1, IKKb and IjBa in IRF-1 promoter regulation. NF-jB and de novo synthesized IRF-1 converge to regulate immunoproteasome gene expression, as evident by the recruitment of both transcription factors to the promoter regions of transporter for antigen processing (TAP)-1, TAP-2, tapasin and the low molecular mass polypeptide (LMP)-2 and LMP-10. Moreover, the RNA interference-mediated knock-down of IRF-1 reduced, whereas inhibition of NF-jB abolished the effects of CD40 on TAP-1 up-regulation. Collectively, these data reveal a novel mechanism of IRF-1 induction by CD40 which ensures that IRF-1 functions concurrently with NF-jB to facilitate immunoproteasome gene transcription.

Inhibition of the ubiquitous transcription factor Yin Yang 1 by phytosteryl ferulates and their therapeutic potentials R. Nagasaka, K. Ohara and H. Ushio Tokyo University of Marine Science and Technology, Tokyo, JAPAN Rice bran oil accepted worldwide contains a lot of phytosteryl ferulates, one of hydroxycinnamic acid derivatives ubiquitously found in plants, compared with other plant oil. We have reported that phytosteryl ferulates inihibited DNA-binding of NF-jB. In this study, we evaluated the effects of phytosteryl ferulates on DNA binding activities of over 300 transcription factors in RAW 264.7 macrophages. It suggested that phytosteryl ferulates would inhibit one ubiquitous transcription factor Yin Yang 1 (YY1) activation. The transcription factor YY1 is known to have a fundamental role in normal biologic processes such as embryogenesis, differentiation, replication, and cellular proliferation. YY1 exerts its effects on genes involved in these processes via its ability to initiate, activate, or repress transcription depending upon the context in which it binds. It is also generally accepted that the transcription factor acts as an initiator of tumorigenesis. Thus, phytosteryl ferulates might prevent metabolic and immunological diseases by NF-jB inhibition and cancer by YY1 repression. The potential clinical significance of the functions will be discussed in this paper, in special the regulation of and the resistance to metabolism and cancer, subsuming complicated cell-cell interactions.

The effects of phytosteryl ferulates on multimeric form of adiponectin secreted from 3T3-L1 adipocytes K. Ohara, R. Nagasaka and H. Ushio Tokyo University of Marine Science and Technology, Tokyo, JAPAN Adiponectin has been postulated to play an important role in the modulation of glucose and lipid metabolisms in insulin-sensitive tissues in mammals. Adiponectin secreted from adipocytes and circulating in blood forms a wide range of multimers from trimers and hexamers to high molecular weight (HMW) multimers, and the ratios among them are closely correlated with insulin sensitivity. Rice bran contains a lot of plant sterols and their ferulic acid esters as compared with other plant oil. It is reported that the phytosteryl ferulates have the LDL cholesterol lowering effect in some mammals. We have recently demonstrated that phytosteryl ferulates enhance adiponectin secretion from adipocytes. In this study, we have investigated the effect of phytosteryl ferulates on the adiponectin multimers secreted from 3T3-L1 adipocytes. Mouse 3T3-L1 cells differentiated to adipocytes were treated with phytosteryl ferulates. The culture media was subjected to SDS-PAGE under non-reducing, non-heat-denaturing conditions, followed by western blotting with anti-adiponectin antibody. Phytosteryl ferulates restrictedly enhanced the HMW multimers secretion of 3T3-L1 adipocytes, while monomer, trimer and hexamer secretions were relatively low, compared to pioglitazone, a PPAR gamma agonist. The results suggest that phytosteryl ferulates might regulate adiponectin multimer formation in part through pathways different from thiazolidine diones. Phytosteryl ferulates might thus prevent and/or ameliorate the type 2 diabetes and its related metabolic syndromes. The family of proteins TSC-22D (Transforming growth factor-beta (TGF-b)-stimulated clone-22 Domain) is constituted by GILZ (Glucocorticoid-Induced Leucine Zipper), TSC-22 (TGF-b)-stimulated clone-22) and THG-1 (TSC-22 Homologous Gene-1). These proteins have both a TSC box and a leucine zipper domain, allowing them to homodimerize or hetero-dimerize. We have shown before that GILZ inhibits FoxO3 activity leading to inhibition of bim expression and apoptosis. As these homologous proteins have the ability to interact, we hypothesized that TSC-22 and THG-1 are able, like GILZ, to inhibit FoxO3 activity. We determined by RT-PCR the expression of GILZ, TSC-22, and THG-1 in different tissues of human origin and in tumour cell lines. THG-1, GILZ, and TSC-22 are present in all the tissues tested. THG-1 mRNA is expressed in all tumour cell lines tested whereas GILZ mRNA and TSC-22 mRNA are deficient in CTLL-2 and Kit 225 T lymphocytes that need IL-2 for their proliferation. The ubiquitous expression of TSC-22D proteins suggests an endogenous role in FoxO3 activity regulation. We then over-expressed TSC-22 and FoxO3 in HL60 cells to measure the transcriptional activity of FoxO3, using a dual-luciferase reporter assay. Results showed that TSC-22 inhibits FoxO3 activity measured using a synthetic IRS promoter composed of three FHRE binding sites. We analysed also the expression of FoxO3 target genes in cells over-expressing TSC-22 or GILZ as a positive control. The following genes were selected: bim, p27, puma, and MnSOD. Preliminary results showed that in CTLL-2 cells, IL-2 deprivationinduced bim was repressed in the presence of TSC-22 or GILZ. Geranylgeranoic acid (GGA) and (R)-2,3-dihydrogeranylgeranoic acid (R-2,3-diGGA) are geranylgeraniol-derived metabolites. We examined their effects on HL-60 cells. The cells were differentiated into neutrophils by the former stimulation like retinoic acid stimulation. In the case of cells stimulated with the latter, neutrophils were not detected, but the formation of lipid droplets was induced. On the other hand, when the cells were cultured in the presence of 0.1% FBS instead of 10% FBS, apoptotic cells were induced not only by GGA stimulation but also with R-2,3-diGGA. In the latter case, when the cells were cultured in the co-presence of a caspase-3 inhibitor, the lipid droplets formation was observed in the cells. These results suggest that GGA and R-2,3-diGGA are extremely different from each other with respect to their effects on HL-60 cells. To further understand the difference, we chemically synthesized several GGA derivatives including 4,8,12,16-tetramethyl-heptadeca-3,7,11,15-tetraenoic acid (homo-GGA). As a result, homo-GGA did not induce the cell differentiation into neutrophils but the lipid droplets formation was induced as observed in the case of R-2,3-diGGA stimulation. In the case of S-2,3-diGGA stimulation, the lipid droplets formation was similarly induced and in this case unusual morphology was observed. These results are discussed in the context of the relationship between structures of GGA derivatives and their effects on cells.

Transcription regulation of the microbial nitril hydratase gene cluster Although the wild-type strain was found to grow on methacrylamide as a sole nitrogen source, the nhpR-disruptant did not grow in the same medium. Furthermore, real-time PCR analysis indicated that the transcription of the eight genes (including nhpR) in the NHase gene cluster was not induced in the nhpR-disruptant on the addition of methacrylamide to the medium, although transcription of these genes in the wildtype strain was significantly induced, demonstrating that nhpR encodes a transcriptional positive regulator in the NHase gene cluster. NhpR was founed to be the first transcriptional regulator of NHase belonging to the XylS/AraC family. The Peroxisome Proliferator-Activated Receptors (PPARs) belong to the nuclear receptor superfamily and are involved in several physiological and pathological processes. Agonists of the a and c subtypes, such as fibrates and glitazones, are currently used to treat patients with metabolic disorders. Therefore, aim of this study was to find novel compounds able to activate both receptors. We analyzed two enantiomeric ureidrofibrate derivates, (S)-1 and (R)-1. By using cell-based reporter assays and co-activator recruitment assays we show that the (R)-1 is a full agonist whereas the (S)-1 is a partial agonist of PPARc. We also provide a molecular explanation for the different behavior as full and partial agonists of PPARc by reporting the crystal structures of PPARc ligand-binding domain respectively complexed with the Rand the S-enantiomer. The analysis of the two crystal structures shows that the different degree of stabilization of the helix 12 induced by the ligand determines its behavior as full or partial agonist. The comparison of the two structures of the complexes with the partial agonist reveals significant differences and is suggestive of the possible coexistence in solution of transcriptionally active and inactive forms of helix 12 in the presence of a partial agonist. Mutation analysis underscores the key role of Gln 286 in the PPARc activity. Gene expression analysis shows that these ligands increase mRNA levels of typical PPARa and c targets. In conclusion, our results identify novel PPARa/c dual agonist and provide the structural basis of their activity on PPARc. Acknowledgements: Supported by a grant from MIUR-COFIN-PRIN2003033405. 

Purine nucleoside phosphorylase gene expression in response to 9-deazaguanine and its derivative in human leukemia and lymphoma cells In insects, ecdysone plays a central role in orchestrating a number of developmental processes. Ecdysone acts through a heterodimeric transcription factor comprising ecdysone receptor (ecr) and ultraspiracle to directly regulate transcription of target genes. Insect ecr gene encodes two or three different isoforms (ecr-A, B1, and B2), which share common DNA binding and ligand binding domains, but differ in N-terminal domains. In Drosophila, ecr-B1 is a key molecule to destruct larval tissues during metamorphosis. Although ecr-B1 has been identified from dipteran, lepidopteran and coleopteran insects, it has not been identified from primitive holometabolous insects, hemimetabolous and ametabolous insects, and thus its functions in these insects remain unknown. To gain insights into molecular evolution of ecr-B1, we performed phylogenetic analysis of ecr-B1 in insects. Sequences comparison of ecr-B1 cloned from various insects including ametabolous and hemimetabolous insects first revealed four conserved motifs at the N-terminal region of ecr-B1. Furthermore, the results suggested that motif-level modification occurred during insect evolution: the N-terminus of holometabolous insect ecr-B1 contains a conserved motif, whereas this motif is lacked in the hemimetabolous insect ecr-B1. Additionally, hymenopteran ecr-B1 contains a conserved N-terminal extension just adjacent to the holometabolous insect-speciifc motif. These motifs might function as binding sites of ecr-B1 isoform-specific cofactors, and the motif-level evolution suggests that modification of ecr-B1 mediated transcriptional regulation occurred associated with the insect evolution. Adipocyte differentiation is a highly complicated process and it includes several signaling pathways. Molecular regulation in the very early stage of adipocyte differentiation is not fully appreciated yet. Several inducible genes at the early stage during pre-adipocyte differentiation have been identified, including the regulator of G-protein signaling 2 (RGS2) gene. RGS2 is a member of the RGS protein superfamily, which primarily acts as a negative regulator in G-protein signaling by enhancing the intrinsic GTPase activity of the G alpha subunit. The aim of this study was to clarify the precise RGS2 induction profile and to evaluate the important DNA transcriptional element(s) mediating RGS2 expression in the 3T3-L1 preadipocyte differentiation. RGS2 mRNA was demonstrated to elevate 1 hour after differentiation initiation and its level remained high until the late stage of differentiation. Meanwhile, the putative 5'-flanking sequence [~3000 base pairs (bp)] of the RGS2 gene was isolated and fused with a luciferase reporter gene. With transient transfection assays, RGS2 promoter activity was provoked 12 hour after inducing differentiation. A sequential deletion and site-directed mutagenesis strategies were adopted to define the critical elements in the RGS2 promoter sequence. The RGS2 promoter could be reduced to 80 bp of the 5'-flanking sequence with the retention of full inducibility by differentiation stimuli. Mutation of a Sp1 site within the 80 bp of RGS2 promoter region significantly blocked the promoter activity. Taken together, our data suggest that RGS2 expression was immediately induced after the initiation of 3T3-L1 preadipocyte differentiation and it remained consistently high through the differentiation process, and the Sp1 site within the 80 bp region appeared as an essential regulatory element for transcriptional activation of RGS2 gene. showed there is also an ARF tumour suppressor pathway that is p53 independent. The ARF protein is localized to both the nucleolar surface and the nucleoplasm. We have analysed the nucleolar protein dynamics of the ARF tumour suppressor pathway using mass-spectrometry-based organellar proteomics and stable isotope labelling, i.e., SILAC. We performed a quantitative analysis of the nucleolar proteome from a human ARF inducible model cell line, called NARF2, and compare this with another ARF inducible cell line, called E6, which lacks functional p53. NARF2 is a stable cell line established from U2OS human osteosarcoma cells. Endogenous ARF expression is prevented in U2OS cells by hyper methylation of the ARF gene promoter region. However, these cells also contain an exogenous, inducible copy of the wild type ARF gene whose expression can be induced in NARF2 cells by addition of IPTG. The E6 cell line was established from the NARF2 cells and expresses the adenoviral E6 protein that deactivates p53. We will present data showing a time course of ARF induction where we use SILAC MS to characterize the responses of over a thousand nucleolar localised proteins, including proteins whose response to ARF is independent of p53.

PP2B-37 Differential expression of hormone receptor-like in 38 (HR38 ) in the honeybee brain depending on the caste differentiation and division of labor of the workers The European honeybee (Apis mellifera L.) is a eusocial insect and the females differentiate into two castes: queens and workers. Workers are sterile and perform various behaviors to maintain colony activities. Young workers (nurse bees) nurse their brood, while older ones (foragers) forage for nectar and pollen. However, the molecular mechanisms underlying the social behavior still remain obscure. To analyze the mechanisms, we used the cDNA microarray to identify a candidate gene, the honeybee homologue of Hormone Receptor-like in 38 (HR38), which encodes an orphan nuclear hormone receptor that mediates ecdysone-signaling. Quantitative RT-PCR revealed that HR38expression is higher in forager brains, when compared to nurse bee and queen brains. Furthermore, HR38-expression was enriched in a subset of mushroom body neurons, a higher center in the insect brain, suggesting its role in expression of forager-specific behaviors. In mosquito, HR38 inhibits transcription of vitellogenin (Vg) gene in the fat body before the blood intake. In honeybee, the hemolymph Vg titer elevates in queens and nurse bees, but not in foragers. It will be interesting to test whether HR38 co-operatively inhibits transcription of the Vg gene in the fat body in foragers. Bacterial LPS has been identified as a major cause of septic shock and stimulates macrophages to produce inflammatory cytokines such as IL-6 and TNF-a. We previously demonstrated the interaction between NF-jB and STAT1 in endotoxin shock model mice, however, it is not fully elucidated the mechanism of the induction of the NF-jB/STAT1 complex. Here we studied the synergistic role of NF-jB and STAT1 in IL-6 production, especially from the viewpoint of phosphorylation. Raw264.7 macrophage cells were stimulated with LPS, and electrophoretic mobility shift assays (EMSA) and the luciferase reporter assay were performed using NF-jB specific probe and reporter plasmid to characterize functionally the in vitro DNA binding. These demonstrated that LPS induced NF-jB activation and IL-6 promoter activity, which are synergistically enhanced by over-expressed STAT1alpha. Furthermore, over-expressed STAT1 with mutation on tyrosine 701 (STAT1mY) also enhanced LPS-induced NF-jB activation. Together with these results, the cross talk between NF-jB and non-phosphorylated STAT1 complex play an important role for the induction of inflammatory cytokine, which causes endotoxin shock. Parkinson's disease (PD) is a common neurodegenerative disorder, characterized by the progressive loss of dopaminergic neurons in the substantia nigra. Even though the cause of PD remains to be answered, several lines of evidence strongly suggest the involvement of oxidative stress and mitochondrial dysfunction. The major mitochondrial defect in PD appears to be associated with inhibition of respiratory chain complex I activity. 1-methyl-4-phenylpyridinium ion (MPP+) has been shown to selectively and potently inhibit complex I of the mitochondrial electron transport chain and induce a syndrome closely resembling PD in cellular model. Okadaic acid is a polyether compound isolated from a black sponge, Halichondria okadai, and is a specific inhibitor of protein phosphatase 1 and 2A. In this study, we investigated the protective effects of okadaic acid on MPP+ induced apoptosis in human neuroblastoma SH-SY5Y cells. Treatment of MPP+ induced the loss of cell viability, condensation and fragmentation of nuclei, which was associated with the elevation of ROS level, the increase in Bax/Bcl2 ratio, and the activation of caspase-3. These MPP+ induced phenotypes were reversed by okadaic acid. These results suggested that the protective effects of okadaic acid on MPP+ induced cytotoxicity and okadaic acid might provide a useful therapeutic strategy for the treatment of progressive neurodegenerative disease. Introduction: This report describes a high-level expression of b-glucosidase gene from Bacillus halodurans in E. coli using T7 expression system, and the effect of codon usage on folding of the product.

Methods: The b-glucosidase gene encoding 447 amino acids, found in the complete genome sequence of B. halodurans C-125 retrieved from NCBI database was amplified by PCR using gene specific primers. The amplicon was ligated into pET-22b and expressed in E. coli BL21-CodonPlus at different temperatures. The effect of secondary structure on translational efficiency was studied by introducing silent mutations in the initial codons of the 5¢-end region.

Results: Native b-glucosidase gene was expressed to over 40% of the total E. coli cellular proteins in soluble form at 37°C, when induced with IPTG or lactose. Intriguingly when the +3 codon TCG in the initial coding sequence 5¢-ATG GTG TCG -3¢ was changed to TCA most of the expressed b-glucosidase was produced at 37°C in insoluble form, although the expression level was quite similar to that in the case of the native sequence. However, when the E. coli transformed with the mutated construct was cultivated at 18°C most of the expressed enzyme was produced in the soluble form. The rate of the protein synthesis at 37°C seems to be too rapid to allow it to fold to a soluble active form in the cells transformed with the mutated construct.

Conclusion: The free energy value (DG°) of the secondary structure in the early part of the coding mRNA, as determined by the mfold webserver, and also perhaps the more preferred serine codon at the +3 position, are the likely factors to explain the above-mentioned results.

Pre-and post-translational study of the CD40 system in children and adolescents with type 1 diabetes mellitus (T1DM): evidence for differences in CD40 mRNA isoforms expressed by peripheral blood mononuclear cells (PBMCS)

Methods: PBMCs'-CD40 of 35 uncomplicated T1DM-patients and 10 healthy controls (HCs) was measured by immunoblotting and CD40 mRNA isoform expression by semiquantitative RT-PCR (and confirmed by sequencing). Plasma-sCD40 was measured by ELISA. CRP, apoB, HbA1c, LDL-C and Lp(a) were estimated per subject.

Results: Patients' PBMCs expressed higher CD40 than HCs (P = 0.082).

T1DM-females showed significantly higher PBMCs'-CD40 than T1DMmales (P = 0.037 Methods: Rat osteoblasts prepared from neonatal rat calvaria were pretreated with low concentrations of nitric oxide (NO) and then exposed to oxidative stress. Cell viability and alkaline phosphatase activity were assayed by colorimetric methods. Oxidative stress, DNA fragmentation, apoptotic cells, translocation of Bax and Bcl-X L , and the mitochondrial membrane potential were quantified using flow cytometry. Analyses of Western and RT-PCR were carried out to evaluate the biosyntheses of Bcl-X L and Bax protein and mRNA. EMSA assay and RNAi were analyzed to determine the roles of GATA-3 in osteoblast survival.

Results: Pretreatment of rat osteoblasts with NO could protect osteoblasts from oxidative stress-induced apoptotic insults. Administration of NO significantly increased the syntheses of Bcl-X L mRNA and protein and its translocation from the cytoplasm to mitochondria. In comparison, NO pretreatment increased Bax protein production and its translocation without affecting the RNA synthesis. Pretreatment with NO increased GATA-3 translocation from the cytosol to nuclei and its binding to the consensus DNA-binding elements. Knockdown of GATA-3 translation significantly decreased NO-involved protection from oxidative stress-induced apoptotic insults.

Conclusions: This study shows that GATA-3 plays a critical role in transducing survival signals in rat osteoblasts through regulation of anti-apoptotic bcl-x L gene expression.

Twelve basic amino acids in a row tend to induce translation arrest, followed by endonucleolytic cleavage and proteasome-mediated nascent peptide degradation in yeast L. Dimitrova 1 , K. Kuroha 1 , T. Tatematsu 2 and T. Inada 1,2   1 Nagoya University, Nagoya, JAPAN, 2 JST, PRESTO, Tokyo, JAPAN Poly (A) tail is an important factor in mRNA stability and translation initiation. Another significant role of poly (A) is its involvement in nonstop mRNA quality control where its translation in a poly Lys sequence leads to translation arrest and destabilization of the protein product (1) . In order to study in detail the mechanism by which poly (A) regulates the above-mentioned events we inserted sequences encoding 12 consecutive amino acids (Lys, Arg, Phe, Glu) between two genes (the first lacking a stop codon). Translation arrest was observed only in the case of poly-Lys and poly-Arg and it was followed by degradation of arrest products by the proteasome. Moreover, deletion of NOT4 ubiquitin ligase, a member of the multifunctional Not-Ccr4 complex, led to significant increase of the arrest product which level remained unchanged when a proteasome inhibitor was added. Northern blot analysis suggests that ribosomal arrest might be followed by an endonucleolytic cleavage at a site upstream to the insertion, with the upstream fragment being rapidly digested and the downstream fragment being blocked by the ribosome stalled at its 5¢end. In conclusion, the translation of positively charged amino acids in a row, induces translation arrest, endonucleolytic cleavage and Not4-mediated nascent polypeptide degradation by the proteasome. This mechanism might be another important step utilized by the cell for the control of translational rate and protein level, respectively. Introduction: During translation initiation, the m 7 GpppG structure (CAP) at the 5¢ end of mRNAs is recognized by eIF4E. Through its interaction with eIF4G the eIF4F complex is formed and recruits the ribosome to the mRNA. Two antigenically distinct eIF4F complexes exist in plants. Early reports evidenced differential expression patterns and CAP-binding affinities for these complexes in different plant species. Particularly, eIF(iso)4F is most abundant in maize non-germinated seeds, whereas both, eIF4F and eIF(iso)4F, are equally present in germinated seedlings. Methods: We tested the in vitro translation activity of eIF(iso)4F and eIF4F on the set of stored mRNAs (non-germinated seeds) and newly transcribed mRNAs (24-hour-germinated seeds). Further, we identified novel components of each complex by mass spectrometry (LC/MS/ MS). Results: eIF(iso)4F accounts for most of the translation activity using as substrate the stored mRNA pool, whereas both complexes are active in translating mRNA from 24-hour-germinated seedlings. The heat shock protein HSP101 was identified in the complexes from non-germinated, but not from germinated seedlings. Lipoxigenase LOX1, containing the conserved YXXXXLU. eIF4E binding motif, was identified in complexes from both 0 and 24-hour-germinated seedlings. Initiation complexes resolved through sucrose gradients revealed that HSP101 preferentially associates with RNPs and 48S complex. In addition, It co-elutes from m 7 GTP-Sepharose column along with the eIF(iso)4F complex.

Conclusions: Novel components of the cap-binding complexes from maize non-germinated and germinated seedlings were identified. HSP101 and LOX1 may contribute to selective eIF(iso)4F-mediated translation of the stored mRNA pool.

Introduction: Environmental stresses, among others heavy metal loading, produce multiple cellular changes that ultimately affect protein structures and functions. To protect against the damage induced, cells respond to toxic metals via activation of metallothionein (MT) gene expression. This induction is mediated by transcription factor MTF-1 (Metal-responsive Transcription Factor), a zinc-dependent MRE (Metal Responsive Element) binding protein. Beside MTs, important target genes of MTF-1 are the Zn-transporter-1 (ZnT-1), gamma-glutamylcysteine synthetase heavy chain (c-GCS hc ), a-fetoprotein (AFP), tear lipocalin, placenta growth factor (PIGF). Methods: The expression pattern of the MTF-1 gene was followed in several tissues (e.g. liver, kidney, muscle, brain and heart) with reverse transcription coupled polymerase chain reactions (RT-PCR).

Results: Under physiological condition the highest mRNA level was found in the brain, comparable to that of b-actin (65%), slightly less in the heart (45%), even less in the muscle (35%). The lowest level was found in the kidney and liver, 25% and 18% of the b-actin mRNA, in the organs most involved in detoxification of heavy metals. In addition, MTF-1 expression was measured under different stress treatment, cold shock and Cd 2+ exposure in liver and in two isolated brain region of carp.

Conclusions: Basal MTF-1 expression showed tissue specificity and under different stress conditions the expressions altered in stress-and tissue-specific manner. Concentration-sensitive sodium channels (Na C ; c: concentration) are present in lung, dorsal root ganglia, peripheral nerve, and circumventricular organs. Na C expression during mouse development in lung markedly changes, but its involvement in physiological roles and the mechanism of expression regulation is not yet known. In this study, we explored expression regulation of Na C in mouse lung. Na C mRNA and protein had low expression at mouse embryonic day (E) 15 in lung. Na C mRNA expression strongly increased from E17 and was highly sustained until mice were mature. On the other hand, although Na C protein level exhibited a similar pattern to Na C mRNA expression between E15 and 5 weeks of age, it decreased from 6 weeks of age unlike the pattern of Na C mRNA expression. The drop in Na C protein from 6 weeks of age to adult suggested that Na C was post-transcriptionally regulated in developmental stages of mouse lung. 3¢ untranslated region (3¢-UTR) of mouse Na C mRNA is 2-kb length containing several poly A sites and AU rich elements that are bound by RNA binding proteins. To explore whether Na C 3¢-UTR regulates Na C protein expression, we performed luciferase reporter gene assay using A549 lung cancer cells that were transfected with vectors containing various length of Na C 3¢-UTR. As a result, luciferase activity was suppressed in Na C 3¢-UTR-dependent manner. Na C 3¢-UTR may be involved in expression regulation of Na C during mouse development in lung.

Identification and analysis of genes that are expressed strongly in the optic lobes in the honeybee brain Introduction: The honeybee (Apis mellifera L.) has excellent visual processing capacities and exhibits various exquisite behaviors based on the visual sense. In the honeybee brain, visual information is processed in the visual center (optic lobes; OLs), which has three layer structures (lamina, medulla and lobula). Little is known, however, about the molecular bases that characterize neural circuits of the OLs. With the aim to identify candidate genes involved in visual information processing, we searched for genes that are expressed strongly in the OLs in the honeybee brain. Methods: We used cDNA microarray to search for genes expressed strongly in the OLs, when compared with other brain regions. Expression patterns of the candidate genes were investigated using in situ hybridization. Results: Forty-five candidate clones were identified by cDNA microarray. In situ hybridization revealed that two of them have unique expression patterns in the honeybee brain. The first one was identified as a homologue of misexpression suppressor of KSR2, which is involved in RAS/MAPK pathway in Drosophila, and was expressed strongly in a cluster of neurons that are located dorsally in the middle layer (medulla) of the OLs. The second one encoded a novel protein with an arrestin domain, and the expressing cells were scattered in the outer layer (lamina) of the OLs, whereas they were spread in the inner layer (lobula).

Conclusions: These findings suggest that the honeybee OL neurons that comprise layer structures can be classified into some clusters and/ or cell populations that have distinct signaling pathways.

Cytoplasmic polyadenylation in translational regulation during meiotic maturation of porcine oocytes P. Karabinova and M. Kubelka Institute of Animal Physiology and Genetics, Libechov, CZECH REPUBLIC Introduction: Meiotic maturation of mammalian oocytes is controlled by time-specific translation of essential mRNAs. The crucial features of this translational regulation are masking/unmasking of mRNAs related with their cytoplasmic polyadenylation. Elongation of poly(A)-tail enables interactions of 3¢UTR-binding proteins and factors associated with 5¢UTR of mRNA and thus initiation of translation. Methods: Specific antibodies were used to observe the expression levels of factors involved in cytoplasmic polyadenylation and initiation of translation -cytoplasmic polyadenylation element-binding protein (CPEB), Aurora-A, poly(A)-binding protein (PABP), PABP-interacting proteins 1 and 2 (Paip1, 2), Maskin -during in vitro maturation of porcine oocytes. For analysis of interactions between 3¢and 5¢UTRbinding proteins, the 7-methyl GTP-Sepharose pull down assay was used.

Results: All studied proteins are expressed during in vitro maturation of porcine oocytes, while expression patterns of PABP, CPEB and Aurora-A differs during the progress of meiosis. The interaction of PABP, Paip1 and Maskin with 5¢UTR-binding proteins occurs during all the crucial stages of meiotic maturation. Conclusions: Our results imply that the interaction between 3¢and 5¢UTR mediated by PABP occurs during meiotic maturation of porcine oocytes, while translation of specific mRNAs is likely inhibited by binding of the inhibitory protein Maskin to the cap-binding protein eIF4E. Early activation of CPEB by Aurora-A is infirmed, as Aurora-A is activated later during meiosis. Introduction: Endometriosis defined as the present of endometrial gland and stroma with in the pelvic peritoneum and other extrauterine site, and it has been well characterized as an estrogen dependent disease. The diagnosis of peritoneal endometriosis at the time of laparoscopy is often made by the observation typically black lesion and numerous subtle appearance of peritoneal endometriosis, atypical lesions. Recently, increasing attention has been drawn atypical form of endometriosis. It has been reported that aromatase is expressed highly in endometriosis and is stimulated by prostaglandin E 2 (PGE 2 ). This result in local production of estrogen, which induces PGE 2 formation and make a positive feedback. The aromatase (CYP19) is well known as a key enzyme specifically involved in the conversion of C19 steroid to estrogen and cyclooxygenase-2 (COX-2) is the key enzyme in the conversion of arachidonic acid to PGE 2 . The objective of this study was to compare he aromatase and cyclooxygenase-2 (COX-2) expression in two different form of peritoneal endometriotic implants, typical and atypical endometriosis. Methods: Normal peritoneum (n = 18), typical (n = 6) and atypical (n = 7) tissues were collected from 21 women with endometriosis. To compare aromatase and COX-2 expression, we quantified expression levels using real-time PCR and immunohistochemical technique.

Results: Aromatase was expressed in a higher in typical endometrial peritoneum but COX-2 was significantly higher in atypical endometrial peritoneum than normal peritoneum and typical endometrial peritoneum.

Conclusions: It suggests that typical endometriosis is more advanced form of endometriosis and atypical endometriosis could be more associated with PGE 2 synthesis and active painful lesions. Methods and Results: Macrophage-like J774 cells were stimulated with LPS (100ng/mL) for 0, 1, 2, and 4 hour. After separation of polysomal, free mRNP, and totall cellular fractions from the cells, their mRNA profiles were investigated using Affymetrix GeneChips. Comparison of the profiles of the fractions suggested total 533 of 19 791 transcripts were translationally modulated after LPS stimulation. Surprisingly, gene-ontology analysis implied that the translationally suppressed genes significantly contained those for the mitochondrial respiratory chain. In fact, RNA blotting analysis represented LPS influenced abundance of transcripts for components in the complexes I, IV and V in free mRNP and polysomal fractions. Moreover, LPS also decreased amounts of total and metabolically labeled COX5a in complex IV without any change of its mRNA level in total cellular fraction. In accordance, the total amounts and activities of complexes I and IV were attenuated by LPS stimulation, followed by decrement of intracellular ATP level. The LPS-mediated attenuation of respiratory chain was not restored by a nitric oxide synthestase inhibitor L-NAME.

Conclusions: Translational suppression critically participates in the LPS-mediated attenuation of mitochondrial oxidative phosphorylation in a nitric-oxide-independent manner in J774 cells. Biochemical analyses of the activities of yeast ribosomes carrying sitedirected mutations in 18S rRNA reveal that helices h18, h34 and h44 of 18S rRNA are crucial in protein synthesis. Mutations G517A (rdn2) and C526A (rdn12A) in helix h18 displayed opposite translational phenotypes; rdn2 caused hyperaccuracy and resistance to paromomycin, an aminoglycoside antibiotic that binds to the ribosomal A-site. In contrast, rdn12A caused infidelity, sensitivity to paromomycin and less efficient enzymatic translocation in the presence of cycloheximide. In helix h34, C1054A (rdn1A) caused infidelity and sensitivity to paromomycin, halved peptidyltransferase activity, an activity of the large ribosomal subunit, and caused less efficient enzymatic translocation; C1054U (rdn1T) caused hyperaccuracy but had no effect on peptidyltransferase activity. Apparently, different substitutions at the same position (C1054) may cause opposite effects. Finally, two mutations in helix h44 of yeast 18S rRNA were investigated. The first, A1491G (rdn15), creates in yeast the same C1409-G1491 base pair as in Escherichia coli and has behaved as an antisuppressor in genetic studies. Ribosomes from rdn15 were errorrestrictive but their peptidyltransferase activity remained unchanged. The second, U1495C (rdnhyg1), was initially isolated as a hygromycin resistant mutation in Tetrahymena thermophila. Ribosomes carrying rdnhyg1 were slightly error prone; they did not affect catalytic activity, but affected translocation, confirming the importance of nucleotide 1495 in the communication between the two ribosomal subunits.

In the presence of paromomycin, the error-restrictive rdn15 became highly error-prone and sensitive to this antibiotic, while mutation rdnhyg1 was relatively resistant. In conclusion, several helices of 18S rRNA participate in the fine-tuning of ribosomal activity.

Upregulation of heme oxygenase 1 as an adaptive response to acrolein in human umbilical vein endothelial cells PP2C-21 c-myc and MDR1 mRNA CRD-BP/IMP1 recognition elements are involved in the regulation of translation CRD-BP/IMP1 is an oncofetal RNA binding protein whose aberrant expression in various malignances associates with aggressiveness. Target sequences of this protein have been identified in a handful of coding and non-coding RNAs, some of them playing a pivotal role in carcinogenesis. The available data indicate that CRD-BP/IMP1 can affect more than one aspect of the post-transcriptional processes of its RNA targets on a transcript and/or cell-type dependent manner. To further investigate the CRD-BP/IMP1 mode of action, the post-transcriptional effects of two cis elements recognized by this protein i.e. the ones present in the coding regions of c-myc and MDR1 (MultiDrug Resistance1) mRNAs, were tested. Fusion of these elements, in frame, within the coding region or in the 3¢Ú TR of a reporter luciferase gene, were constructed and transiently transfected in the 293T cell line. Luciferase activity and protein levels, as well as luciferase mRNA levels and stability were determined. The normalized luciferase activity/ mRNA ratio was used as measure of translatability. Both elements when placed either in frame within the coding region or in the 3¢UTR, had little or no effect upon the reporter gene mRNA stability. On the other hand, when placed within the coding region, the c-myc element caused a 50% decrease in translatability while the MDR1 element nearly annihilated translation. On the contrary, these two elements increased reporter gene translatability when present in the 3¢UTR.

These data indicate that both the c-myc and MDR1 CRD-BP/IMP1 binding elements play a role in the regulation of translation of both genes.

Analysis of physiological state of workers in queenless colonies of the European honeybee T. Nakaoka, H. Takeuchi and T. Kubo University of Tokyo, Tokyo, JAPAN Introduction: The honeybee (Apis mellifera L.) is a eusocial insect and the females differentiate into two castes: queens (reproductive caste) and workers (labor caste). In addition, the workers shift their labors from nursing their brood (nurse bees) to foraging for nectar and pollen (foragers) according to their age after eclosion. When the queen is lost from the colony, however, some workers become 'laying workers' whose ovaries develop to lay eggs. Here we investigated whether the physiology of laying workers is more similar to that of nurse bees or foragers by examining the hypopharyngeal gland (HPG) and the hemolymph vitellogenin titers. Methods: In normal colony, nurse bees have well-developed HPGs that synthesize 'major royal jelly proteins' and high vitellogenin titers, whereas foragers have shrunken HPGs that synthesize 'alpha-glucosidase' and low vitellogenin titers. We measured the size of HPG acini and analyzed protein expression pattern of HPG and hemolymph in queenless workers using SDS-PAGE and Western blotting. Results: In queenless colonies, workers with developed ovaries (putative laying workers) tended to have more developed HPGs and synthesized major royal jelly proteins, whereas workers with shrunken HPGs tended to have undeveloped ovaries and synthesized alpha-glucosidase. Furthermore, the workers with developed ovaries had higher vitellogenin titers than nurse bees, whereas those with undeveloped ovaries had lower vitellogenin titers.

Conclusions: These findings indicate that the physiology of laying workers is similar to that of nurse bees, but opposite that of foragers, suggesting close relation between mechanisms that determine physiology of laying workers and nurse bees. European honeybee (Apis mellifera L.) is a social insect and the workers show highly advanced social behaviors, such as age-related division of labor (age-polyethism) and usage of dance communication. The neural/molecular basis of the honeybee social behaviors, however, remains largely unknown. Honeybee brain is characterized by a welldeveloped higher-order center, mushroom bodies (MBs), whose proportion in the worker alters depending on the division of labor. With the aim to understand the neural/molecular basis underlying the honeybee social behaviors, we performed proteomic analysis to search for proteins expressed selectively in the MBs in the worker brain. Four protein spots were detected in a MB-selective manner by 2-dimentional electrophoresis. These were identified, by LC-MS/MS analysis, as farnesoic acid O-methyltransferase (FAMeT) and juvenile hormone diol kinase (JHDK), which are the enzymes involved in JH biogenesis and metabolism, respectively, and two calcium-binding proteins: calreticulin and reticulocalbin. In situ hybridization analysis revealed that the expression of famet is restricted in the cells localized outer bottom of the MB calyx, while that of jhdk is inside of the MB calyx. Since JH accelerates division of labor of the workers, these results suggest that MBs might be a target of JH effect and different MB cell types are involved in JH biogenesis/metabolism. In addition, the MB-selective expression of calreticulin and reticulocalbin suggest that neural plasticity based on the calcium-signaling is enhanced in the MB neurons, which might be related to the brain functions involved in worker behaviors. Aberrant activation of human epidermal growth factor receptor 2 (HER2) in breast tumors results in increased metastasis and reduced survival. Trastuzumab, a humanized monoclonal antibody to HER2 is indicated for patients whose tumor exhibit an amplified copy number and/or overexpression for HER2. The mechanisms of trastuzumab action are not fully understood and resistance to this treatment is frequent. Trastuzumab effect is related in vitro to the PI3K/Akt pathway, which is linked to translational control. Additionally, in some reports activation of HER2 pathway is associated with translation of several genes including CXCR4, VEGF and c-src. Our goal is to evaluate the role of trastuzumab in the regulation of protein synthesis in HER2overexpressing breast tumor cell lines. We demonstate that the trastuzumab-dependent inhibition of the PI3K/Akt pathway is correlated to the phosphorylation state of a major translation initiation regulator, the eIF4E-BP1 protein. Moreover, trastuzumab treatment leads to a sequestration of eIF4E by its inhibitor eIF4E-BP1, as determined by cap affinity pull-down assays. This results in the disruption of the translation initiation eIF4F complex and therefore in the translational deregulation of several genes. Translationally-regulated genes are identified by using real-time qPCR and Northern Blot on mRNAs isolated from polysomal fractions. In addition, we showed that eIF4E-BP1 phosphorylation was associated with the response to trastuzumab treatment in human breast tumor specimens, indicating that it can be a diagnostical target for treatment orientation. Comprehension of trastuzumab translational regulation and identification of target genes may help us to design therapeutic strategies based on translation mechanisms. a variety of functions inside cells, such  as transcriptional regulation, chromosome replication, protein translocation and degradation, apart from their more traditional role in protein expression. These RNA molecules that never get translated into proteins are called non-coding RNAs (ncRNAs). Their increasing importance calls for an increasing demand for bioinformatics tools that will allow us to detect them. We perform a genome-wide computational screening for conserved structures and transcriptional signals in Corynebacterium glutamicum, a gram-positive bacterium, important in biotechnology, due to its glutamic acid production capabilities. With this search we test the functionality and reliability of ncRNA finding tools.

Methods and Results: The role and functionality of ncRNAs relies on their 2D structure, therefore from an evolutionary point of view it is important to look for structure conservation as well as sequence. We created whole genome alignments of C. glutamicum strains and closely related species and we performed a genome-wide scan for ncRNA using RNAz, a relatively new ncRNA finding algorithm. We also use a more direct approach by applying INFERNAL on the C. glutamicum.

The results of both methods were compared. We also used bioinformatic tools such as PromScan and TransTerm to search for promoters and terminators, respectively. We identified approximately 300 potential ncRNAs. Some predictions had already been registered in ncRNAs databases while others show concordance with microarray data. Discussion: We suggest a bioinformatics method for finding non-coding RNAs in bacteriaof the genus Corynebacterium. The results are promising and the method could be in the future applied to other bacterial species. Human papillomaviruses (HPVs) are involved in the pathogenesis of cancer of the cervix (CaCx). HPVs are also associated with a subset of squamous cell carcinoma of the head and neck (SCCHN). Interactions between viral and cellular genes are important for the progression of HPV-associated cancers. We analyzed the expression of cellular miR-NAs in HPV-16 positive cervical cell lines and tissues. Using the Ambion MirVana microarrays, we found that 3 miRNAs were overexpressed and 24 underexpressed in cervical cell lines containing integrated HPV-16 DNA compared to the normal cervix. Interestingly, microRNA-218 (miR-218) was specifically underexpressed in cell lines, cervical lesions and cancer tissues containing integrated HPV-16 DNA as compared to both the HPV-negative cell line C-33A and the normal cervix. Expression of the HPV-16 E6 oncogene in transfected cells reduced miR-218 expression, and conversely, RNA interference of E6/ E7 oncogenes in an HPV-16 positive cell line increased miR-218 expression. One of the predicted targets of miR-218 is the epithelialspecific gene LAMB3 which is involved in cell migration and tumorigenicity. The 3¢ untranslated region of LAMB3 mRNA contains a putative target site of miR-218, and exogenous expression of miR-218 in HPV-16 positive cell lines decreased LAMB3 expression at both RNA and protein levels. We have also identified differentially expressed miRNAs in HPV-positive and HPV-negative SCCHN cell lines and tissues. Our findings demonstrate specific regulation of cellular miRNAs in the presence of HPVs and its oncogenes, and our results may contribute to a better understanding of the molecular mechanisms involved in HPV-mediated carcinogenesis.

Real-time RT-PCR quantification of mature microRNA let-7a expression in non-small cell lung cancer (NSCLC) In the early stages of Drosophila spermatogenesis the microRNA machinery is necessary for germ stem cell maintenance. The stem cell daughters divide mitotically before entering meiosis to produce mature sperm. These mitotic sperm progenitors produce mRNAs that remain untraslated until the onset of meiosis, an event in which miRs could potentially play a role. We are studying whether particular miRs act during Drosophila spermatogenesis and reveal their expression pattern. Methods: Transgenic Drosophila lines carrying EGFP-3¢UTR fusions act as sensors for miRs that recognize the specific 3¢UTR. We studied the expression of sensors responsive to 4 specific miRs in larval and adult testes.

Results: Based on sensor analysis, miR-2 and miR-13 (K-box family) as well as miR-7 and bantam were found to be enriched at the apical testis tip. This area contains somatic niche cells, germline stem cells, as well as their mitotic progeny. As sperm progenitors progress towards meiosis, EGFP expression is strongly induced, suggesting a downregulation of all four miRs.

Conclusions: A set of miRs was found to act during the early spermatogenesis stages and silence EGFP sensor transgenes. However, as progenitors enter meiosis, these miRs appear to be inactivated.

Micro-RNAs expressed in human microvascular endothelial cells Ribonuclease P (RNase P) is a ubiquitous enzyme that endonucleolytically cleaves all precursor tRNA transcripts to produce the mature 5¢ ends. RNase P from Dictyostelium discoideum is comprised of an essential RNA and seven probable protein subunits (dPop1, DRpp14, DRpp20, DRpp25, DRpp29, DRpp30 and DRpp40) as revealed by sequence similarity. We present herein the cloning, overexpression and preliminary characterization, as well as molecular modeling of DRpp29. Purified recombinant DRpp29 was used for the production of rabbit polyclonal antibodies. Immunobiochemical analysis revealed that DRpp29 indeed participates in the enzyme's architecture. In order to address this protein's role, we investigated DRpp29-RNA interactions within the ribonucleoprotein complex. Therefore, we employed electromobility shift assays, but we could not detect specific binding of DRpp29 either to the RNase P RNA or the pre-tRNA substrate. Reconstitution experiments of DRpp29 and M1 (RNA subunit of E. coli RNase P demonstrate that this hybrid complex is sufficient for 5?cleavage of pre-tRNAs in agreement to the exposed positively charged surface area on the modeled DRpp29 structure. Reconstitutions assays performed with increasing concentrations of MgCl 2 demostrated that the optimum ionic requirement was at 10 mM Mg 2+ . Moreover, kinetic analysis of the heterologous holoenzyme was performed and the kinetic constants were estimated. Interestingly, DRpp29 is not sufficient to form catalytically active complexes with the RNA subunit from D. discoideum. Hydrophobic cavities revealed on the surface of this protein suggests its potential implication in protein recognition. It will be our future goal to understand the role of this protein in the RNase P enzyme functional core.

Studies on ribonuclease P (RNase P) from the ethanologenic bacterium Zymomonas mobilis Ribonuclease P (RNase P) is a ubiquitous enzyme that endonucleolytically cleaves all precursor tRNA transcripts to produce the mature 5¢ ends. Most forms of RNase P enzymes are ribonucleoproteins. RNase P activity has been identified in bacteria, archaea, and eucarya and in subcellular organels. In vitro, the RNA subunit of the bacteria as well as the relevant subunit of some archaea is catalytically active in the absence of protein at high ionic conditions. Here we report, the partial purification and characterisation of RNase P from the ethanologenic bacterium Zymomonas mobilis. The enzyme was purified using anion exchange chromatography. pH and temperature optima were determined as 8 and 37 o C respectively. The holoenzyme shows optimal activity in the presence of 100 mM NH 4 Cl and 25 mM MgCl 2 . Inhibition of Z. mobilis RNase P by proteinase K and micrococcal nuclease strongly indicates that the activity requires both protein and RNA components. We also characterized both the RNA and the protein subunit of the holoenzyme. The gene which encodes the RNA subunit of the bacterial RNase P was cloned in the appropriate vector and expressed in Escherichia coli cells. The expected catalytic activity of the RNA subunit was explored by screening of temperature, pH, and ionic requirements. Furthermore the protein subunit of the enzyme was cloned and overexpressed in a heterologous system. In order to study the structural and functional attributes of the enzyme, reconstitution experiments have taken place using the RNA and protein subunit from Z. mobilis as well as from E. coli. 3 - ions. Carbonic anhydrases are ubiquitous metalloenzymes present in prokaryotes and eukaryotes that are encoded by five evolutionarily unrelated gene families, namely a-CAs, b-CAs, c-CAs, d-CAs and e-CAs. In mammals, 15 a-CA isozymes or CA-related proteins have been described with different catalytic activity, subcellular localization and tissue distribution. The hCAII enzyme is the target for drugs, such as acetazolamide, methazolamide and dichlorphenamide for the treatment of glaucoma. However, since the specificity of sulphonamides against CA enzymes is low, the adverse effect could be encountered during the treatment of the related diseases. Therefore, it is important to elucidate the inhibition mechanism of the enzyme in order to develop more specific novel inhibitors. In this work, we plan to identify some amino acids that may be involved in catalytic centre of hCAII enzyme. PCR based site-directed mutagenesis strategy has been performed replacing Asn67 to a hydrophobic amino acid, Isoleucin and Leu204 to a hydrophilic amino acid, Serin. The related mutations have been confirmed by sequence analysis. The mutant proteins were expressed in E coli by inducing IPTG. The expressed proteins were purified by sepharose 4B-Ltyrosine-sulphonamide affinity chromatography. The hydratase, esterase activity and inhibition manner of the mutant enzymes were compared to the wild type enzyme. HSA (human serum albumin) is a major circulatory protein of wellknown structure having 585 amino acids. HSA binds a wide variety of endogenous ligands and some drugs such as warfarin, diazepam, ibuprofen, salicylate and so on. In this study, the interaction between HSA and a new synthesized anti-cancer drug (2, 2(-bipyridin butylglycinato Pd(II) nitrate) have been investigated by fluorescence quenching and CD techniques at different temperatures of 27 and 37°C. By the analysis of fluorescence intensity, it was observed that Pd(II)-complex has a strong ability to quench the intrinsic fluorescence of HSA through a static quenching procedure. The number of binding site and the association binding constant of Pd(II)-complex were calculated at 27 and 37°C. From DH and DS˚values, it can be concluded that hydrophobic interactions may play an important role in the binding of Pd(II)-complex to HSA. The distance (r) between donor (Trp of HSA) and acceptor (Pd(II)-complex) was obtained according to the Forster's theory of non-radiation energy transfer at different temperatures. Far-UV-CD results represented that Pd(II)-complex induced decreasing in content of a-helical structure of protein.

From above results, it can be concluded that the blood carier protein of HSA could bind and transfer of this new anti-cancer drugs, but the stability of the protein decreased upon the interaction with this complex.

Free and calixarene encapsulated sinomenine binding by human serum albumin as determined by HPLC procedure Retention behaviour of alkaloid sinomenine -derived from traditional Chinese medicinal plant Sinomenium acutum Rehdder and Wilson and indicating a range of bioactivities including immunomodulation, antiinflammation, arthritis amelioration -has been determined in the narrow-bore isocratic and gradient HPLC-DAD systems employing calix[6]arene-, calix[8]arene-as well as tert-butyl calix[4]arene-bonded stationary phases for preliminary evaluation of inclusion process of this compound by hydrophobic cavities of peptidomimetic surface of cyclic oligophenoles. Formation of the 1:1 inclusion host-guest complexes between each calixarene-type stationary phase and sinomenine were studied with MM + and AM1 molecular modelling methods. Next, the freeze-drying process was developed for preparation of sinomenine/ calix[6]arene complex and influence of the processing technique on the drug content followed by dissolution behaviour was studied in detail by HPLC. Using an immobilized human serum albumin (HSA) stationary phase the in vitro binding affinity of free and calixarene encapsulated sinomenine to HSA were determined by HPLC-DAD assay and compared with results achieved with in vivo equilibrium dialysis technique. The analysis of the obtained complexes and trajectories has shown that substrate nitrogen atom is always sorbed near Trp84(82) benzol moiety. Substrate orientation and complexes dynamics properties depend on protonation state of the enzymes catalytic residues. It has been concluded that sorption of a substrate molecule in ChE active site is conditioned by the mooring of the substrate nitrogen near Trp84(82). The active serine must be deprotonated and the active histidine must be double protonated such that the ligand is properly placed near serine for the reaction to proceed.

Study of the interaction of Helix pomatia agglutinin with anticancer drug roscovitine V. Bogoeva and G. Russev Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia, BULGARIA

Our previous study has shown that a snail lectin Helix pomatia agglutinin (HPA) has a dual function and besides its carbohydrate binding property, can accommodate hydrophobic ligands as: arylaminonaphthalene sulfonate TNS and adenine. We have also found that this lectin binds hydrophobic physiological ligands as: steroid hormones with high affinity of kDa (1.9-2.4 lM) which has suggested an important biological role of HPA. Steroids may represent the relevant physiological ligands of HPA, occupying the quaternary structured based 'hydrophobic' site, located in the central cavity of the oligomeric molecule. The present investigation analyses the interaction of HPA with the hydrophobic anticancer drug roscovitine, a purine analogue, applied in clinical studies. Binding of HPA to roscovitine is accompanied by a significant enhancement of the Trp fluorescence (up to 50%), characterizing the conformational changes within lectin molecule. The value of the dissociation constant kDa (4.1 ± 0.2) for roscovitine is similar to the previously calculated kDa (1.9-5.4 lM) for steroids and adenine which shows high affinity binding for this ligand. The hyperbolic shape of the binding curve for roscovitine-HPA complex indicates one high affinity site for this ligand, suggested to occupy the previously identified 'hydrophobic' site in the protein molecule. Since HPA has already been applied as a cancer marker, recognizing primary tumours in tissue sections, our results suggest this lectin might be viewed as a potential drug carrying molecule which could be exploited for targeted delivery, and to be useful in anticancer therapies.

Structural comparison of l-opioid receptor selective peptides and peptidomimetics A. Borics and G. To´th Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, Szegeb, HUNGARY Structural determinants of binding to the l-opioid receptor -an important target in analgesia -attracts great scientific attention. Many natural and synthetic peptides and peptidomimetics were shown previously to bind to the l-opioid receptor selectively but there is no consensus about what structure is responsible for such biological activity. No high resolution structure of this receptor is available and the binding site of ligands is not exactly known despite numerous sitedirected mutagenesis studies. This suggests that the determination of structural aspects of l-opioid activity should focus on the ligands. l-Opioid ligands with similar affinity and selectivity should possess at least one common structural feature in which they differ from other ligands of different affinity and selectivity. Comparative structural analysis of such ligands, considering adequate representation of binding conditions may reveal key features of bioactivity. In this study ten l-opioid receptor ligands, DAMGO, Tyr-W-MIF-1, morphiceptin, endomorphin-1 and 2 and their analogues, possessing different affinity and selectivity were examined using molecular dynamics. Conformational preference of these molecules was determined in aqueous environment along with structural properties previously proposed to be important for binding. No structural trend was observed in correlation with previously measured bioactivities. It was observed, that increased backbone flexibility do not have a negative impact on receptor affinity and selectivity, which supports the idea of induced fit binding. Results, however, suggest that binding does not occur on the surface of the receptor, which condition is modeled by the aqueous environment. 

, a sympathomimetic beta adrenergic receptor agonist used to treat asthma, and its dimethylcarbamate ester were studied as inhibitors of human erythrocytes acetylcholinesterase (AChE; EC 3.1.1.7) and plasma butyrylcholinesterase (BChE; EC 3.1.1.8). The aim was to determine dissociation or rate constants and to compare the selectivity of the two related enzymes as well as of native BChE variants (U, A and F). Results: Homozygous usual BChE (UU) has the highest affinity for isoproterenol (i.e. the lowest dissociation constant, K i = 0.47 mM) while AChE has the lowest affinity (K i = 2.5 mM). The dissociation constants for the homozygous atypical BChE (AA), fluoride-resistant (FF) and heterozygote UA were 1.6, 0.99 and 0.72 mM, respectively. Dimethylcarbamate ester of isoproterenol inhibited usual BChE 90 times faster than AChE, while the inhibition rate constants of BChE variants ranged from 0.021 minute -1 lM -1 for AA to 0.20 minute -1 lM -1 for UU BChE. Conclusions: Both inhibitors are more potent for BChE than AChE, but carbamoylation by dimethylcarbamate of isoprotenerol is more selective. Since isoprotenerol is the leaving group of the ester and a product of cholinesteraseres carbomoylation, the ester could be used as a prodrug because slow hydrolysis of dimethylcarbamate of isoproterenol by plasma BChE could ensure a slow release of isoproterenol in the organism.

The fluorescence study of covalent conjugation of peptide epitopes of foot-and-mouth disease virus (FMDV) with anionic polyelectrolytes and bovine serum albumin B. Mansuroglu, S. Acar Derman, Y. Budama Battal, K. Kizilbey and Z. Mustafaeva Department of Bioengineering, Yildiz Technical University, Esenler-Istanbul, TURKEY Water-soluble protein-Polyelectrolyte (PE) complexes as functional biopolymer systems represent a specific class of polymer-PE compounds that have important applications in various areas. Synthetic PEs have been widely used to modify proteins via covalent attachment, increasing the immunogenicity of antigenic peptides. Fluorescence techniques have recently been used to study peptide-protein or peptide-PE conjugates. From the fluorescent emission shift of tryptophan residues in protein or peptides, it is possible to localize the interaction between protein and PE at certain protein domains. In this study, we investigate covalent binding mechanism of FMDV synthetic peptides 135-161 amino acid residues with copolymers of acrylic acids and N-vinylpyrolidone (VP\AA) and Bovine Serum Albumin (BSA), depending upon the weight concentration ratio of components by fluorescence and HPLC methods. From the analysis of peptide-BSA and Peptide-PE conjugates, it is possible to discuss the mechanism of the conjugate formation and structure of forming particles. According to the fluorescence analysis results, free peptide which containing Trp residue, gives fluorescence at k max 353 nm, free BSA which containing two Trp gives fluorescence at k max 339 nm. However, after the conjugation reaction it is observed that products k max decreased and it is characterized as blue shift of k max . This indicates that when conjugates formed, Trp is completely isolated from aqueous solution.

The study of the interaction of bovine serum albumin with anionic polyelectrolytes by fluorescence method Y. Budama Battal and Z. Mustafaeva Department of Bioengineering, Yildiz Technical University, Istanbul, TURKEY In this study, the interaction of Bovine Serum Albumin (BSA) with anionic polyelectrolyte (polyacrylic acid, PAA) have been examined and the formation of interpolymer-protein complexes based on the variation of the fluorescence aspects of the tryptophan amino acid present in protein molecules have been researched. It has been determined that water soluble and insoluble polymer-protein complexes have been formed depending on the initial molar ratios of the components and the pH values. BSA-PAA polycomplexes have been synthesized at values of pH = 4.0-7.0 and at initial molar ratios and have analyzed by different methods. The polycomplexes have been examined using UV-VIS Spectrophotometer, Steady State Flourescence Spectrometer and HPLC System. The soluble complexes were characterized by the structure of particles in which protein molecules are densely covered by the shelf of a polymer coil. The fluorescence method is applied to the water soluble PAA-protein mixtures, the water solubility of the system has been researched by turbidimetric method depending on the polymer/protein ratio and the pH value of the mixture, and insoluble systems have been determined. Aggregation of intrapolymer complexes has been found depending on the ratio and pH value. kmax and Imax values of tryptophan in the protein molecule change according to the ratio and pH. These systems have been researched in detail using fluorescence method and new information has been gathered. Dioxygenases catalyze the ring cleavage of aromatic compounds in the alkylphenol degradation pathway, thus are responsible for the catabolism of the alkylphenols in aerobic bacteria. To date, crystal structures of previously reported dioxygenases showed a similar active-site, but revealed the structural differences in the substrate specificity. Therefore, the structural studies of bacterial dioxygenase LapB (for long-chain alkylphenols) will lead to detailed information about the mechanism of phenolic moiety ring cleavage and the substrate-recognition system for LapB. To elucidate the functional features of LapB, we successfully expressed, purified and crystallized the enzyme from Pseudomonas sp. Strain KL28. The enzyme was expressed as the C-terminal His-tagged protein in Escherichia coli, followed by purification using immobilized metal affinity chromatography. The molecular mass of the purified LapB was estimated to be about 140 kDa by Superdex 200 gel filtration chromatography, corresponding to a homotetramer. LapB was crystallized using the hanging-drop method, and its structure was determined by the molecular replacement using the 2.3 Å resolution data which were collected at Pohang Accelerator Laboratory, Korea. The crystal belongs to a space group P2 1 2 1 2 1 with the unit-cell parameters a = 94.9, b = 97.0, c = 133.4 Å , a, b, c = 90°, and four monomers per asymmetric unit. We are now in the middle of determining the structure in a complex with various in vitro substrates.

Role of the C-terminus in the secondary structure stability and unfolding properties of apolipoprotein E4 

The investigation of FBPase and aldolase structure with atomic force microscopy (AFM) Introduction: Muscle fructose 1,6-bisphosphatase (FBPase) and aldolase are the enzymes indispensable for the glyconeogenesis. The both enzymes are globular homotetramers of molecular mass 37 kDa (FBPase) and 41 kDa (aldolase). FBPase strongly interacts with aldolase what results in formation of the FBPase-aldolase complex enabling the glyconeogenesis to proceed [1] . The primary aim of the present paper was the investigation of the proteins' surfaces, essential for the complex formation and adsorption onto a mica surface. Methods: The rabbit muscle aldolase and the recombinant forms of the human muscle FBPase expressed in E. coli (the wild type and the truncated form with the deletion of 10 C-terminal amino acids) were purified and imaged at pH 7.4 and 6.3 using Nanoscope IIIa (Veeco) working in the tapping mode.

Results: The single molecules and their complexes immobilized on the freshly cleaved mica were clearly visible for the different protein concentrations. The highest structural resolution (of about 5 nm) was observed for the truncated form of FBPase. The effect of pH on the protein properties was also investigated. Introduction: SHP-2, a protein tyrosine phosphatase, encoded by the PTPN11 gene, mediates cell signalling by growth factors and cytokines acting via the RAS/MAP kinase pathway. The enzyme is normally inactive and is activated when connected to other tyrosine phosphorylated proteins. SHP-2 mutations leading to continuously active enzyme, were found in Noonan Syndrome and certain leukemias. In the present study, seventeen 2-thiazolylimino/heteroarylimino-5aryliden-thiazolidin-4-ones divided, according to the heteroarylimino group, into three series of derivatives bearing thiazole-, benzothiazole-and benzoisothizole rings, were tested for SHP-2 inhibitory action. Results and discussion: Eleven out of seventeen compounds had Ki values lower than 54.54 lM whereas four of them had Ki values lower than 12.85 lM. Compounds of the benzothiazole series were more active than thiazole and benzoisothiazole analogues. Docking studies revealed that hydrophobic interactions and hydrogen bond formation stabilize enzyme-inhibitor complex. In all cases, stable complex formation was favoured by the placement of a hydrophobic group in the vicinity of aminoacids Tyr62, Glu361, Arg362, Lys364, Ser365, Lys366 and Ser460. This hydrophobic group may be a, preferentially methoxy substituted phenyl group in the thiazol series, adamantane in the adamantanyl-thiazolyl-derivative or the benzothiazolyl group in the benzothiazole series. Hydrogen bond interactions between aryliden substituents or groups of the backbone of the compounds and enzyme aminoacids further increase stability. Lys 280, Tyr62 and Arg 278 are the aminoacids mainly involved in this process. Conclusions: Docking results explain well the inhibitory action of the new compounds and may be useful for future design of more inhibitors of this series.

Structural, biochemical and computational studies of a Bacillus cereus deacetylase: activity and inhibition Introduction: Bacillus cereus is an opportunistic pathogenic bacterium closely related to Bacillus anthracis, the causative agent of anthrax in mammals. A significant portion of the B. cereus chromosomal genes are common to B. anthracis, including genes which in B. anthracis code for putative virulence and surface proteins. B. cereus provides thus a convenient model organism for studying proteins potentially associated with the pathogenicity of the highly infectious B. anthracis. The BcZBP protein of B. cereus is encoded from the bc1534 gene which has three homologues to B. anthracis. The protein exhibits deacetylase activity. However, neither the specific substrate of the BcZBP nor the biochemical pathway have been conclusively identified. Results and conclusions: BcZBP has been crystallized and its structure has been determined at 1.8 Å resolution. Structural and biochemical data provide evidence that (i) the protein binds zinc through a conserved catalytic triad which characterizes zinc-dependent hydrolases, (ii) the biologically active form is a spherical hexamer, (iii) the active site is formed at the level of the complete hexamer, (iv) the protein exhibits deacetylase activity with N-acetyl moiety of the N-acetylglucosamine, diacetylchitobiose and triacetylchitotriose. A computer program was developed to search the Cambridge data base for putative inhibitors. Approximately 100 compounds fulfilled the requirements and they will be tested for BcZBP inhibition.

Insights into the role of oligomeric state on the biological activities of crotoxin B: a tumor growth inhibitor and analgesic toxin from rattlesnake venom M. R. Fontes 1 , D. Marchi-Salvador 1 , A. J. Magro 1 , L. C. Correa 1 , C. Oliveira 2 and A. Soares 2 1 UNESP -Instituto De Biocieˆncias, Botucat, BRAZIL, 2 USP -FCFRP, BRAZIL Introduction: Crotoxin B (Cro) is a basic PLA 2 found in the Crotalus durissus terrificus venom and it is one of the subunits which constitute the crotoxin. This heterodimeric toxin, main component of the C. d. terrificus venom, is completed by an acidic, non-toxic and non-enzymatic component (crotoxin A), and it is related to important envenomation effects, as neurological disorders, myotoxicity and renal failure. Despite of its harmful effects, Cro has been tested successfully as inhibitor of tumor growth and as an efficient analgesic substance. Although the crotoxin was the first animal toxin to be crystallized (1938) , no crystal structure is available for crotoxin, crotoxin A or B. Methods: The crystal structure of a novel tetrameric complex formed by two dimers of crotoxin B isoforms (CB1/CB2) was solved at 2.2 Å resolution. The solvation, dissociation free energy variations were calculated and dynamic light scattering and electrophoresis experiments were performed in order to test if tetrameric assembly is stable in solution.

The results suggest that this assembly is stable in solution and Ser1 and Glu92 residues found respectively in the isoforms CB1 and CB2 are essential to the oligomerization of toxin. The most probable sites related to the neurotoxic activity are exposed on the surface. Conclusions: This arrangement may increase the neurotoxic activity of this protein, helping to anchor the molecule to its substrate located at the pre-synaptic membrane. Deeper structural and functional studies with this enzyme can lead to new and effective drugs. Zinc binding domains, or zinc fingers (ZnFs), form one of the most numerous and most diverse superclasses of protein structural motifs in eukaryotes. Although our understanding of the functions of several classes of these domains is relatively well developed, we know much less about the molecular mechanisms of action of many others. Myelin transcription factor 1 (MyT1) type ZnFs are found in organisms as diverse as nematodes and mammals and are found in a range of sequence contexts. MyT1, one of the early transcription factors expressed in the developing central nervous system, contains seven MyT1 ZnFs that are very highly conserved both within the protein and between species. We have used a range of biophysical techniques, including NMR spectroscopy and data-driven macromolecular docking, to investigate the structural basis for the interaction between MyT1 ZnFs and DNA. Our data indicate that MyT1 ZnFs recognize the major groove of DNA in a way that appears to differ from other known zinc binding domains. The gene BH3554, of 747 pb, from Bacillus halodurans C-125 was cloned, sequenced and overexpressed into Rosetta Escherichia coli strain. The deduced protein was a 32 kDa monomeric esterase with high homology to carboxylesterases from Bacillus thuringiensis (86% identity) and Geobacillus kaustophilus (85% identity). The sequence analysis showed that the protein contains a catalytic triad formed by Ser93, Asp192 and His222, and the Ser of the active site is located in the conserved motif Gli 91-X-Ser93-X-Gli 95 included in most esterases and lipases. The enzyme showed remarkable stability at high pH, against denaturing agents, like organic solvents, detergents and urea. The protein catalyzed the hydrolysis of p-nitrophenyl (p-NP) esters with different acyl chain lengths, confirming the esterase activity. The best substrate was p-NPAcetate (C 2 ) with Km and Vmax values of 0.26 mM and 1541 mM/minute, respectively. Phenylmethanesulphonyl fluoride (PMSF), a serine inhibitor, and the organophosphate Paraoxon, strongly inhibited the esterase activity, whereas Eserine had no effects on the activity, this data showed that the Serine-esterase belongs to the B-esterase group. The structure was modelled and the topological diagrams compared. Acknowledgment: This work was supported by MEC-FEDER (BIO2004-00439/BIO2007-62510), Fundacio´n-Se´neca (00608/PI/04), and DGI-Consejerı´aEducacio´n-CARM (09BIO2005-01-6470). J.N.F. has a predoctoral grant from MEC-FPI. S.M.G. has a predoctoral grant from Fundacio´n-Se´neca.

A statistical coupling and crystallographic study of Fe/Mn superoxide dismutases F. Bachega, L. Bleicher, M. Navarro, A. P. Araujo and R. Garratt Instituto de Fisica de Sao Carlos/Universidade de Sao Paulo, Sa˜o Paulo, BRAZIL Introduction: Parasite FeSODs have been proposed as drug targets for the development of alternative chemotherapy. This is based on their role in parasite defense against host-generated free radicals as well as experimental data such as antisense RNA experiments. Knowledge of their 3D structures and their comparison with human MnSOD are essential for any serious attempt to implement such ideas. A better understanding of the determinants of metal specificity is therefore also pertinent to this question. Methods: The 3D structures of FeSODs from T. cruzi, T. brucei and P. falciparum were solved using standard crystallographic techniques. A full sequence alignment of all family members was derived from the Pfam database using ClustalX and used for Statistical Cluster Analysis. DDG values were calculated, clustered and mapped onto the structure of the T. brucei enzyme. Results: Minor differences are observed at the entrance to the active site which might be exploitable for selective inhibitor design. SCA suggests that different groups of residues are statistically coupled in FeSODs and MnSODs. These surround the conserved core and converge on Gln72 (145 in MnSODs) which is known to be related to metal specificity. Conclusion: We suggest that the SCA clusters observed are involved in fine tuning the active site and related to the determination of metal ion specificity.

PP3A-29 RNA is required for the dimerisation of the yeast eukaryotic translation initiation factor 5A P. Gentz, G. Blatch and R. Dorrington Rhodes University, Grahamstown, SOUTH AFRICA Introduction: Eukaryotic translation initiation factor 5A (eIF5A), conserved throughout all eukaryotes and archaebacteria, is the only protein known to contain the unique amino acid, hypusine. Hypusinated eIF5A is essential for cell viability and is proposed to facilitate translation of mRNAs encoding products required for progression through the G1-S phase. While crystal structures of eIF5A homologues are available, there is limited structure-function information on hypusinated eIF5A. We describe here the biochemical characterisation of yeast eIF5A to determine the oligomeric state of the native protein and relate this data to its in vivo function. Methods: Gel filtration, native PAGE and cross-linking experiments were used to determine the oligomeric state of purified native eIF5A. Site-directed mutagenesis was used to identify critical residues involved in maintaining the oligomeric state and the biological activity of these mutants was tested by complementation assays in an eIF5A-knockout yeast strain. Homology models were used to predict potentially important residues involved in RNA binding and in maintaining the oligomeric state of yeast eIF5A. Results: Native eIF5A exists as a dimer in solution and dimerisation is dependent on hypusination and RNA binding. We propose that dimerisation is required for eIF5A function. A sequential two-step dimerisation model, involving hypusine in the formation of a loose dimer (step one) and a tight dimer configuration requiring RNA (step two) will be presented. Conclusions: This is the first direct biochemical evidence that native eIF5A is a dimer and that dimerisation requires RNA binding. These findings provide important insight into the functional relevance of hypusine. In-vitro assembly of vimentin intermediate filaments (IFs) proceeds from tetrameric complexes to mature IFs in three distinct stages: (i) tetramers laterally associate into 16 nm-wide unit-length-type filaments (ULFs); (ii) ULFs longitudinally anneal into short, immature filaments; (iii) these radially compact yielding mature, 10 nm-wide IFs. Here, we probe this assembly mechanism using a new approach: near UV circular dichroism (CD) spectroscopy is employed to identify new interactions of the aromatic amino acids emerging at each assembly stage. In particular, the CD signal of ULFs does not change as compared to that of the soluble tetramers exhibiting two major peaks at 285 and 279 nm. Upon elongation, two changes occur in the CD signal: (i) The band at 279 nm, reflecting the transition of the tyrosines from the ground state to the first vibrational mode of the excited state is lost and (ii) a new band appears at 291 nm. These changes take place at different time scales. The disappearance of the 279 nm signal denotes a fast process occurring within the first minute of assembly, compatible with an increased rigidity of the tyrosines during elongation of the ULFs. The rise of the 291 nm band represents a slower process with a lifetime of~25 minute, indicating the emergence of a new electronic species. Potential candidates are phenolates, created by the deprotonation of the tyrosines' phenyl rings as a consequence of their new localization in a negatively charged environment. The appearance of these tyrosine-binding polar protein pockets documents molecular rearrangements during the radial compaction.

The a IIb b III inhibitors from snake venom: analyzing the RGD-peptides/disintegrin structure-activity relationship [1, 2] . This receptor is inhibited by snake venom molecules named disintegrins [3] . Recently, we characterized the inhibitory profile of jarastatin (JAST) and jararacin (JARC), two disintegrins from Bothrops jararaca venom, on human platelet aggregation induced by ADP (IC 50 of 281 nm and 110 nm, respectively) and thrombin (IC 50 89.5 nm and 29 nm, respectively). Studies using six cyclic peptides synthesized based on these disintegrins RGD hairpin loop also revealed no activity against ADP-induced assays and an inhibitory profile at lM range on thrombin-induced assays. In this work our purpose was to identify the role of RGD loop and others disintegrins regions to the biological profile of JARC and JAST. Methods: Therefore we used a molecular modeling program (Swiss Model 3.7) for constructing their homology models and their complexes with the aIIbb3 crystal structure. Results: Interestingly JARC and JAST conserved their 3D-structure compared to others snake venom disintegrins crystal structures whereas the cyclic peptides present a displacement of the RGD region. Our docking results showed interactions between the disintegrins and a IIb b III (i.e. Asp 51 -Lys 125 ) including through the N-terminal region (i.e. Thr 11 -Glu 157 ). As expected JARC presents more interactions (n = 14) with the integrin than JAST (n = 12) whereas the cyclic peptides showed less interactions (n = 3-10) only with the integrin b III subunity. Conclusion: These results reinforced the requirement of further disintegrins interaction regions (i.e. N-terminal) for a IIb b III recognition besides the RGD sequence as suggested by the experimental results. 

The role of tyrosine 90 in regulation of Src kinase activity and its interaction with signaling partners Z. Gerndtova, J. Brabek, M. Novotnu¨, D. Roesel, M. Sztacho and P. Folk Department of Cell Biology, Charles University, Prague, CZECH REPUBLIC Introduction: The protooncogenic kinase c-Src can be activated by displacing the intramolecular interactions of its SH2 and SH3 domains. We have shown previously that SH3 domain continues to exert negative regulatory effect on the kinase activity even in the absence of the pY527-SH2 domain interaction. New site of tyrosine phosphorylation on Src pTyr-90 was recently discovered and found to be highly enriched in the Src transformed cells. Tyr-90 is a conserved residue in ligand binding region of the SH3 domain. Methods: Phosphomimicking Y90E and non-phosphorylable Y90F mutants of SH3 domain of Src were introduced in both wild-type and activated Y527F mutant of Src and analyzed for impact on kinase activity, ligand binding and capacity to transform rat fibroblasts. Effects of Y90E and Y90F mutations were further analyzed using homology modeling. Results: Y90E mutation in Src SH3 domain resulted in greatly reduced binding of key signaling partners of Src, namely: FAK, CAS and Stat-3. Moreover Y90E mutant of c-Src exhibited elevated autophosphorylation Y416 site in the activation loop of kinase domain to the extent of SH2-activated mutant c-Src Y527F. The same effect was not however observed for c-SrcY527/Y90E double mutant confirming our previous results that SH3-linker interaction is not critical for regulation of SH2-activated Src. The homology modeling shows that Y90E mutation leads to a reorganization and a partial loss of hydrophobicity of the second SH3 ligand-binding pocket. Conclusions: Our results suggest that phosphomimicking mutation Y90E elevates kinase activity of Src and at the same time results in reduced ligand binding. These findings suggest that Src has the capacity to undergo more extensive autophosphorylation and structural changes than previously suggested, with implications for regulation of substrate selection and kinase activity.

Comparison of subunit exchange of aA-crystallin and its chaperone action in crowded system A. Ghahghaei University of Sistan and Baluchestan, Zahedan, IRAN a-Crystallin, a member of small heat shock protein (sHsp) family, is comprised of aA and aB subunits and acts as a molecular chaperone by interacting with unfolding proteins to prevent their aggregation. The aA-crystallin homopolymer consists of 30-40 subunits that are undergoing dynamic exchange. In vivo, a-crystallin elicit its chaperone action in a crowded cellular environment (e.g. in the lens). In vitro, inert molecular crowding agents (e.g. dextran) are often used to mimic crowded conditions. In this study, it was found that a-crystallin and aA-crystallin are poorer chaperones in the presence of dextran. Using fluorescence resonance energy transfer, it is shown that the aA-crystallin subunit exchange rate strongly increases with temperature. Binding of reduced ovotransferrin to aA-crystallin markedly decreases the rate of subunit exchange, as does the presence of dextran. In addition, in the presence of dextran the effect of reduced ovotransferrin on decreasing the rate of subunit exchange of aA-crystallin is greater than in the absence of dextran. Under the conditions of molecular crowding, the aA-crystallin subunit exchange rate is not temperature-dependent. The exchange rate of aA-crystallin subunits correlates with its chaperone efficiency, i.e. in the absence of dextran, the variation in chaperone ability of aA-crystallin increases with temperature whereas in the presence of dextran the temperature dependence of the chaperone ability of aA-crystallin is eliminated.

Synthesis and anticancer activity of hydrazino-peptoids based on substance P C-terminal region hexapeptide P. Glezakos, X. Stahtea, Y. Sarigiannis, N. Karamanos and G. Stavropoulos Department of Chemistry, University of Patras, Patras, GREECE Introduction: Small synthetic peptides, based on substance P C-terminal fragment, increase the secretion of tumor necrosis factor a (TNFa) and prevent the proliferation of several cancer cells. We have also shown the antiproliferative activity of tri-and tetra-peptoids in breast and prostate cancer cells. The aim of this study was to incorporate D-Trp in the sequence of the synthesized peptides and to evaluate their effect on cancer cell proliferation. Methods: All the syntheses were carried out stepwise by SPPS, using the Fmoc/Bu t methodology on the solid support 2-chlorotrityl chloride resin and DIC/HOBt as coupling reagent. All analogs were purified (HPLC) and identified (ESI-MS). The highly invasive breast cancer epithelial MDA-MB-231 and HT-29 colon cancer cells of high tumorigenity were cultured in absence or presence of hydrazino-peptoids for 48 hours, in presence and abscence of serum and cell proliferation was documented by WST-1 method. Results: We report the synthesis of a series of hydrazino-peptoids, analogs of SP 5-10 hexapeptide containing peptoid residues like HN-N(R)CH 2 CO, NPhe and NAla, and also D-Trp and Tic. 1. Glp 1 -NPhe 2 -Gly 3 -[NH-N(Bzl)-CH 2 -CO] 4 -D-Trp 5 -Leu 6 -OH 2. Glp 1 -NAla 2 -Gly 3 -[NH-N(Bzl)-CH 2 -CO] 4 -D-Trp 5 -Leu 6 -OH 3. Glp 1 -Tic 2 -Gly 3 -[NH-N(Bzl)-CH 2 -CO] 4 -D-Trp 5 -Leu 6 -OH All peptides significantly inhibited the proliferation of MDA and HT-29 cells in a dose-dependent manner in absence of serum, even at low concentrations. However, in presence of serum only the peptoids 2 and 3 inhibited the cell proliferation of MDA, whereas none inhibited HT-29 cells. Conclusions: Hydrazino-peptoids act as potent antiproliferative agents in breast and colon cancer cells and further studies are warranted to evaluate their inhibitory effects in the view of the new analogues.

A double role for a strictly conserved serine: further insights into the dUTPase catalytic mechanism Ser72 at the active site of the Escherichia coli dUTPase has been mutated to an alanine and the properties of the mutant investigated. The serine is absolutely conserved among the monomeric and trimeric dUTPases and has been proposed to promote catalysis by balancing the negative charge at the oxygen that bridges the a-and b-phosphorus of the substrate. In all reported complexes of dUTPases with the substrate analog a,b-imido-dUTPAEMg, the b-OH of the serine is indeed hydrogen bonded to the a,b-bridging nitrogen of the analog. However, in the complex of the Asp90 -> Asn mutant dUTPase with the natural substrate dUTPAEMg, the serine b-OH points in the opposite direction and is hydrogen bonded to Asn84 at the bottom of the pyrimidine pocket. Here we show that the replacement of the b-OH by hydrogen reduces k cat from 5.8 to 0.008 s -1 , but also k -1 , the rate of substrate dissociation, from 6.2 to 0.1 s -1 (K M = 6 10 -9 M). We conclude that the serine b-OH exercises both ground state (GS) destabilization and transition state (TS) stabilization, effects not usually linked to a single residue. We argue that the b-OH destabilizes the GS by imposing conformational constraints on the enzyme, and that TS stabilization depends on a rotation of the serine side chain, which not only relieves the constraints but brings the b-OH into a position where it can electrostatically stabilize the TS.

PP3A-36 Improved recognition, tracking and handling of protein crystals for high-throughput crystallography M. Groves EMBL, Hamburg, GERMANY

The availability of high-throughput crystallization and imaging robotics has provided a major advance in the types of biological samples that can be analyzed by X-ray crystallography. Lower sample biomass and ever decreasing crystallization volumes have resulted in a huge increase in the number of crystallization experiments and a concomitant increase in the number of images of crystallization experiments that need to be analyzed to determine a successful experiment. While, significant research has been done in the field of automated crystal detection, advances are still required in order to identify protein crystals in the increased number of crystallization images produced. We have previously shown that the addition of low concentrations (> 9 mM) of a non-covalent dye (1,8-ANS) to high-throughput crystallization experiments has no discernable effect upon the success of crystallization experiments and significantly increases the fluorescent signal of protein crystals, aiding the identification of successful crystallization trials [1] . In this poster we will describe developments in the method as applied to the automated tracking and handling of protein crystals on beamline BW7B at the EMBL Hamburg Outstation. Future directions will also be discussed. Since the fluorescence enhancement of polarity-sensitive dyes has been useful in identifying hydrophobic sites on a number of targets, changing in intrinsic fluorescence of protein and extrinsic fluorescence of Nile red after exposing enzyme at range of pH values was investigated. Results showed that Nile red dye fluorescence at alkaline pH were higher than acidic pH values. In the other hand, it can be concluded that the protein at alkaline pH has more accessible hydrophobic patches relative to acidic pH. Intrinsic fluorescence studies at different pH represented noticeable alteration in tertiary structure of protein. Respectively, Far-UV-CD studies showed that the native protein has a-helix structure while at higher pH a transition from a-helix to b-structure was appeared. Moreover, at lower pH the content of a-helix structure was increased. These results suggested that various pH induced significant structural changes in choline oxidase. Method: In this study, we have generated C-terminal truncated mutants by serial truncations. The quaternary structure of the enzyme was analyzed using both sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation.

Results: Global analysis of the combined results showed that truncation of C-terminus from 306 to 300 had no appreciable effect on the quaternary structure, and the enzyme remained catalytically active. However, further deletion of Gln-299 or Arg-298 drastically decreased the enzyme activity to 1%-2% of wild type (WT), and the major form was a monomeric one. Detailed analysis of the point mutants of these two residues and their nearby hydrogen bond partner Ser-123 and Ser-139 revealed a strong correlation between the enzyme activity loss and dimer dissociation. Conclusion: The monomeric SARS-CoV main protease was enzymatically inactive and this enzyme provides an ideal model system to study an enzyme activity regulated through quaternary structure.

Structural and functional characterizations of the non-histone-like chromatin proteins in archaea C.-H. Hsu Department and Institute of Agricultural Chemistry, Taipei, TAIWAN Organisms growing at elevated temperatures face the challenge of maintaining the integrity of their genetic materials. Archaea possess unique chromatin proteins for gene organization and information processing. Several small DNA-binding proteins, such as Sso10b1, Sso10b2, Sso7c4 and Sso7d from hyperthermophilic organism Sulfolobus solfataricus was found and has been proposed to play a regulatory role in gene transcription and chromatin formation, however, very little is known regarding the structural and functional relationship of these proteins. Therefore, the structure determination of these proteins was carried out using multi-dimensional nuclear magnetic resonance (NMR) technique and X-ray diffraction method. Furthermore, gel retardation and EMSA were applied to examine their DNA binding lengths and specificities. Introduction: There are two distinct families of EF-hand calcium binding proteins with regard to their functionality: calcium sensors and calcium buffers. These families also have distinct structural characteristics, which are underlined here by several scaling laws: the protein backbone length with the interval of residues, the surface area with the radius of the probe molecule rolled onto it, the radius of gyration with the residues number and the surface area with the molecular weight. Methods: Within this paper we calculate the radius of gyration, the surface area, the protein backbone length for the studied proteins and using the fractal geometry concepts we determine the corresponding scaling laws for each class of proteins. Results: The fractal dependences with regard to the proteins backbone lengths indicate dissimilar mechanisms responsible for their local folding. Different scaling properties of the radius of gyration with the residue number show that the shapes of these proteins are quite unlike. The proteins surface area scales, on the one hand, with the radius of the probe molecule and on the other hand, with the molecular weight revealing different strengthens of the intermolecular interactions for the two classes of the investigated proteins. Conclusion: We have pointed out that statistical scaling laws may be employed in describing structural features of proteins at both local and global levels. Furthermore, they can be of great help in novel function prediction methods that do not rely on sequence or fold similarity to other known proteins.

PP3A-43 RRE motif in Erm, its role in 23S rRNA methylation based on structure and its implication in drug development

The University of Suwon, Whasuag City, REPUBLIC OF KOREA Introduction: Erm proteins mediate one or two consecutive methylation reaction to produce the N 6 -mono or dimethyl adenine of specific nucleotide (A2058) to reduce the affinity of MLS (macrolide-lincosamide-streptogramin B) antibiotics, thereby microorganisms escape from antibiotics action. ErmSF from Streptomyces fradiae has extra long Nterminal end region (NTER) in which one 60RRE62 motif is contained as a part of a-helix because it is unstructured but peptide from R58 to V81 assumes a secondary structure of a-helix. Methods and results: To define the role of RRE motif, site directed mutagenesis of each amino acid was performed to get much lowered MIC of each mutant. NTER could be truncated up to A59 (NTTE59) to show only 30% of activity relative to wild type enzyme but one more truncation of R60 almost destroyed the enzyme activity and surprisingly enough, another truncation of R61 presumably recovered the enzyme activity to carry out monomethylation reaction to exhibit elevated lincomycin resistance. Site-directed mutagenesis of 60RRE62 in NTTE59 showed similar antibiotic resistance profile.

Conclusions: Even though it is not quite sure because any complex structure of Erm and 23S rRNA has not been determined, RRE motif could presumably locate the methylatable adenine correctly by interacting with nucleotide close to methylatable adenine or directly with it, if it maintains the orientation in space in a-helical context. So, sequentially truncated mutants of RRE motif in NTTE59 and itself are thought that it positioned the amino acids of RRE motif sterically and electronically for lowered activity, monomethylation and dimethylation, respectively. This subtlety could be exploited for inhibitor development.

Solution structure and Ca 2+ -channel blocking and antifungal activities of a novel insect peptide, diapause-specific peptide Introduction: Diapause-specific peptide (DSP), derived from the leaf beetle, inhibits Ca 2+ channels and has antifungal activity. DSP acts on chromaffin cells of the adrenal medulla in a fashion similar to that of x-conotoxin GVIA, a well-known neurotoxic peptide, and blocks Ntype voltage-dependent Ca 2+ channels. However, the amino acid sequence of DSP has little homology with any other known Ca 2+ channel blockers or antifungal peptides. In this paper, we analyzed the solution structure of DSP by using two-dimensional 1 H nuclear magnetic resonance and determined the pairing of half-cystine residues forming disulfide bonds. Results and discussion: The arrangement of the three disulfide bridges in DSP was distinct from that of other antifungal peptides and conotoxins. The overall structure of DSP is compact due in part to the three disulfide bridges and, interestingly, is very similar to those of the insect-and plant-derived antifungal peptides. On the other hand, the disulfide arrangement and the three-dimensional structure of DSP and GVIA are not similar. Nevertheless, some surface residues of DSP superimpose on the key functional residues of GVIA. This homologous distribution of hydrophobic and charged side chains may result in the functional similarity between DSP and GVIA. Thus, we propose here that the three-dimensional structure of DSP can explain its dual function as a Ca 2+ channel blocker and antifungal peptide [1] . As part of the Center for Structures of Membrane Proteins (CSMP) our aim is the structure determination of membrane proteins of both bacterial and human origin. Membrane proteins are currently the targets for~40% of all therapeutic drugs, therefore the structures are expected to provide invaluable information for rational drug design. Our initial target genes are all 25 Escherichia coli membrane kinase receptors, and a handful of human membrane proteins. At least two different constructs are prepared for each target, one as a fusion with Mistic (a novel protein that allows for expression of eukaryotic proteins in E. coli), as well as a non-misticated one. We studied the effect of Mistic on protein expression and membrane integration levels of the E. coli histidine kinase receptor family. We find that Mistic fusion greatly increases the overall yield, targets the cargo proteins more efficiently to the membrane and may even replace the signal sequence, while the proteins retain kinase activity. The proteins with the highest expression levels have been selected for large-scale preparation and crystallization screening. Prior to high-throughput crystallization screening a rapid and accurate evaluation of the oligomeric homogeneity of the samples, and the detergent-to-protein ratio in Protein-Detergent Complexes (PDC) is necessary. For this purpose we use an analytical HPLC system in combination with refractive index and static light scattering detectors, and NMR spectroscopy to characterize detergents free in solution and in PDC. Our crystal optimization approaches include automated detergent screening and crystallization under oil. Currently we have obtained crystals for four targets.

Inhibition studies on mutant Phe91Asn human carbonic anhydrase I (HCA I) gene It's known that sulfonamides which are used in glaucoma treatment to reduce inner eye pressure in glaucoma, inhibits hCAI isoenzymes as well as hCA II remarkably. Thus, the aim of our study is to get mutant HCA I enzymes which have low affinity to sulfonamides. In this study hCA I gene was cloned into PGEMT vector from HL60 (Human acute myeloid leukemia cell line) by RT-PCR strategy and subsequently subcloned into pET21a(+) expression vector. Phe91 hydrophobic residue was changed into more hydrophilic Asn residue with PCR based site directed mutagenesis using specific primers. After the expression of wild type and mutant HCA I enzymes in E. coli were purified by specific Sepharose 4B-L-Tyrosine affinity gel, hydratase and esterase activities measured. Inhibition manner of these enzymes by Sulphonilamide and acetozolamide widely used for the treatment of glaucoma was investigated.

New contact site in coiled coil region of human fibrin beta-chain taking part in protofibril lateral association S. Komisarenko, E. Lugovskoy, P. Gritsenko, I. Kolesnikova and N. Lugovskaya Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv, UKRAINE Introduction: The transformation of fibrinogen into fibrin by thrombin gives rise to structural rearrangements in the molecule leading to the exposure of the polymerization or contact sites taking part in fibrin intermolecular binding. We obtained monoclonal antibodies (mAbs) to various parts of fibrinogen-fibrin molecule(s) during its transformation into polymeric fibrin and used mAbs as molecular probes to study mechanisms of fibrin polymerization. Methods: Human fibrin desAABB in 2 M urea was used as an antigen. The epitope for mAb in fibrin molecule was localized using ELISA and immunoblot analysis with various fibrin(ogen) fragments. Turbidity analysis and transmission electron microscopy were used to study the effect of mAbs and their Fab-fragment on fibrin polymerization. Results: The epitope for mAb FnI-3C proved to be localized in the fibrin fragment Bbeta118-134, which is situated in the coiled coil region of the molecule. MAb FnI-3C and its Fab-fragment inhibited polymerization of fibrins desAA, desAABB and fibrin formed in fibrinogen +thrombin reaction at equimolar ratio of mAb or Fab to fibrin(ogen). This testifies the blocking of polymerization or contact site(s) by this mAb and its Fab. The turbidity analysis and electron microscopy showed that this mAb and its Fab retarded the stage of fibrin protofibril lateral association. Previously other authors found several amino acid residues localized in coiled coil region of fibrin molecule: Bbeta-Ser111, Bbeta158-160 and BbetaArg166, which participated in fibrin protofibril lateral association. Conclusion: We suggested that coiled coil fibrin fragment Bbeta118-134 may be fibrin contact site taking part in protofibril lateral binding.

Design and intracellular delivery of enzymes to enhance metabolic activation of prodrugs and improve anticancer therapy Methods: NAs, such as AZT, ganciclovir (GCV), or AraC and gemcitabine, are phosphorylated by different kinases. The rate-limiting reaction is often the first, and in some cases the second phosphorylation step. Through crystal structure analyses of various enzyme-nucleotide complexes, in combination with kinetic measurements, we wish to identifiy variants that are significantly more active and selective than wildtype. Using enzyme-monoclonal antibody-conjugates we target specific cell types.

Results: (1) A mutant of human TMP kinase phosphorylates AZTMP up to 200-fold more efficiently. We evaluated this enzyme/prodrug combination as a novel means to induce cell death in T cells and to kill erythroid leukemia cells in a mouse tumor model. (2) An engineered human deoxycytidine kinase shows improved activation of the prodrugs AraC and gemcitabine. Remarkably, this enzyme is highly active in phosphorylating NAs of the non-physiological stereochemical L-configuration, such as 3TC (lamivudine) and TRO (troxacitabine), that are less toxic in vivo and biologically more potent than the corresponding D-enantiomers. Phosphorylation of Ser-74 up-regulates catalytic activity about 10-fold.

Conclusions: Suicide enzyme therapy may become an even more promising strategy to establish control over the fate of cells transduced with integrating viral vectors, and direct protein transduction may lead to the development of novel schemas in nucleoside prodrug-dependent cancer chemotherapy. Introduction: Tuberculosis is the leading cause of death in the world from a bacterial infection disease. Difficulties related with unusual structure of Mycobacterium tuberculosis cell wall, which is intricate structure of polysaccharides, proteins and lipids, and is a major determinant of virulence for the bacterium. Understanding of the details of key components of the cell wall has resulted in identification of several points potentially susceptible to therapeutic attack. An example is furnished by the glycosyltransferases, which are part of the critical polysaccharides synthetic machinery. Methods: With using bioinformatics tools, several therapeutically famous glycosyltranferases (GTs) have been identified and genes rv3782 and rv0225 were amplified and cloned into the different expression vectors in order to provide their expression, purification and characterization. The task was complicated by the nature of glycosyltransferases, which are often unstable, hydrophobic and hard to isolate and purify. Therefore, the performance of various expression systems was explored in order to identify the best experimental routes.

Results: The extensive optimization covers up preparation of different recombinant proteins, expression systems and conditions testing (expression in ArticExpress cells etc.), using renaturation processes, and finally leads into target GTs preparation that allowed preliminary crystallization trials. Conlusion: Understanding the structure/functional relations of glycosyltransferases is important for designing ways of their inhibition, a potential point in tuberculosis treatment.

Deciphering the sugar preference rulescomputer-assisted protein engineering of bacterial lectin PA-IIL The increasing bacterial resistance to antibiotics poses significant threat in treatment of infectious diseases. This calls for designing antibiotics with improved permeation properties using a bottom-up approach, based on the knowledge of molecular mechanisms regulating resistance. Bacterial outer membrane contain general diffusion porins, such as OmpF, which constitute the main path of diffusion for antibiotics. A common mechanism by which bacterial strains become resistant is to introduce mutations modulating the permeation of the porins. Methods: To study the diffusion of antibiotics through OmpF at a molecular scale, we performed molecular dynamics (MD) simulations, combined with the metadynamic algorithm that accelerate rare events. Results: We simulated the wild type porin OmpF as well as variants with substitutions. We found that a mutation on the residue D113 (a residue at the constriction region) changes drastically the Ampicillin diffusion in terms of strength and localization of minima on the associated free energy map. Then led by these results, we used an antibiotic that potentially interact less with this residue, such as penicillin-G, and predicted the expected diffusion pathway. Conclusion: We further extended this investigation to other antibiotics, such as carbencillin and even fourth generation of cephalosporins, which are the most frequently prescribed class of antibiotics in the world. These findings are in good agreement with experimental results obtained by our collaborators (liposome swelling assays and electrophysiology) and demonstrate how theory and experiments can be combined to reveal the mechanism of antibiotic diffusion and designing potent antibiotics.

Synergistic substrate binding in peptide phosphorylation reaction by cAMP-dependent protein kinase catalytic subunit A. Kuznetsov and J. Jarv Institute of Chemistry, University of Tartu, Tartu, ESTONIA

In the protein kinase A (PKA) catalyzed reaction of peptide phosphorylation the c-phosphate group of ATP is directly transferred to the phosphorylatable serine of peptides, and for this reaction simultaneous binding of both substrates should occur in distinct binding sites of the enzyme. Recently it was shown by NMR spectrometry [1] that binding of AMP-PNP at the ATP binding site allosterically changed the enzyme affinity for peptide substrate, and vice versa, peptide binding affected nucleotide binding. In this report we observed the same allosteric (or 'synergistic', as both ligands are substrates) effects in the PKA catalyzed peptide phosphorylation reaction. Simple kinetic procedure was elaborated for this analysis, based on application of the secondorder rate constants of this bisubstrate enzyme reaction [2] . The study involved a series of peptide substrates of diverse structure and different binding effectiveness. It was found that peptide structure had significant influence on the allosteric effect, thereby governing the binding effectiveness of both substrates as well as the observed rate of the overall reaction. It can be pointed out that mutual influence of substrates on their binding effectivenss might regulate activity of a monomeric bisubstrate enzyme by analogy with cooperativity known for multimeric enzymes. Many proteins undergo conformational changes as they shift between active and non-active states. Molecules that interfere with these changes are important biological reagents. In this study, we put forward an innovative in silico approach for identifying peptides that have the potential to block conformational changes of selected target proteins. These peptides are derived from the target protein and are designed to inhibit intra-molecular interactions that are important for the protein's function. The applicability of our approach is demonstrated through two antagonistic peptides. One peptide is derived from secreted Clusterin, a chaperon protein that evokes chemotherapy resistance in cancer cells. A second peptide is derived from an HSP, which is involved in inflammation. The clusterin-derived peptide binds to its target on the parent protein, and induces growth arrest when applied to cancer cell lines. Furthermore, it significantly enhances the cytostatic activity of the chemotherapeutic agent Taxol in a synergetic way, as revealed by in vitro studies with breast, lung and prostate cancers cell lines. The HSP-derived peptide induces up to 90% decrease in the level of various inflammation-related cytokines when applied to blood cells derived from the myeloid lineage that were stimulated with LPS or anti-CD3 mAb. These two peptides are promising anti-cancer and antiinflammatory lead compounds and their in silico discovery paves a new path for the discovery of numerous novel therapeutic peptides to a wide range of indications. In this lecture, I will describe the details of the computational method, and extensive biological data that supports it. *These authors contributed equally to this work. Methods: We compared computational interaction models with measurements of antibacterial and haemolytic activity, Small Angle X-Ray and Surface Plasmon Resonance data, and structure predictions by Circular Dichroism (CD). Results: All peptides were active against Escherichia coli (Gram negative) and Staphylococcus carnosus (Gram positive) bacterial cultures, but the haemolytic properties against human red blood cells were found to be poor and indicated the peptides' selectivity. CD studies of the peptide secondary structure confirmed the prediction of peptide helicity. The antibacterial activity can be correlated with a change of the hexagonal phase transition temperature of 1-palmitoyl-2-oleoyl-snglycero-3-phosphoethanolamine (POPE) as determined by Small Angle X-ray Scattering (SAXS). The calculated peptide membrane affinity is not related in linear way with the antibacterial activity. The reason for this might be aggregation as shown by surface plasmon resonance.

Conclusions: The inverse hexagonal phase transition temperature was increased by the peptides and this promotes a positive curvature of the membranes. We assume that this curvature finally leads to the disruption of the model membranes. In summary an over all helical structure, electrostatic and hydrophobic parameters as well as strong amphipaticity are good measures to describe antibacterial peptide interaction. Human mitochondrial NAD(P) + -dependent malic enzyme (m-NAD-ME) is a malic enzyme isoform with dual-cofactor specificity, ATP sensitivity and substrate cooperativity. In the present study, since the inhibitory effect of ATP is more pronounced on m-NAD-ME than on other ME isoforms, we investigate the possible role of Lys346 on the cofactor specificity, malate cooperativity and ATP sensitivity of the enzyme. Our kinetic data clearly indicate that the K346S mutant enzyme has shifted its cofactor preference from NAD + to NADP + . Furthermore, the cooperative binding of malate becomes remarkably less in the human m-NAD-ME after mutation of Lys346, and the sigmoidal phenomenon that appeared in WT was not prominent in Lys346 mutations, suggesting the importance of Lys346 in malate cooperativity. Although these Lys346 mutations changed the cooperative binding of malate to the enzyme, they could also be activated by fumarate and the cooperative effect can still be abolished by fumarate, implying that the allosteric property was still held in these mutants. Lys346 in the human m-NAD-ME plays a critical role in governing ATP sensitivity. Mutation of Lys346 to Ser or Ala causes the enzyme to be much less sensitive to ATP, similar to c-NADP ME. Substitution of Lys to Arg did not change the ATP sensitivity of the enzyme. The dissociation constants of ATP for K346S and K346A are increasing, while for K346R, they are similar to those of WT. Thus, ATP sensitivity is proposed to be determined by the electrostatic potential involving the positive charge of side-chain Lys346.

Characterization of two glycolytic enzymes from Giardia lamblia as target for drug design The most common non-bacterial diarrhea in human is caused by the parasite Giardia lamblia. The incidence of giardiasis worldwide may be as high as 1000 million cases. The drug of choice for treating the giardiasis is metronidazole, a 5-nitroimidazole derivative. Although metronidazole is effective, it exerts strong side effects in the host, and there is evidence that strains resistant to metronidazole are existent. This underlines the need for developing drugs that are effective against giardiasis. In this regard, it is noted that G. lamblia lacks oxidative phosphorylation because it possesses mitosomes instead of mitochondria. Thus, in G. lamblia, the main source of ATP is the glycolytic pathway suggesting that the enzymes of the glycolysis can be potential targets for drug design. Fructose-1,6-bisphosphate aldolase (FBPA) and triosephopsphate isomerase (TIM) are two glycolytic enzymes representing key steps on this pathway. FBPA catalyzes the reversible cleavage of D-fructose-1,6bisphosphate producing R-glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP). TIM catalyzes the reversible isomerization between GAP and DHAP. We characterized the recombinant FBPA and TIM of G. lamblia and used several compounds trying to inactivate both. The effect of these compounds over the enzyme activity, and cell viability were tested. Results showed a specie specific effect over TIM of cysteine-reactive compounds though FBPA was not affected. The cysteine at position 222 of TIM is the main target of these compounds. One of these compounds had citotoxic effect on trophozoites of G. lamblia, through inactivation of TIM and low effect on human cells. 

Reconstituted high density lipoprotein particles: a promising medium for high-resolution NMR investigations of membrane proteins and membrane-active peptides. The reconstituted high density lipoprotein particles (rHDLs) are nanoscale phospholipid bilayers wrapped around by a dimer of apolipoprotein-A1. In contrast to the commonly used for high-resolution NMR investigations spherical micelles and small elliptical bicelles, the rHDLs contain a patch of planar lipid bilayer (160 lipid molecules per particle). rHDL has a disc-like shape with diameter of about 10-12 nm and thickness of~4 nm. The theoretical calculation of the rotational diffusion tensor for rHDLs indicates isotropic motions of these particles, providing the application of high-resolution NMR spectroscopy of the peptides and proteins embedded into their bilayer. In principle, the rHDLs could provide superior membrane mimicking characteristics and enhanced sample stability when compared with conventional detergent-based membrane mimetics; therefore they may be a very attractive environment for NMR investigations of membrane proteins.

To test the applicability of high-resolution NMR methods for studies of membrane proteins and membrane-active peptides embedded into rHDLs, the uniformly 15 N-labeled peptide Antiamoebin-1 was incorporated into anionic rHDL/DOPG particles. This sample exhibits a 1 H-15 N-TROSY spectrum very similar to the Antiamoebin-1 spectrum in neutral DMPC/DHPC bicelles and slightly different from spectra in anionic micelles (LMPG, SDS). All expected 13 backbone HN resonances were observed in this spectrum, but the signals were significantly broader ( 1 H line-width 40-60 Hz in rHDLs vs. 30 Hz in bicelles). The topology of the peptide in the bilayer of the rHDL was determined using the lipid-soluble relaxation probe technique. Obtained results prove the applicability of rHDLs for high-resolution NMR studies. Cellular tetrapyrroles such as porphyrins and hemes are extremely reactive and poorly soluble under physiological conditions. The protein p22HBP is a heme-binding protein that has also strong affinity towards other cellular tetrapyrroles. The structure of p22HBP has been recently determined by NMR [1] and the molecular recognition mechanism of several cellular tetrapyrroles (Hemin, Protoporphyrin IX, Coproporphyrin 1/III, Uroporphyrin I/III and Bilirubin IX) investigated by modelling methodologies is reported here. The flexible docking screening of the tetrapyrroles was done with Autodock and was targeted to a specific protein region identified previously by NMR studies. Next, molecular dynamics simulations were carried out for 10 ns in a fully hydrated system using the GROMOS force field and RESP atomic charges for the tetrapyrroles. We show that the binding site of p22HBP is able to recognize other tetrapyrroles other than Hemin. We propose that Hemin iron (III) may interact by two axial methionines at position 63 and 176. The binding affinity tetrapyrrols towards p22HBP were estimated by free energy calculations that follows the order: Protoporphyrin IX < Hemin < Coproporphyrin I = Coproporphyrin III < Uroporphyrin I < Bilirubin IX < Uroporphyrin III. These results show that p22HBP is able to bind with different degree of affinity most of the anabolic and catabolic intermediates of the heme. Tetrapyrroles are stabilized by strong electrostatic effects due to lysine 177 arginine 56 located in the edges of the binding pocket. Introduction: Crossing the plasma membrane is a prerequisite for intracellular targeted drug delivery. Cell penetrating peptides (CPPs) are known to transport cargos attached to it into cells primarily by endocytosis. However, confinement of biomolecules into endosomes limits their use for intracellular targeting. We developed a novel cysteine-rich peptide that has the ability to enter directly into the cytosolic compartment of the cell. The factors affecting cytosolic distribution of peptide are reported. Methods: Uptake of fluorescently labeled peptide was assessed in NIH-3T3 mouse fibroblasts. Effect of varying the labeling concentration, time course, changing incubation temperature from 37°C to 4°C, etc. on intracellular distribution was observed by fluorescence spectroscopy and microscopy. Results: Our novel peptide exhibited mainly cytosolic localization along with some vesicular uptake in cells at a concentration as low as 2.5 lM. Diffused appearance was visible earliest after 4 hours. A reduction in vesicular uptake was observed on incubation at 4°C, however cytosolic uptake was almost unaffected, indicating the presence of an additional non-endosomal pathway. Due to higher stability, d-form of most of the well studied cationic CPPs (like Tat, arginines, etc.) is reported to be taken up better than the l-form. Our peptide differs as the l-form shows most efficient cytosolic uptake while the d-form and retro-inverso form exhibited especially reduced cytosolic diffusion.

Conclusions: A novel cysteine-rich peptide has been found that is directly taken up into cytosol largely, avoiding endosomal entrapment. Therefore, it has the potential to be used as CPP for efficient cytosolic drug delivery for intracellular targeting.

Structural analysis of lipocalin-type prostaglandin D synthase complexed with biliverdin by multi-dimensional NMR and small-angle X-ray scattering Introduction: Lipocalin-type prostaglandin D synthase (L-PGDS) is abundantly expressed in the central nervous system and male genitals of various mammals, and catalyzes the isomerization of PGH 2 to produce PGD 2 , acts as a neuromodulator in the central nervous system. On the other hand, L-PGDS is a member of the lipocalin superfamily, has the ability to bind a variety of hydrophobic ligands such as bile pigments and retinoids. However, the complex structures of L-PGDS with ligands have not been revealed. In this study, we investigated the interaction of L-PGDS with biliverdin (BV) which has a higher binding affinity to L-PGDS by using multi-dimensional NMR and small-angle x-ray scattering (SAXS).

In NMR experiments, we determined the solution structure of L-PGDS by means of multi-dimensional NMR and distance geometry calculations. L-PGDS possessed the typical lipocalin fold, consisting of an eight-stranded b-barrel and one a-helix.

Upon the titration of BV, chemical shift changes were induced at the strand C, D, E, F and G in 1 H-15 N HSQC spectra. In SAXS experiments, from the linear extrapolations of R g 2 to infinite dilution, R g s of L-PGDS and L-PGDS/BV were calculated to be 19.22 Å and 17.89 Å , respectively. L-PGDS became compact by approximately 1.3 Å in radius after binding BV. These results, taken together, indicate that BV enters the large cavity of the b-barrel, which triggers a conformational change such that the whole molecule of L-PGDS becomes compact; and thus the ligand is tightly held inside the L-PGDS molecule. Introduction: Cytochrome c is a mitochondrial protein fundamental to the cell's respiratory process and apoptosis. Considering that apoptosis can be triggered by the photodynamic action of drugs, it was investigated the photochemical effect of phenothiazines on cytochrome c structure. Methods: Cytochrome c was submitted to UV-irradiation in the presence of phenothiazine compounds: phenothiazine (PTH), thioridazine (TR), trifluoperazine (TFP) and fluphenazine (FP), in different aggregation states modulated by the concentration of the drugs. Results: In the monomeric form, photoexcited TR, TFP and FP promoted cytochrome c damage evidenced by Soret band shift from 409 to 405 nm assigned to the conversion of heme iron to the high spin form (cytc405). The spin change was attested by EPR and MCD measurements. MALDI-ToF mass spectrometry of cytc405 revealed oxidation of Met65, Met80 and Tyr74. In high concentrations of drugs, the irradiation promoted the formation of stable cation radicals in the phenothiazine aggregates that was unable to oxidize cytochrome c. However, low and high concentrations of PTH promoted significant damages in cytochrome c under irradiation. Cytochrome c damage could be among the causes of cell viability decrease when irradiated in the presence of these drugs. Conclusion: For TR, TFP and FP, the aggregation stabilizes the cation radical and prevents the access of the drugs to the heme crevice. PTH, being a small molecule, probably forms less stable aggregates that did not prevent the access of monomers into cytochrome c structure and allows the attack of the photochemically-generated cation radical to the amino acid residues. Acknowledgement: Supported by FAPESP, CNPq and FAEP-UMC. Introduction: Leishmania major, Trypanosoma cruzi, and Trypanosoma brucei collectively cause disease and death in millions of humans widespread in developing countries. There are no vaccines and the few drugs available are inadequate due to high toxicity and resistance. Recently, the flavin containing enzyme dihydroorotate dehydrogenase (DHODH) has been suggested to be a potential target for drug development against trypanosomatids. DHODH catalyses the conversion of L-dihydroorote (DHODH) to orotate, the fourth step and only redox reaction in the de novo pyrimidine biosynthetic pathway. Furthermore, DHODH has been described as a soluble fumarate reductase playing a role in connecting succinate/fumarate metabolism to the de novo pyrimidine biosynthesis. Results: We have solved the crystal structure of both Trypanosoma cruzi and Leishmania major DHODHs in the apo-form and in complex with orotate and fumarate. Structure-based sequence alignment between T. cruzi, L. major and human DHODH combined to site directed mutagenesis and kinetic studies have been used to guide the selection of target sites for drug discovery. The regions, comprising residues 87-94 and 196-219, are fully conserved in tryapanosomatid DHODH sequences and have been identified to share low sequence and structural similarity to the human enzyme. In addition, those regions have been found to provide structural and catalytic stability for the parasitic enzymes. Conclusions: In the present work we have suggest potential sites to be further exploited for design of highly specific inhibitors for DHODHs as an important tool for the development of anti-trypanosomatid drugs. Acknowledgements: to FAPESP for financial support.

Structural insights underlying glyceraldehyde-3phosphate dehydrogenase iodoacetate irreversible inhibitor: crystallography, kinetics studies and molecular mechanism R. V. C. Guido, T. L. Balliano, A. D. Andricopulo and G. Oliva Centro de Biotecnologia Molecular Estrutural, Instituto de Fysica de Sa˜o Carlos, Universidade de Sa˜o Paulo, Sa˜o Paulo, BRAZIL

The structural and chemical basis involved in molecular recognition and binding affinity remains a major challenge in biochemistry and drug design. A key strategy to overcome this limitation is the integration between kinetics studies and crystallography. One of the most prevalent parasitic illnesses in Latin America is Chagas' disease, caused by the parasite Trypanosoma cruzi. An attractive target for the development of novel inhibitors is glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme from the parasite glycolytic pathway. Iodoacetic acid (IAA) is a known inhibitor that inactivates GAPDH by modifying the catalytic cysteine residue. The previously proposed mechanism of inactivation consists of preventing the formation of the GAPDH-NAD complex. However, the molecular determinants underlying the inhibitory mechanism and binding mode remain unknown. The IAA time-dependency and irreversible properties were evaluated by incubating GAPDH with IAA. Crystallization assays were conducted on purified enzyme pre-incubated with inhibitor. Analysis of the structure shows that the modified cysteine is hydrogen bonded to the NAD mediated by a water molecule. Additionally, the delocalized p-electrons from IAA form a p-interaction with NAD. The IAA biding mode suggests that the cofactor plays central role for the inhibitor binding stabilization and the mechanism of inhibition involves preventing the formation of the thiohemiacetal intermediate and the hydrogen transfer between cysteine and NAD during the dehydrogenation step. The results suggest an alternative mechanism of inactivation and elucidate the structural determinants for binding, thus providing insights on molecular modifications aimed at improving affinity. Instead, the protein can binds to membranes, which makes it possible that it recognizes some more general features of the poles. A real fact is that there are two highly conserved residues (R18 and G19) that seem to constitute a polar target determinant. DivIVA secondary structure prediction and in vitro experiments show a coiled-coil protein that can oligomerize in 'doggy-bone' shaped oligomers. But how the protein interacts with other proteins or even with the membrane is still poorly understood. We have undertaken attempts toward the determination of the crystal structure of DivIVA from B. subtilis getting good diffracting data up to 3.5 Å . Also we have developed in vitro liposome binding experiments and electron microscopy analysis to try to understand the way DivIVA can interact with itself, with other proteins (MinD, RacA or SpoOJ) and with the membrane.

Optimising recombinant production of recalcitrant proteins prone to aggregation: the case of papillomavirus E6 oncoproteins A. Ould M'hamed Ould Sidi, K. Zanier and G. Trave Ecole Supe´rieure de Biotechnologie de Strasbourg, Illkirch, FRANCE E6 is a 150-residue oncoprotein composed of two homologous zincbinding domains. It is produced by the human papillomaviruses (HPV) responsible for various tumors including cervical cancer. E6 forms with cellular ubiquitin ligase E6AP a complex that targets the tumor suppressor p53 for degradation. E6 also targets more than 40 other cellular proteins implicated in cell proliferation, apoptosis or adhesion. The structural study of E6 had been hindered for 20 years because it is difficult to express under a native soluble form. We first produced separately the two domains of HPV E6, which turned out to be well folded. We solved the NMR structure of the C-terminal domain, and the structural analysis of the N-terminal domain is under way. However, the full-length HPV E6 remained largely insoluble. We therefore explored several strategies aimed at obtaining soluble samples of full-length E6. 1/ Fusing E6 to the C-terminus of MBP (Maltose Binding Protein) combined with low temperature expression was found to help for solubility and folding of E6; 2/ high-speed ultracentrifugation was utilised to eliminate soluble aggregated particles of MBP-E6 fusions, 3/ systematic checks of monodispersity were performed on all MBP-E6 preparations using light scattering measurements performed in a fluorimeter; 4/ site-directed mutagenesis was performed on nonconserved surface exposed cystein residues in order to prevent intermolecular disulfide bonds; 5/ hydrophobic surface exposed residues were mutated in order to decrease self-association; 6/ we explored the production of alternative E6 proteins from animal papillomaviruses other than HPV possibly presenting a better biochemical behaviour.

The extracellular domain of nicotinic acetylcholine receptor a7-subunit: studying the effects of modifications on the characteristics and pentameric formation Nicotinic acetylcholine receptors (nAChRs) belong to the superfamily of ligand-gated ion channels. Their function is mediated by the binding of specific ligands, responsible for the channel opening and cell communication.

The nAChRs exist in hetero-or homopentameric form. Each subunit consists of an N-terminal extracellular domain (ECD), four transmembrane ahelices and a cytoplasmic loop. The human a7 nAChR forms homopentamers and is known to be implicated in many diseases, therefore presents an attractive drug target. Aiming to proceed to structural studies, several mutants of the human a7-ECD were designed based on the structure analysis of the homologous molluscan acetylcholine-binding protein and Torpedo a1 nAChR-ECD respectively and expressed in the supernatant of the yeast Pichia pastoris. One mutant was identified to fulfill the criteria for initiation of crystallization trials, which resulted in the production of small crystalls. Yet, further improvements are probably needed in order to obtain high quality crystals of the ECD. We thus introduced novel modifications, including changes in the glycosylation status of the expressed protein or different location of various tags. From size exclusion chromatography and dynamic light scattering, it was indicated that the glycosylation, the positioning and the nature of the tag were catalytic for improved protein folding. This observation was further supported by the improvement in the ligand binding affinity of the new mutants. The above improvements, and the enhanced yield and solubility of the new a7 ECD mutant allow us to proceed to the production of improved crystals for high resolution studies. Organophosphorus (OP) nerve agents manifest their toxicity by inhibiting acetylcholinesterase (AChE). Reactivation of nerve agent-inhibited AChE by nucleophilic compounds such as oximes (2-PAM, MMB4, HI-6 and HLo-7) is the most important approach to treating nerve agent poisoning. Since oxime-induced reactivation of OP-inhibited AChE depends on the source of enzyme as well as the structures of OP and oxime, no single oxime is effective against all nerve agents. Therefore, the goal of this study is to design and discover a broad-spectrum oxime for its universal use in the treatment of OP poisoning. Although a number of crystal structures of AChE in complex with reversible inhibitors, or nerve agents, or oximes are reported, the determination of X-ray crystal structure of enzyme-OP-oxime conjugate is extremely difficult because of the fast chemical reaction between oxime and nerve agent-inhibited AChE. Therefore, we employed molecular modeling and simulation methods to build the 3-D structures of nerve agentinhibited human AChE-oxime complexes, using the available crystal structures of AChE. Our modeling studies were guided by our experimental data on the reactivation of sarin or soman-inhibited wild-type and mutant human AChEs by these oximes. These models reveal the position, orientation, and the interactions of 2-PAM, MMB4, HI-6 and HLo-7 with soman-or sarin-inhibited human AChE and amino acid residues critical for reactivation. This information was used for generating a pharmacophore and queries from the pharmacophore were used to identify potential reactivators from our 'in-house' database of small organic molecules. Gating the ion-permeation pathway in potassium channels is associated with conformational changes in activation and inactivation gates. We have prepared proteoliposomes with a purified recombinant potassium channel displaying the KcsA pore and the extracellular pore entrance of a voltage-gated Shaker Kv1.3 channel (KcsA-Kv1.3). Shifting the intracellular pH from 7.5 to 4.0 transiently activates and subsequently inactivates the channel. We investigated structural alterations correlated with gating transition from closed to inactivated states in a membrane setting using solid-state NMR spectroscopy in direct reference to electrophysiological and pharmacological experiments. We observed that at rest the inactivation gate of the KcsA-Kv1.3 channel is opened and the activation gate closed. By contrast, the inactivated channel displayed an opened activation and a closed inactivation gate. Importantly, the selectivity filter of inactivated KcsA-Kv1.3 adopts a nonconductive structure. A remarkably similar structural change within the selectivity filter was induced by binding a pore-blocking tetraphenylporphyrin derivative to the KcsA-Kv1.3 channel. The results establish a structural link between inactivation and inhibition of a K+ channel. Thus, we obtained a channel structure where both activation and inactivation gate were closed. Based on our elucidation of detailed KcsA-Kv1.3 structures with different gate conformations we propose a simple gating model where the two gates within the pore domain are either in an off (0) or on (1) position. Furthermore, we showed that conformational changes near or at the inactivation gate have an important influence on the affinity of potassium channel blockers encluding the well-studied scorpion toxins.

Characterization of anti-angiogenesis protein (SCP1) from cartilage A. Rabbani-Chadegani and S. Abdossamadi Department of Biochemistry, Institute of Biochemistry and Biophysics, University of Tehran, Tehran, IRAN Angiogenesis is the process of forming new capillary blood vessels from preexisting vasculature and it is now believed that it is obligatory for the growth and progression of solid tumors. Shark cartilage is considered as a rich natural source of angiogenesis inhibitors. In this study, by using gel filtration and preparative gel electrophoresis, we have isolated a protein from shack cartilage designated shark cartilage protein 1 (SCP1) with a molecular weight of 13.7 KDa and neutral pI. The bioassay of the protein on microvessel sprouting collagen-embedded rat aortic rings showed a high anti-angiogenesis activity. Circular dichroism spectrum of the protein represents a main negative band at 220 nm which is characteristics of helix structure. The protein contains 28% helix, 20% b-sheet and 40% of the molecule is random coil. Also fluorescence spectroscopy shows that the maximum fluorescence emission intensity is observed at 305 nm which matches with tyrosine emission intensity.

Oligomerization status directs the overall activity regulation of the Escherichia coli class Ia ribonucleotide reductase Ribonucleotide reductase (RNR) is a key enzyme for the synthesis of the four DNA building blocks. Class Ia RNRs contain two subunits, denoted R1 (a) and R2 (b). These enzymes are regulated by nucleotides binding to two allosteric sites on the R1 subunit, termed the overall activity and specificity sites. ATP and dATP can bind to both allosteric sites whereas dTTP and dGTP bind exclusively to the specificity site. The overall activity in class Ia RNRs is generally considered to be regulated by ATP activation and dATP inhibition. By using gas-phase electrophoretic mobility macromolecule analysis (GEMMA), we found that the Escherichia coli RNR formed an a 4 b 4 complex in the presence of dATP and an a 2 b 2 complex in the presence of ATP, dTTP or dGTP. The results from studying two known mutants of the E. coli R1 subunit (N238A and H59A) suggested that a 4 b 4 octamer formation is required to enable dATP inhibition. ATP could also induce an inhibited a 4 b 4 complex but only when used in combination with dTTP/ dGTP. The formation of this complex is therefore determined by a cross-talk effect between the two allosteric sites. In conclusion, the E. coli RNR is regulated by cycling between an active a 2 b 2 complex and an inhibited a 4 b 4 complex. This regulation/oligomerization makes the E. coli enzyme different from the mammalian RNR which is always stimulated by ATP and cycles between active and inactive a 6 b 2 complexes. In both species, the ATP-induced complexes dissociated easily and seemed therefore to be more flexible than the tight dATP-induced a 4 b 4 and a 6 b 2 complexes. Introduction: Xylella fastidiosa is a phytopathogen that causes economically important diseases in wide range of plants including citrus variegated chlorosis, a major threat to citrus industry. In this work, it is studied the stationary phase survival protein SurE from this microorganism, named XFSurE. SurE is a phosphatase whose physiological role and reaction mechanism are not yet completely known. In Escherichia coli, SurE, named EcSurE, acts as nucleotidase and exopolyphosphatase, and it is thought to be involved in stress response. EcSurE dephosphorylates diverse intracellular substrates, especially nucleoside monophosphates in order to maintain all (ribo)deoxynucleotide pools required to RNA, DNA synthesis and other pathways [1] . Methods: The protein XFSurE was expressed using pET29a vector in E. coli BL21(DE3) and purified by nickel metal affinity chromatography. Secondary structure contents and some low resolution parameters were obtained by dichroism circular and SAXS (Small-Angle X-Ray Scattering) analysis, respectively. Functional assays were performed against various phosphorylated substrates. Results: The purified XFSurE has a beta-sheet predominance conformation, a tetrameric organization in solution and a radius of gyration around 37 Å . XFSurE possesses the nucleotidase activity with preference for purine nucleoside monophosphates, especially 3'-AMP and 5'-GMP at neutral pH in the presence of a divalent metal cations (preferentially Mn 2+ ). In addition, some crystals were obtained in three conditions in preliminar crystallization assays, which may lead to its structural determination. There is some evidence that elevated levels of homocysteine and S-adenosylmethionine (SAM) are causal risk factors for several neurological disorders. However the signalling pathways, which control homocysteine metabolism in neural cells, are not fully understood. Ras is a central molecular switch of intracellular regulation and stress response and through interactions with downstream effector molecules regulates the survival and apoptosis of neural cells. To demonstrate the specificity of Ras-elicited effects on the activity of methyl cycles, wild-type pheochromocytoma PC12, mutant oncogenic rasH gene (MVR) expressing PC12 pheochromocytoma and normal c-rasH stably transfected M-CR3B cells were incubated with the N-nitro-L-arginine methyl ester (L-NAME), and manumycin, (inhibitors of nitric oxide synthase and farnesyltransferase, respectively). We have found that L-NAME significantly changes the SAM/SAH ratio in both MCR and MVR cells. Moreover, these alterations have reciprocal character; in the MCR cells, the SAM/SAH ratio was raised, whereas in the MVR cells this ratio was decreased. We conclude that depletion of endogenous NO with L-NAME increased the production of SAH only in cells with mutated oncogenic RasH, possibly through enhancement of production of reactive oxygen species (ROS). Oxidative stress can increase cystathionine beta-synthase activity that switches methyl cycles from remethylation into transsulfuration pathway to maintain the intracellular glutathione pool (essential for the redox-regulating capacity of cells) via an adaptive process.

The b-hairpin containing membrane-active antimicrobial peptides: from the spatial structure in membrane-mimicking media to mechanisms of action The structure of several b-hairpin containing membrane-active antimicrobial peptides belonging to two classes (arenicins and cyclotides) was investigated in membrane-mimicking media using NMR and CD spectroscopy. NMR investigation shows that in water solution arenicin-2 (21-residue cationic peptide from marine polychaeta Arenicola marina) adopts significantly twisted b-hairpin conformation, stabilized by one disulfide bond. CD spectroscopic analysis indicates that arenicin-2 strongly binds to SDS and DPC micelles and to anionic lipid vesicles (POPE/POPG) in contrast to zwitterionic vesicles (POPC). NMR study revealed the formation of asymmetric arenicin dimers upon incorporation into the DPC micelle. Two monomers of arenicin-2 are aligned parallel to each other by the N-terminal strands of the -hairpin (CN››NC type of association). PAGE analysis indicated that in environment of anionic SDS micelles the arenicin-2 undergoes further oligomerization and form tetramers. The spatial structure of ternary complexes peptide/Mn 2+ /DPC micelle was studied for two cyclotides: kalata-B1 and kalata-B7 (29-residue weakly cationic cyclic peptides from African perennial plant Oldenlandia affinis). The both cyclotides are monomeric in DPC micelles, share identical spatial scaffold (triple-stranded b-sheet, formed by b-hairpin and third short distorted strand, stabilized by three knot-forming disulfides) and possess similar divalent cation coordination site near invariant Glu3. In spite of the overall structural homology both cyclotides demonstrate different topology of peptide/micelle complexes. SDS-PAGE analysis and vesicle titration experiments indicated that in environment of anionic detergents or lipids kalata-B7 might undergo oligomerization and form trimers or tetramers. The obtained results point to the 'carpet/toroidal-pore' as the main mechanism of arenicins and cyclotides action.

Characterization of butyrylcholinesterase in a state of lipoprotein disorders K. Siskova, I. Paulikova and L. Bezakova Faculty of Pharmacy Comenius University, Bratislava, SLOVAKIA Introduction: Butyrylcholinesterase (3.1.1.8., BChE) has long been known as a detoxifying enzyme for playing a crucial role in the metabolism of xenobiotics. In recent years we have been investigating various sources (species, tissue), different molecular forms and changes in BChE activity under physiological and pathological conditions. During the observation of BChE activity in dyslipoproteinemic pathological diseases we found it especially interesting, that the activity of BChE correlated with the levels of blood lipoproteins. Surprisingly, after treatment with iso-ompa (specific inhibiotor of BChE) the levels of lipoproteins rectified. Following this, we were interested in the whole picture of BChE behaviour under various dyslipidemic conditions. Methods: Distinct animal models of metabolic lipoprotein disorders were created by dividing rats in three groups: diabetes mellitus (DM), hyperlipidemia (HL), combination DM + HL. Streptozotocin (80 mg/ kg) and high-fat diet were used to develop DM and HL, respectively. After 2 weeks the animals were sacrified. BChE activity was measured by the Ellman method (Ellman G.L. et al., 1961) and molecular forms of BChE were identified by the means of native gel electrophoresis. Results: Altered BChE activity in dependence on the kind of tissue in all of studied models was reported. Moreover, modifications in the spectrum of molecular forms in contrast to control animals were described. Conclusion: We provide characterization of BChE on intertissue and interorgan level in a state of several metabolically distinct lipoprotein disorders.

Correlation of P450c21 modelling structural analyses for disease-causing protein variants and clinical form of congenital adrenal hyperplasia Introduction: Deficiency of steroid 21-hydroxylase is the most common cause of congenital adrenal hyperplasia (CAH). CYP21A1P pseudogenederived mutations, gene deletion and conversion are responsible for 95% of the cases. New and rare mutations are observed in the remaining mutated alleles. Clinical manifestations depend directly on the structurefunction relationship of mutant proteins. The rare R426C mutation, and the S113F, A265V, P267L and V358I novel mutations were identified for the first time in Brazilian patients. Methods: In order to test structural variations of CYP21A2 mutations a PDB-1DT6-based model was used, alterations in protein structure for these mutations were estimated in silico by Sting Millenium (EMBRAPA). Each enzyme variant was evaluated by comparing changes in polarity, distances to heme group and steroid binding site, and heme surface accessibility. The conservation of each aminoacid residue was compared by alignment to human, murine, canine, ovine, porcine, and bovine CYP21 sequence. Results: A265V and P267L mutations are not conserved residues whereas the aminoacid changes cause relevant structural changes in Hhelix end region because L267 residue loses the hydrophobic interaction with Q270. The S113, V358 and R426 are very conserved residues and F113, V265 and C426 substitutions resulted in drastic enzyme conformation changes. The predictions obtained for R426C in this model were similar to structural data in the literature. Conclusion: Hence, this study indicates structural analysis as a powerful tool for predicting effects of new mutations on the enzyme activity when biochemical functional analysis are not available, therefore molecular conclusions can expanded order to confirm clinical diagnosis.

Organic moiety and central metal are both critical players of anticancer activity of metallocene dihalides in human breast and colon cancer cells X. Stahtea 1 , N. Karamanos 1 and N. Klouras 2 1 Laboratory of Biochemistry, University of Patras, Patras, GREECE, 2 Laboratory of Inorganic Chemistry, University of Patras, Patras, GREECE Introduction: Metallocene dihalides, which are cyclopentadienyl complexes with general formula R 2 MX 2 (where R=g 5 -C 5 H 5 , g 5 -CH 3 C 5 H 4 , g 5 -SiMe 3 C 5 H 4 etc.; M=Ti, Zr, Hf, V, and Nb; and X=halogen), are highly effective agents against Ehrlich ascites tumor cells and lymphocytic leukaemia. The aim of this study was to evaluate the anti-tumor activity of the various metallocene dihalides and particularly their effects on cell proliferation of human breast and colon cancer cells. Methods: The compounds used were of the general formula R 2 MCl 2, where for compound [1] there was R= g 5 -C 5 H 5 and M=Ti, for [2] R= g 5 -C 5 H 5 and M=V, for [3] R= g 5 -CH 3 Introduction: Synthetic peptides of small length, based on Substance P C-terminal fragments, increase the secretion of tumor necrosis factor a (TNF-a) and prevent the proliferation of several cancer cell lines. Tri-and tetra-peptides show antiproliferative activity against breast and prostate cancer cells. The aim of this study was the synthesis of a series of peptoid analogs of hexapeptide of the C-terminal region containing residues like D-Trp and Tic and the peptoid residues NHN(R)CH 2 CO and NPhe and Nala. Moreover we stydy their anticancer effect. Methods: Syntheses were carried out stepwise by SPPS on 2-chlorotrityl chloride resin and DIC/HOBt as coupling reagent. Breast cancer epithelial MDA-MB-231 and HT-29 colon cancer cell lines were cultured in absence or presence of hydrazino-peptoids for 48 hours, in presence and abscence of serum and cell proliferation was documented by WST-1 method. Results: In order to study the influence of D-Trp on the antiproliferative activity against cancer cells, D-Trp was incorporated in positions 4 and 6. Purification and identification was performed by HPLC and ESI-MS, respectively. Nicotinic acetylcholine receptors (nAChRs) are ligand-gated cation channels, located mainly on postsynaptic membranes, responsible for cell communication through neurotransmitters. Seventeen different subunits compose homo-or heteropentamers and the stoichiometry gives the features of the individual receptor. The nAChRs composed of a4 and b2 subunits (a4b2-nAChR) are the predominant subtype in the central nervous system, they are mainly responsible for addiction to nicotine and are thought to be implicated in Alzheimer's and Parkinson's diseases; this makes them possible targets for drug design. This aim would be facilitated by the knowledge of the structure of their ligand-binding domains. Our aim is to produce properly folded domains of this nAChR for structural studies. To this purpose, wild type and mutated forms of a4 and b2 extracellular domains (ECDs) of the human nAChR were expressed in Pichia pastoris. Size exclusion chromatography and dynamic light scattering, showed an improvement in solubility when the a4 and b2 ECDs are coexpressed. To further enhance the formation of pentamers, we linked the ECDs from the individual subunits with an (AGS) 8 24-peptide. In addition to the observed increased solubility of the linked ECDs compared with the individually expressed or coexpressed a4-with b2-ECDs, the formation of dipentamers can be suggested in which (a4) 2 (b2) 3 pentamer is linked to the (a4)3(b2)2 pentamer. We conclude that functional assembly of the ECDs is possible and will be further investigated prior to the initiation of crystallization trials.

Cloning and expression of plant ornithine delta-aminotransferase Ornithine d-aminotransferase (OAT, EC 2.6.1.13) is a pyridoxal-dependent enzyme catalyzing the transamination of L-ornithine. In plants, OAT has been found for example in pea and wheat. The physiological role of OAT is related to proline and arginine metabolism. Proline is known to have an important role in the adaptation of plant cells to drought and salinity stress. In this work, a cDNA coding for pea OAT was obtained by reverse transcription of pea seedling mRNA followed by PCR amplification. A PCR product of about 1500 bp, whose identity was verified by DNA sequencing (GenBank acccesion number EU414030), was cloned and expressed in E. coli to obtain C-terminally 6xHis-tagged recombinant protein. To achieve expression, the cDNA was digested by NcoI and XhoI, ligated into the pET28b+ plasmid vector and then used for transformation of NEB Express Competent E. coli cells. The cells were cultured in a liquid medium, harvested by centrifugation and disrupted for isolating the soluble cytoplasmic fraction. Recombinant OAT was partially purified by chromatography on HIS-select affinity gel under native conditions. Basic molecular and kinetic properties of the recombinant enzyme were characterized. SDS-PAGE demonstrated a molecular mass value of 50 kDa. Activity assay was performed using a spectrophotometric method, which is based on the formation of a colored compound by the reaction of pyrroline-5-cyrboxylate with ninhydrin. A specific activity value of 0.3 nkat/mg was measured in the lyzate, which increased to 5.6 nkat/mg after the affinity purification. Acknowledgement: Supported by the grants MSM 6198959216 and GACR 522/08/H003.

Identification of novel tyrosinase inhibitors by structure-based computational approach Tyrosinase (EC 1.14.18.1) is known to be a key enzyme for melanin biosynthesis that leads to pigmentation of the skin. At the present day, many tyrosinase inhibitors have been evaluated in cosmetics and pharmaceuticals because they become important to prevent hyperpigmentation of the skin. However, many of these inhibitors suffer from limitations, such as low activities, high cytotoxicities, insufficient skin penetration and low stabilities in formulations. In this study, we tried to identify novel tyrosinase inhibitors that can overcome those limitations by structure-based computational approach. (i) We constructed homology models of mushroom and human tyrosinases based on the crystallographic coordinates of Streptomyces castaneoglobisporus tyrosinase. (ii) We estimated our chemical virtual library using structurebased virtual screening. (iii) Small molecules that showed high scores were examined inhibitory activities to both mushroom and human tyrosinases by in vitro assay. As a result, we identified a unique Com-pound23 as a novel tyrosinase inhibitor. The Compound23 showed inhibitory effects on mushroom and human tyrosinases in a dose dependent manner, and the ID 50 values were established to be 0.1 lM and 2.0 lM, respectively. Therefore, these results indicate that our screening system is useful for discovering new tyrosinase inhibitors. We believe that the optimized molecules from Compound23 have potentials to become cosmetics and pharmaceutical agents.

Directed mutations of oligomerization interface of TyrRS cytokine-like C-domain result in its low aggregation propensity F. Tereshchenko and A. Kornelyuk Institute of Molecular Biology and Genetics, Kiev, UKRAINE Introduction: Tyrosyl-tRNA synthetase (TyrRS) catalyzes specific attachment of tyrosine to the acceptor stem of cognate tRNA. Mammalian cytoplasmic TyrRS is composed of two structural modules: N-terminal catalytic core and cytokine-like C-terminal domain separated by unstructured linker. The C-domain belongs to a class of partially unfolded proteins, with transiently disordered regions between amino acids 95 and 123. One of the major obstacles in doing biochemical analyses of the isolated C-domain is its tendency to aggregate. This property is typical for the protein with transiently disordered regions. Methods: Docking of C-domain with itself followed by structural analysis of the interface has shown that three amino acids may play key role in aggregate formation. In vitro analysis confirmed low aggregation propensity of TyrRS triple mutant E120K, N150P and M152E. We have compared 10 ns molecular dynamic trajectories (GROMACS) of wild-type C-domain with its computationally engineered triple mutant at four temperatures (310, 315, 321 and 328K). Results: The secondary structure content of mutant protein increases with temperature rise -yet another typical feature of intrinsically disordered aggregating proteins. Correlation analysis shows that the mutant also exhibits much lower motions amplitude. We have shown previously that wt C-domain starts transition into molten globule at 328K. Conclusions: We hypothesize that since process of aggregation is often preceded by formation of molten globule (hydrophobic residues are getting exposed to solvent), an overall stabilization of mutant secondary structure and decrease in motions amplitude may preclude it from exposing more hydrophobic patches, transiting into molten globule, and, therefore, aggregating.

Antimicrobial peptides (crocosins) from freshwater crocodile, (Crocodylus siamensis) white blood cell extracts Introduction: Most researches to date have focused on searching the antimicrobial peptides without bringing resistance. This report describes a new antibacterial peptides (Crocosins) purified from leukocyte extracts of freshwater crocodile, and characterized for their functions. Methods: Crocosins were purified by RP-HPLC. The primary structure, The MIC of these peptides toward two bacterial strains and their hemolytic activity toward human RBC were determined. The kinetic of bacterial killing were characterized by SEM. Results and conclusion: The Crocosin II and III exhibit antibacterial activity toward Salmonella typhi and Staphylococcus epidermidis. The MICs of Crocrosins against bacteria were 0.125 and 0.033 lg/ll respectively. Crocosin II shows no significant destroy RBC but Crocosin III displays high toxicity. The N-terminal of both peptides were found to be blocked for sequencing, amino acid composition was used to determine the partial sequence. Crocosin II contains high mole% of Ile and Crocosin III shows high mole% of Glu. The SEM images of bacteria exposed to Crocosins provide the evidence that membrane is the main target of the bactericidal of these peptides. This study shows that freshwater crocodile leukocyte extracts contains broad spectrum antimicrobial peptides and encourage further investigation of the therapeutics potential of these peptides.

Preparations, properties, and active-site structure of Escherichia coli ethanolamine ammonia-lyase Adenosylcobalamin (AdoCbl)-dependent ethanolamine ammonia-lyase (EAL) catalyzes the conversion of ethanolamine to acetaldehyde and ammonia. The eutB (a) and eutC (b) genes in the eut operon of E. coli were cloned and expressed at high levels. The purified apoEAL existed as a a6b6 complex. At high concentrations, it formed inactive precipitates which were reactivated by a dithiothreitol treatment. Limited proteolysis of the enzyme by trypsin led to the removal of the N-terminal 28 amino acid residues from the b subunit without loss of enzyme activity. Several kinds of EALs that lack the N-terminal 28 residues of b subunit and contain a His 6 -tag were expressed and purified by a Nichelate affinity column, and their properties studied. When the eutB and eutC genes were placed under the control of tac or T7 promoter in this order, subunit compositions of the purified enzymes were imbalanced, but the imbalanced expression was much improved when the order of genes was reversed. Km values for substrate and AdoCbl as well as inactivation properties in the absence of substrate and in the presence of substrate analog 2-aminopropanol were not affected, while the susceptibility to SH inhibitors was lowered, by the N-terminal truncation and C34S mutation. EALs with truncated b subunit and His6tag were not precipitated/inactivated at high concentrations. A trun-cated enzyme was crystallized in complex with cyanocobalamin and ethanolamine, and the structure of EAL was analyzed by X-ray crystallography. The mechanism of action of EAL is discussed on the basis of its active-site structure. Introduction: Burkholderia sp. MBA4 produces a haloacid permease, Deh4p, which mediated the uptake of haloacid as a growth substrate. Deh4p contains 552 residues with a mass of 59.4 kDa. Structural prediction of Deh4p suggested the presence of 11 or 12 transmembrane helices. Most of these predictions suggested that the N terminus is located in the cytoplasm. In this study we reported the determination of the topology of Deh4p by using a PhoA-LacZ dual-reporter system. Methods: PhoA is an alkaline phosphatase which only works in the periplasm while beta-galactosidase (LacZ) is an enzyme that works in the cytoplasm. DNA fragments encoding various lengths of deh4p were amplified by PCR and fused in-frame with a phoA-lacZ cassette. These recombinant plasmids were transformed and the fusion proteins expressed in E. coli using a weak constitutive promoter. Enzyme activities of PhoA and LacZ were determined to localize the whereabouts of the reporter and thus the authenticity of the transmembrane domains. Results: The expression of the membrane proteins did not affect the growth of the cells. More than 36 constructs were made and transformed into E. coli. The PhoA/LacZ enzyme activity ratios for these clones have been determined using PNPP and ONPG. The results showed that the first two and the last two predicted transmembrane domains were absent. Conclusions: The current results suggested that there are at most eight transmembrane domains found in Deh4p. The N-and the Cterminals were located in the cytoplasm and a large loop was exposed in the periplasm.

Evolution of a molecular switch by a single mutation surmounts the chloride ion dependent mechanism of ACE Angiotensin Converting Enzymes (ACEs), are a major target for cardiovascular therapies that consist an evolutionary conserved family of zinc metalloproteases. The last years were landmarks for the structure based knowledge of ACEs offering new details at the molecular level. However, important questions concerning the different mechanisms of the chloride ion selectivity among ACEs remain open. On the basis of pK(A) calculations and homology modelling we suggest a model for the ion (in)dependent mechanism of ACEs. We validate our model through site-directed mutagenesis. We suggest that ACEs from different organisms may have evolved unique ionic dependencies to cope with the diverse environments they encounter.

A new docking and screening method based upon bio-informatics H. Umeyama, M. Takeda-Shitaka, D. Takaya, K. Kanou and G. Terashi Kitasato University, Tokyo, JAPAN Introduction: Many disease-protein targets have been found form biochemical experiments. Druggable compounds which inhibit or activate those significant protein targets must be researched rapidly. Many researches are using in-silico screening program such as DOCK, Auto-Dock and GOLD using classical mechanical potentials. We report the new method which uses simulated annealing based on bio-informatics on the protein-ligand flexible docking. Methods: We use docking engine based upon bioinformatics such as protein structure prediction and effective usage for many X-ray or NMR structures of protein-ligand complex. An amino acid sequence of the target protein which is query sequence are aligned in the filter of CE Z-Score 3.7 for biological similarity significant with the PDB database with some BLAST alignment methods. In the stage of liganddocking, fingerprint (FP) is used as the basis set in the bio-informatics based simulated annealing. FP is composed of two, three or four atoms. Moreover this FP includes the information of atom-type such as used in Sybyl atom-type [[REF=]] and bond-type. Next FP alignment score is defined to determine the docking pose of the ligand to be the most stable one. Results: We could make the docking and screening method based upon bio-informatics newly. The accuracy is almost similar to the GOLD and GLIDE docking methods most famous, and higher than the DOCK, and AutoDock methods famous. Conclusions: A new docking and screening method based upon bioinformatics were made, and this method covers new docking poses different from classical mechanical potential methods.

Compact packing of lipocalin-type prostaglandin D synthase induced by binding of lipophilic ligands Introduction: We measured small-angle x-ray scattering (SAXS) of lipocalin-type prostaglandin D synthase (L-PGDS) and that of two other lipocalins, b-lactoglobulin (bLG) and retinol-binding protein (RBP), to clarify their conformational changes induced by binding of small lipophilic ligands, such as all-trans-retinoic acid (RA), bilirubin (BR) and biliverdin (BV). Methods: SAXS data were collected at the BL40B2 in the Spring-8 synchrotron radiation facility (Hyogo, Japan). Scattering profiles in the small-angle region were analyzed by Guinier's approximation for monodispersive systems and then, the radius of gyration and the forward scattering intensity were calculated. Molecular structures of the polypeptides were predicted by applying the ab initio structure determination program. Results and discussion: The radius of gyration was estimated to be 19.4 Å for L-PGDS, 18.8 Å for L-PGDS/RA, 17.3 Å for L-PGDS/BR and 17.8 Å for L-PGDS/BV complexes. Alternatively, the radius of gyration of bLG and RBP (20.3 and 26.2 Å , respectively) was almost the same before and after the binding of RA. Moreover, the ab initio model of L-PGDS and three complexes showed not only that the overall structure of molecule was globular in shape, but also that complexes have become more compact than L-PGDS upon ligand binding. All SAXS data suggested that the L-PGDS molecule had the structural flexibility to accommodate different kinds of lipophilic ligands. Such flexibility of the L-PGDS molecule must be a main factor for the broad selectivity of L-PGDS.

Structural studies of natural killer cell receptor family NKR-P1 Introduction: Natural killer cells are able to directly kill tumour cells without previous antigen sensitization. NK cell receptors are divided in two structural groups: immunoglobuline and C-type lectin. From the latter group, first identified signalling molecule was named NKR-P1 (also known as CD161 or KLRB1). Several members of this receptor gene family are known now from rodents, but only single one was found in human. As there is no crystal structure of any of NKR-P1 receptor molecules yet, hope is we can shed light onto these closely relative but functionally divergent proteins. Methods: Cloning via standard protocols, recombinant expression, HPLC, analytical ultracentrifugation, dynamic light scaterring, dropcoating deposition Raman spectroscopy, mass spectrometry, X-ray crystallography. Results: We cloned lectin domains of rat and human NKR-P1 receptors into pRSETB vector and recombinantly produced them in E. coli ArcticExpress expression system. Extensive HPLC purification was followed by characterization by DLS, AUC, DCDR and MS techniques. Crystallization trials were set up in hanging drop arrangement. Conclusions: We successfully cloned desired genes and were able to produce them as soluble proteins which preparation and characterization is shown. Crystallization is currently in progress. Vascular endothelial cells (ECs) are constantly exposed to blood flowinduced shear stress. Shear flow, which is atheroprotective, stimulates endothelial nitric oxide synthase (eNOS) activity. Recent study suggests that nitric oxide (NO)-mediated S-nitrosylation (-S-NO) is a key posttranslational modification of proteins. The effect of eNOS-derived NO on redox-sensitive protein tyrosine phosphatase (PTP) in ECs was examined. Using Biotin Switch method, a flow dependent S-nitrosylation of PTPs including PTP1B, PTEN and SHP2 was observed in ECs after the onset of flow. ECs transiently transfected with PTP containing catalytic cysteine mutant (Flag-SHP2 C459S or HA-PTP1B C215S ) abolished the flow-induced S-nitrosylation, indicating that this redox-sensitive Cys is the vulnerable target of NO. ECs under flow, similar to NO donor (SNAP) treatment, reversibly inhibited the SHP2 activity via this S-nitrosylation. ECs pretreating with an eNOS inhibitor (L-NAME) significantly suppressed this flow-induced S-nitrosylation in SHP2. In contrast, ECs pretreating with DPI, an inhibitor to NADPH oxidase (NOX), or SiRNA to NOX2/NOX4 to decrease intracellular ROS significantly enhanced flow-induced S-nitrosylation of SHP2. Consistently, ECs exposed to H 2 O 2 alone decreased the S-nitrosylation of SHP2. Our results indicate that major PTPs in ECs under flow are predominantly S-nitrosylated at catalytic Cys residue. NO and ROS appear to compete for the catalytic site in PTP. We conclude that S-nitrosylation of PTP is a key posttranslational control of PTP activity in ECs under flow. This S-nitrosylation may protect PTP from ROS-induced irreversible oxidation and thus play an important role in controlling signaling responses in ECs. Introduction: The deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus was directly evolved for enhancement of penicillin G (PenG) expansion into phenylacetyl-7-aminodeacetoxycephalosporanic acid (G-7-ADCA), an important precursor for industrial manufacture of cephalosporin. The current industrial synthesis of 7-ADCA includes complicated chemical expansion of the five-membered thiazolidine ring of PenG into the six-membered dihydrothiazine ring of G-7-ADCA, followed by enzymatic removal of side chain. Since the chemical ring expansion is expensive and polluting, the development of alternative biosynthetic methods has been studied for decades. Methods: A two-step screening was developed to select desirable mutants from hydroxylamine random mutagenesis, error-prone PCRbased random mutagenesis and DNA shuffling. Results: The S. clavuligerus DAOCS was directly modified for enhancement of PenG expansion for industrial production of 7-ADCA. Fifteen point mutations with 1.4-to 14.2-fold increases in the k cat /K m ratio were obtained by site-directed and random mutagenesis. Subsequently, DNA shuffling was carried out to screen possible combinations of these substitutions. Three mutants with increased activity were found. Conclusions: SC59, SC14 and SC63 mutants with increased k cat /K m ratios of 41-, 51-and 60-fold were obtained using directed evolution. SC63 showed the highest increase on binding of PenG (22-fold) with 2.8-fold improvement on conversion rate. SC14 showed the best improvement on conversion rate (6-fold) with 9-fold increased binding affinity. SC59 showed 14-fold and 3-fold improvements in K m and k cat values. This study provides not only promising enzymes for cephalosporin biosynthesis, but also some unpredictable combination of mutations, which reveal new insights into the structure-function relationship of this protein.

Identification of hot spot residues in XIAP-peptide complexes using molecular docking and MM-GBSA scoring The X-chromosome-linked inhibitor of apoptosis (XIAP) found to be widely expressed in the human cancer cell lines, and apoptotic resistance was found to correlate with the expression levels of XIAP in the human cancer cells. Recent studies suggested that potent XIAP antagonists that bind its BIR3 domain may have promising potential as a new class of anti-cancer drugs. However, because the binding interfaces are modulated by protein-protein interactions (PPI), it is difficult to develop effective drug compounds to inhibit these processes. Identification of hot spot residues can be a good first approach in a PPI targeted drug lead design, because it is known that binding free energy of PPI is mainly from amino acid residues in hot spots. Here we try to identify hot spots by per-residue binding free energy decomposition for PPI using Molecular docking and MM-GBSA (Molecular Mechanics-Generalized Born-Solvent Accessibility) scoring. To evaluate the performance of our approach, 37 peptide antagonists of XIAP, whose dissociation constants are known, were considered. The correlation coefficient (R 2 ) between experimental binding free energy and MM-GBSA scoring is 0.585. This result shows that our approach could predict accurate binding modes and binding free energies. On the decomposition analysis, K299, G306, T308, D309, E314, and W323 were revealed to contribute < -3.5 kcal/mol to the effective energy. Therefore, we propose these amino acids as hot spot residues on the XIAP binding interface. The efficient identification of hot spot residues using the computational approach may be useful to the development of PPI modulator drugs.

Three isoforms of complement properdin factor P in trout: cloning, expression, gene organization and constrained modeling M. Chondrou 1 , A. Papanastasiou 1 , G. Spyroulias 2 and I. Zarkadis 1 1 Department of Biology, School of Medicine, University of Patras, Patras, GREECE, 2 Department of Pharmacy, University of Patras, Patras, GREECE Introduction: Properdin is a plasma glycoprotein and the only known naturally occurring positive regulator of the complement system, stabilizing the alternative pathway convertase (C3bBb). In order to elucidate the molecular evolution of properdin factor P (pfc), here we report the cloning and characterization of three gene isoforms of properdin in rainbow trout (Oncorhynchus mykiss). Methods and results: The predicted polypeptide sequences of trout properdins pfc1, pfc2 and pfc3 (447, 449, and 447 amino acids, respectively) share 78-90% identity to each other showing the highest identity score (47%) with their zebrafish ortholog protein. The six TSR domains of the three trout properdin isoforms seem to adopt the folding pattern of human thrombospondin 1 TSP-1 domains, where each TSP-1 domain forms an antiparallel three-stranded structure that consists of alternative stacked layers of Trp and Arg residues from respective strands capped by disulfide bonds on each end. The trout pfc2 and pfc3 genes are arranged in nine and ten exons, respectively, which span 3.5 kb of the genome. Liver, in contrast to the expression profile of the properdin gene in mammals, is the main source of the trout properdin mRNA transcripts. In a phylogenetic analysis, trout pfc1, pfc2 and pfc3 genes are clustered with their orthologs from other teleost species. Conclusion: This is the first report of three separate genes coding for properdin factor P in a vertebrate species. The trout pfc proteins resemble to the 'domain' architecture of mammalian ortholgs, while, in contrast to the mammalian properdins, trout pfc genes are expressed mainly in liver.

Human muscle fructose 1,6-bisphosphatase (FBPase) is highly sensitive toward calcium ions due to Glu69: comparative study of the Glu69 localisation in vertebrate muscle FBPases M. Zarzycki and A. Dzugaj Institute of Zoology, University of Wroclaw, Wroclaw, POLAND Introduction: Two FBPase isozymes: liver (regulatory enzyme of gluconeogenesis) and muscle (regulatory enzyme of glyconeogenesis) were found in vertebrates. Recently we have found that unlike liver isozyme, muscle FBPase is highly sensitive toward inhibition by calcium ions due to Glu 69 . In the muscle isozyme the presence of two acidic residues (Asp 68 , Glu 69 ) interacting with calcium might stabilize the loop 50-70 in the inactive position, whilst in the liver isozyme the position of 69 is occupied by Gln. In the present paper we have attempted to answer the question if the other vertebrate muscle FBPases contain also Glu in position 69. Methods: Chemical modification, site-directed mutagenesis, BLAST program. Results: Comparative study of available FBPase sequences among 11 mammalian species revealed high sequence homology of the loop 50-70. The 69 residue is exclusively occupied by glutamic acid in muscle isoenzyme and glutamine in liver FBPase. The high homology of the loop 50-70 was also observed in chicken, snake and frog FBPases: containing Glu 69 in muscle and Gln 69 in liver isozymes. In some fish species (e.g. Tetrarodon, Gastrosteus) Glu is substituted with Asp in muscle isozyme. Nevertheless it confirms that an acidic residue is indispensable for calcium binding. Conclusions: Hypothetically speaking during evolution the replacement of Gln to Glu dramatically changed the kinetic properties of the new isoenzyme, making the muscle FBPase more sensitive toward inhibition by calcium and enabling regulation of glyconeogenesis by calcium. In Gram-negative bacteria membrane-protein channels, also known as porins, constitute the entry point of various classes of antibiotics. With the advent of bacterial resistance to antibiotics, porins are becoming a prime target. The outer membrane porin F (OmpF) is the major outer membrane protein component of Escherichia coli and bacterial strains resistant to antibiotics showed either underexpression or mutations of OmpF. To study antibiotic translocation at a molecular scale, we performed molecular dynamics (MD) simulations combined with the metadynamics algorithm. This recently designed algorithm overcomes the time scale problem of classical MD by accelerating some reaction coordinates. We compared the following methodologies: modeling (i) OmpF in its monomeric or trimeric form and (ii) membranes as surrounding detergent molecules or as lipid bilayers. Furthermore, we considered antibiotics of different structural, chemical and physical properties (penicillins, fluorokinolones, cephalosporines). Earlier MD studies on the OmpF trimer reported structural differences between each monomer. Differences arising from some key mutations were also investigated. However, the influence of such differences on antibiotic translocation is not clear. To answer this question we performed a careful structural analysis of our simulations in the different conditions stated above. We reveal the reciprocal influence of each monomer and the role of single residues and loops L2-L3. We also calculated the cross sectional surface area at the pore constriction zone and determined the preferred binding sites for the antibiotics. Overall, our results are in good agreement with experiments. This work directly benefits the design of new antibiotics with improved transport properties. Introduction: Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine to form phosphatidic acid and choline. PLD activity is thought to be involved in a broad range of physiological responses, including rapid responses such as secretion and superoxide generation, as well as in long-term responses such as proliferation, differentiation, and immune response. CKII is a ubiquitous serine/threonine kinase and plays a critical role, not only in cell growth and proliferation, but also in anti-apoptosis. Methods: CK2 activity was assessed using a specific peptide substrate. Gene expression was investigated by reverse transcription-PCR and Western blotting analysis. Protein-protein interaction was examined by immunoprecipitation and yeast two-hybrid assay.

Results: The overexpression of PLD downregulated CKII activity in NIH3T3 cells. The same results were found with catalytically inactive point mutants of PLD, indicating that the catalytic activity of PLD is not required for PLD-mediated CK2 inhibition. The reduction in CK2 activity in PLD-overexpressed NIH3T3 cells was due to reduced protein level, but not mRNA level, of the CK2b Subunit. This PLDinduced CK2b degradation was mediated by ubiquitin-proteasome machinery. The C-terminal domains of PLD interacted with the N-terminal domain of CK2b. The colocalization of PLD and CK2b was observed in the cytoplasm of NIH3T3 cells, especially in the perinuclear region. Conclusion: PLD can downregulate CK2 activity through the acceleration of CK2b degradation by ubiquitin-proteasome machinery.

Post-translational activation of IAP(s): a conserved mechanism from Drosophila to mammals Although IAPs are best known for their ability to bind and neutralise caspases, they also fulfil signalling function that are independent of caspases such as ubiquitin-dependent survival signalling, cell migration and receptor-mediated NF-kB activation. IAPs harbour a C-terminal RING finger domain, which provides them with E3 ubiquitin-protein ligase activity. Recent data suggest that IAPs are not constitutively active as E3s but require activation through post-translational modification. Thus, the E3 activity of the Drosophila IAP DIAP1 is regulated through phosphorylation by DmIKKe (Drosophila IKK-related kinase). Once phosphorylated DIAP1 is activated, the protein is selfubiquitylated and degraded through the proteosome. Consequently, active caspases can take part in non-apoptotic signalling events [1] . Using large scale affinity purification coupled to mass spectrometric analysis, we have identified TWINS, a regulatory subunit of the the serin-threonine phosphatase PP2A, as a DIAP1 interaction partner. We are currently testing whether PP2A antagonises DmIKKe-mediated activation of DIAP1's E3 ligase activity. Thus, the anti-apoptotic activity of DIAP1 may be under the tight regulatory control of both DmIKKe and PP2A. Moreover, we have also found that the mammalian IAP, c-IAP1, specifically interacts with PR55, the mammalian orthologue of Drosophila TWINS. Thus, our preliminary results suggest that the activity of c-IAP1, like the one of DIAP1, is under the control of kinases and phosphatases. Given the evolutionarily conservation of this regulatory circuit, it is likely that this post-translational modification fulfils an important regulatory function. It is now well established that proteins labeled with ubiquitin (Ub) may lead to proteasomal degradation. Ubiquitination also controls the function and intracellular localization of a wide variety of proteins. Small ubiquitin-like modifier (SUMO) involve in various cellular processes, such as DNA damage repair, nuclear transport, chromosome segregation, signal transduction, cell division, stress response, oncogenesis, inflammatory response in mammals and the regulation of flowering time in plants. Some reports have shown that reactive oxygen species may trigger the ubiquitination system, and the levels of SUMO conjugates increased substantially following exposure of animal cells or plants to heat shock, ethanol and hydrogen peroxide. Here we report that hydrogen peroxide and arsenite caused dramatic inductions of ubiquitination and sumoylation respectively in cells. In order to set up a suitable study system, we have generated cell lines stably expressing HA-tagged Ub, HA-tagged SUMO1 and FLAG-tagged SUMO2. Using S5a-conjugated agarose beads or immunoprecipitation with antibodies against SUMO1 and SUMO2, molecules targeted by Ub and SUMOs were purified from hydrogen peroxide or arsenite-treated cells respectively. Samples were further processed by USP2 or SENP1 for removing Ub or SUMO and subjected to SDS-PAGE, 2D-PAGE and Mass Spectrometry analysis. Because the Ub conjugates and SUMO conjugates induced by cytotoxic stress and genotoxic stress in cells have not been systematically studied, this new approach using ubiquitin-specific protease and SUMO-specific protease reveals a specific way for refining the data and eliminating the non-specific binding caused by the conventional affinity chromatography. The Par polarity complex consisting of the evolutionarily conserved proteins mPar3, mPar6, and atypical PKCs (aPKCs) regulates cell polarity in many cell types including neurons. In neurons, the establishment of polarity is directed by the sequential activity of the GTPases Rap1B and Cdc42. Rap1B is initially present in all neurites of unpolarised neurons, but becomes restricted to the tip of a single process where it specifies axonal identity. Here we show that mPar3 is required for the establishment of neuronal polarity and links the E3 ubiquitin ligase Smurf2 to Kinesin-2. Smurf2 ensures that neurons extend only a single axon by initiating the degradation of inactive Rap1B through the ubiquitin/proteasome system. Its interaction with mPar3 is required to localize Smurf2 to growth cones and restrict Rap1B to a single axon. Interfering with the binding of mPar3 to Kinesin-2 or of Smurf2 to mPar3 and knockdown of mPar3 by RNA interference (RNAi) disrupt the establishment of neuronal polarity through the failure to restrict Rap1B to a single neurite. Introduction: Schizosaccharomyces pombe Btb3p, which has two ankyrin repeats and two BTB/POZ domains at the N-and C-terminals, respectively, functions as a putative substrate-specific adaptor for Pcu3p-based ubiquitin ligase. BPOZ-2, which is involved in the growth suppressive effect of PTEN, has a high degree of amino acid sequence homology to that of Btb3p. Since the proteins with the BTB/POZ domain have been considered to function as the substrate-specific adaptors for CUL3-based E3 ligases, we speculated that BPOZ-2 is a human counterpart of Btb3p and functions as a substrate-specific adaptor for CUL3-based E3 ligase. Methods: Interactions between proteins were examined by immunoprecipitation, GST pull-down, and yeast two-hybrid assays. We identified the substrates of BPOZ-2 by the yeast two-hybrid screening of a human thymus cDNA library using BPOZ-2 as the bait. The in vivo ubiquitination assay was performed by transfecting several combinations of plasmids together with His-tagged Ub vector into 293T cells.

After precipitating ubiquitinated proteins with Ni 2+ beads, the precipitants were analyzed by immunoblotting.

Results: BPOZ-2 associates with CUL3 through its BTB/POZ domains to be ubiquitinated by the CUL3-based E3 ligase and degraded by the 26S proteasome. TdT gene was isolated as a BPOZ-2 binding protein using a yeast two-hybrid system. TdT, BPOZ-2 and CUL3 form a ternary complex and co-localize within the nucleus. BPOZ-2 promotes the ubiquination and degradation of TdT through the CUL3-based 26S proteasome system. Conclusions: BPOZ-2 is a human counterpart of Btb3p and a substrate of CUL3 ligase. TdT is the substrate of the BPOZ-2 ⁄ CUL3 ligase complex.

Mechanism of ubiquitin-independent proteasomal degradation of human thymidylate synthase Thymidylate synthase (TS) is a well-recognized chemotherapeutic target, and inhibitors of the enzyme are broadly used in cancer treatment. TS levels inversely correlate with sensitivity of cells to TS inhibitors; therefore, we have focused on degradation of the enzyme as a determinant of its intracellular concentration. We have shown that TS degradation is mediated by the proteasome in a ubiquitin-independent manner, and is regulated by the unstructured N-terminal domain of the enzyme. We hypothesized that the disordered nature of the N-terminal domain, perhaps promoted by the region's high Pro content, along with a free, non-acetylated N-terminus are necessary for recognition by, and entry into, the proteasomal chamber. Using 2D-gel electrophoresis and MALDI-TOF-MS, we analyzed the acetylation status of the N-terminal residue of both wild-type and several mutant forms of TS. We observed that unstable TS polypeptides (i.e., the wild-type enzyme and the P2A and P2G mutants) have a free, non-acetylated Nterminus; in contrast, stable polypeptides (i.e., all other substitutions at Pro2) are N-alpha-acetylated. Deletion of the proline-rich regions within the N-terminal domain of the wild-type enzyme led to 3-fold stabilization of the polypeptide, while conversion of all Pro residues to Ala had no appreciable effect. Finally, we determined that the N-terminus is the proteasome entry site for degradation, and that proteolysis occurs from the N-toward the C-terminus. Our results shed light into the mechanism of ubiquitin-independent degradation of TS, and provide possibilities to modulate the enzyme's intracellular concentration that might lead to enhancing therapeutic efficacy of TS inhibitors.

PP3B-9 X-ray structure of the Skp1-Fbs1 glycoprotein complex Introduction: Ubiquitin-mediated proteolysis plays a regulatory role in a number of diverse cellular processes and involves the selective destruction of short-lived functional proteins. The ubiquitin ligase complex SCF Fbs1 , which contributes to the ubiquitination of glycoproteins, is involved in the endoplasmic reticulum-associated degradation (ERAD) pathway. Misfolded proteins and unassembled protein complexes that fail to assume their functional states in the endoplasmic reticulum are subjected to ERAD, which involves retrotranslocation into the cytosol and degradation by the ubiquitin-proteasome system. Fbs1/Fbx2, a member of the F-box protein family, recognizes highmannose oligosaccharides. To elucidate the structural basis of SCF Fbs1 function, we determined the crystal structures of the Skp1-Fbs1 complex and the sugar binding domain (SBD) of the Fbs1-RNaseB complex at 2.4 and 2.7 Å resolutions, respectively. Results: The structure of the Skp1-Fbs1 complex illustrates a different class of F-box proteins within the SCF ubiquitin ligase model. The mechanistic model indicated by the structures appears to be well conserved among the SCF ubiquitin ligases. The structure of the SBD-glycoprotein complex indicates that the SBD primarily recognizes Man3GlcNAc2. The surface areas occupied by Man3GlcNAc2 and the protein portion of the substrate are 349 and 165 Å 2 , respectively, suggesting that Man3GlcNAc2, but not the protein in RNaseB, defines the interaction with Fbs1. SCF Fbs1 is a functionally unique molecule that recognizes the innermost Man3GlcNAc2 in N-glycans as a marker of denatured proteins. Our results provide a mechanistic basis for the recognition and ubiquitination of various glycoproteins by SCF Fbs1 .

Ribosomal protein S6 kinase 1 interacts with and is ubiquitinated by ROC1 ubiquitin ligase Ribosomal protein S6 kinase (S6K) is involved in the regulation of cell growth and cellular metabolism. The activation of S6K in response to diverse extracellular stimuli is mediated by multiple phosphorylations coordinated by the mTOR and PI3K signalling pathways. Downregulation of S6K signalling occurs through the actions of S/T phosphatases (PP2A and PP1) and tumor suppressors (TSC1/2 and PTEN). The overexpression of S6K1 in various cancer cell lines and tumors, and the oncogenic potential of its splicing form has been reported. The increased expression of S6K1 has been linked to gene amplification and translational upregulation of S6K1 transcript. We have recently found that both forms of S6K are modified by ubiquitination. Following these findings, we demonstrate here that S6K1 associates specifically with ubiqutin ligase ROC1 in vitro and in vivo. The interaction was initially identified in the yeast two-hybrid screening and further confirmed by pull-down and co-immunoprecipitation assays. Furthermore, the overexpression of ROC1 leads to an increase in S6K1 ubiquitination. Consistent with this observation, we showed that the steady-state level of S6K1 is regulated by ROC1, since downregulation of ROC1 by specific siRNA promotes stabilization of S6K1 protein.

The results suggest the involvement of ROC1 ubiquitin ligase in S6K1 ubiquitination and subsequent proteasomal degradation. Also deregulation of the ubiquitination/proteasomal degradation pathway may regulate the level of S6K1 in cells. Malfunctioning of the ubiquitin/ proteasome proteolytic pathway has been implicated in the development of cancer and other human pathologies. The connection between the ubiquitination-mediated turnover of tumor suppressor PTEN, which is a key component of the PI3K/mTOR signaling pathway, and tumor progression has been recently demonstrated. A similar mode of regulation might be relevant for the regulation of S6K signaling in cancer cells.

Tyrosinase degradation by the proteasome is stimulated with an algae extract in human melanocytes Introduction: Tyrosinase is a key enzyme for melanin synthesis. A dysregulation in its activity leads to melanin accumulation in the skin and problems with skin staining such as lentigos. The proteasome is responsible for tyrosinase degradation. In a previous study [1] we have shown that a polyunsaturated fatty acid rich algae extract from Phaeodactylum tricornutum had a stimulating effect on proteasome peptidase. Methods: The human melanocyte cell line MNT-1 was treated with Phaeodactylum tricornutum for 24 hours. Proteasome and tyrosinase activities and amounts as well as melanin concentration were monitored before and after treatment. Results: In this study, we have found that the algae extract can activate the proteasome, but does not increase its quantity in the treated human MNT-1 melanocytes. This proteasome stimulation was associated with an increased tyrosinase degradation and a decreased melanin accumulation. In addition, immunoprecipitation experiments showed that tyrosinase was less ubiquitinated suggesting that its degradation by the proteasome was more efficient. Conclusions: These results indicate that stimulation of the proteasome could serve as a new strategy for modulating skin pigmentation and for preventing melanin accumulation in lentigos. Introduction: Pex5p recognizes newly synthesized peroxisomal matrix proteins in the cytosol and transports them to the peroxisomal membrane where it participates in a network of protein-protein interactions with components of the docking/translocation machinery. These interactions ultimately result in the insertion of Pex5p and the translocation of the cargo-protein across the organelle membrane. After this step, Pex5p is exported and becomes available for new cycles of transportation. Recently, it was suggested that yeast Pex5p needs to be monoubiquitinated to be exported from the membrane. In this work, we addressed whether this step also occurs in mammals. Methods: To determine if mammalian Pex5p is ubiquitinated, we used an in vitro import assay comprising a rat liver postnuclear supernatant and 35 S-labeled Pex5p/Pex5pC11S. GST-ubiquitin was included in some experiments to facilitate the identification of ubiquitinated species. Results: We demonstrate for the first time that ubiquitin is involved in Pex5p-mediated import pathway in mammals. Two different kinds of ubiquitination could be observed. One of these is thiol-sensitive and involves conjugation of ubiquitin to Cys11 of Pex5p. This ubiquitination is required for Pex5p export back into the cytosol. Conclusion: Our data reveals the existence of an ubiquitin-conjugation cascade acting on mammalian Pex5p during its transient passage through the importomer. Here, Pex5p is ubiquitinated via a thiol-ester bond at Cys11, an unusual type of ubiquitination that may be more common in nature than previously considered.

Lysine 63-linked polyubiquitin chain serves as a targeting signal for the 26S proteasome Y. Saeki, T. Kudo, A. Toh-e and K. Tanaka Tokyo Metropolitan Institute of Medical Science, Tokyo, JAPAN Recruitment of substrates to the 26S proteasome for their destruction usually requires covalent attachment of the Lys48-linked polyubiquitin chain. In contrast, modifications with the Lys63-linked polymeric chain and/or monomeric ubiquitin are known to function in proteasomeindependent cellular processes. However, the ubiquitin chain-type specificity for the proteasomal targeting are still poorly understood. Using mass spectrometry, we show here that Rsp5, a ubiquitin-ligase in budding yeast, catalyzes entirely the formation of homopolymeric Lys63linked ubiquitin chains in vitro. Unexpectedly, the 26S proteasome degraded well the Lys63-linked polyubiquitinated substrate in vitro. In addition, while the membrane-anchored transcriptional factor Mga2-p120 is modified by both Lys48-and Lys63-linked polyubiquitin chains in a steady state, the level of Lys63-linked chains is significantly increased by proteasome inhibition in vivo. Furthermore, chase experiments revealed that Mga2-p120 processing was retarded by overproduction of Lys63-mutated ubiquitin in vivo. Our results suggest that Lys63-linked polyubiquitin chains may function in an alternative proteasomal targeting signal. The proteasome is a multicatalytic protease responsible for the breakdown of intracellular proteins. We have previously described in vitro and in vivo proteasomal S-glutathionylation and in vitro deglutathionylation by glutaredoxin 2 (Grx2). Moreover, proteasomal multicatalytic activity was proved to be modulated by both mechanisms. The thioloxidoreductase Grx2 is able to enter the catalytic core to deglutathionylate the proteasome by an ubiquitin-independent mechanism and concomitantly degraded. Since the S-glutathionylated redox state of the proteasome was more effective to degrade Grx2 as well as oxidized proteins, the purpose of this study was to analyze protein fragments generated by the proteasome at different redox states taken Grx2 as model. Rates of degradation were evaluated by SDS-PAGE and the peptide products through mass spectrometry by comparing DTTreduced and partially S-glutathionylated proteasomal preparations. Results revealed that the products of degradation differed according to the proteasomal redox state. Results accumulated thus far indicate that S-glutathionylation seems to be an important post-translational modification of the proteasome. Our hypothesis is that proteasomal redox modulation may have a significant functional role during oxidative stress and antigen presentation since higher eukaryotic proteasomes are also redox modulated. In normal cells, p53 is present at low concentration due to its rapid turnover via the ubiquitin-proteasome system and the role of the ubiquitin ligases Mdm2, COP1, Pirh2, TOPORS and CHIP. The aim of our project was to determine whether p53 is labeled for ubiquitylation in senescent cells by specific ligases. We used HFL-1 fibroblasts and observed reduced protein levels of p53, which was accompanied by a decrease in protein levels of Mdm2 and a slight decrease in the RNA and protein levels of COP1 in senescent cells. In contrast, protein levels of Pirh2 and TOPORS were not altered, but there was a slight increase in RNA and protein levels of CHIP. In senescence caused by partial inhibition of the proteasome, p53 ligases followed similar expression patterns as in natural replicative senescence. Since Nutlin-3 has been established as a specific inhibitor of the p53-Mdm2 interaction, we were then interested in identifying the effect of this inhibition on the other ligases of p53. Pre-treatment of young and senescent cells with Nutlin-3 stabilized p53 leading to induction of its transcriptional targets, Mdm2 and p21. After removing the inhibitor, p53 was rapidly decreased, while no significant change was observed for COP1, Pirh2, CHIP and TOPORS, suggesting that Mdm2 is a central key molecule to the regulation of p53. In conclusion, a differential effect of ubiquitin ligases on p53 regulation has been observed, and more direct interactions are currently under investigation.

Role of ubiquitin C-terminal hydrolase-L1 in mammalian oocyte A. Susor, A. Pavlok, L. Jelinkova and M. Kubelka Institute of Animal Physiology and Genetics, Libechov, CZECH REPUBLIC Introduction: Ubiquitin C-terminal hydrolase-L1 (UCH-L1) is a member of family of deubiquitinating enzymes. The ubiquitin-proteasome system regulates many cellular processes via specific protein degradation. Proteomic analysis of oocytes demonstrated that UCH-L1 was one of the most abundant oocyte proteins during meiotic maturation [1] . Methods: To study role of UCH-L1 in the process of fertilisation in bovine model we used the specific inhibitors of this enzyme, UCH-L1 inhibitor (isatin O-acyl oxime derivate) and inhibitor 16 [3-Amino-2keto-7H-thieno(2,3-b)pyridin-6-one derivate]. To further investigate the UCH-L1, we analysed the oocytes by SDS/ Native PAGE, Western blotting, immunocytochemistry/confocal microscopy and oocytes undergo in vitro fertilization. Results: The level of ubiquitinated proteins and monoubiquitin in oocytes treated with inhibitors is increased in comparison with control. Treated eggs have significantly higher rate of polyspermy in in vitro fertilization assays (46% vs. 13%). UCH-L1 is localized at cytoplasmatic membrane in both control and treated oocyte during meiosis. Conclusion: Our results unveil that the UCH-L1 is essential in fertilization process where contribute to the block of polyspermy in mammalian eggs.

The substrate competition toward the tissuebound semicarbazide-sensitive amine oxidase (SSAO) purified from rat lung G. Ucar and S. Yabanoglou Department of Biochemistry, Faculty of Pharmacy, Hacettepe University, Ancara, TURKEY Introduction: Semicarbazide-sensitive amine oxidase (SSAO), which catalyzes the oxidative conversion of primary amines, was suggested to play a scavenger role in the case of overproduction of many endogenous/exogenous amines. There is limited data concerning the substrate competition towards SSAO. Methods: Substrate competition toward the purified SSAO from rat lung by Cibacron Blue 3GA-agarose and Concanavalin A-Sepharose 4B affinity chromatographies were evaluated by kinetic studies. Results: Oxidation of benzylamine by purified SSAO was obeyed at only low concentrations. High substrate inhibition was detected with benzylamine. Oxidation of methylamine by purified SSAO obeyed to the Michaelis-Menten behavior, and no high substrate inhibition was detected for the oxidation of SSAO by MA. The ability of some biogenic amines such as tyramine, kynuramine, methylamine, dopamine, serotonin (5-HT) and phenylethylamine to interfere with the metabolism of benzylamine by SSAO was determined. Kynuramine was found to be the most potent inhibitory amine which behaved as a non-competitive inhibitor. The rest of the amines tested for rat lung SSAO inhibited benzylamine metabolism in a decreasing order as methylamine, dopamine, serotonin and phenylethylamine. These amines behaved as competitive inhibitors of BA metabolism by rat lung SSAO suggesting that they possibly compete with the substrate for the active site of the enzyme. Tyramine did not show any inhibitory effect on the oxidation of benzylamine by rat lung SSAO. Conclusions: It was shown that all the amines tested, except tyramine, inhibited the benzylamine oxidation by rat lung SSAO suggesting that there is a competition between the endogenous amines towards SSAO. Introduction: Rodents and insectivores carry hantaviruses some of which cause human diseases: either hemorrhagic fever with renal syndrome (HFRS) or hantavirus pulmonary syndrome (HPS). It was recently reported that the cytoplasmic tail of envelope glycoprotein Gn of pathogenic but not of apathogenic hantaviruses are proteasomally degraded.

Results: Here, we show that the Gn tail of the apathogenic Tula hantavirus (TULV) is also degraded through the ubiquitin-proteasome pathway. We also report that the Gn tails of TULV form aggresomes in transfected cells when treated cells with proteasomal inhibitors. Conclusion: These results suggest that the degradation upon aggregation of Gn tails may be a result of a general cellular response to accumulation of miss-folded protein and might be used by hantaviruses to control maturation of virus particles.

The TRIP12 HECT domain alone is sufficient to function as the minimal ubiquitin ligase for the ubiquitin fusion degradation pathway J.-B. Yoon and Y. Park Yonsei University, Seoul, KOREA Introduction: Ubiquitin (Ub) fused linearly to the N-terminus of a protein is efficiently removed by deubiquitinating enzymes (DUBs) in the cell. However, the Ub moiety rendered noncleavable by DUBs acts as a signal for degradation of the fusion protein. Genetic and biochemical analyses have revealed key enzymes involved in UFD pathway in yeast, including the E3 Ub ligase. Human E3 Ub ligase for the UFD pathway has not been studied so far. Methods: To investigate the role of TRIP12 in the UFD pathway, expression of TRIP12 was blocked by transfection of TRIP12-specific siRNAs into HeLa cells, and the stability of the UFD substrate was evaluated. In addition, the ubiquitination of UFD substrate was reconstituted with purified recombinant proteins. Results: Knockdown of TRIP12 expression stabilized the UFD substrate but not the N-end rule substrate. Expression of siRNA-resistant TRIP12 rescued the defect in the UFD pathway. Surprisingly, the TRIP12 HECT domain alone reversed suppression of the UFD pathway in TRIP12 knockdown cells. In an in vitro ubiquitination assay system, TRIP12 HECT domain mediated ubiquitination of the UFD substrate in conjunction with E1, E2, and E4 enzymes.

The results indicate that that TRIP12 is the human E3 Ub ligase responsible for the UFD pathway in vivo and in vitro, and that the TRIP12 HECT domain alone is sufficient to function as the minimal Ub ligase for the UFD pathway. Introduction: In the present study, the response of aB-crystallin to hydrogen peroxide (simulating oxidative stress) and calpain inhibitors was investigated. The high expression levels of this sHsp in cardiac muscle underline its significant role in preservation of homeostasis under adverse conditions. Methods: H9c2 cardiac myoblasts were used as our experimental model since their response reflects the signal transduction pathways activated by stress conditions in the myocardium. We performed immunoblot analysis parallel to immunofluorescent and immunoprecipitation experiments. Results: In H 2 O 2 -and calpain inhibitors (ALLN, ALLM)-treated H9c2 cells, the mechanism regulating aB-crystallin phosphorylation was found to involve primarily intracellular free calcium levels and subsequently the p38-MAPK signalling pathway. Phosphorylated aBcrystallin was demonstrated to be co-localized with tubulin, potentially preserving cytoskeletal architecture. Prolonged calpain activity repression ultimately led to apoptosis confirmed by PARP fragmentation and chromatin condensation. However, the apoptotic pathways activated by ALLM and ALLN differed, indicating the diverse transduction mechanisms triggered. Furthermore, an anti-apoptotic role for phospho-aB-crystallin was verified by confirmation of its interaction with pro-caspase 3, hindering its cleavage and subsequent activation in ALLM-treated H9c2 cells. Conclusions: Collectively, our findings highlight aB-crystallin fundamental role as an effector mediating cardiac cells early response to stressful stimuli potentially compromising their survival.

A. Alberti, P. Karamessinis, M. Peroulis, K. Kypreou, P. Politis and A. Charonis Biomedical Research Foundation of the Academy of Athens, Athens, GREECE Introduction: Type 2 diabetes is associated with pancreatic b-cell dysfunction and insulin resistance. Hyperglycemia is a major contributor of b-cell dysfunction, a phenomenon known as 'glucose toxicity'. Methods and results: We examined differential regulation of the b-cell proteome in cultured murine TC-6 pancreatic beta cells, under low versus high glucose conditions, mimicking 'glucose toxicity'. Two-D gel electrophoresis, image analysis software and MALDI-TOF-TOF Mass Spectrometry analysis led to identification of 45 differentially expressed spots, corresponding to 20 proteins. Among these, ERp46, a novel endoplasmic reticulum resident protein related to protein disulfide isomerase, was further studied. This protein is highly expressed in cells producing proteins exhibiting disulfide bonds as is the case with insulin in pancreatic beta cells. Immunoblotting analysis revealed a decrease in the expression of ERp46 under high compared to low glucose conditions. Since cell lines do not perfectly mimic the primary bcell physiology, pancreatic islets were isolated for further studies. Immunoperoxidase staining in pancreatic sections revealed high expression of ERp46 in islets compared to exocrine tissue. This observation was further confirmed by immunoblotting analysis. Furthermore, isolated murine islets cultured under conditions of high glucose demonstrated a down-regulation in ERp46 expression, compared to low glucose. Most importantly, inhibition of ERp46 expression by siRNA in cultured beta cells led to a significant decrease of insulin content monitored by immunofluorescence and Western blotting. Conclusion: These data suggest that ERp46 may be a crucial component of the 'glucose toxicity' phenomenon, playing an important role in the pathogenesis of diabetes. Besides their anticoagulant activities, they have been reported to possess cholesterol lowering and radical scavenging properties. In this study, lipid lowering activity of 7,8-dihydroxy-3-(3-methylphenyl) coumarin (DHMC) which was synthesized by our department was shown in hyperlipidemia induced female Sprague Dawley rats. We investigated the protein changes in liver tissues with proteomics technology-two dimensional gel electrophoresis (2-DE). We isolated protein samples from the hyperlipidemia induced (n = 5) and control (n = 5) rat livers. 2-DE was carried out with an immobilized pH gradient strip in the first dimension and by polyacrylamide gel electrophoresis in the second dimension. The 2D PAGE revealed that the liver protein profile was intensified in the range of pI: 5,5-9, Over 1000 polypeptide spots were resolved by the PDQuest scanning and projection program. We observed that the protein profile of the hyperlipidemia induced group was changed, intensity of some of the spots decreased and some of them increased. These proteins were excised and identified by MALDI-Tof analysis and compared to Matrix-Science protein data base. Their molecular weights and pI values were determined.

The tPA/plasminogen system in TSEs: friend or foe? Introduction: Transmissible Spongiform Encephalopathies (TSEs) are invariably fatal neurodegenerative diseases, for which no effective treatment is yet available. The prion is the common pathogen of all TSEs and its major component is the abnormal isoform (PrP Sc ) of the cellular prion protein (PrP C ). Tissue plasminogen activator (tPA), is a serine protease involved in the dissolution of blood clots, through the activation of plasminogen to active plasmin. tPA also plays a role in the CNS and is involved in neurodegeneration and neuronal plasticity. Numerous reports have linked the tPA/plasminogen system to TSEs and PrP. We have previously shown: that tPA expression and activity are enhanced in TSE-affected animals, that PrP C but not PrP Sc can be cleaved by plasmin, and that tPA -/and plasminogen -/mice display shorter survival intervals than wild type (wt) mice upon challenge with the infectious material (Xanthopoulos, et al. Neurobiol. Dis. 2005; 2: 519-527). To gain a better understanding of the relationship between the tPA/plasminogen system and TSEs, we have challenged transgenic mice overexpressing tPA in their neurons. Methods: Transgenic mice, which overexpress tPA in their neurons were generated and characterized for tPA gene expression level and enzymatic activity, as well as PrP C expression. These mice have been challenged with the infectious agent. Results/conclusions: tPA activity in the brains of transgenic mice was found 20% higher than in wt mice, whereas PrP expression was normal. The mice were successfully challenged with the infectious material. Results from this bioassay will shed light on the relationship between TSEs and the tPA/plasminogen system. Methods: For this purpose, cUNG, hUNG, and UNG inhibitor (UGI) were expressed and purified in large scale nearly to homogeneity by different chromatographic techniques using AKTA purification system. Then the enzymes were characterized biophysically using two different calorimetric analysis methods: Differential Scanning Calorimetry (DSC) and Isothermal Titration Calorimetry (ITC), in order to determine overall stability/flexibility, and to determine protein-protein or protein-DNA interaction energetics, respectively. Results: DSC analysis showed cUNG has almost 9°C lower in melting temperature than hUNG indicating less stability of cUNG than its mesophilic counterpart. The finding supports cold adaptive characteristics of cUNG suggested by previous studies. ITC experiments also revealed lower K d and higher enthalphic contribution for free energy of binding of cUNG with UGI than that of hUNG, in support of cold adaptive behaviour of cUNG. Conclusion: cUNG has higher activity than hUNG at the cost of low thermal stability as indicated for many cold adaptive enzymes. Introduction: Muscle acetylcholine receptor (AChR), formed by five homologous subunits (a 2 bcd or a 2 bed), is the major autoantigen in the autoimmune disease myasthenia gravis (MG), in which pathogenic autoantibodies bind to, and cause loss of functional AChRs in the neuromuscular junction. Our aim is to construct the heteropentamers a 2 bcd and a 2 bed of the human AChRs, consisting only of the extracellular domains (ECDs). Such molecules should retain the complete antigenic structure of the AChR and thus should allow us to optimize a novel therapeutic approach we are currently developing, which involves the specific depletion of the pathogenic autoantibodies from MG sera. Methods: The ECDs used were mutants which contained the hydrophilic Cys-loop from the homologue acetylcholine binding protein (AChBP), which was previously observed to improve the expression yield and solubility of the ECDs. In order to achieve the construction of the pentameric ECDs, we linked the ECDs in pairs (a-c, a-e, a-d, bd or vice versa) using a 24-residue flexible linker [(AGS)8] and expressed them in Pichia pastoris.

The results showed that the linkage between two subunits is efficient and SDS-PAGE confirmed that they appeared at the expected molecular weight (60 kDa). Successful ligand-binding experiments with a-bungarotoxin and other ligands (d-tubocurarine and carbamylcholine) suggested proper protein folding.

The results confirmed that it is feasible to proceed to the construction of the heteropentameric human AChR ECDs using the linked subunit-ECDs, and that these molecules are promising material for future structural and therapeutic studies.

Proteolytic processing of the b-amyloid precursor protein (APP) in membranes of the human neuroblastoma SH-SY5Y cells Introduction: Deposition of b-amyloid peptide, derived from the altered processing of APP, is an invariant feature of Alzheimer's disease. APP, substrate for three different proteases, can undergo two different proteolitic pathways: the non-amyloidogenic pathway involving a-and c-secretases, and the amyloidogenic pathway involving b-and c-secretases. Both APP and secretases are membrane-bound proteins, but data concerning their membrane distribution yielded diverse results. Indeed, studies have been often performed using APP-and/or secretase-overexpressing cells and the membrane protein overexpression might lead to altered interactions with membrane lipids. Here we report the proteolitic processing of APP in membranes of non-transfected SH-SY5Y cells.

Methods: Membrane fractions were isolated by a 5-30% linear sucrose gradient fractionation in the presence of Triton X-100. Every fraction was characterized for its content in lipids and proteins: lipids (cholesterol, sphingomyelin, glycosphingolipid) were analyzed by HP-TLC and proteins (flotillin, actin, transferrin receptor, APP and its proteolitic fragment, a-, b-and c-secretases) by immunoblotting with specific antibodies.

Results: Membrane fraction analyses show the existence of two distinct pools of APP: whereas the major pool is in the lipid rafts where there is also the fragment b-CTF and b-and c-secretases, the major pool is in 'non-raft' membrane regions where there are also localized a-CTF, a-and c-secretases.

Conclusions: These data are consistent with the concepts that APP amyloidogenic processing occurs in raft domains in contrast to non-raft localized non-amyloidogenic process. Moreover, they indicate nontransfected SH-SY5Y cells as valid cellular model to unravel how access of these enzymes to APP is regulated. Research Group on Brain Aging (EA 3808) University of Poitiers, Poitiers, FRANCE, 2 Department of Geriatrics, Poitiers University Hospital, Poitiers, FRANCE, 3 Department of Neurology, Poitiers University Hospital, Poitiers, FRANCE Introduction: Two major pathways, PKR (double-stranded RNAactivated Protein Kinase) and mTOR (mammalian Target of Rapamycin) could be involved in the neuronal death observed in Alzheimer's Disease (AD) marked by memory disturbances and cognitive dysfunctions. Effectively, there is an activation of PKR and a sustained reduction of phosphorylated mTOR in different cellular, animal models of AD and in brain and lymphocytes of AD patients. However, the molecular link between PKR and mTOR going by way p53, Redd1 and TSC2 remains unknown in AD. Methods: Human neuroblastoma cells (SH-SY5Y) and rat primary neuronal cultures (E17) were incubated with 20 lM amyloid-beta peptide . Clinical study was also conducted in lymphocytes of AD patients. By using PKR siRNA and a specific PKR inhibitor, these three factors were analyzed by western blotting, immunocytochemistry and RT-PCR. Results: The upregulation of PKR and the downregulation of mTOR were associated with an increase of phosphorylation of p53, a transcriptional activation of Redd1 gene and a decrease of phosphorylated TSC2 expression in cellular models. PKR siRNA and specific PKR inhibitor decreased the transcriptional activation of Redd1. Clinical study showed also an increase of p53 and Redd1 expression in lymphocytes of AD patients with significant correlation with activated PKR. Conclusions: These results demonstrate that Redd1 could represent molecular link between PKR and mTOR pathways and a possible target for new therapeutic strategies in AD. In recent years, great interest has been generated related to characterization of protein folding and unfolding, as well as in developing small molecule inhibitors of protein aggregation. In the present work, thermal aggregation of alpha-amylase from Bacillus amyloliquefaciens (BAA) was studied at 65°C in phosphate buffer pH 6.9 .Various techniques including turbidity measurements, intrinsic fluorescence, farand near-UV, circular dichroism spectroscopy, and light scattering were employed to characterize the aggregation process. Acidic and basic pH conditions were investigated in order to study the effect of protein net charge. While aggregation was significantly reduced at pH 2, it was observed at greater extents upon increasing pH toward pI of the protein (6.2). Light scattering results showed that the two phases of nucleation and elongation in the process remain similar in these pH values. No aggregation could be observed in the pH range of 8-12, apparently due to repulsive electrostatic interactions. Proline, lysine, tryptophan, and tyrosine (5-30 mM) significantly reduced aggregation, with proline being the most effective. Addition of aspartic acid and glutamic acid enhanced aggregation, again supporting involvement of electrostatic interactions in the aggregation process.

Mutations in APC gene and constitutive activation of Wnt-pathway Methods: Genomic DNA was purified from peripheral blood leukocytes and the complete coding sequences of APC gene, including splice junctions, were amplified using PCR, followed by sequencing. Multiplex ligation-dependent probe amplification (MLPA) was also used in order to identify genomic rearrangements of APC gene. Additionally, the identified non-synonymous variants were investigated using the Exonic Splicing Enhancers (ESEs) software for their interference to correct splicing. Results: Analysis of the APC gene revealed thirteen germline mutations in twenty families matching the criteria for Familial Adenomatous Polyposis. Five of these mutations are described for the first time (novel). Further investigation of several synonymous or non-synonymous variants of unknown significance revealed that many of them may interfere with alternative splicing of the APC gene. In particular the silent variant p.T1493T, found to interfere with exonic ESEs sequences, resulting in aberrant splicing. Conclusion: The identification of germline mutations within the APC gene patients with family history, along with the complete characterization of the effect of rare missense variants in sporadic colorectal cancer cases, will provide the in-depth understanding of mechanisms underlying colorectal tumorigenesis.

Misfolding of heterologously-produced eukaryotic translation initiation factor 5A P. Gentz, G. Blatch and R. Dorrington Rhodes University, Grahamstown, SOUTH AFRICA Introduction: The function of eukaryotic translation initiation factor 5A (eIF5A) is dependent on hypusine, which is formed by the posttranslational modification of a highly conserved lysine residue (Lys51 in yeast) involving deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). DHS, DOHH and hypusinated eIF5A have become targets for anti-cancer therapies, particularly as eIF5A is thought to play a role in the translation of specific mRNAs encoding proteins involved in the onset of the G1-S phase in the cell cycle. Structural data of eIF5A and archaeal homologues has been derived from crystals produced from heterologously-expressed unhypusinated eIF5A. Methods: Affinity chromatography was used to purify heterologouslyproduced eIF5A (EceIF5A). The oligomeric state of EceIF5A was determined using gel filtration, native PAGE and cross-linking experiments. Site-directed mutagenesis of specific residues in EceIF5A was used to identify residues involved in maintaining the oligomeric state. The biological activity of these mutants was determined in a yeast complementation system. Results: EceIF5A exists as a dimer in solution. The formation of this dimer is dependent on the highly conserved Cys39 residue, but not Lys51, which is required for dimerisation of the native protein in yeast. These and other results indicate that eIF-5A produced in E. coli is misfolded with important implications for the interpretation of current structural data. Conclusions: Evidence of misfolding in heterologously produced eIF5A implies limited information from crystal structures derived from this protein. There is a need for structural data on hypusinated eIF5A to understand its function in vivo.

Correlation between protein misfolding, immunity and neurodegeneration Methods: AFM was used for characterization of amyloidogenic species, as also die mapping. Abs were determined using ELISA and Dotblot analysis in patient sera with diagnose Alzheimer's disease and Parkinson's disease. Results: Comparative investigations of sera antibodies (Abs) production to precisely characterized various species ranging from oligomers to protofilaments and fibrils of disease specific proteins (Ab (25-35) , asynuclein) and abundant proteins (human lysozyme, insulin) in progressive Alzheimer's disease and Parkinson's disease were performed. We have revealed specific correlations between nature of misfolded protein species, Abs production to them and early outset of disease. Conclusion: These studies provide new insights on the role of immune networks that protect neuronal cells from damage and apoptotic death during progressive neurodegeneration and usage of data to new therapeutic approaches targeted against misfolding diseases.

PP3C-15 DRPLA aggregates are highly dynamic J. Hinz and Z. Ignatova Department of Cellular Biochemistry, Max-Planck-Institute of Biochemistry, Martinsried, GERMANY Introduction: The molecular basis of slowly progressing hereditary neurodegenerative disease dentatorubralpallidoluysian atrophy (DRPLA) is associated with a mutation that abnormally increases inframe the number of glutamine residues in the atrophin-1 over a threshold value of 30-40 repeats. Methods: Using fluorescence imaging of cells and FRAP live imaging we study the dynamic localization and mobility of the aggregates in HEK293 and differentiated and undifferentiated N2a cell line. The behaviour of the full length atrophin-1 with polyQ-stretch in the nonpathological (Q = 14) and pathological (Q = 71) length (FL14, FL71) and with truncated NLS sequence (NLS14, NLS71) fused downstream the GFP protein was examined. To quantitatively assess the partitioning of the aggregate species between the detergent-labile and detergentresistant fraction we perform fractionation assays followed by an immunodetection. Results and conclusions: nNLS71 forms readily hyperfluorescent loci, even at the early time points of expression with a clearly defined SDS-resistance pointing out to an early appearance of the fibrillar phenotype. However, these early species are less densily packed with a higher degree of mobility within the aggregate loci. The FL71 expressing cells show a mixed population of cytoplasmic and nuclearly localized aggregates, whereas prolonged expression results in mainly cytoplasmic localization and fusion to one large aggregate. For the non-pathological variants nNLS14 and FL14 we observed in addition to cells with homogenous GFP fluorescence in substantial fraction of cells (and intriguingly in all differentiated cells) hyperfluorescent foci. The latter showed a fast recovery of the fluorescence and lack of insoluble fraction after fractionation suggesting spatial localization within the cytoplasm. Introduction: Carbonic anhydrases (CA, EC 4.2.1.1) are a family of metalloenzymes catalyzing the hydration of CO 2 to bicarbonate and a proton. a-Cas are involved in essential cellular functions such as pH regulation, respiration, gluconeogenesis, lipogenesis, ureagenesis, tumor progression. So far, 15 catalytically active carbonic anhydrase isozymes are described in mammals. hCA XII is one of the transmembrane human carbonic anhydrase isoforms. hCA XII is present in many normal tissues and overexpressed in some tumors. Aim: The aim of this study was to investigate CA XII expression in HT 29 colon carcinoma cell line upon cytokine (TNF-a, TGF-b). Methods: The expression of CA XII in colon cells was detected by real-time fluorescent quantitative PCR. Results: There was no significant difference in the expression level of CA XII between cytokine treated and non-treated colon carcinoma cells. While TNF-a treatment increased proliferation TGF-b had a significant anti-proliferative effect in cells.

Conformational changes of blood plasma albumin in humans exposed to ionizing radiation Introduction: Albumin is the most abundant of the proteins in the blood plasma and acts as a transport carrier of endogenous and exogenous ligands poorly soluble in water. Dynamics of membrane proteins play a prime role in most functional characteristics of cells. Aim: The aim of work was to determine the several aspects of patients blood plasma albumin alterations related to the biophysical modifications of cell membrane. Methods: For the study 54 Latvian residents, who participated in accident cleaning-up works during 1987-1988 were selected during April 2006-June 2007. We registered the spectral characteristics of probes ABM (derivative of aminobenzanthrone) and ANS in lymphocytes suspension and blood plasma, determine the membrane anisotropy and plasma albumin auto-fluorescence. Results: Screening of ABM labeled blood plasma samples revealed that in 83% of patients (Group 1) the fluorescence zone is shifted (compared to the spectrum for healthy donors) by 20-50 nm to the short wave region (maximum 630-600 nm). The 17% of patients (Group 2) had a fluorescence maximum at 650 nm corresponding to the fluorescence observed in healthy donors. Patterns of spectra are associated with membrane anisotropy and conformational changes of blood plasma albumin which is more pronounced in Group 1. Results in Group 1 are like to N-F transition of albumin. The shift is accompanied by increasing ABM fluorescence intensity two times. It may be explained by an increase of albumin binding sites capacity. Human plasma albumin has 2-3 binding sites for ANS, different in affinity for this probe. The fluorescence from these binding sites has a different quantum yield and relatively large degree of polarization (i.e., higher mobility of bounded ANS, higher accessibility of water to these binding sites). Obtained hydration of tryptophanyl region in albumin molecule is interpreted as indole group transition into the buried region, inaccessible to water (closing of a structural slit). There is a strong correlation between all investigated parameters. Conclusion: Results shown that the cell membranes, first of all for their proteins, are significant targets of ionizing radiation. It may be concluded that membrane fluorescence characteristics (e.g., anisotropy changes) are representative for radiation induced membrane alterations and albumin carrier function changes.

Starmaker is an intrinsically disordered protein Introduction: Aim of the present study was to investigate the effects of Polyscias filicifolia Bailey biomass tincture on activities of aminoacyl-tRNA synthetases (ARS) under anoxia. Methods: Isolated pig hearts were perfused under normoxic and anoxic conditions. The activities of aminoacyl-tRNA synthetases in preparations of total ARS were measured by an initial rate of tRNA aminoacylation. Results: The activities of alanyl-, seryl-glutamyl-and leucyl-tRNA synthetases after 20 minutes anoxia had increased by 67-77% compared to the control. The tincture of Polyscias filicifolia Bailey biomass did not affect the activities of these aminoacyl-tRNA synthetases in norm. However, after 20 minutes anoxic perfusion with buffer containing Polyscias filicifolia Bailey, activities of alanyl-, seryl-glutamyl-and leucyl-tRNA-synthetases reached control values. The activity of inorganic pyrophosphatase, which is found in total ARS preparations, after 20 minutes anoxia increased by 87% compared to the control. However, after 20 minutes anoxic perfusion with buffer containing Polyscias filicifolia Bailey, the activity of inorganic pyrophosphatase reached control values. Conclusions: Alteration in the activity of inorganic pyrophosphatase is one of the reasons causing changes in functional activity of ARS under anoxia. One of the possible mechanisms of Polyscias filicifolia Bailey biomass tincture protective action on the activities of ARS may be the effect on inorganic pyrophosphatase which is important in regulation of aminoacyl-tRNA synthetases activities.

Studying the mechanism of amyloid formation by apomyoglobin mutant forms Amyloidogenesis and misfolding are general fundamental problems of medicine and biophysics, the solution of these problems may provide effective treatment for many diseases. A lot of proteins, even not involved in diseases, is known to be able to form amiloid-like structures similar in final fibrils architecture. This fact suggests, that formation of cross-b structure is a common property of a polypeptide chain under appropriate conditions. Apomyoglobin is a convenient model for amyloid formation study due to the folding of this protein is well known. Based on prediction of our laboratory theoretical group, we chose two residues (V10 and L115) and replaced them with alanine and phenylalanine. For identification of amyloids we used CD spectroscopy and electron microscopy. Kinetics of amyloid formation was monitored by ThT binding and turbidity, rate constants were calculated. We found, that rates of amyloid formation by mutant proteins with different substitutions at the same position are approximately equal, but they vary from for apomyoglobins with substitutions at 10th and 115th positions. Our data suggest that the rate of apomyoglobin amyloid formation is affected by the position of a substitution, but not hydrophobicity of amino acids at the same position. 

Interactions between prion proteins and molecular chaperones by the example of ovine prion variants VRQ and ARR, and chaperonin GroEL Results: To clarify the role of chaperones in the development of prion diseases, the interaction of the chaperonin GroEL with two ovine prion isoforms VRQ M ARR has been studied. On the first step the ability of prions to bind chaperonin GroEL was proved using chaperonin immobilized on sepharose and differential scanning calorimetry method. It was shown that prions compete with other denatured proteins in binding to chaperonin GroEL. This was also confirmed by decrease of efficiency of the GAPDH chaperon-assisted folding in the presence of prions. On the second step the chaperonin GroEL effect on thermoaggregation and the aggregate size of VRQ and ARR was studied. Thermoaggregation of prions was observed by monitoring the turbidity of prion solution at 320 nm. The size of aggregates was estimated by the dynamic light scattering analysis. Obtained results demonstrate that chaperonin GroEL (without or in presence of Mg-ATP and cochaperonin GroES) increase the level of prions thermoaggregation. Chaperonin GroEL provoke further association of partially aggregated prions, having strong influence on the aggregates size. Introduction: Hepcidin is a 25-aa cysteine-rich peptide present in plasma and urine. It is produced in the liver and regulates iron homeostasis. The aim of this study was to produce functional recombinant hepcidin and to develop a specific immunoassay for its quantification in human sera. Methods: Hepcidin-His was expressed in P. pastoris and further purified by Ni 2 -NTA affinity and size exclusion chromatography. The function of the recombinant peptide was tested by antimicrobial and labile iron pool (LIP) assay. Its ability to bind and downregulate its receptor ferroportin was tested by Western blot, cross-linking pull-down assay and confocal microscopy. A polyclonal antibody generated in rabbits was used in competitive ELISA assay for hepcidin quantification in human sera.

Results: Hepcidin-His was bactericidal and active in modulating cellular iron homeostasis. It consistently bound to ferroportin causing its internalisation and promoting an increase in the levels of the LIP and a decrease in the expression levels of transferrin receptor 1 in macrophages. A competitive ELISA assay was developed that could detect 5 ng/ml hepcidin with a linear detection range of 20-20 000 ng/ml. This represents the first ELISA-type immunoassay for hepcidin peptide quantification in sera. Discussion: Our results suggest that recombinant Hepcidin-His generated in P. pastoris faithfully recapitulates the functional properties of native peptide and represents a powerful tool for the development of an ELISA assay for its quantification in human sera.

Investigation of the correlation between FKBP12 and APP processing/AD pathogenesis One of the pathological hallmarks of Alzheimer's disease is the presence of insoluble extracellular amyloid plaques, whose major constituent, the Ab peptide, is derived from the amyloid precursor protein (APP) by two sequential proteolytic cleavages, which also generate the APP intracellular domain (AICD). We have previously demonstrated that AICD interacts with FKBP12, a peptidyl-prolyl cis-trans isomerase (PPIase) ubiquitous in nerve systems. This interaction was interfered, in a dose-dependent manner, by FK506, whose neuroprotective effect has been suggested to be correlated with its FKBP12 PPIase inhibitory activity and may be separated from its immunosuppressive effect mediated via calcineurin inhibition. The exact mechanism(s) of FK506's neuroprotective activity, however, still remains unclear. FKBP12 has been shown to be expressed at a higher level in areas of pathology in the brains of neurodegenerative disease patients, suggesting its involvement in the pathogenesis of those diseases. Our preliminary results revealed that FKBP12 overexpression increased the levels of CTFb but not CTFa, and therefore resulted in a higher CTFb/CTFa ratio. The increase in the b-secretase cleavage products was reverted by adding FK506. These results support our hypothesis that FKBP12 may participate in the regulation of APP processing in a fashion similar to Pin1, and FK506 may partially induce a neurotrophic activity either by hindering the interaction between FKBP12 and AICD, or by inhibiting the PPIase activity of FKBP12 directly.

The carbohydrate binding module family 21 contains a unique segment directing nano-fibril formation Methods: Site-directed mutagenesis, circular dichroism (CD) spectroscopy, thioflavin T (ThT) fluorescence assay, transmission electron microscopy (TEM), and atomic force microscopy (AFM) were the methods of choice since they have been applied to study fibril formation in vitro.

Results: In the present work, a C-terminal segment of SBD was identified to play an important role in the fibril formation process. Sitedirected mutagenesis study also revealed that several charged residues were essential for assembly of the fibril-like structures. A synthetic peptide representing the C-terminal 14 residues of an SBD variant was further demonstrated to act as a fibril-forming segment of nano scale, where terminal charges and two characteristic motifs served as key fibril-forming determinants.

Conclusions: Our results represent the first study regarding fibril formation in a fungal SBD, along with its chemical and biophysical requirements. The uncovering of the role of terminal charge pairs associated with special motifs may shed some light on the mechanisms mediating fibril formation. These results may have implications for novel regulatory functions of CBM21-containing proteins. Method: We use DNA microarray analysis to compare alternative mRNA expressing in the hippocampi of AD mouse model and wild type control. Primary neuronal culture was used to confirm the role of these identified proteins in neurodegeneration and to dissect the molecular mechanism of these alternations.

Result: Striking alternation in the expression of some genes of extracellular matrix protein was revealed in AD mouse model and in primary neuronal culture. Our preliminary data indicated these proteins can prevent Abeta-mediated cell death. Molecular mechanism of the protective effect of these extracellular matrix proteins are under investigation.

Conclusion: These results identify collagen VI as an important component of the neuronal injury response and reveal its neuroprotective potential. Introduction: The MC4R is a G-protein coupled receptor involved in the central regulation of food intake and energy homeostasis. Eighty percent of childhood obesity-related MC4R mutants are retained intracellularly, probably via the quality control system acting on misfolded receptors. Thus, rescuing cell surface targeting and functionality of these mutant receptors could have therapeutic value. Methods: Cell surface targeting and functionality of wild-type (WT) and mutant (S58C, R165W and P299H) MC4R were assessed by flow cytometry and by measuring cAMP production. The exit of the receptors from endoplasmic reticulum (ER) and their maturation were monitored by Bioluminescence Resonance Energy Transfer (BRET) to follow the interaction between MC4Rs and calnexin and by pulse chase labelling assays.

Results: Cell permeable MC4R selective ligands have been tested and were able to restore cell surface expression and signalling activity of S58C and R165W MC4R mutants. Those compounds, according to their mode of action, are described as pharmacological chaperones (PC). The effect of MC4R-PCs on the maturation of the MC4R mutants monitored by BRET suggests an action downstream of the calnexin-MC4R interaction, most likely at the level of the Golgi apparatus. Using metabolic labelling and co-immunoprecipitation assays, we are currently investigating the steps and kinetics of the PC-promoted MC4R maturation process. Conclusion: MC4R selective ligands act as PCs to rescue intracelullarly-retained MC4R mutants by acting downstream of the ER-resident protein, calnexin. A better understanding of PCs's mode of action could help design novel therapeutic approaches not only for EOMO but also for other conformational diseases resulting from protein misfolding.

Mutations relocating the ER-resident protein torsin A to the nuclear envelope

Institute of Mother and Child, Warsaw, POLAND Torsin A (TOR1A) belongs to the torsin family of AAA+ proteins and is an endoplasmic reticulum (ER) lumenal glycoprotein, mutated in early-onset torsion dystonia type 1 (DYT1). A deletion of a single glutamic acid (delE302/303) near the C-terminus of this protein is the only known disease-causing mutation. It promotes the formation of intracellular membrane inclusions and relocalizes torsinA to the nuclear envelope. Similar relocalization has also been demonstrated for the E171Q mutation within a conserved Walker B motif, but not for the K108A mutation within the Walker A motif, although both conserved motifs are required for efficient ATP binding and hydrolysis by the AAA+ proteins.

To elucidate the relationship between the nature of molecular defect and the protein's tendency to relocalize to the nuclear envelope, we created a series of TOR1A constructs, containing mutations affecting different conserved regions of torsinA. Besides the two previously studied mutations (delE302/303 and E171Q), only the C-terminal truncation at position 296 (296X) and two mutations within the sensor 1 motif (N208Q, N208A) lead to significant redistribution of torsinA from ER to the nuclear envelope. No such relocation was found for mutations affecting several other conserved regions. We hypothesize that the Walker B and sensor 1 motifs, both involved in ATP hydrolysis, cooperate with the C-terminal tail when binding and/or releasing a substrate in the nuclear envelope. Such binding may arrest the protein at this location in case the conformational change associated with the ATP hydrolysis cannot be completed. Acknowledgement: Supported by grant PBZ-KBN-122/P05/01-11.

On the amyloid fibril formation of apo-yeast alcohol dehydrogenase induced by alcohols Protein aggregation currently receives considerable attention with respect to the significance of amyloid-related diseases as well as stability and delivery of protein drugs. Amyloid related disorders, including Alzheimer's disease, involve the aggregation of normally soluble proteins into insoluble amyloid fibrils (amyloidogenesis) and proceeds through intermediates with debatable mechanism and some common morphological features. Alcohols have been shown to induce the fibril formation in prion and Alzheimer's disease peptides in vitro. The amyloid formation of non-disease-related proteins via partial denaturation with alcohols has been little studied. In the present study, amyloid formation of structural Zn 2+ -depleted yeast alcohol dehydrogenase (apoI-YADH) was studied in an aqueous solution of four alcohols (methanol, ethanol, iso-propanol and tert-butanol). The apo-protein was obtained by removing the structural zinc with the catalytic zinc remaining on the enzyme. The amyloid formation of the enzyme was monitored by using Congo red test and thioflavin T (Th T) fluorescence assay, which are widely used for specific detection of amyloid fibrils. Fibrilogenesis occurs following a conformational transition to a b-sheet rich structure as evidenced by far-UV circular dichroism (CD) spectroscopy. Transmission Electron Microscopy (TEM) confirms the presence of regular amyloid-like fibrils with long, unbranched and often twisted structures of few nanometers in diameter. The amyloid-inducing ability of alcohols increased with increasing carbon number (hydrophobicity) and obeyed a concentration dependent manner in the order of: 20% < 40% < 60% < 70% < 80%. Therefore, the relative strength of the water-alcohol interaction is considered to reflect the amyloid formation in the aqueous alcohol through destabilization of apoI-YADH. The similarity in properties of amyloid in different amyloidoses suggests that principles learnt in this study would be of fundamental relevance for the design of effective therapeutic agent's against amyloid related disorders.

Homocysteine thiolactonase/esterase activity of paraoxonase 1 in heart failure patients Introduction: Plasma homocysteine (Hcy) is a risk factor for cardiovascular disease and stroke and predicts mortality independently of traditional risk factors in patients with coronary artery disease. Hyperhomocysteinemia exerts its adverse effects, in part through Hcy metabolite, homocysteine thiolactone (HcyTL). HcyTL is a highly reactive compound which has the ability to modify proteins, decreasing their physiologic activities and having toxic effects on cells. Recently it was shown that paraoxonase 1 (PON), an enzyme with antioxidant and antiatherogenic properties, also has homocysteine thiolactonase (HTLase) activity, HcyTL being its physiologic substrate. We presumed that PON1 esterase/HTLase activities play an important role in heart failure (HF), a complex syndrome in which oxidative stress and inflammation are implicated in disease evolution. Our objectives were to determine PON1 esterase/HTLase activities and to correlate both activities with two inflammatory markers myeloperoxidase (MPO) and lipoprotein-associated phospholipase A2 (PLA2-LDL Introduction: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is one of the most abundant cellular proteins. Many works have appeared concerning the participation of GAPDH in the development of neurodegenerative diseases. Several authors described a reduction of GAPDH activity in Alzheimer's disease, despite its overexpression, which could occur to compensate the decrease of activity. Results: We investigated the interaction of soluble and aggregated forms of beta-amyloidy1-42 (Ab), with different forms of GAPDH to clarify whether the certain forms of GAPDH are directly involved in the formation of amyloid structures or they adsorbed to the existing amyloid aggregates. Besides, we studied the effect of Ab on the aggregation of GAPDH and on the spontaneous and chaperonin-assisted renaturation of the enzyme. We revealed no interaction between Ab and the native form of GAPDH. Ab prevented the spontaneous reactivation of GAPDH as a result of its interaction with the enzyme. Surface Plasmon Resonance method allowed demonstration of binding of the non-aggregated form of Ab to the denatured GAPDH, while the aggregated Ab does not interact with the nonnative forms of GAPDH.

Our results indicate that Ab itself neither inactivates nor unfolds GAPDH, but efficiently binds to GAPDH denatured by different factors. Denaturation of GAPDH can be caused by oxidative stress, since this enzyme is sensitive to oxidation. Oxidative stress can result in the oxidation and denaturation of GAPDH, and then soluble Ab species bind to the denatured GAPDH forms, yielding insoluble aggregates. The goal of this project is to identify proteins modulating ataxin-1, -3 and -7 misfolding, as these molecules may prove to be therapeutical targets for SCA diseases. To achieve this goal, we are studying the effect of aggregation modifiers and ataxin-interacting proteins in ataxin aggregation/toxicity. Materials and methods: Yeast two-hybrid (Y2H) experiments were used to identify ataxin-interacting proteins. Aggregation modifiers and ataxin-interacting proteins were cloned into yeast and mammalian expression vectors using the Gateway technology. Cell based assays were used to measure ataxin toxicity/aggregation. Results and conclusions: Using a proteomic approach, we have identified 67 ataxin interacting proteins. Combining our results with bibliographic data, we generated an ataxin interaction network that comprises 249 molecules. In order to identify proteins modulating polyQ toxicity, 35 characterized ataxin interactors and 145 aggregation modifiers were selected. These proteins are co-expressed with wild type or polyQ ataxin-1, -3 and -7 in yeast and cell lines. The role of hit molecules, modifying ataxin aggregation/toxicity in cell models, will be further studied in in vivo experiments with transgenic flies overexpressing mutant ataxins.

Structure-function study of histone H1 in normal and diabetic rats R. Rahmanpour, S. Z. Bathaie and A. Jafarnejad Clinical Biochemisty Department, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, IRAN Introduction: Chronic hyperglycemia increases the non-enzymatic glycation of inter-and intracellular proteins in diabetes mellitus. It is well established that the function of a protein depends on its native structure; hence, glycation can induce structural and functional alterations in a variety of proteins and these modifications could be involved in the pathogenesis of some complications of diabetes. Histone H1, a basic protein and susceptible to glycation, is characterized as a packing agent of chromatin and general controlling factor of gene expression. We aimed to study the effect of glycation on H1 structure and function.

Method: In this study Male Wistar rats were divided into two groups. One group was injected intraperitoneally with streptozotocin (50 mg/ kg body); thereafter only rats with blood glucose levels ‡270 mg/dl were included in our experiments as diabetic group. Histone H1 was extracted from liver of normal (N-H1) and diabetic rats (D-H1) by salt-method of extraction. The structural analysis was performed by fluorescence and circular dichroism (CD).

Result: Results indicated that: intrinsic fluorescence, a-helical and random coil contents of D-H1 is lower than N-H1; in addition, D-H1 is denatured at lower concentration of SDS in comparison to N-H1. The results of functional analysis assayed by UV-spectroscopy (percent of precipitation of DNA by H1) and CD showed that complex formation is decreased in D-H1 in comparison with N-H1. Conclusion: Finding data indicated significant changes on Histone H1 from diabetic rat, which because of its important role on chromatin structure-function could be the reason for some diabetic disorders.

Formation of small soluble aggregates may be responsible for irreversible thermal denaturation of yeast hexokinase B Conclusions: Taking various findings on the influence of osmolytes and chaperone-like agents on YHB thermal denaturation together, it is proposed that the purely conformational transition of YHB is reversible, and irreversibility is caused by the aggregation reaction following the initial unfolding process. Thermoinactivation studies indicated that the process was irreversible at temperatures greater than 35°C, in spite of reversibility of thermal unfolding at temperatures up to 55°C. It is suggested that deamidation of a critical Asn or Gln residue(s) may play a determining role in this process.

Cell biology of disease-associated mutants of the proteolipid protein P. Roboti, L. Swanton and S. High Faculty of Life Sciences, University of Manchester, Manchester, UK Introduction: More than a hundred mutations in the myelin integral plasma membrane proteolipid protein (PLP) are associated with dysmyelinating diseases of varying severity. While point mutations in PLP transmembrane domains cause severe disease, very few such mutants result in a mild phenotype. How mutation-dependent misfolding of the PLP protein is recognised at the cellular level, how these various mutants are processed, and the mechanism(s) by which they cause disease are unknown.

Method: Immunofluorescence and pulse-chase studies were performed in order to compare the intracellular transport and degradation of two transmembrane mutants of PLP stably expressed in HeLa cells.

Results: The severe PLP-A242V mutants are retained in the endoplasmic reticulum (ER), causing the formation of vacuolar structures typical of an ER overload with misfolded proteins. Mild PLP-G245A mutants are present in the ER, but with significant amounts also accumulating in late endosomal/lysosomal compartments. Pulse-chase assays show that PLP-G245A is degraded faster than PLP-A242V, and the degradation of both mutants takes place by a process sensitive to proteasome inhibition. Notably, a UPR is induced by the severe mutants, but not by the mild PLP mutants.

The retention of PLP mutants in the ER contributes to disease pathology, most likely via the induction of the UPR, which may modulate disease severity.

Influence of structural state of sHSP for aggregation of amyloid beta Introduction: Small heat shock protein (sHSP) is a molecular chaperone that normally forms large oligomers (9-50) in their native state. Under stressed conditions, the oligomer dissociates to prevent protein aggregation. Although sHSPs have been found colocalized with misfolded cellular proteins such as amyloid beta (Ab), it remains to be demonstrated how the assembly states of sHSP influence on Ab aggregation. In this study, we have examined Ab aggregation in the presence of Schizosaccharomyces pombe sHSP (SpHSP16.0) which forms 16mer at room temperature and dissociates into dimer at higher than 40°C. Methods: Ab42 monomer was incubated with SpHSP16.0 at 50 or 30°C for 24 hours, and then amyloid aggregates were characterized.

Result: TEM analysis showed that amyloid formation was prevented at 30°C (16-mer), whereas fibrillar structures were observed at 50°C (dimer). ThT dose not bind to this fibril, suggesting that this fibrillar structure differs from the normal amyloid fibril. The toxicity, evaluated by MTT methods, of this fibrillar Ab aggregate to PC12 cells was significantly low, which is explained by weak affinity of the fibril to the cell using immunohistochemical study. Conclusion: Our data indicates that 16-mer of SpHSP16.0 inhibits amyloid beta fibrillation, and dimer induces formation of low toxic fibril structure. Many human small heat shock proteins (sHsp) undergo phosphorylation catalyzed by a number of different protein kinases and phosphorylation might affect their structure and functions. Human small heat shock protein with molecular weight 22 kDa (HSP22, HspB8) contains two Ser residues (Ser24 and Ser57) in consensus sequence RXS that are recognized and effectively phosphorylated by protein kinase A in vitro.

In order to determine the sites of phosphorylation and to analyze effect of phosphorylation on the structure and properties of HSP22 we obtained point mutants (S24D, S57D and S24,57D) mimicking phosphorylation at putative sites phosphorylated by protein kinase A. Mutation S24D did not affect, whereas mutations S57D or S24,57D prevented phosphorylation of HSP22 by protein kinase. This means that Ser57 is the primary site of phosphorylation. Phosphorylation (or mutation) of Ser57 (or Ser24 and Ser57) resulted in the changes of the local environment of tryptophan residues and increased HSP22 susceptibility to chymotrypsinolysis. Mutations mimicking phosphorylation decreased dissociation of HSP22 oligomer at low concentration without affecting its quaternary structure at high protein concentration. Mutations S24D, S57D, and especially S24, 57D were accompanied by decrease of chaperone-like activity of HSP22 if insulin or rhodanese were used as substrates. If protein kinase A phosphorylates HSP22 in vivo, this might modify physiologically important properties of this protein in the cell. Acknowledgement: This investigation was supported by Russian Foundation for Basic Science.

Roles of calpain in survival signaling J. Takano and T. Saido RIKEN Brain Science Institute, Saitama, JAPAN eCalpain-calpastatin proteolytic system f is composed of two calciumactivated proteases (milli-calpain and micro-calpain) and their endogenous inhibitor protein (calpastatin). Although abnormal activation of calpains has been implicated in pathogenesis of neurodegenerative diseases, such as Huntington fs and Alzheimer fs diseases, cataract, muscle dystrophy, brain or cardiac ischemia, recent studies suggest that this system may play important roles in cell death, cell differentiation and memory formation under physiological conditions. To clarify the roles of calpain, we reduced calpain activity by genetic manipulation or inhibitors treatment. Reduction of calpain activity resulted in apoptotic cell death. We detected caspase-like activity in this cell death. Although calpain has been known as a cell death effector so far, our results indicate that m-calpain may play a role as a life saver of cells in a faint stress like placenta ischemic condition. Introduction: The MLH1 and MSH2 proteins are the main components of the MutL and MutS complexes, respectively, which recognize and repair mispairings and insertion/deletion loops occurring during the S phase of DNA synthesis. Germline mutations in the MLH1 and MSH2 genes may cause loss of mismatch repair activity. Methods: Genomic DNA was extracted from colorectal cancer patients' peripheral blood following standard protocol. The complete coding sequences of MLH1 and MSH2 genes, including splice junctions, were amplified using PCR, followed by sequencing. Multiplex ligation-dependent probe amplification (MLPA) was also used in order to identify genomic rearrangements of MLH1 and MSH2 genes.

Results: In a total of sixteen families subjected to genetic testing, three nonsense mutations in exons 5, 7, 13 of the MSH2 gene, a deletion of the exon 6 of the MLH1 gene, as well as a splice site alteration in the MLH1 gene, leading to exons 9 and 10 skipping, have been identified. Conclusion: There is growing list of evidences that post-translational thiolation of human immunoglobulin free light chains have influence of their physiological properties (kidney filtration, blood concentration, deposits formation). This is the first quantitative large scale investigation of kappa FC thiolation process.

Human ER-60 prevents fibril formation of amyloid beta Introduction: Fibril formation of amyloidÒ ?peptides (Ab) is central to the pathogenesis of Alzheimer's disease, and occurs during transport of amyloid precursor protein from the endoplasmic reticulum to the trans-Golgi network. Hence, the intracellular quality control mechanisms by several chaperones should prevent intracellular aggregation of Ab in the healthy brain. To explore this possibility, we determined whether an ER chaperone, ER-60/ERp57 works as an intracellular chaperone of Ab in vitro.

Methods: The aggregation of AÒ? in vitro was determined with thioflavine T. The conformational change of AÒ was monitored by CD spectroscopy. Recombinant ER-60 and its fragments were expressed in E. coli expression system and purified. Binding analysis was carried out with a BIAcore 2000.

Results and Conclusions: ER-60 inhibited both spontaneous and seed-dependent fibrillogenesis of amyloidÒ (1-40) and (1-42) by preventing the conformational change of these peptides from randomcoiled to Ò-sheet-rich structures, even at a molar ratio of 1:60 to 1:100 ER-60/amyloidÒ peptide ER-60 also disaggregated part of the amyloid b (1-40) fibril to the monomer in a temperature-dependent manner. By surface plasmon reasonance analysis, it was shown that ER-60 immobilized on the sensor chip bound to AÒ (1-28), AÒ (1-40), and AÒ (1-42), weakly to AÒ (1-16), but not AÒ (12-28) or AÒ (25-35), indicating that ER-60 recognized the N-terminal region of residues 1-28 of AÒ. Chaperone activity for amyloid Ò peptide and interaction to AÒ peptides was recovered in b and b¢ domains among a, b, b¢, and a¢c domains of ER-60.

Investigation of structure-toxicity relationships in alpha-synuclein, a protein implicated in Parkinson's disease Parkinson's disease (PD) affects over four million people worldwide. The alpha-synuclein protein (a-syn) causes PD if it is overexpressed or contains certain point mutations. The a-syn monomer is intrinsically unstructured, but can adopt either a beta-sheet conformation (fibrillar a-syn is found in PD brain inclusions) or an alpha-helical conformation, which mediates vesicle trafficking. It is composed of three regions: (i) the N-terminus adopts an alpha-helical structure upon membrane binding; (ii) the middle hydrophobic region (NAC) promotes fibrillization; (iii) the C-terminus is a negatively charged random coil. To study how each of these three regions affect a-syn localization and toxicity, constructs with extended or truncated N-termini, NAC regions, and Ctermini respectively were made and their effects on yeast cells were studied. Overexpressed a-syn strongly inhibits yeast growth; it localizes at the plasma membrane and forms cytoplasmic inclusions. An a-syn variant with an extended N-terminus was found to be more toxic to yeast than a variant with a truncated N-terminus. The former is prone to form a-helical structure, whereas the latter is prone to form beta-sheet fibrils in vitro. Increased alpha-helical structure propensity, which is associated with tighter membrane binding, therefore, promotes a-syn toxicity. In line with this result, cytoplasmic variants are generally less toxic than membrane bound variants. Constructs with altered NAC regions display intermediate toxicities, whereas a-syn variants with elongated or truncated C-termini are highly toxic to yeast. When expressed individually, each protein region exhibits low toxicity, suggesting that long range or multiple interactions are involved in a-syn toxicity. Introduction: Acetaminophen is a widely used analgesic/antipyretic drug that produces renal damage, especially in proximal tubular cells.

In those cells, cytotoxicity is commonly preceded by changes in the organization of the actin cytoskeleton and alterations in cell adhesion, suggesting that these processes are important for cell survival. In tubular cells, accumulation of Heat Shock Proteins (HSPs) produces cellular tolerance to a variety of adverse conditions; therefore, we evaluated the role of HSPs in the acetaminophen-induced cytoskeletal changes and cell death using LLC-PK1 cells.

Methods: LLC-PK1 cells were exposed during 24 hours to different concentrations of acetaminophen (0.3-40 mM) and cell viability was estimated by the neutral red accumulation assay. Changes in the actin cytoskeleton were evaluated through F-actin labeling and fluorescence microscopy. The levels of HSP27 and HSP70 were quantified by Western blot analysis. In order to induce HSPs expression, some cultures were exposed to thermal stress (43°C) for 1 hour. Results: Acetaminophen did not modify the basal expression of HSP27 or HSP70, but induced cell death at concentrations >5 mM. Subtoxic concentrations of acetaminophen produced alterations in the actin cytoskeleton, decreasing the content of stress fibers. The induction of HSP27 and HSP70 by thermal stress was associated with an increase on the stability of the actin cytoskeleton and with tolerance against acetaminophen cytotoxicity. Conclusions: These data show that the actin cytoskeleton participates in the cytoprotective effect produced by HSPs induction. Further studies will focus on the participation of focal adhesion proteins. Acknowledgment: Supported by Conacyt-Mexico.

Evaluation of the preventive effect of Salvia miltiorrhiza on DMN-induced rat liver fibrosis model using functional proteomics Herein, we like to discover the effective products of roots from Salvia for curing or protecting from liver diseases. The hypothesis behind this study is to explore the different components in Salvia miltiorrhiza that may elicit specific cellular responses and signaling pathway to suppress hepatocellular carcinoma and reverse liver fibrosis.

We have performed comprehensive delineation of altered protein expression with or without Salvia miltiorrhiza treatment on DMNinduced liver fibrosis using proteomic platform. Forty-four proteins were found to change significantly in expression levels. of note, stress 70 protein, Cu/Zn SOD, thio-specific antioxidant protein, GST and ERp29 were correlated with regulating oxidative stress and scavenging free radicals. These results indicate that Salvia miltiorrhiza could be a useful therapeutic agent for ROS-induced hepatocarcinogenesis.

Immunization with PrP and recombinant PrP fusion proteins and its possible immunoprophylactic use against TSEs Introduction: Immunization against prions has been attempted in several animal models. However, the low immunogenicity of this molecule in wild type (wt) animals has hampered the attempts for active immunogenization. In this study we evaluate the immunogenicity of various forms of murine recombinant prion protein (mrPrP) in wt mice and their possible protective role in an animal model of transmissible spongiform encephalopathies (TSEs). Methods: Murine prion protein (PrP) was cloned and expressed, either alone, or fused to the bacterial heat shock protein DnaK. The recombinant proteins were administered to groups of C57Bl/6J mice, along with the corresponding controls. The immunization scheme consisted of priming and two boosts, after which the mice were challenged intraperitoneally with the RML scrapie strain and observed for the appearance of clinical symptoms. When the mice reached terminal stage they were sacrificed and their tissues prepared for immunohistochemistry and Western Blotting (WB). Splenic lymphocytes were harvested and lymphocyte proliferation assays, as well as cytokine expression profile in the presence of mrPrP, were performed. Results/conclusions: ELISA and WB analysis indicate that the elicitation of humoral immune response against mrPrP, even in wt animals, occurred with one of the antigens, but this regime did not protect the mice against TSEs. Immunization with a different form of mrPrP lead to a statistically significant prolongation of the survival interval for the immunized versus the control mice by 27 days, although no antibodies against mrPrP were detected. In this latter case, the protection was probably mediated through cellular immunity. Introduction: A key event in transmissible spongiform encephalopathies (TSEs) is the conversion of the soluble, protease-sensitive glycosylated prion protein (PrP C ) to an abnormally structured, aggregated and partially protease-resistant isoform (PrP Sc ). Both PrP isoforms bear two potential glycosylation sites, and thus in a typical western blot with an anti-PrP antibody three distinct bands appear, corresponding to the di-, mono-or unglycosylated forms of the protein. The relative intensity and electrophoretic mobility of the three bands are characteristic of each TSE and have been used to discriminate between these.

Methods: In the present study we devised a novel protocol for the enrichment of PrP Sc from brain homogenates of TSE affected tissues, which produces PrP Sc with the required purity and yield and then used lectin-based one and two dimensional western blotting, followed by densitometric analysis, to examine the possibility that in addition to these differences, there are also variations in composition within sugar chains carried by PrP Sc purified from subjects affected with different TSEs, a novel. Results/conclusions: Our findings indicate that despite similar sugar moieities are found on PrP Sc purified from different TSEs, the relative abundance of sugars varies, depending on TSE type. These results reinforce the idea that PrP glycosylation may be important for the transmission of TSEs between different species, as well as for the appearance of some of the characteristic traits of TSE strains. Furthermore, they suggest that lectin based western blotting could be useful as an additional assay for the discrimination between the various TSEs. Introduction: The BRCA1 protein contains a tandem of two BRCT domains at its C-terminal region. Several proteins interact with this region and collaborate with BRCA1 in important functional pathways, such as DNA repair and transcriptional regulation. These interactions are disrupted by BRCA1 mutations that affect the BRCT repeats. The majority of cancer-associated germline BRCA1 mutations result in truncated products lacking one or two of the BRCT domains. Such mutations confer severe susceptibility to breast and/or ovarian cancer.

We have previously shown, after screening a cohort of highly affected Greek families, that mutations in the BRCT domain are dominant in the BRCA1 mutational spectrum of breast and/or ovarian cancer patients. Here, we aimed to determine the frequency of each of the five recurrent BRCT-domain mutations in consecutive cases of breast cancer patients. Mutation screening methods employed were quantitative multiplex PCR of short fluorescent fragments (QMPSF), multiplex ligation-dependent probe amplification (MLPA), and DNA sequencing as a final confirmation step. Results: In a total of 785 random breast cancer patients, 27 (3.44%) carried one BRCA1 BRCT domain germline mutation. 26% of those were complex genomic rearrangements not detectable by routine screening methods. The break points of the three genomic rearrangements (0.9% frequency) were mapped and one of them was proven to produce no transcript. A combination of biochemical and genetic data was used to evaluate the pathogenicity of some missense variants.

Conclusions: Germline mutations in the BRCT domain of BRCA1 are the main cause of breast cancer predisposition with hereditary etiology in the Greek population.

Cystatins proved as a good model to study amyloidogenesis. Morphological, structural and biochemical data have been gathered on amyloid-fibril formation by human stefin B (cystatin B) and some of its mutants. Here, based on more kinetic measurements, we propose an improved model for the reaction. The observed kinetics follow the nucleation and growth behaviour. The minimal kinetic scheme that best fits measurements of changes in CD and thioflavin T fluorescence as a function of protein concentration and temperature includes nucleation (modeled as N I irreversible transitions with equivalent rates (k I ), which fitted with N I = 64), fibril growth and non-productive oligomerization, best explained by an off-pathway state with a rate-limiting escape rate. Three energies of activation were derived from global fitting to the model, and independently through the fitting of the individual component rates. Nucleation was found to be a first-order process within an oligomeric species with an enthalpy of activation of 55 ± 4 kcal/mol. Fibril growth was a second-order process with an enthalpy of activation (27 ± 5 kcal/mol) half that of nucleation, and which is indistinguishable from that of tetramer formation by cystatins, which involves limited conformational changes including proline trans to cis isomerization. The escape rate of the off-pathway oligomeric state has a high enthalpy of activation (96 ± 5 kcal/mol) characteristic of a substantial degree of unfolding. The observed transitions are consistent with the current model of stefin B fibrils where the helix is displaced and proline 74 is in a cis conformation. Mammalian peroxisomal membrane protein, Pxmp2, with monomeric molecular mass 22 kDa (also known as PMP22 protein) belongs to a family of integral membrane proteins with unknown function. Here we show that disruption of Pxmp2 encoding Pxmp2 leads to partial restriction in peroxisomal membrane permeability to solutes in vitro and accumulation of peroxisomal metabolites (e.g. uric acid) in body fluids (blood and urine) of mice. Multiple channel analysis of peroxisomal preparations isolated from liver of wild-type mice reveals presence of two pore-forming components with conductance in 1 M KCl of 1.3 and 2.5 nS, respectively. One of these components (with conductance of 1.3 nS in 1 M KCl) was lost in Pxmp2-/-mice indicating that Pxmp2 may be a pore-forming channel. This was confirmed by expression of a functional recombinant Pxmp2 in insect cells and using native Pxmp2 purified from mouse liver. The Pxmp2 channel, with an estimated diameter of 1.4 nm shows weak cation selectivity and no voltage dependence. Hence, Pxmp2 is the first peroxisomal channel identified, and its existence suggests that the mammalian peroxisomal membrane is permeable to small solutes while transfer of 'bulky' metabolites, e.g., cofactors (NAD/H, NADP/H, and CoA) and ATP, requires specific transporters.

The pyruvate carboxylase tetramer -a complex machine Conclusions: Acetyl CoA activates the enzyme by binding to a pair of subunits on one face of the tetramer and facilitates the adoption of a configuration of these subunits such that the biotin carboxylaation occurs in its own BC domain but pyruvate carboxylation by the resultant carboxybiotin occurs in its partner subunit's CT domain. The asymmetric configuration of subunits on the two faces of the tetramer implies that the enzyme exhibits half-of-the-sites reactivity.

The role of CesAB chaperone on T3S secretion Expression of the oncogenic fusion tyrosine kinase Bcr-Abl is the cause of chronic myelogenous leukemia (CML). Imatinib (Gleevec) is inhibiting Bcr-Abl, but relapse due to acquired drug resistance remains problematic. Although Bcr-Abl is an extensively studied protein, the key protein interactors and critical pathways necessary and sufficient for oncogenicity have not been identified. As a starting point we purified endogenous Bcr-Abl complexes, analyzed their composition by LC-MS/MS and identified a set of seven stoichiometric core interactors. We created recombinant epitope standards and antibodies for the seven Bcr-Abl core interactors and were able to determine the proportion of Bcr-Abl-associated versus 'free' protein and interaction stoichiometry within the complex, as well as the absolute protein copy numbers per cell. The seven core complex constituents interact with Bcr-Abl in a 0.5:1-4:1 stoichiometry. In addition, we have generated a comprehensive dataset based on tandem affinity purification using the seven core interactors as bait following LS-MS/MS analysis and generation of network models. Furthermore, reciprocal interactions were confirmed by co-immunoprecipitations. We are currently investigating effects of tyrosine kinase inhibitors on the composition and integrity of this Bcr-Abl core complex as well as the functional consequences on its signaling network. This should enable us to identify the key Bcr-Abl interactors and signaling pathways and to provide a rationale for combinatorial pharmacological intervention in CML.

The role of loop of L22 in the ribosome function Introduction: L22 protein of the large ribosomal subunit consists of one globular and one elongated domain. In order to further investigate the role of this loop of L22 from E. coli in the ribosome function reconstituted ribosomes with the wild type L22 as well as its truncated forms mentioned below were used for performing poly (Phe) synthesis in vitro.

Methods: The wild type L22 as well as three truncated forms, namely EryL22 missing the amino acids 82-84, loop1L22 missing the amino acids 82-95 and loop2L22 missing the amino acids 82-98 were cloned in pET29c vector that provides 6His at the c-terminal of the proteins, purified in Ni-NTA column, and used in the total reconstitution experiments.

Results: The activity of the reconstituted 50S subunits was tested via poly(Phe) synthesis after the addition of the 30S subunit. The pmol Phe produced by pmol of ribosome gave interesting results. Namely for wtL22 the value was 8.8 while for L22loop1 truncated form that lacks 14 amino acids (from 82 to 95 ) the observed value was 15.3. On the contrary the truncated form Ery missing only three amino acids as well as the L22loop2 missing 17 amino acids exhibited 2.3 and 6.3 pmolPhe/pmol ribosomes values.

Conclusions: These results point out that the area of the loop1 may not to be essential for ribosome assembly and activity, and the higher activity of these mutant ribosomes, in comparison to the other mutants and even the wtL22 reconstituted ribosomes, instigates more speculations about the role of this area in the EcL22. Single stranded DNA binding proteins (SSBs) are ubiquitous and fundamental for a wide variety of DNA metabolic processes including DNA replication, recombination, transcription, repair and in the recruitment of repair proteins to sites of DNA damage. SSB proteins from the three domains of life are evolutionarily conserved and utilise oligonucleotidebinding (OB) domains for DNA binding. In eukaryotes, the major SSB, Replication Protein A (RPA), is a heterotrimer. We describe a second human SSB (hSSB1), with a domain organisation closer to the archaeal SSB than to eukaryotic RPA. We show the first biochemical characterisation of hSSB1 and demonstrate that it is phosphorylated by ATM kinase in response to DNA double strand breaks (DSBs) [1] . This phosphorylation event is required for DNA damage-induced stabilisation of hSSB1. Upon induction of DNA damage, hSSB1 accumulates in the nucleus, forming distinct foci that co-localise with known repair proteins. We show that depletion of hSSB1 abrogates the cellular response to DSBs. Cells deficient in hSSB1 exhibit enhanced genomic instability coupled with a diminished capacity for DNA repair. We propose the novel hSSBs are functional homologues of the bacterial and archaeal SSB family of proteins, placing them centrally in the DNA repair pathway. Like many early participants in the damage response pathway, hSSB1 may be involved in tumorigenesis and may affect the response of patients and tumours toward cancer therapies. is an ADP-ribosylating putative toxin identified in the virulent strain MC58 of N. meningitidis serogroup B by computational approach.

Methods: NarE was expressed in E. coli as a C-terminal His-tag fusion protein and purified on nickel columns in anaerobic conditions (rNarE). Results: Enzymatic assays have shown that rNarE links the ADPribose unit from b-nicotinamide adenine dinucleotide (NAD + ) to small guanidine compounds like agmatine at a rate of 6.7 ± 0.87 nmoles/ hour with the simultaneous release of nicotinamide (Nam). We have noticed that addition of Fe 3+ in the mixtures of enzymatic assays enhanced 3-fold the incorporation of radioactive ADP-ribose, while the presence of o-phenanthroline, a specific iron chelator, drastically reduced the enzymatic activity. These results together with the presence of four Cys spaced as residues of iron-sulfur-cluster motif, prompted us to confirm the presence of this structure using the EPR technique, a method that evidences the presence of paramagnetic ions and identifies the type of coordination in which they are involved. EPR experiments carried out combining rNarE with FeCl 3 evidenced the presence of an iron center sourrended by four Cys residues. Further, EPR signal obtained at g = 4.3 indicates the presence of a single Fe 3+ atom in a distorted sulfur ligands field. In order to confirm the presence of the Fe-S cluster Cys mutated forms of rNarE have already obtained and used for enzymatic assays. Conclusion: Our experiments confirmed, as the rNarE enzymatic activity is strictly dependent by the presence of iron coordinated with the four cysteines.

Cdc37 is localized at the cell surface and is possibly involved in cancer cell invasion Results: The hrpG gene was cloned in an expression vector for protein overproduction in E. coli. The protein was classified on the basis of its sequence as a typical class I chaperone. Solution studies of the full-length HrpG and its deletion mutant were performed using gel filtration, Small Angle-X-ray Scattering (SAXS) and Circular Dichroism (CD). These revealed considerable deviations from the properties of a typical class I chaperone, as exemplified by the AvrPphF ORF1 protein of P. syringae which was used as a positive control in all experiments. A search for HrpG interaction partners through native electrophoresis revealed two T3SS proteins. Subsequent construction of bicistronic expression vectors, co-production and co-purification experiments helped characterize these interactions and revealed two further binding partners.

Conclusions: HrpG of P. syringae was found to be a highly soluble component of the Hrp1 T3SS which lacks the typical structure of a T3SS class I chaperone. HrpG was found to interact with a crucial export substrate and a core component of T3SS. The latter indicates an unknown function in T3SS substrate export regulation.

Targeting signal domain analysis of Pex3p: topogenesis and functional role in peroxisomal sorting of Pex3p Helicobacter pylori is bacterium colonizing in the stomach of almost half of the world population. Infection of H. pylori would increase the risk of developing gastric cancer, gastritis and duodenum cancer. Functional flagella would enhance colonization and adhesion to gastric mucosal cells which are essential in infection. The regulation and formation of flagella involves about 40 genes including genes of interest, fliS and Hp1076. FliS is an export chaperone in the bacterial cytosol to prevent pre-mature polymerization of flagellin molecules before export. Hp1076, a hypothetical protein with unknown function, is under the regulation of rpoN promoter in flagellar system. Previous yeast twohybrid analysis of H. pylori has showed an interaction linkage between FliS and Hp1076. Herein, GST-tagged FliS and His-tagged Hp1076, were over-expressed in Escherichia coli expression system and purified. Their interaction was demonstrated by glutathione and Ni-NTA sepharose pull-down assays, gel-filtration, co-expression and co-purification methods. By constructing different fragments of Hp1076, a region consisting of residues 21-172 of Hp1076 was mapped to be indispensable for interaction. The specific interaction residues will be further identified by site-directed mutagenesis. At present, protein crystals of FliS/Hp1076 were obtained and the crystallization condition is being optimized. Results obtained will provide the first insight to molecular interaction of FliS and Hp1076 in flagellar assembly pathway.

PP3D-13 Arrangements for the regulated processing of alphaviral nonstructural polyprotein Semliki Forest virus (SFV) is a type member of Alphavirus genus, which produces its replicase proteins in a form of ns-polyprotein precursor P1234 and their maturation is performed in a temporally controlled manner by papain-like protease activity of nsP2 protein. The data obtained in our study provided evidence that so-called macrodomain (170 aa) of nsP3 protein is used for precise positioning of a substrate recognition sequence at the catalytic center of the protease and this process is coordinated by exact amino-terminal end of the N-terminal domain of nsP2 protein, thus representing the unique regulation mechanism used by alphaviruses. Although maturation of alphaviral replicase was always considered to be an attractive model for studies of fine mechanisms of proteolytic processing, it can now be concluded that even in such a seemingly simple system the efficiencies and the character of the cleavages performed by the same protease are obviously different and all three sequentially organized processing events are regulated by use of different mechanisms, only some of which are common for both SFV and SIN. Experimental data also disclosed the involvement of viral RNA in the regulation of the processing of 2/3 site suggesting the multimeric nature of the alphaviral replication complex residing on a viral RNA in a specifically ordered manner. Based on a newly available data a model for replicase complex formation was proposed, which may apply for replicases of several positive-strand RNA viruses.

The COG complex dynamically assembles on the Golgi membrane to regulate intra-Golgi trafficking R. D. Smith and V. Lupashin University of Arkansas for Medical Sciences, Little Rock, AR, USA

The Conserved Oligomeric Golgi (COG) complex is a putative vesicle tethering factor that interacts with Golgi SNARE and Rab molecules and orchestrates retrograde intra-Golgi transport. The COG complex consists of eight subunit (COG1-8) and is partially co-fractionates with the Golgi membrane. We have used siRNA depletion, quantitative immunoprecipitation and live fluorescent microscopy of HeLa cells that stably express CFP and YFP-tagged COG subunits to uncover intracellular dynamic of the COG complex. siRNA-induced knock-down of Lobe A subunits Cog3p and Cog4p induced rapid accumulation of COG complex dependent (CCD) intra-Golgi vesicles and consequent Golgi ribbon breakdown, while the knock-down of Lobe B subunits Cog6p, Cog7p and Cog8p did not cause significant vesicle accumulation. Double knock-down of Cog3p and Cog7p restored accumulation of CCD vesicles. Quantitative immunoprecipitation experiments with affinity-purified antibodies to Cog3, 4, 6 and 8p revealed that soluble cytoplasmic COG complex is a stable hetero-octamer, while >60% of membrane-bound COG subunits exist as distinct COG sub-complexes. Live cell microscopy of HeLa cells stably expressing CFP-Cog6 and YFP-Cog3 revealed significant diversity in molecular dynamic of Lobe A and Lobe B sub-complexes. CFP-Cog6 was associated with rapidly moving small vesicles that most likely correspond to the intra-Golgi trafficking intermediates. We propose that the transient re-association of two COG sub-complexes is the initial stage in intra-Golgi vesicle tethering. Acknowledgement: Supported by NSF grant MCB-0645163.

A comparative study on the ability of camel and bovine b-caseins to suppress the heat aggregation of yeast alcohol dehydrogenase apo-forms b-casein is among the most abundant proteins of mammalian milk. The protein met with the less ordered secondary structure and the lack of tertiary structure accounts for its high flexibility and the stability against heat denaturation. There are similarities between b-casein and other known chaperones in their tendency into micelle-like aggregate. This was a starting point to evaluate whether b-casein play any chaperone-like activity. Prevention of thermal aggregation of substrate proteins by molecular chaperones is a commonly method for in vitro assay of their activity. In the present study, after preparation of apoI-YADH (yeast alcohol dehydrogenase, EC.1.1.1.1), by removal of structural zinc and apoII-by depleting both structural and functional zinc atoms from the protein, both forms exposed to heat aggregation at 50°C for 20 minutes in citrate sodium 20 mM pH 7.0. The heat aggregation between b-casein and apo-YADH was followed by turbidity development at 360 nm. The used b-caseins (bovine, camel), exhibited a considerable role to decrease the extent of thermal aggregation in apoforms of YADH. Camel b-casein was more active than their bovine counterpart. In addition, camel b-casein showed high ability on recovering the catalytic activity of apoI-YADH after heat induced unfolding. Investigation of conformational changes by fluorescence spectroscopy with ANS, as a common extrinsic fluorescence probe, revealed both milk b-caseins caused a remarkable reduction in the surface hydrophobicity of apo-YADHs. The obtained results are in agreement with the common features of molecular chaperones. Besides, our results may provide a good opportunity for studying more details of camel milk b-casein chaperone-like properties.

Insights into the assembly of ORC, the DNA replication initiator complex in Saccharomyces cerevisiae M. Moreno-Del Alamo, A. Sanchez-Gorostiaga, A. Serrano, A. Prieto and R. Giraldo Centro de Investigaciones Biologicas -CSIC, Madrid, SPAIN Introduction: The origin recognition complex (ORC) is a six-subunits (Orc1-6p) protein that leads the assembly of the highly processive machinery responsible for DNA replication in any eukaryotic cell. We had previously found that Orc4p interacts with chaperones of the Hsp70 family [1] . Methods and Results: By means of biochemical, proteomic and bioinformatics approaches we have characterized a hydrophobic sequence motif in Orc4p as the main target for Hsp70. We have generated mutants in this motif and studied them both in vivo and in vitro.

In a haploid S. cerevisiae strain, we have replaced ORC4 by each of its mutant alleles. Two of them exhibit a thermosensitive phenotype. We have further characterized their cell cycle (FACS) and also studied the effect of the mutations in the firing of the ARS1 origin of replication (2D gel electrophoresis). We have developed a novel multisubunit expression system in E. coli, in order to map intersubunit interactions mediated by Orc4p. After affinity purification of the different ORC subcomplexes we have found that ORC assembly is altered by the mutations. Introduction: Regulation of synaptic vesicle protein levels is critical for neuronal function. The Adaptor Protein complex-3 (AP-3) and BLOC-1 sorting machinery regulate synaptic vesicle protein targeting from endosomes. The ubiquitous AP-3 isoform and BLOC-1 complex mediate lysosomal delivery of membrane proteins, while the neuronal AP-3 isoform forms a subset of synaptic vesicles [1] . Recent proteomes of AP-3-derived synaptic-like microvesicles [2] or synaptic vesicles [3] revealed the presence of lysosomal membrane proteins and lysosomal targeting machinery in these organelles. These results suggest that the two AP-3s may recognize and competitively sort similar cargo proteins to either a synaptic vesicle or lysosomal fate. Introduction: Histone deacetylase (HDAC) function has been found to be impaired in certain cancer cells. This may explain the differential sensitivity of cancer cells to HDAC inhibitors (HDACIs). It is therefore important to know the response of different cell types to HDACIs being used as antineoplastic drugs. In this presentation, the time-dependent kinetics of the HDACI, trichostatin A (TSA), in the induction of apoptosis and core histone H4 acetylation are compared between two leukemic cell lines, K562 and Molt-4. Methods: Molt-4 (lymphoblastic) and K562 cells (erythroleukemic) were incubated with TSA (500 ng/ml) for 1, 3, 6, 9, 12, 15, 18, 21 and 24 hours. Histones were separated in an acetic/urea/Triton-X100 gel system and histone H4 acetylation levels were quantified. Apoptosis was monitored by PARP and DFF45 cleavage as well as by Annex-inV/PI staining and subG1 DNA content. Hydrogenases are metalloenzymes that catalyze reversible reduction and oxidation of molecular hydrogen and are present mostly in prokaryotes. Our previous results demonstrate an autocatalytic mechanism during the reaction cycle of HynSL hydrogenase from purple photosynthetic bacteria, Thiocapsa roseopersicina although the nature of the autocatalyst is still a question. By analyzing spatial patterns of hydrogenase reaction in a thin layer reaction chamber we observed the dependence of reaction propagation speed (front velocity) on both enzyme and substrate concentrations. Front velocity increased on increasing the enzyme concentration and decreased on increasing the substrate concentration. The experiments give further evidence for the autocatalytic nature of hydrogenase reaction and also suggest the role of the enzyme as autocatalyst. As HynSL has different conformers, the autocatalytic reaction could be an interaction of inactive and active enzyme forms (prion type autocatalysis). On the basis of the experimental results we set up a model that explains the experimental findings. The experiments were performed with purified enzyme. It is still a question if the hydrogenase functions autocatalytically in vivo. Considering that the expression of hydrogenase enzyme is a very difficult process requiring lots of accessory proteins and that the enzyme in its active form is extremely sensitive towards oxygen, it seems to be reasonable to save enzyme molecules in inactive forms in the cell and activate them only at appropriate time and under proper conditions. Further experiments have to be performed in order to demonstrate this assumption.

Solution structure of the bacteriophage lambda major tail protein, gpV insights into viral tail assembly The bacteriophage lambda tail acts as an excellent model for the study of macromolecular complex assembly. Greater than 80% of the tail's mass is made up of the product of gene V (gpV). gpV exists in a monomer-dimer equilibrium; but upon encountering its binding partner, gpV forms hexameric rings. Thirty-two hexameric rings polymerise to form the viral tail tube. To improve our understanding of not only phage function and propagation, but to provide a starting point to answering some of the fundamental questions surrounding the assembly and function of supramolecular structures, we have chosen to study gpV.

Methods & Results: gpV was previously known to be comprised of two distinct domains. Using limited protease digestion, N-terminal sequencing, and in vivo complementation studies, the structural and functional boundaries of the two domains have been delineated. Using NMR spectroscopy, the structures of individual N-terminal (NTD) and C-terminal domains (CTD) have been solved. While the CTD has an immunoglobulin-like fold, the NTD has an unclassified fold containing several unstructured regions. To determine the functional surfaces of gpV, several point mutations have been tested for their ability to complement in vivo. The mutants Y59A and D61A/D62A have a dramatic effect on gpV activity. Interestingly, the D61A/D62A double mutant also displays a dominant negative phenotype suggesting it lies on the top surface of the gpV hexamer.

Conclusions: The NMR structure of gpV, together with mutagenesis and functional studies, has provided significant insight into the selfassembly and polymerization of the bacteriophage lambda tail. Introduction: In this communication we present a novel cell-fractionation procedure which results in enrichment of cellular proteins that participate in functional complexes. This extraction procedure is designated 'SoTRIs' (Solubilisation of TritonX-100 Resistant Interactors) and follows mild Triton X-100 extraction.

Methods: Mass spectrometry analysis was performed on SoTRIsextracts from NCIadr cells (i) resolved on one-and two-dimensional SDS-PAGE gels, and (ii) straight-from-the-liquid. Immunoblotting was employed to verify selected proteins. Physical association between proteins was examined by affinity chromatography. Results-Conclusions: A collective list of 350 known proteins was generated by mass spectrometry analysis. Most interestingly, among the SoTRIs-extracted proteins are proteins resistant to solubilisation with other mild extraction procedures (such as nuclear matrix, hydrophobic, trafficking proteins, etc). Gene Ontology (GO) analysis revealed an unbiased distribution of SoTRIs-proteins between cell compartments, with the exception of depletion in the soluble fraction. Furthermore, SoTRIs-extract is enriched in proteins that are physically associated with four major macromolecular structures: (i) chromatin, (ii) cytoskeleton, (iii) ribonucleoprotein and (iv) cell and subcellular membranes. To examine functional association, we performed affinity chromatography where SoTRIs-extracts were incubated with conjugated-on-beads peptides that are known to participate in theses macromolecular structures, and assemble functional clusters of proteins. Immunoblotting revealed that different clusters of proteins are assembled with, during different biological processes. This evidence shows that the SoTRIs extraction procedure is enriched in the functional fraction of proteins that participate to macromolecular complexes. Ongoing experiments will demonstrate the type of post-translational modifications that SoTRIs-extracted proteins bear, that enable them to participate in functional complexes. Rotation of helical filaments attached to BFM propels bacteria towards nutrients and optimal temperature. Ion flow through the stator generates torque (turning force) that is applied to the rotor by as yet unknown mechanism. The stator is believed to be attached to the cell wall peptidoglycan layer through the periplasmic domain of MotB (MotB-C). The first crystal structure of the latter is presented. Methods: MotB-C crystals have been obtained using polyethylene glycol as a precipitant. The structure has been determined by a MAD experiment using an ytterbium derivative and refined using X-ray diffraction data to a resolution of 1.6 Å . Co-crystallization with N-acetylmuramic acid yielded crystals of the complex the structure of which has been determined at 2.3 Å resolution.

Results: The structure contains a central b-sheet composed of four strands flanked by three a-helices on one side. Gel filtration linked to MALLS experiments demonstrate that MotB-C is a dimer in solution.

Analysis of the crystal structure suggests that this dimer is formed through association of the b-sheets of two subunits into a single extended b-sheet. Two large loops at one end of each b-sheet accommodate the binding site for N-acetylmuramic acid.

Conclusions: MotB-C functions as a dimerisation domain. Modelling of the (NAM-NAG) polymer into the 37-Å long shallow grove provides an insight on how MotB-C can insert itself into peptidoglycan pores anchoring the stator unit to the cell wall.

Structural bases of signalling through the PII protein: the structure of the complex of PII protein with its cyanobacterial target protein PipX P II protein is a signalling protein which signals nitrogen/carbon abundance, interacting with numerous protein targets. The structure of two complexes of P II protein, with the ammonium channel AmtB and with the enzyme acetylglutamate kinase, have been described. We now report the structure of the complex with the cyanobacterial transcriptional regulator PipX. P II and PipX from Synechococcus elongatus strain PCC 7942 were mixed and concentrated by ultrafiltration. Crystals grew after several months in hanging drops. X-ray diffraction yielded maps at 3.2 Å . Phasing was accomplished by molecular replacement using P II as template. The PipX model was built after density averaging, applying non-crystallographic symmetry and solvent flattening. The complex consists of one P II trimer of 112-residue subunits associated to three PipX 89-residue subunits. Each PipX subunit has a 6-stand antiparallel beta sandwich formed by four-strand and 2-strand (beta hairpin) layers, and a C-terminal two-helix hairpin. The beta hairpins of the three subunits form a continuous layer that provides a flat surface for interaction with the flat surface of P II. A PipX trimer having a trefoil shape sits on the PII trimer. Each PipX subunit contacts two P II subunits, with one T-loop of P II embedded between two PipX subunits. The C-terminal helix is mobile and both helixes are mainly polar and exposed. The structure explains the regulation of PipX-PII complex formation, and allows hypotheses to be made on ways of PipX action. Acknowledgement: Grant BFU2004-05159 of the Spanish Ministry of Education and Science.

Rebuilt and characterization of hetero-oligomeric group II chaperonin complexes of Thermococcus sp. strain KS-1

Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Tokyo, JAPAN Thermococcus sp. strain KS-1 (T. KS-1), express two different chaperonin subunits, a and b, for folding of its proteins. The composition of subunits in the hexadecameric double ring changes with the temperature. The b-subunit was significantly more abundant with increasing temperature. The recombinant b subunit oligomer exhibits higher thermostability and affinity to the co-chaperone, prefoldin, than that of a subunit. However, the hetero-oligomer was not studied yet because of difficulty in building hetero-oligomers with fixed order and composition. In this study, we built the hetero-oligomers of T. KS-1 chaperonin with various composition and order by linking two or four a and b subunits. The hetero-oligomer containing a and b subunits alternately could capture an acid denatured GFP and enhance refolding of it in ATP dependent manner. However, the efficiency was almost same as the b homo-oligomer irrespective of the presence of four a subunits in a ring. The arresting activity and conformational change ability of the hetero-oligomer was similar to those of the homo-oligomers. Interestingly, affinity of the hetero-oligomer with prefoldin is almost between a and b homo-oligomer. Existence of four b subunits in the ring increases the affinity with prefoldin. Although the thermostability of the hetero-oligomer similar to that of a homo-oligomer, existence of b subunit in the hetero-oligomer likely to be needed to increase affinity interaction prefoldin. Because the prefoldin consists of six tentacles and T. KS-1 chaperonin has eight monomers per ring, we think that interaction between prefoldin and group II chaperonin is asymmetric. 

Developing predictive dynamic models of complex intracellular networks for neurological disease To this end, a comprehensive multidisciplinary strategy has been established combining functional genomics, proteomics and bioinformatics. Methods and Results: Using a mouse model of induced hippocampal sclerosis associated with focal epilepsy, dynamic expression analyses are conducted at different time points. Proteomic databases are being used along with advanced microarray and proteomics platform systems to investigate protein-protein interactions and regulation networks, identify and validate biological targets in complex intracellular pathways. The first phase involves whole genome and proteome analysis, integrating biological and statistical data in order to functionally annotate genes and proteins. Using Affymetrix microarrays, 2D-DIGE and MALDI/TOF-TOF, we are evaluating whole genome and proteome expression profiles bringing to light critical new pathways and molecular targets implicated in neurodegeneration. Discussion: VALAPODYN develops a dynamic and quantitative analysis method for new therapeutic targets through MIN dynamic models and specifically addresses the systems biology of complex cellular pathways and transcriptional networks. Novel predictive dynamic models will be validated by testing the selected drug targets on innovative in vivo and in vitro models of CNS pathologies. VALAPODYN will provide a cutting-edge highly accurate in silico tool for identifying novel and effective therapeutic targets in a faster, more efficient and more economical way than it is possible today.

Characterization of PilP, a protein required for the assembly of Pseudomonas aeruginosa type IV pili Introduction: Pseudomonas aeruginosa is a ubiquitous environmental bacterium and opportunistic pathogen which causes severe systemic infections in immunocompromised individuals and chronic -eventually fatal -lung infections in persons with cystic fibrosis. P. aeruginosa establishes infections using its type IV pili (T4P) and the pilMNOPQ operon has been identified as being essential for T4P assembly. While PilQ is the putative outer membrane (OM) secretin through which the pili exit the cell, the function(s) of PilM/N/O/P proteins are currently unknown. Here, we characterize the predicted lipoprotein PilP.

Methods & Results: Heterologous expression of PilP identified a stable protein fragment that N-terminal sequencing revealed to be a deletion of the first 71 residues (D71). CD and 15 N, 13 C-NMR spectroscopy reveals that the D71 fragment is well ordered with primarily b-sheet secondary structure and the 3-D structure determination of this fragment using NMR spectroscopy is in progress. Currently 90% of the chemical shifts have been assigned and the preliminary 3-D structure shows a b-sandwich-type fold. Phenotypic characterization of PilP transposon mutants using our specific antibodies shows that loss of PilP reduces the levels of PilN/O and PilQ multimers, with little effect on levels of PilM. Sucrose gradient membrane fractionation experiments localize PilP to the inner membrane with PilN and PilO. Conclusions: Our data suggest that PilP is part of the pilus assembly complex and interacts with proteins in both the inner (PilN/O) and outer membranes (PilQ). PilP may have chaperone-like properties and be important for bridging the periplasm.

A lipid mass spectrometry investigation on the effect of non-bilayer forming amphiphiles to the phospholipid metabolism of HeLa cells Introduction: Non-bilayer forming, or Type I amphiphiles have been shown in vitro to inhibit the catalytic activity of the rate-determining and regulatory enzyme of lipid metabolism CTP: phosphocholine cytidylyltransferase (CCT, EC 2.7.7.15) [1] . Membrane stored curvature elastic stress has been suggested as a hypothesis of regulation of CCT activity [2] . This investigation was undertaken to study in vivo the effect of two Type I amphiphiles, hexadecyl phosphatidylcholine (HDPC) and hexadecyl ammonium bromide (CTAB) to phospholipid metabolism in a transfected GFP-HeLa cell line. Methods: HeLa cells were synchronised at the G1/S barrier by Lmimosine, and then harvested every three hours for the first 21 hours post synchronisation. A Blight Dyer total membrane lipid extraction was performed. The lipid composition of membranes was quantified by electrospray ionisation mass spectrometry using non-physiological lipids as internal standards. Cells were also sized using optical microscopy image analysis at 21 hours post synchronisation. Results: HDPC was considerably more potent in inhibiting new phosphocholine (PC) synthesis than CTAB. Both drugs changed the acylation patterns for endogenous and new PC, as well as endogenous phosphoethanolamine head group classes. Also, the presence of both drugs in the culture shifted cell population distribution towards smaller cell diameters.

The two Type I amphiphiles inhibited CCT and in consequence phospholipid metabolism as predicted by our hypothesis. Therefore, our results have provided with important insight on the effect of membrane compositional diversity to phospholipid metabolism in HeLa cells. Introduction: Chromogranin A (CgA) is the major protein secreted from catecholamine storage vesicles of chromaffin cells in the adrenal medulla. One of its proteolytic cleavage peptides by plasmin called catestatin inhibits secretagogue-stimulated catecholamine release. In this report, we investigated a role of CgA in the plasminogen/plasmin activation process via the tissue plasminogen activator (tPA) in neuroendocrine processing of prohormones like CgA itself. The proteins involved in this proteolytic processing have been reported to reside within the secretory vesicles and could play a significant role in the local release of neurotransmitters in neuroendocrines.

Methods: Proteolytic activity of plasmin were measured using fluoregenic substrates in the presence of either the fibrin fragment, DESA-FIB Ò -X control, or the recombinant human CgA (rec hCgA). Fluorogenic products were monitored in a fluorospectrometry. Results: At a concentration of 45 lM, DESAFIB Ò -X could reduce Michaelis-Menten constant (K m ) of the plasminogen/plasmin activation via tPA at the neutral pH from 208 lM (control without an enhancer) to 0.323 lM. The catalytic efficiency (K cat /K m ) of the reaction increased from 0.011/lM/s (control) to 7.166/lM/s. The K m values of the activation process via tPA reduced from 208 lM to 0.307 lM when the rec hCgA was used at 40 nM concentration, while the catalytic efficiency (K cat /K m ) increased from 0.011/lM/s to 7.540/lM/s. Conclusions: These data suggest that the rec hCgA demonstrated a far better enhancer property for plasminogen/plasmin activation process via tPA than the control (DESAFIB Ò -X) at the neutral pH by approximately 3 orders of magnitude. This study revealed a novel regulatory mechanism of neurotransmitter release by the plasminogen/plasmin activation via tPA with the CgA enhancer in neuroendocrine system. 

Identification and functional significance of a brain AQP4/ Na+, K+-ATPase/mGluR5 macromolecular complex The water channel aquaporin-4 (AQP4) is expressed in astrocytes, which play a major role in clearing the narrow extracellular space (ES) between neurons and astrocyte endfeet from K + and glutamate released during neuronal activity. Compelling evidence suggests that AQP4 may play a role in the K + clearance. We recently reported that glutamate, acting on the mGlu5 receptor, regulates AQP4 water permeability. We have now shown, using GST pull down assays, that AQP4 interacts with Na + , K + -ATPase, the pump responsible for active transport of K + , as well as with the mGluR5 receptor. Extensive mutation studies revealed that the amino acid residues Trp30 and Thr31 are the critical sites for interaction. Substitution of Trp30 and Thr31 to Ala decreased or abolished the interaction, while substitution of Thr31 to Asp appeared to enhance the interaction, suggesting that phosphorylation of Thr31 residue may enhance interaction. Using rubidium uptake and astrocyte water permeability measurements, we found that group 1 mGluR agonist DHPG increased both Na + , K + -ATPasemediated K + transport and AQP4-mediated water transport. We created mathematical model of K + transport across plasma membrane in the ES. This model predicts that AQP4-mediated water transport will facilitate K + clearance and stabilize the membrane potential. Thus we have demonstrated a multiprotein K + transporting microdomain in the astrocyte that includes AQP4. This microdomain is functionally regulated by glutamate and serves to optimize the clearance of K + from the ES. Low mobility group proteins are abundant and heterogeneous class of non-histone proteins which have been less investigated than other nonhistone proteins. LMG 160 is a fraction of LMG proteins purified in this lab and recognized as a ribonucleoprotein (RNP) particle of nuclear matrix with an inhibitory effect on transcription in vitro. In this study the effect of various concentration of Sodium chloride, Urea and Guanidine-HCl (G-HCl) on the intact LMG 160 and RNase treated LMG 160 one investigated using fluorescence spectroscopy. The results show that in the NaCl and Urea condition, the fluorescence emission intensity is decreased and RNase treated LMG 160 represents higher structural changes compared to intact protein.

In the presence of G-HCl the effect is dose dependent thus at low concentration (< 0.5 M) of the denaturant reduces the fluorescence emission intensity of the intact and RNase treated protein, whereas the emission intensity is decreased at high concentration of G-HCl (> 4 M). From the results it is concluded that the most quenching is achieved at tyrosine position and the RNA moiety of LMG 160 is essential in the entire structure and function the LMG 160 protein.

Multiple levels of clusterin regulation following proteasome and lysosome inhibition ApoliporoteinJ/Clusterin (CLU) is a secreted glycoprotein with numerous physiological roles. The aim of our work was to examine the effect of proteasome or lysosome inhibition on CLU expression and to determine whether those proteolytic pathways are implicated in CLU degradation. Complete, as well as partial proteasome inhibition increased both mRNA and protein levels of CLU in U-2 OS and WI38 cells. Since transcriptional elements of HSF-1 and AP-1 are found in CLU promoter, we sought to identify whether CLU mRNA up-regulation after proteasome inhibition is mediated by one of these transcription factors.

Pre-treatment of cells with a HSF-1 inhibitor abolished both CLU mRNA and protein induction in U-2 OS cells; whereas, in WI38 cells, although it prevented CLU mRNA up-regulation it further increased CLU protein levels. The AP-1 inhibitor did not abolish proteasomeinhibition-mediated CLU up-regulation in either cell line. Furthermore, pulse-chase experiments revealed accumulation of the intracellular CLU protein form upon proteasome inhibition, while a CLU-specific antibody immunoprecipitated additional higher molecular weight bands after proteasome inhibition that may correspond to ubiquitinated CLU. Lysosome inhibition led to elevated levels of CLU protein but did not induce any changes in the RNA level. Addition of a CLU antibody to the cell medium resulted in reduced protein levels of the secreted form intracellularly, suggesting that secreted CLU is endocytosed and targeted for degradation at the lysosome. These findings indicate that CLU up-regulation relates to both positive transcriptional regulation by HSF-1, after proteasome inhibition, and post-translational accumulation due to reduced proteasomal and lysosomal degradation.

The chance of gender dependency of oxidation of brain proteins in aged rats Introduction: The effect of aging on brain-protein oxidation is well documented. However, none of the findings of studies specify the effect of gender.

The purpose of this study is hopefully to clarify the ambiguity raised in preliminary reports as to gender dependency of oxidative damage in brain proteins.

Methods: In the current study, we investigated the relation between protein hydroperoxide levels (P-OOH) and other oxidation parameters such as protein carbonyl (PCO), total thiol (T-SH), advanced oxidation protein products (AOPP), and nitrotyrosine (NT). Our study also covered other oxidative stress parameters, such as 4-hydroxyalkenal (4-HNE), malondialdehyde (MDA), and the redox index (RI) in brain tissue of the male and female aged rats.

Results: P-OOH, 4-HNE, T-SH, and P-SH levels of male rats were significantly higher than in the female rat group. On the other hand, PCO, AOPP, NT, MDA, and RI levels were all found to be not different.

We suggest that increased T-SH and P-SH levels found in our study may point to an adaptive reaction to oxidative protein damage reflecting P-OOH, 4-HNE overproduction. We are of the conviction that the increased T-SH and P-SH that we have determined in aged male rats may be a limiting factor in propagation of protein oxidation, as the PCO, AOPP and NT levels in the brain tissue were unchanged. It has thus been found that gender indeed affects the oxidation of brain proteins and thus it is aging; though the extent of the underlying mechanisms affecting brain aging and its etiology are still obscure.

Evaluation of free radical metabolisms in carbontetrachloride cirrhosis in rat liver: antioxidant effect of stobadine Introduction: Carbon tetrachloride is used for experimental liver chirrosis.

[1]. During metabolization, its initial unstabil free radical metabolite called trichloromethylperoxyde was occured. These free radicals cause lipid peroxidation of cell membrane and at the end cell degeneration occurs. Liver fibrosis and chirrosis may occur at the end of this progressive process. Superoxide radical is converted hydrogen peroxide and oxygen by superoxyde dismutase. Therefore, H 2 O 2 is transformed to water and oxygen by glutathione peroxidase and catalase [2] Stobadine is one of the antioxidant agent that prevent lipid peroxidation and protein oxydation in oxydative stres. [3] .

Methods: Control (free diet), stobadine (three times in a week, diluted in 0.5% Avicel solution, orally), CCl 4 (three times in a week, diluted in 1/10 olive oil, intraperitoneally), stobadine and CCl 4 groups (described above) each included 10 rats, were designed for 8 weeks. Tissues were homogenized and gross extracts were then centrifugated. Supernatants enzyme activities of MDA and catalase were evaluated. SOD and GSH-Px are evaluated from chloroformethanol-treated supernatants. Formalin fixed-paraffin embedded sections were stained with Masson thrichrome technique evaluated then histologically. Introduction: Apolipoprotein E (apoE), a major lipid transport protein of 299-aa, has three common isoforms (apoE2, apoE3, apoE4) in humans. ApoE4 is a major risk factor for Alzheimer's disease (AD), a neurodegenerative disorder characterized by overproduction and aggregation of amyloid peptide beta (Ab) in brain. Proteolytic bioactive carboxy-terminal truncated apoE4 fragments have been found in brains of AD patients. Results: We examined the effect of two carboxy-terminal truncated apoE4 forms, apoE4[D(186-299)] and apoE4[D(166-299)], which result by proteolysis in AD brains, on amyloid precursor protein (APP) processing and Ab levels in HEK293 and human neuroblastoma SK-N-SH cells transiently transfected with human APP. Lipid-free apoE4-165 led to reduced Ab levels in both cell lines, while it had no effect on sAPPa levels as compared to WT apoE4 or apoE4-185. The decrease in Ab levels by apoE4-165 cannot be attributed to cellular cholesterol levels which were not affected following incubation of cells with WT or truncated apoE4 forms. Incubation of cells with exogenously added Ab in the presence of WT or truncated apoE4 forms showed that apoE4-165 stimulates greatly the reduction of extracellular Ab levels as compared to WT apoE4 or apoE4-185. Furthermore, binding studies, by using intrinsic fluorescence quenching and Elisa, also showed different affinity for the binding of apoE4-165 to Ab as compared to WT apoE4 or apoE4-185. Conclusion: Our findings suggest that potential interactions of aminoterminal region 1-165 of apoE4 with Ab may affect the extracellular Ab levels and may influence the pathogenetic mechanisms that lead to AD.

Heterogeneity of the redox state of commercial human serum albumin products S. Era, Y. Matsuyama, T. Terada and T. Minami Graduate School of Medicine, Gifu University, Gifu, JAPAN

Albumin has been widely served as nutrients for tissue cultures in laboratory field and as plasma expander for blood transfusion in clinical field. Human serum albumin (HSA) is a mixture of human mercaptalbumin (HMA, reduced form) and non-mercaptalbumin (HNA, oxidized form) (HNA-1; mixed disulfide with cysteine, HNA-2; more higher oxidation product), i.e., a protein redox couple. As commercial HSA products are manufactured from large-scale pooled blood, those may have a different degree on protein redox state. By using a convenient HPLC system for the clear separation from HSA to HMA, HNA-1 and HNA-2, we examined the redox state of HSA products from various sources. HSA products were obtained from Sigma Co. (product No. A1653, A9511, A1887, A8763 and A3782) and from Calbiochem Co. (product No. 12666, 126658 and 126654). The A1653 from Sigma is the product corresponding to Cohn Fraction V, which a starting material from pooled sera. In the case of A1653 (lot No. 86K7540), values (%) for HMA, HNA-1 and HNA-2 were 37.5, 51.9 and 14.6%, respectively. In contrast, corresponding values for the final product (A3782; lot No. 78H7603), which prepared from A1653 through lyophilized and defatted processes, were 13.0, 38.7 and 48.3%, respectively, and it was contained dimer remarkably. These results suggested that the heterogeneity of redox state of various kinds of commercial HSA products appears to occur during manufacturing process of HSA from large-scale pooled blood. This is especially important in studies where these products are used to interact with other biological materials in both laboratory and clinical fields.

Proteomic study of differential protein expression in stable and unstable human atherosclerotic plaque Introduction: Atherosclerosis is a form of chronic inflammation characterized by the accumulation of lipids and fibrous elements in medium and large arteries. The rupture of the atherosclerotic plaque is the predominant underlying process in the pathogenesis of acute coronary syndromes and peripheral vascular disease. The aim of this work is identification, by means of a proteomic approach, of differentially expressed/retained proteins in stable and unstable carotid endarterectomy segments. Methods: We analyzed 19 stable and 29 unstable plaques obtained by carotid endarterectomy. Surgery samples were histologically classified as described previously [1] . 300 lg of extracted proteins were separated by IEF (pH 4-8) and subsequently by SDS-PAGE (10%T, 3%C). 2D images were analyzed by using PD-Quest software and proteins identified by MALDI TOF MS analysis. Results: We identified 95.6% of total extractable proteins resolved by 2D electrophoresis: about 1/3 constitutive and about 2/3 filtered. The results from differential analysis allowed us to identify a panel of nine proteins that are differently represented in stable and unstable plaque extracts.

Conclusions: These proteins are indicative of a more pronounced inflammatory and oxidative status in unstable lesions respect to stable ones. Such differential expression profiles in advanced lesions could help in understanding, at molecular level, the mechanisms leading to plaque destabilization. Acknowledgements: This work was supported by Fondazione Banco di Sardegna, Italy. Normal human fibroblasts undergo replicative senescence due to both genetic and environmental factors. The proteasome is the major cellular proteolytic machinery, consisted of both a-regulatory and b-catalytic subunits, responsible for the degradation of both normal and damaged proteins. Alterations of proteasome function have been recorded in various biological phenomena including aging and replicative senescence. Loss of proteasome activities and function has been reported upon replicative senescence and oxidative stress. In contrast, proteasome activation extends lifespan in human fibroblasts. The aim of this study was to identify natural compounds and extracts that enhance proteasome activity and may exhibit anti-aging properties. Specifically, we studied the effect of several compounds and extracts derived from greek plants, on proteasome activity of HFL-1 human embryonic fibroblasts. Out of the several compounds tested, we have identified one that caused the following characteristics to the exposed cells: a) a significant increase of all three proteasome activities namely, chymothrypsin-like, thrypsin-like and peptidylglutamylpeptide-hydrolyzing activity, b) increased RNA and protein expression levels of proteasome subunits, c) increased amounts of assembled proteasome, d) decreased intracellular levels of reactive oxygen species (ROS). The results so far indicate the beneficial effects of this compound on human fibroblasts. The induction of proteasome through natural compounds provides new insights in anti-aging and therapeutic strategies.

Isolation and characterization of mammalian specific enzyme for racemized proteins, D-aspartyl endopeptidase T. Kinouchi and N. Fujii Research Reactor Institute, Kyoto University, Osaka, JAPAN Introduction: The accumulation of D-isomers of aspartic acid (D-Asp) in proteins during aging has been implicated in the pathogenesis of Alzheimer's disease, cataracts and arteriosclerosis. D-Asp containing a synthesized in vitro forms an abnormally folded b-sheet and fibril, and is cytotoxic. Here, we sought to identify an unknown proteolytic system specific for the D-Asp-containing protein, namely D-aspartyl endopeptidase (DAEP). Methods: We developed an assay system for DAEP activity using the synthetic D-Asp containing substrate, Succinyl-D-Aspartic acid a-(4methyl-coumaryl-7-amides) (Suc-D-Asp-MCA). Suc-D-Asp-MCA is hydrolyzed and the fluorescence of liberated aminomethylcoumarin is measured at kex = 380 nm and kem = 460 nm. Results and Discussion: Mammalian liver was shown to have the highest DAEP activity among tissues, but DAEP activity was not detected in E. coli, S. cerevisiae, and C. elegans. The molecular weight of the purified mouse DAEP was determined by gel filtration to be 600 kDa. The properties of purified DAEP were as follows: K m was 0.12 mM, optimal pH was 8.5 (at 37°C), and the specific activity was increased by a factor of two following addition of 3 mM divalent cations (Mg 2+ , Ca 2+ , Sr 2+ , Ba 2+ , Mn 2+ ), but was strongly inhibited by Zn 2+ . A specific inhibitor for DAEP (benzoyl-L-Arg-L-His-D-Asp-CH 2 Cl) was also synthesized and applied to identify the components of DAEP by affinity labeling. MALDI-TOF MS analyses revealed glutamate dehydrogenase 1 as a catalytic subunit of DAEP components. DAEP would control levels of the noxious racemized proteins in the long life span of mammals.

Ionizing radiation provokes premature senescence in human lung fibroblasts that enhance the growth of malignant lung epithelial cells in vitro and in vivo A. Papadopoulou and D. Kletsas Laboratory of Cell Proliferation and Ageing, Institute of Biology, National Centre for Scientific Research 'Demokritos', Athens, GREECE Cellular senescence is considered to be a potent anti-cancer mechanism. However, it has been proposed that senescent stroma cells may enhance the growth of adjacent malignant epithelial cells. Exposure of tumours to repeated low doses of c-irradiation is a common treatment regime in several tissues. However, the effect of this stress to the neighboring stromal cells and the interaction of the latter with cancer cells have not been adequately investigated. In this study, we have exposed human lung fibroblasts to repeated subcytotoxic doses of 4Gy of c-irradiation. We have found that a single dose immediately activates a DNA damage response, as shown by the activation of the ATM/Chk2/ p53/p21 WAF1 axis, leading to cell cycle arrest. After a series of 4Gy doses (total dose approx. 50 Gy), followed by cell subculturing, cellular senescence was accelerated, as shown by morphological alterations, growth arrest, p21 WAF1 and p16 INK4a upregulation and senescenceassociated b galactosidase staining. Next, we studied the effect of these prematurely senescent cells on the growth of three human malignant lung cell lines. Medium conditioned by young and prematurely senescent cells has no major effect on the proliferation of all these cell lines. However, in co-culture studies we have found that the growth of cancer cells was strongly enhanced when cultured on senescent cells. In addition, in immunocompromised (SCID) mice c-irradiation-induced senescent cells, similarly to replicative senescent fibroblasts, intensely promoted cancer cells to form tumors. These findings support the idea that replicative-or stress-induced-senescence may contribute to cancer growth. Acknowledgements: Supported by KES!. Introductions: Peroxiredoxins (Prx) are ubiquitously distributed peroxidases that can be divided into 1-Cys and 2-CysPrx groups, based on the number of conserved cysteine residues that participate in their catalytical cycle. Prx have been described to be strictly dependent on thiols (RSH), but recently we have shown that ascorbate (vitamin C) also reduces 1-CysPrx, but not 2-CysPrx, from several taxonomic groups. We are analyzing which structural features of 1-CysPrx can be important for the ascorbate-peroxidase activity. In addition, we are studying the couple sulfenic acid/ascorbate in the regulation of other proteins, such as GAPDH.

Methods: We are studying these properties using site-directed mutagenesis, activity assays and crystalography. Results: Reduction by ascorbate is partly related to the fact that the oxidized form of 1-CysPrx is a stable sulfenic acid (Cys-SOH) instead of a disulfide. In addition, a histidine residue conserved only in the active site of 1-Cys Prx is required. The molecular modeling studies followed by mutagenesis and activity assays suggested the importance of histidine and threonine residues to stabilize the ascorbate-protein interaction. Histidine could be important not only structurally but also during the catalysis mechanism through an acid-base reaction needed for dehydroascorbate release from 1-CysPrx active site.

Conclusions: The identity of the biological reductant of most 1-CysPrx is unknown. Ascorbate is a candidate for this role and possibly in other proteins sulfenates, since it is abundant and widely distributed reductant.

Immune response in healthy ageing: immunosenescence versus immunodeficiency Results: The depletion of superoxide levels after 24 hours of treatment was correlated with a significant increase of SOD activity; however after 48 and 72 hours the superoxide levels increased as the SOD activity returned to the control one. The decrease of catalase activity was directly proportional with the duration of the exposure. The citochemical studies showed the absence of cell division in the intoxicated cells, nuclear disorganization and an increase of micronuclei. Caspase 3 expression was negative in the intoxicated cells.

Conclusions: According to our results, the DON induced-production of ROS caused DNA disorganization and blocked the caspase 3 mediated apoptosis.

Isoaspartate content and isoform alteration of cyclodextrin glycosyltransferase from Paenibacillus sp. RB01

Introduction and objective: The isoforms of cyclodextrin glycosyltransferase (CGTase) from the thermotolerant Paenibacillus sp. RB01 showed identical mass with different pI values (1). Three isoforms, two major (isoform I and II) and 1 minor (isoform III) were observed on native-PAGE. The aim of the present work is to investigate the cause of isoform formation, following our deamidation hypothesis. Methods: The CGTase gene from Paenibacillus sp. RB01 was cloned into pET19b vector and expressed in E. coli BL21(DE3). The enzyme was purified by starch adsorption and DEAE-cellulose column and the isoforms were separated on an FPLC mono Q column. The isoform I (2.5 mg/ml) was incubated in either deamidation buffer pH 6.0 or 9.0 (2) at 37°C for 0, 3, 7, 15 and 30 days, then kept at )80°C until use. The sample enzyme was quantitatively detected for isoaspartate content by the ISOQUANT Isoaspartate Detection Kit using HPLC method. The isoform pattern was investigated by IEF, native and SDS-PAGE.

The cloned enzyme showed the same multiple forms and biochemical properties as in wild-type. The isoform I was successfully isolated from other isoforms by FPLC. The isoaspartate content when incubated in buffer pH 9.0 was higher than in buffer pH 6.0. The longer the incubation time, the higher the amount of isoaspartate detected. The isoform pattern was shown to be changed as analyzed by native and IEF-PAGE. Isoform I was decreased while isoform II and III were significantly increased at pH 9.0 after 3 and 15 days of incubation, respectively. We have previously reported that acetylcholinesterase (AChE) expression is induced during apoptosis. Since the c-Jun NH2-terminal kinase (JNK) has been implicated in mediating AChE expression during anticancer drug-induced apoptosis, in this study, we examined its role in the regulation of H 2 O 2 -induced AChE expression using the 293T cell line. We show that H 2 O 2 increases AChE expression via transcriptional activation through JNK, since the JNK inhibitor SP600125, but not the extracellular signal-regulated kinase (ERK) pathway inhibitor PD98059 or p38 kinase inhibitor SB203580, attenuated H 2 O 2 -induced AChE expression and its promoter activity. Over-expression of HA-JNK increases H 2 O 2 -induced AChE expression and its promoter activity, whereas the dominant negative mutant form of JNK suppressed H 2 O 2 -induced AChE expression and promoter activity. Mutation analysis indicates that the major response elements for JNK in the AChE promoter are the AP1-like element (TGAGTCT) site, located within the -1565/-1569 region of the AChE promoter, and the ATF2 element (CCACGTCA), within the -2185/-2177 region. The AP1-like element binds to the transcription factors, c-jun and ATF2, while the ATF2 element binds mainly ATF2. Taken together, our results strongly suggest that H 2 O 2 induces AChE expression via the JNK/AP1/ATF2 signaling pathway.

Identification on membrane and characterization of phosphoproteins using a phosphate binding tag We have developed direct on-membrane mass spectrometric phosphoprotein identification using the chemical inkjet printing technology, which enabled to microdispense on microscale region (at a few hundred picoliter) in combination with a MALDI-TOF MS and established an effective rapid profiling approach contacting of detection with a phosphate binding tag. Reagents at sub-nanoliter volume levels can be microdispensed for on-membrane PMF analysis, allowing digestion of only a tiny region within a protein spot without cross-contamination between proximate proteins. Using this method, we analyzed human epidermoid carcinoma cell lysates of A-431 cells stimulated with epidermal growth factor, and identified six proteins with intense signals upon affinity staining with the phosphate binding tag. It was already known that these proteins are phosphorylated, and our new approach proved to be effective at rapid profiling of phosphoproteins. Furthermore, by MS/MS analysis after in-gel digestion of the corresponding spots on the 2-DE gel to the rapid on-membrane identifications, we successfully characterized a phosphorylation site at Ser-113 on prostaglandin E synthase 3.

Spin labeling EPR studies of spectrin at the tetramerization site C. Antoniou and L. Fung Department of Chemistry, University of Illinois at Chicago, Chicago, IL, USA Spectrin, a major cytoskeletal protein in a wide-range of cells, consists of ab subunits that form functional tetramers. The affinities for the association of the a subunit with the b subunit vary among different spectrin isoforms. The K d for the erythroid spectrin is in the micromolar range, whereas that for the non-erythroid (such as brain) spectrin is in the nanomolar range. A structural and mechanistic understanding of the affinity differences may provide means to understand pathological tetramerization in either erythroid or non-erythroid spectrin. We have used recombinant model proteins of the first 368 amino acids of erythroid a-spectrin that include the region responsible for tetramer formation, to study the association mechanism at the tetramerization site. An a-spectrin protein, SpaI-1-368, which consists of a partial structural domain of the N-terminus of a-spectrin and a b-spectrin protein, SpbI, which consists of a partial domain of the C-terminus of b-spectrin, were used. Positions 16 to 20 and 46 to 52 that are in an unstructured conformation in SpaI-1-368 were spin labeled for EPR studies. The EPR spectra were obtained with and without SpbI. Our results indicate that these regions undergo a conformational change, from unstructured to helical. In addition, isothermal titration calorimetry measurements on the association of each spin labeled protein with SpbI provide information on the effects of spin labeled cysteine replacement on the association affinity. We propose a mechanism for the tetramer formation of erythroid spectrin as well as an explanation for the difference in the affinities at the tetramerization site of the different isoforms. Acknowledgement: Supported by funds from UIC, AHA and NIH. Introduction: Pulchellin belongs to the class of Ribossome Inactivating Protein (RIP) type 2. It is well known that RIPs A chain retrontraslocation is driven by partial protein unfolding and interaction with the ERAD machinery, suggesting that the A-chain hydrophobic C-terminal is exposed allowing its recognition by chaperons or RE membrane interaction. This study investigated the interaction of pulchellin A-chain (r-PAC) C-terminal with biomembrane model. Methods: rPAC-2 and 4 isoforms and two mutants, rPAC 244 and rPAC 239 (lacking 7 and 12 aminoacids from C-terminal region) were produced and proteins were purified by affinity chromatography. Interaction of the proteins with DPPC was studied using Langmuir Monolayers as biomembrane model by measuring kinetics of adsorption and surface elasticity (E). Results: rPAC injection promoted an increase in surface pressure (p) with time for DPPC monolayers, corresponding to its insertion in the membrane model. This increment was proportional to the r-PAC concentration up to 5 ng/ml where no steady state was observed in 500 seconds. Same results was observed for rPAC-24 Faculty of Physics, 4 whereas r-PAC-239 attained a constant p in less than 100s. Dilatational elasticities (E) duplicated in the r-PAC and r-PAC-244, but only about 1.4 times higher than E value obtained for DPPC monolayer in the presence of r-PAC-239.

The results supported rPAC insertion in a membrane model by the C-terminal region. r-PAC or r-PAC -244 presented a relatively slow insertion whereas the r-PAC-239 is faster inserted but with a lower effect in the monolayer, as attested by the p and E values. Introduction: Satureja hortensis has been been shown to have medicinal value, but research on its basic biochemistry is still very scarce. The purpose of this investigation was to study the enzymatic properties of flavocytochrome b 2 , in S. hortensis leaves. Methods: Leaves were homogenized in phosphate buffer 0.01 M, pH 7.4, centrifuged at 3000 g for 10 minites, then at 35 000 g for 30 minutes. The clear supernatant ('extract') was used in our studies. Flavocytochrome b 2 activity was measured by following the reduction of potassium ferricyanide in the presence of lactate and EDTA. Assays were performed in a spectrophotometer equipped with temperature controller. Polyacrylamide gel electrophoresis of the extract was performed under non-denaturating conditions and followed by activity staining in the presence of lactate and tetrazolium blue. Results: pH activity profile indicated optima at pHs 8.0 and 9.5. With potassium ferricyanide as the varied substrate, K m (micromolar) and V max (micromolar/minute/mg prot) were, respectively, 23 and 0.3 at pH 8.0, and 40 and 0.65 at pH 9.5. The enzymatic activity increased with increasing temperature, exhibiting two peaks which were, for pH 8.0, at 40°C, with 200% activity and at 80°C with 300% activity, and, for pH 9.5, at 30°C with 150% activity and at 80°C with 250% activity compared to control. Malate was a competitive inhibitor of the activity. Electrophoresis revealed two distinct bands.

Conclusions: Data showed that at least two cytochrome b 2 isoenzymes were present in S. hortensis leaves and that the enzymes were stimulated at temperatures as high as 80°C. Introduction: Since the formation of drug-protein complexes was identified as a potential cause for cellular damage and death, monitoring drug-protein interactions has become a major concern. Acriflavine (3,6-diaminoacridine), an antiseptic and fungicide, also induces apoptosis and necrosis in yeast. Catalase, a vital enzyme actively involved in the defense against oxidative stress, catalyzes H 2 O 2 dismutation. In this study, the interaction between acriflavine and catalase was investigated.

Methods: Mixtures of catalase and various concentrations of acriflavine (3-60 millimolar) were incubated for 2 hour at 30°C in phosphate buffer 0.1 M, pH 7.0, and then electrophoresed in 1% agarose gels at 60 V for 1 hour. Their fluorescence emission spectra obtained upon excitation at either 280 or 297 nm were recorded between 310 and 450 nm. Their far-UV circular dichroism spectra were recorded between 200 and 260 nm. All spectra were taken at 25°C in 0.1 M phosphate buffer, pH 7.0.

Results: Incubation of catalase with increasing concentrations of acriflavine led to a lost of activity and to a progressive retardation of the enzyme's migration through 1% agarose gels. Fluorescence studies showed that acriflavine caused a progressive quenching of the fluorescence due to tryptophan and tyrosine residues. Far-UV circular dichroism spectra showed that a progressive increase in the random coil fraction and a progressive decrease in the alpha-helical content took place in the presence of acriflavine.

Conclusions: The present study shows that acriflavine binds to catalase, forming a complex that is not dissociated during electrophoresis and that alters the enzyme's structure and function.

Interaction with negatively charged lipids modifies the secondary structure of the Bax C-terminal domain and produces changes in its insertion into the membranes Apoptosis is a process of controlled and programmed cell death. Its regulation is due to various factors, and among them the Bcl-2 family proteins play an important role. Bax is one of these proteins and it has a pro-apoptotic function. The C-terminal domain of Bax shows an amphipathic character and it has been demonstrated that during the apoptosis it binds to the outer mitochondrial membrane. Fourier transform infrared spectroscopy (FTIR) was used to study the secondary structure of peptides which imitate the amino acid sequences of the Cterminal domain of the pro-apoptotic protein Bax (Bax-C) when incorporated into different lipid vesicles with compositions such as EYPC, PI, POPG and a mixture of EYPC/POPG. Infrared spectroscopy results show that while the b-sheet components are predominant in membrane-free Bax-C secondary structure as well as in presence of EYPC vesicles, the peptide changes its conformation in presence of negatively charged membranes depending on the lipid composition and their molar ratio. The negative charges in the model membrane surface cause a marked change from b-sheet to a-helix secondary structure. Moreover, using the attenuated total reflection infrared spectroscopy (ATR-FTIR), we have investigated the orientation of a-helix structures of Bax-C with respect to the oriented bilayer normal when it is incorporated into the same model membranes. The orientation of Bax-C in model membranes was also affected by negatively charged lipids, so that the presence of POPG reduced the insertion angle from 50 in EYPC vesicles to 27 in POPG vesicles. Introduction: G-proteins, through their interaction with G-Protein Coupled Receptors (GPCRs), act as switches for signal transduction from the extracellular space into the cell. The stimulation of GPCRs leads to the activation of G-proteins, which then activate several effector molecules that lead to many kinds of cellular and physiological responses. Methods: In order to collect recent data concerning interactions between G-proteins and effectors in gpDB, we performed an extensive literature search. The initial sequence information for effectors was retrieved from the UNIPROT database. Annotations regarding the interaction between G-proteins and effectors, the effect of the particular interaction and the corresponding references were appended manually in a spreadsheet. The data has been organized on the basis of a relational model and is stored in a PostgreSQL database system. Results: GpDB currently contains data concerning 410 G-proteins, 2795 GPCRs and 1510 effectors. The data is classified in classes, families, subfamilies and types for G-proteins and GPCRs, while for effectors in families, subfamilies and types. The information on the interaction between G-proteins and effectors is presented in each entry of both of these molecules and also at the subfamily level for G-proteins and at the type level for effectors. Conclusions: GpDB does not aim at being a universal resource for GPCRs, but will be acting complementary to the existing GPCRDB database regarding GPCRs, providing information on the coupling preferences of G-proteins to GPCRs and the interaction between G-proteins and effectors according to the reported data in the scientific literature. Introduction: Transmembrane proteins constitute approximately 20-30% of fully sequenced proteomes and they are responsible for a wide variety of cellular functions. However, due to the difficulty of obtaining crystals of transmembrane proteins suitable for crystallographic studies, biochemical and computational methods are routinely used to determine their topology. Biochemical methods include: techniques of gene fusions, using enzymes such as alkaline phosphatase, b-galactosidase, b-lactamase, and various fluorescent proteins, detection of post-translational modifications such as glycosylation, phosphorylation and biotinylation, cysteine-scanning mutagenesis, proteolysis methods and epitope mapping techniques. Methods: We performed an extensive literature search (up to January 2007), in order to find studies that reported the use of such biochemical methods for the determination of the topology of transmembrane proteins. The information was evaluated manually and the data are organized in a Mysql database using PHP for creating the web interface. Results: We collected 900 articles regarding 1417 transmembrane proteins. Specifically, 346 articles refer to gene fusions of various reporter proteins, 120 refer to glycosylation scanning, 9 refer to phosphorylation scanning, 10 refer to biotinylation scanning, 88 refer to cysteine scanning, 214 refer to epitope mapping and 113 refer to proteolysis experiments.

Conclusions: The information we acquired regarding the proteins' topology is of great importance as we show here that incorporation of experimental information for several sites of the protein sequence, into the predictive algorithms improves the prediction performance and the reliability of prediction. The database will be a valuable tool for training and testing prediction methods and is available at http://bioinformatics.biol.uoa.gr/ExTopoDB/.

Proteomic analysis of superathletes' plasma as a model of severe, sustained physical stress High-throughput mass-spectrometry-based proteomics help with the identification, cataloguing and quantification of proteins in complex biological processes. In this study, we employed the Spartathlon race (246 km in <36 hours) as a model of severe physical stress and investigated proteomic alterations in the plasma of athletes obtained at the start-(Athens) and finish-point (Sparta), as well as 2 days after the race ended. Twelve sets of athletes' plasma were analysed by 2D gelelectrophoresis and the differentially expressed proteins were identified by MALDI-TOF-MS. Peptidomic analysis revealed extensive proteolysis at the finish-point, partially recovered 2 days after the race ended. Thirty proteins were differentially expressed in the plasma of the athletes obtained at these 3 time points. At the finish point, three unknown function proteins of were overexpressed, as well as amyloid A, complement component 4A -a local mediator of inflammation-and creatine kinase M-type -an serum marker of myocardial infarction playing a role in energy transduction in tissues with large energy demands. Moreover, hemoglobin alpha-and beta-subunit levels decreased at the finish point, while haptoglobins (HPT and HP proteins) were overexpressed 2 days after the race ended. Haptoglobins combine with free plasma hemoglobin, preventing loss of iron through the kidneys and protecting them from damage, while making this protein accessible to degradative enzymes. By these preliminary results we conclude that Spartathlon athletes' plasma proteomics reveal in an integrated fashion, major changes in circulating stress-related proteins, some of as of yet unknown function. Acknowledgement: Funded partly by Hellenic Endocrinology Society.

Intermediates, which are observed during denaturation by different agents, are one and the same thermodynamic state Bacterial protein seretion is catalyzed by the SecYEG protein conducting channel complexed with the SecA ATPase motor. SecA bound to SecYEG undergoes nucleotide-driven conformational changes termed 'insertion-deinsertion' cycles. These changes occur concomitantly and are apparently coupled with the segmental transfer of the preprotein polymer into and across the membrane through the SecYEG preprotein conducting channel. High resolution structures of the archaeal protein-conducting channel and the bacterial SecA motor as well as several mutations that affect SecYEG function have been identified however none of the above addresses the questions of how these subunits assemble to form the functional translocase or which regions of SecYEG are essential for binding to SecA. We have studied the SecA-SecYEG interaction using peptide arrays and combined it with quantitative biophysical measurements, mutagenesis and functional in vitro and in vivo assays. This approach identified several regions on SecYEG that interact with SecA and led to a number of important conclusions: the SecA binding surface on SecYEG is multivalent and requires the contribution of several low affinity sites. The identified sites are mainly located on cytoplasmic loops. Several of the identified binding sites are essential for translocase catalysis. Association of SecA to SecYEG is regulated by temperature and the presence of nucleotide. Collectively these data provide the first, to our knowledge, residue level dissection and dynamics of the SecYEG -SecA interaction.

Analysis of structure-function relationship of the key DNA repair and recombination factor Rad51

Introduction: Rad51's function is dependent on its proper structure which enables interactions with partner molecules as well as DNA, in order to maintain genome integrity through the accurate repair of DNA double strand breaks. Occupation of Rad51 functional sites by factors as BRCA2 and p53 might directly modulate recombination processes and consecutively cellular pathways like cell cycle control and apoptosis, depending on the DNA repair outcome. Therefore, we focus on the analysis of structure-function relationship of Rad51, translated in complex formation with such partner molecules involved in regulation of this cellular network. Consequently, a number of Rad51 mutants, designed to modify Rad51's binding to BRCA2, were created and analyzed. Methods: Cells culture, transfection, cell cycle analysis, immunofluorescence, confocal microscopy and FRET analysis methods were applied. Results: Expression of Rad51 mutants was initially detected in the cell cytoplasm, resembling the pattern of the Rad51 wild type (wt) protein.

Unpredictably, after about 15 hours of expression, mutant forms migrated to the cell nucleus, forming filament-like structures. Moreover, cell cycle analysis versus wt protein showed a G1 arrest/delay, following the translocation of the mutant protein from the cytoplasm to the nucleus. FRET experiments indicate direct interaction between Rad51m5 and m6 with wt protein, possibly suggesting hetero-complex formation.

Conclusions: Single amino acid residue's substitution of Rad51, designed to modify Rad51 structure in the area interacting with BRCA2 and p53 tumor suppressors, remarkably alters both the subcellular location of the protein and the cell cycle progression of the expressing cells, implying modification of Rad51 wt's function.

Gelatinases: enzymatic activity as potential biomarker in multiple sclerosis Objective: Gelatinases (MMP-9 and MMP-2) are enzymes which degrade components of the extracellular matrix. Their activity is the result of a balance between two processes: activation of proenzymes and inhibition by Tissue Inhibitors (TIMPs). Multiple Sclerosis (MS) appears associated to an unbalance MMP-2 and MMP-9. MMP-9 is present in three different forms: the inactive proenzyme (92 kDa), a 'long' (82 kDa) active form lacking in the N-terminal propeptide, and a 'short' (66 kDa) active form, lacking N-and C-terminal. TIMP-1 binds with high affinity to the C-terminal domain, the binding between 'short' MMP-9 and TIMP-1 seems unlikely. The aim of this study is to verify the presence of the 'short' active form of MMP-9 in vivo and its potential role as marker of disease in MS. Materials and Methods: MMP-9 and MMP-2 activity was measured using commercially Activity Assay Systems. The levels of gelatinases were, also, measured by zymography on gelatin-copolymerized gels. Introduction: Electron Spin Resonance (ESR) has been experiencing a moment of resurgence in structural studies of protein and lipid-protein interactions since the introduction of site-directed spin labeling (SDSL) methodology, where a nitroxide side chain is selectively introduced at specific sites using site-directed mutagenesis. In this work, we present results obtained from SDSL studies on the interaction of dihydroorotate dehydrogenase (DHODH) with mixed vesicles. DHODH catalyzes the fourth step in the de novo pyrimidine nucleotide synthesis pathway.

Results: ESR spectra of labeled phospholipids introduced in vesicles show that the protein binds to mixed as well as pure detergent micelles. A second less-immobilized component is clearly observed in the spectra of phospholipids chain labels 5-and 10-PC, which is absent when other labels, such as headgroup label DPPTC and 16-PC, are used. The appearance of such component is attributed to the formation of a defect in the micelle hydrophobic region. SDSL studies were performed on residues Y2, F5, H19, and F21, which are located at the N-terminal extension of the protein. Their ESR spectra were characterized in terms of protein local backbone fluctuations, and indicate a more disordered structure of the N-terminal extension than predicted from the enzyme structure.

Conclusions: To the best of our knowledge, this is the first report showing direct evidences concerning the binding of class 2 DHODH to membrane systems and its implication in protein function. Also, this is the first site-directed spin labeling study developed in Brazil. Acknowledgments: FAPESP, CNPq. Tissue transglutaminase (TG2) catalyzes the Ca 2+ -dependent posttranslational modification of proteins via formation of isopeptide bonds between glutamine and lysine residues of proteins. The substrate specificity of TG2 was studied extensively at sequence level but no clear consensus sequence was determined so far. Using the TG2 substrate proteins listed in TRANSDAB Wiki (http://genomics.dote.hu/wiki/ index.php/) database we examined the tertiary structure of those substrates where the modification site by TG2 was known. We observed a slight preference of TG2 for glutamine and lysine residues situated in turns while we could find only several substrate glutamines and no substrate lysines situated in beta sheet and bend regions. The spatial environment of substrate and non substrate glutamine and lysine residues was also examined and using logistic regression analysis we could find dedicated amino acids having role in glutamine and lysine substrate selection of TG2.

Phosphorylation-mediated unfolding of a KH domain regulates KSRP localisation and mRNA decay by 14-3-3 binding The AU-rich-element (ARE)-mediated mRNA degradation activity of the RNA-binding K-homology Splicing Recognition Protein (KSRP) is regulated by phosphorylation of a serine within its N-terminal KH domain (KH1). In the cell phosphorylation promotes the interaction between KSRP and 14-3-3f protein and impairs the ability of KSRP to degrade RNA targets. Here we examine the molecular details of this mechanism. We report that phosphorylation leads to the unfolding of the structurally atypical and unstable KH1, creating a site for 14-3-3 binding. Using this site 14-3-3 discriminates between phosphorylated and un-modified KH1, driving the nuclear localization of KSRP. 14-3-3-KH1 interaction regulates the mRNA decay activity of KSRP by sequestering the protein in a separate functional pool. This study demonstrates how an mRNA degradation pathway is connected to extra cellular signaling networks by reversible unfolding of a protein domain. Methods: Phage display technology offers a very efficient way to purify and characterize proteins by displaying them on the surface of the bacteriophage virion. In this study, HBe antigen was genetically fused to the minor capsid protein gp3 and displayed on the surface of filamentous phages. To assay the activity of the phage displayed HBe antigen, HBe-phages were tested by ELISA and used for immunization of BALB/c mice. Results and Conclusion: To obtain HBe phages, HBV E gene was cloned into phagemid vector and transferred into E.coli TG1. Four clones was randomly selected and controlled by colony PCR. According to the PCR result, colony3 was rescued by M13 K07 phage and tested by ELISA for their affinity to Hbe antibodies. As the ELISA results were positive, BALB/c mice were immunized with colony3phages. The immune response was detected with ELISA from blood samples and positive response was obtained. These results showed that HBe antigen can be displayed on phage surface without loss of activity and that phages can be effectively used as HBe antigen carrier for the production of monoclonal antibodies. Venom is a rich source of biochemically active enzymes, proteins, peptides and low molecular weight compounds. The peptides in the venoms of scorpions are particularly interesting for pharmaceutical, industrial and protein engineering purposes because of their remarkable diversity, molecular targets, and their flexible loops despite rigid scaffold. The molecular targets are ion channels, acetylcholine receptors, acetylcholine esterase, membranes, coagulant and anticoagulant pathways and metaloproteases which are inhibited or activated with high selectivity and affinity by these peptides. In this report, sequences of the peptides with high similarity to the peptides BeI2, BeM9, BeM10 and BeM14 from Mesobuthus eupeus eupeus scorpion venom were found by Multiple Sequence Alignment by doing BLAST search. The templates were selected and pair wise alignment was carried out by CLUSTALW to achieve best score for alignment. Tertiary structures of these four peptides were predicted by homology modeling from the template structures after several steps of energy minimization. Our results suggest that long chain peptides BeI2, BeM9, BeM10 and BeM14 have affinity for sodium channels. Then, the secondary structure elements in modeled structures were recognized from their atomic coordinates and H-bond pattern by STRIDE. All the peptides have a/ b structure.

PP3-21 L-pyruvate kinase inhibition by phosphopeptide Arg-Arg-Ala-Ser(Pi)-Val-Ala, resembling the phosphorylated N-terminal domain, responsible for cooperativity of the enzyme I. Faustova and J. Ja¨rv Institute of Chemistry, University of Tartu, Tartu, ESTONIA Pyruvate kinase (ATP-pyruvate 2-O-phosphotransferase, EC 2.7.1.40, PK) catalyzes the last step of glycolysis, transferring the phosphoryl group from phosphoenolpyruvate (PEP) to adenosine diphosphate (ADP) and producing pyruvate and ATP. Activity of one subtype of PK, called L-type pyruvate kinase (L-PK), is controlled by phosphorylation at the N-domain of the enzyme. This phosphorylation takes place at Ser(12) residue, flanked by peptide sequence Arg(9)-Arg(10)-Ala(11)-Ser(12)-Val(13). As we have shown previously, this phosphorylation induces cooperative behavior of L-PK, as the nonphosphorylated enzyme is not regulated cooperatively. In this study we investigated influence of phosphopeptide Arg-Arg-Ala-Ser(P i )-Val-Ala, added into the reaction mixture, on catalytic properties of the nonphosphorylated L-PK. It was found that this phosphopeptide inhibited the enzyme activity by reducing the V value and increasing the K m value, but was unable to induce cooperative behavior of L-PK. This means that the enzyme phosphorylation cannot be imitated by this short phosphopeptide, although reduction of the enzyme activity can be observed also in the case of L-PK phosphorylation. Some implications of the data are discussed with reference to the mechanism of cooperative regulation of the phosphorylated L-PK.

A few glycolytic enzymes as chaperonin TRiC substrates. Purification and thermodynamic analysis of TRiC complex that mediates protein folding in the eukaryotic cytosol. It is known that TRiC assist folding of certain proteins like actin, tubulin, neurofilament and few others, but its physiological substrate spectrum is still poorly defined. Probability of TRiC to prevent a protein thermoaggregation is unknown.

Methods: TRiC isolation: ammonium sulfate precipitation, pelleting into a sucrose cushion and heparin-sepharose chromatography. Chaperonin analyzing: SDS-and native blue electrophoresis, dynamic light scattering, mass spectroscopic analysis, precipitation with denaturated glyceraldehyde-3-phosphate dehydrogenase (GAPDH), reactivation, differential scanning microcalorimetry, measurement of thermoaggregation kinetics.

We used published methods of TRiC isolation and create simple method of TRiC purification from ovine testis that includes only three steps. The obtained preparation was homogeneous and identified as TRiC by definition of molecular weight, size of particles and identification of seven subunits with mass spectroscopic analysis. Chaperonin binds guanidine hydrochloride denaturated forms of GAPDH and lactate dehydrogenase (LDH). Chaperonin-assistant reactivations of these enzymes were performed, but only reactivation rate of LDH was increased. Analyzing of chaperonin dependence on thermoaggregation of GAPDH showed that TRiC does not bind thermodenaturated forms of GAPDH and does not prevent the thermoaggregation.

Conclusions: New simple method of TRiC purification from ovine testis was developed. TRiC binds guanidine hydrochloride denaturated GAPDH and LDH but folds only second of them. TRiC does not bind thermodenaturated GAPDH and does not prevent its thermoaggregation. HACL1 is a homotetrameric matrix protein which is transported into peroxisomes after recognition of its targeting signal PTS1. So far, HACL1 is the only known peroxisomal TPP-dependent enzyme in mammals.

Methods: In this study we investigated whether mutations in the highly conserved TPP-binding domain of HACL1 have an effect on enzyme activity, subcellular localization and tetramerization. To pursue this goal, we mutated the residues in the positions 455 and 456 of the human HACL1, generating the mutants D455S, D455R and the double mutant D455S/ S456R.

Results: Based on our results, it appeared that the mutations did not affect the targeting, as observed by the punctate peroxisomal pattern for the N-His 6 -tagged fusion proteins in transfected CHO cells. However, enzyme activity was completely abolished in comparison with wild-type HACL1.

Further studies with purified control and mutated N-His 6 -HACL1 fusions, overexpressed in yeast and subjected to gel filtration, allowed us to conclude that mutation in the TPP-binding site did not affect the oligomerization of the enzyme.

Our results show that the binding of TPP is essential for HACL1 activity but not for peroxisomal targeting and correct folding of the protein.

Since mammalian peroxisomes are considered as a TPP-containing compartment and HACL1 is the only known peroxisomal TPP-dependent enzyme, it remains to be established whether the transport of TPP into the peroxisome is dependent on HACL1 translocation. 

Identification of amino acid residues essential for binding of calmodulin in C-terminal region of TRPC6 Methods: The specified sequence was subcloned into pET42b and used as a template for site directed mutagenesis. There were performed mutations of several amino acid residues that could potentially disrupt calmodulin binding. These residues were chosen on the basis of three-dimensional computer model. Fusion proteins were expressed in E. coli and purified by affinity chromatography and gell filtration. The homogeneity was confirmed by SDS-PAGE and mass spectrometry. The ability of binding of recombinant proteins to calmodulin was tested by fluorescent anisotropy measurements using Calmodulin Alexa Fluor 488 dye.

Results: TRPC6 binds directly to calmodulin via its C-tail with a one to one stoichiometry in a calcium-dependent way. Our results show that amino acids R852, K856, I857A, K859, R860, K863 and R864 participate in calmodulin binding on C-terminus of TRPC6. The dissociation constants were determined.

Conclusions: We identified amino acid residues participating in binding of calmodulin in C-tail of TRPC6 channel using in vitro binding assays. Methods: To dissect structure-function relationships in the NAT signature motif, we use Cys-scanning mutagenesis of YgfO, the xanthine permease of E. coli, followed by site-directed alkylation and extensive functional analysis of selected mutants in right-side out membrane vesicles.

Results: Q324 and N325 are irreplaceable for high-affinity xanthine uptake:

single-Cys N325C is partially protected by substrate against labeling with N-ethylmaleimide (NEM); single-Cys Q324C is highly reactive with NEM but not substrate-protectable, as replacement of Q324 leads to impaired binding.

Site-directed alkylation with the membrane-impermeant MTSESshows that the motif residues are highly accessible to solvent and the sequence region Conclusions: The NAT motif sequence region contains two conserved, irreplaceable residues that are close or at the periphery of the purine binding site (QN) and an alpha-helical face of residues that are exposed to solvent and participate in dictating purine specificity (TGxxSxxV). Introduction: Hepcidin is a small, 25 amino acids peptide, produced in the liver as a preprohormone (84 aminoacids), containing eight cysteines enrolled in four disulfide bridges. Discovered in early 2000's [1, 2] , this iron regulting 'hormone' control iron concentration in the body by inhibiting intestinal iron absorption and iron recycling by macrophages. Recently T. Ganz and coworkers have demonstrated that hepcidin was capable of inducing ferroportin internalisation and degradation, ferroportin being the unique iron exporter in mammals. Hence in the presence of hepcidin, ferroportin is destroyed, iron is trapped inside the cell and circulating iron concentration is decreased [3] . Our project was to get a reliable source of hepcidin and prohepcidin for cellular assays. Not only the production is not simple because of the four disulfide bridges, but also hepcidin is extremely prone to aggregation and its peptidic synthesis yield is very poor. Methods: We produced Hepcidin and prohepcidin using a Thioredoxin fusion protein based E. Coli expression system that does not require any renaturation step. Recombinant peptides were caracterized using Ellman's regeant, ESI and SELDI-ToF mass spectrometry and their biological activity was tested for their ability to induce ferroprotin degradation in macrophages. Results and Conclusion: We achieved the purification of active recombinant hepcidin (mouse and human) and prohepcidin (mouse). Hepcidin 61 amino acids precursor releases hepcidin upon furin cleavage. We demonstrate that the activity of prohepcidin towards FPN is only related to its capacity to generate hepcidin. Introduction: In the last years, a number of models to study amyloid diseases (Alzheimer, Parkinson, Huntington and prions) based on nonpathogenic proteins have been developed. We report here our work towards the establishment of a fully bacterial model system to study intracellular protein amyloidogenesis. It is based on the N-terminal WH domain of RepA, an initiator of DNA replication of the pPS10 plasmid of Pseudomonas. RepA dimers (transcriptional repressors) undergo a structural transformation upon binding to origin DNA sequences (iterons), resulting in monomerization (initiator of replication). Such conformational change in WH1 implies a-helix into b-strand conversion. Methods and Results: We have found that RepA-WH1 forms different amyloid assemblies in vitro (spheroids and fibres) depending on its interaction with different DNA sequences (iteron and operator, respectively). This feature is shared with the mammalian prion protein, PrP. We have identified an essential motif in the protein responsible for amyloid cross-aggregation when isolated as a peptide. This has allowed us to design point mutants with enhanced or reduced amyloidogenicity [1] . We have also identified a small ligand molecule competing with DNA binding to RepA-WH1, thus inhibiting the assembly of amyloids [2] . Conclusions: Our results validate the concept that DNA can promote protein assembly into amyloids and reveal that the binding sites of effector molecules can be targeted to inhibit amyloidogenesis. We are now developing a system for the expression in E.coli of WH1 mutants in different genetics backgrounds. Tryptophanase (Trpase) from Esherichia coli is a pyridoxal phosphate (PLP) -dependent tetrametic enzyme with a molecular weight of 210 kD. We studied the reversible cold inactivation and dissociation of E. coli Trpase and its Y74F, C298S and W330F mutants. The incubation of E. coli Trpases at 2°C for 20 hours resulted in a significant loss of activity and enhanced the dissociation into dimers. Rewarming at 25°C restored their initial activity and the re-association of the dimers into the tetrameric active form. We found also that at 25°C, all apo forms of Trpase markedly (about 70%) dissociated into dimers and further upon cooling to 2°C (about 90%). We determined the crystal structures of the two mutants, Y74F and C298S in their apo form, at 1.9Å resolution and compared them to the known structure of holo, wt P. vulgaris Trpase in order to unravel the mechanism of reversible cold dissociation of Trpase. structural analysis revealed that the mutants in their apo form are found in an 'open' conformation compared to the 'closed' conformation of P. vulgaris in its holo form. Thus, the cold inactivation and dissociation of E. coli Trpases is primarily affected by PLP release. The main cause for the enhanced loss of activity of the three mutants is presumably due to the reduced size of the side chain of the amino acids. This prevents the tight assembly of the active tetramer, making it more susceptible to the cold driven changes in hydrophobic interactions which facilitate PLP release. Introduction: Campylobacter strains are recognized as the major agent of bacterial diarrhoea worldwide. Their virulence depends mainly on extracytoplasmic proteins, many of which contain two or more cysteine residues and achieve the final structure as a result of disulfide bridges formation, catalyzed by Dsb proteins. We identified and characterized a novel subfamily of disulfide oxidoreductases, denoted DsbI. The DsbI N-terminal domain contains five transmembrane helixes and its periplasmic C-terminal domain is predicted to achieve a b-propeller structure, rare for bacterial proteins. The aim of the present work was to analyze Campylobacter jejuni DsbI sequence-structure-function relationships.

Methods: Camphylobacter jejuni dsbI knock-out strains were constructed by allelic exchange, resulting in disruption of dsbI gene with chloramphenicol resistance cassette. A comparative model of the DsbI N-terminal catalytic domain was constructed by means of bioinformatics methods using DsbB crystal structure (pdb code: 2hi7) as the template. The role of DsbI conserved amino acid residues was analyzed by site-directed mutagenesis of dsbI, followed by Western blot and GGT functional test.

Results: Sequence alignment and modeling of the DsbI allowed us to determine conserved residues that are likely significant for the enzymatic activity. They were replaced with glycine/alanine and mutant versions of dsbI were subsequently used for complementation of dsbI knock-out strain. DsbI crucial amino acid mutations led to malfunction of the protein (GGT test).

Conclusions: It was shown that some of the DsbI conserved amino acids are crucial for enzyme activity. The results of the complementation experiment support the role of DsbI in disulfide bridge formation.

Partial purification of the ubiquitin-conjugating enzyme(s) acting at Cys11 of mammalian Pex5p Introduction: The peroxisomal cycling receptor Pex5p recognizes newly synthesized peroxisomal matrix proteins transporting them into the organelle. After cargo release, Pex5p is exported back into the cytosol for further transportation cycles. Monoubiquitination of yeast and mammalian Pex5p at a conserved N-terminus cysteine residue is required for the export step. Whereas in yeast Pex4p is the ubiquitinconjugating enzyme (E2) mediating this thiol-sensitive ubiquitination, the identity of this enzyme remains unknown in mammals. Our aim was the isolation of the mammalian Pex4p counterpart(s).

Methods: An in vitro rat liver system was used to assess the E2 mediating the ubiquitination of Pex5p in subcellular fractions, and to monitor its distribution throughout the purification process. The E2(s) purification was performed using a method previously described [1] , with some modifications. Mass spectrometry analysis was done in the purest fraction.

Results: The finding that peroxisomes supplemented with E1 are competent for Pex5p ubiquitination at Cys11 only when supplemented with cytosol, led us to hypothesize that the E2 enzyme could be cytosolic. A cytosolic protein fraction was subjected to affinity chromatography using ubiquitin-sepharose, and the eluate incubated with DE52. The ubiquitinating activity was found in the DE52-unbound fraction. SDS-PAGE analysis of this fraction revealed two bands, where five ubiquitin-conjugating enzymes were identified by mass spectrometry. Conclusions: We developed a protein purification strategy to purify the enzymes mediating the peroxisome-dependent ubiquitination of the conserved cysteine of mammalian Pex5p. Introduction: Amyloid fibril deposition, resulting from protein misfolding and assembly, is usually associated with several diseases called amyloidoses. However, recent evidence suggests that functional amyloids also exist in humans, invertebrates, fungi and bacteria, following our suggestion for the existence of natural protective amyloids. Silkmoth chorion is the major component of the eggshell, a structure with extraordinary physiological and physical properties, which protects the oocyte and the developing embryo from environmental hazards. Here, we review recent evidence on several silkmoth chorion protein synthetic peptide-analogues and on silkmoth chorion itself, in our search to document the function of silkmoth chorion as a natural protective amyloid.

Methods: Silkmoth chorion protein synthetic peptide-analogues and silkmoth chorion were studied utilizing electron microscopy, Congo red binding, X-ray diffraction, ATR FT-IR and FT-Raman spectroscopy and modelling.

Results: All silkmoth chorion protein peptide-analogues produce amyloid-like fibrils, under a great and diverse variety of conditions by selfassembly mechanisms, which strongly suggests that in chorion proteins, the amyloidogenic conformation is, apparently, the native conformation. The studies of silkmoth chorion itself verify this finding.

We present convincing evidence that silkmoth chorion is indeed a natural protective amyloid.

Theoretical studies of silkmoth chorion protein interactions -insights into amyloid fibril structure It is largely proteinaceous and due to the properties of its constituent proteins, it protects the oocyte and the developing embryo from environmental hazards. Our lab has previously shown that several peptideanalogues of the central conservative domains of the A and B classes of chorion proteins form, under a variety of conditions, fibrils with amyloid characteristics. This led us to propose that silkmoth chorion is a natural protective amyloid. Although amyloid fibrils have been associated with many diseases (amyloidoses), it seems that several organisms take advantage of the inherent ability of proteins and peptides, to form such natural protective amyloids, and, our proposal set the basis for the discovery of several functional amyloids. Methods: In order to investigate the fibrillogenesis mechanisms and the structural properties of amyloid fibrils, in conjunction with experimental data, comparative modeling was used to construct structural models of both the conservative parts of chorion proteins as well as entire proteins. Models of antiparallel b-pleated sheets and left-handed parallel b-helices where constructed for all available silkmoth chorion proteins. Their packing interactions were investigated theoretically, applying protein docking techniques.

Results: It seems that chorion proteins (folded as antiparallel b-pleated sheets) pack in a parallel or antiparallel fashion, with a packing distance of 10Å and form dimeric filaments ca. 31Å in diameter.

Conclusions: These studies, along with experimental data, may enlighten our understanding of chorion helicoidal architecture and may shed some light to the underlying molecular structure of amyloid fibrils.

Cks1-mediated multisite phosphorylation by Cdk1 The master regulator of the eukaryotic cell cycle, the protein kinase Cdk1, triggers the two major steps of the cell cycle -the S-phase and the mitosis. In budding yeast, the S-phase is switched on when the Cdk1 signal overcomes its G1/S threshold value and multiphosphorylates the G1/S inhibitor Sic1 at six phosphorylation sites. At the G2/M barrier, the mitotic cyclin-Cdk1 complexes trigger the onset of the mitosis by phosphorylating the Cdk1 inhibitor tyrosine kinase Swe1 at multiple sites on its N-terminus and this phosphorylation primes a secondary phosphorylation of Swe1 by polo-kinase Cdc5. The multiphosphorylation of both Sic1 and Swe1 leads these inhibitors to the SCF-mediated destruction. In the present study, we describe the role of Cks1, a phosphoadaptor subunit of cyclin-Cdk1 complex, in the mechanism of multisite phosphorylation of Sic1 and Swe1. Using sitedirected mutagenesis, we have studied the kinetic mechanism of phospho-docking by cyclin-Cdk1-Cks1 complex and found that a minor level of initial phosphorylation of Swe1 initiates a very fast Cks1mediated cooperative propagation of multisite phosphorylation resulting in a highly phosphorylated state of Swe1 protein. Secondly, we have found that contrary to the existing models, the Cks1 plays a critical role in the dynamics of Sic1 phosphorylation by Cdk1. Based on the obtained data, we propose a novel mathematical model for the ultrasensitive G1/S switch.

What does determine the degree of compactness of a calcium binding protein? Introduction: The EF-hand calcium binding proteins (CaBP) may exist either in an extended or a compact conformation. This conformation is correlated with the function of the CaBP. Usually calcium sensors are extended and calcium buffers are compact, hence the interest to predict the form of the protein starting from its sequence. Method: In this study we used two different procedures, one that already exists in the literature, the SOSUIdumbbell algorithm, mainly based on the charges of the two EF-hand domains, and the other is a novel procedure that is based on the linker average hydrophilicity (LAH). The linker consists of the residues that connect the domains.

Results: The two procedures were tested on 17 known-structure CaBPs and then applied on 59 unknown-structure centrins. The SOSUIdumbbell yielded the right conformations for 15 of the knownstructure proteins and predicted that all centrins should be in a closed form. The LAH procedure discriminated well between the extended and non-extended forms of all the known-structure CaBPs and it reflected well the phylogenetic classification of centrins.

The LAH criterion is a simple and powerful means to discriminate between extended and non-extended forms of CaBPs.

What is remarkable is that only few residues that constitute the linker (between 3 and 12 in our tested sample of proteins) are responsible for the form of the CaBP, showing that this form is mainly governed by short-range interactions. Introduction: Cell Penetrating Peptides (CPPs) are potential tools for the intracellular delivery of wide range of cargos. Though the exact translocation mechanism is still unknown, endocytosis is the most prevalent uptake mechanism seen for highly cationic peptides. Release from endosomes for colocalization of cargo/drug and target in the cytoplasm is the major hurdle of targeting approaches. Therefore, there is a need of vectors capable of transferring cargo molecules directly into the cytoplasm. Herein we focus on the development of a novel CPP derived from Crotamine (polypeptide in venom of rattle snake) which shows an efficient uptake at low concentrations (£2.5 lM) and cytosolic distribution along with vesicular uptake. Methods: Series of peptides were synthesized by Fmoc strategy, introducing mutations in Cro (27-39) (proposed CPP sequence in Crotamine). All were labeled with fluorescein isothiocyanate at the N-terminal. SAR studies were done by substitution and/or deletion of amino acid residues in the sequence observing the uptake behaviour by fluorescence spectroscopy and microscopy.

Results: Amongst 61 synthesized peptides one of shorter length was showing the best intracellular delivery and cytosolic distribution. Replacing or deleting cysteines had negative impact on internalization.

Results also show the involvement of tryptophans in cellular uptake indicating along with cationic amino acids the importance of each residue in this optimized sequence.

Conclusions: SAR studies identified a peptide showing aside of endosomal uptake also an efficient delivery into the cytoplasm. Thus, this peptide might prove useful for efficient transmembrane delivery of agents directed to cytosolic targets.

In vitro radiosensitivity of Fanconi anemia fibroblasts measured by gamma-H2AX foci The key pathological hallmark of Alzheimer's disease (AD) is deposition of amyloid-beta peptide (Ab) in the brain. There is evidence that binding of Zn2+ and Cu2+ ions to the Ab may contribute to the Ab oligomerization and toxicity. Cu2+ and Zn2+ form complexes with Ab peptides in vitro; however, the published metal-binding affinities of Ab vary in an enormously large range. The interactions of Cu2+ and Zn2+ with monomeric Ab40 were studied using intrinsic Ab fluorescence and competitive metal-selective fluorescent dyes. Cu2+ formed a soluble 1:1 complex with Ab40, whereas buffer compounds acted as competitive copper-binding ligands. Buffer independent conditional KD for Cu(II)-Ab40 complex at pH 7.4 was equal to 0.035 lmol/l. We also demonstrated that the initial weak Ab40 complex with Zn2+ (KD = 60 lmol/l) partially aggregated during zinc titration experiments and underwent a transition to a more tight complex with KD = 2 lmol/l [1] . Further studies of the transformation of the initial metal ion complexes of Ab42 into amyloid fibrils showed that both metal ions are tightly bound in the forming peptide aggregates, however, these aggregates show lower fluorescence in the presence of Thioflavine T and altered morphology monitored by TEM. Interaction of both Zn2+ and Cu2+ ions with Ab peptides may occur in brain areas affected by Alzheimer's disease and Zn2+-induced transition in the peptide structure might contribute to amyloid plaque formation. The enzyme catalyzes the reduction of dihydrofolate to tetrahydrofolic acid, a biochemical reaction vital for the cell. DHFR has been thoroughly studied in the past, but its adaptation mechanisms at low temperatures still remain unknown. Methods: In order to clarify the adaptation mechanisms of the enzyme at low temperatures, sequence analysis of 10 psychrophilic DHFRs (psyDHFRs) was performed along with the construction of structural models applying comparative modeling techniques. Optimization of the models at 277°K utilizing AMBER84 force fields and Monte Carlo minimization algorithms was performed. Structural evaluation and exhaustive analysis of the derived models in comparison to their mesophilic counterparts was also carried out.

Results: Analysis reveals that psyDHFRs share a weak hydrophobic core and surfaces with less positively charged residues and more highly hydrophobic ones, such as Val and Ile, compared to non-psychrophilic DHFRs. Furthermore, ionic interactions were calculated to be fewer in number in comparison to mesophilic homologous structures. psyDHFRs have a relatively poor optimal hydrogen bonding network, an alteration that provides the enzyme with plasticity and flexibility.

Conclusions: All the derived results lead to the conclusion that DHFRs from psychrophilic bacteria are adapted to function at low temperatures by possibly evolving global flexibility. This is further supported by several similar studies on other enzymes. Introduction: Horseradish peroxidase (HRP) belongs to the superfamily of heme-containing plant peroxidases that catalyze the hydrogen peroxide-mediated oxidation of a wide range of substrates. Isoenzyme C (HRPC) is the most abundant and best known isoenzyme of HRP, used in countless investigations. Acriflavine which is a member of the aminonacridine family induces petite mutation in the yeast Saccharomyces cerevisiae and kinetoplast lost in trypanosomidae. It is also an effective agent to produce apoptosis and necrosis but little is known on its interaction with proteins, and especially, enzymes. In this study, we investigated the effect of acriflavine on HRPC.

Methods: HRPC activity was measured by following the H 2 O 2 -dependent oxidation of o-dianisidine at 460 nm. pH activity profile was performed using a citrate-phosphate-borate buffer system. Spectrophotometric and fluorescence studies were perfomed at optimum pH, with HRPC in the presence of various acriflavine concentrations. Results: The pH activity profile showed that pH 4 was optimum for HRPC. When 65 micromolar acriflavine and HRPC were mixed and incubated at room temperature for 0, 10, 20, 30, 45, 60 minutes, the enzymatic activity was increasingly inhibited so that only 2% activity remained after 60 minutes incubation. Spectrophotometric studies showed that acriflavine induced a blue-shift and a change of chromicity in the HRPC Soret absorption band at 405 nm. Furthermore, fluroscence studies showed that addition of acriflavine to HRPC resulted in increasing emission due to the enzyme's single tryptophan residue upon excitation at 297 nm. Conclusions: Results showed that acriflavine altered HRPC conformation and catalytic activity.

Structural and biochemical characteristics of thioredoxin reductase from Dictyostelium discoideum Introduction: Thioredoxin reductase (Trr) belongs to a flavoprotein family of pyridine nucleotide-disulfide oxidoreductase. Trr catalyses the transfer of the reducing equivalent from NADPH via FAD to the redox-active disulfide, which then can reduce oxidized thioredoxin. The reduced thioredoxin serves as an electron donor for the reduction of disulfide bonds in various proteins. Methods: Trr and Trr mutants were overexpressed and purified in Escherichia coli. Biochemical properties of Trr were analyzed by sitedirected mutagenesis, spectral analysis and X-ray crystallography. Results: The crystal structure of Trr revealed that the redox-active disulfide formed between Cys-144 and Cys-147 was juxtaposed to the isoalloxazine ring of FAD. The mutation at Cys-144 or Cys-147 resulted in complete loss of thioredoxin-reducing activity and changed spectral properties. Regardless of the presence of NADPH, C144S and C144A mutant showed the spectral characteristic of the reduced flavin. In contrast, C147S mutant retained the feature of the oxidized flavin even with NADPH and showed the strong fluorescence quenching, indicating that C147S is restricted to the FO conformation. Conclusions: Our results indicate Dictyostelium discoideum Trr undergoes the FO/FR conformational change for its catalysis and the point mutation at each active site cysteine affects the conformational equilibrium. Also Dictyostelium Trr and its mutants in cysteine are expected as a model to elucidate the mechanism of the transition between the FO and FR conformation of thioredoxin reductase. Translationally controlled tumor protein (TCTP), also known as IgEdependent histamine-releasing factor (HRF), has both extra-and intracellular functions. The histamine releasing ability of recombinant HRF (rHRF) was found to be much less than that of HRF isolated from activated mononuclear cells (HRFmn), raising a question regarding the identity of the histamine releasing protein moiety and suggesting that the active form of extracellular TCTP may be different from intracellular TCTP. We therefore conducted studies directed at identitying the active moiety in rat recombinant TCTP (RrTCTP). We found that some modified RrTCTPs increases IL-8 secretion from BEAS-2B cells more effectively than the original protein. The modified RrTCTP contains intermolecular disulfide bonds unlike the original RrTCTP, suggesting that the former exists as polymers. Site-directed mutagenesis at two cysteine residues, Cys 28 and Cys 172 , indicates that the intermolecular disulfide bond between Cys 172 and Cys 172 is critical for the IL-8 release ability of the modified RrTCTP. We conclude that the biological activity of extracellular TCTP results from the dimerization of TCTP through the intermolecular disulfide bonds.

Study on the evolution of the peroxisomal proliferation machinery in yeast and human cells Peroxisomes are vital single membrane-bound organelles present in all eukaryotic cells. Their function is mainly associated with lipid metabolism and they enclose hydrogen peroxide-generating and degrading enzymes. Peroxisomes are highly versatile organelles that quickly adjust their shape, size, number and protein content according to the cellular requirements. In humans, defects in peroxisome biogenesis lead to devastating diseases such as the Zellweger syndrome. Over 30 proteins, the peroxins, are involved in peroxisome biogenesis. In the yeast Saccharomyces cerevisiae, peroxins of the PEX11-family, namely PEX11, PEX25 and PEX27, participate in peroxisome proliferation. Indeed, yeasts lacking PEX11 present fewer and larger peroxisomes than wild type cells. Orthologues of the yeast PEX11-proteins, PEX11a, b, c and PEX11-1 to -5 have been identified in human and plants, respectively. We sought to analyze the regulation of peroxisome proliferation through the PEX11-protein family in yeast and human cells. Here we show co-expression studies in human cells using DsRed-SKL as peroxisomal protein marker. We analyzed the effect of ectopic expression of HsPEX11a, b and c as well as ScPEX11, ScPEX25, ScPEX27 and AtPEX11-1 and -2 in human cells on peroxisome size, number and shape. We also performed functional tests in yeast pex11-mutants. Our analyses suggest that although the peroxisome proliferation mechanism is conserved from yeast to man some species-specificity does exist regarding the function of PEX11 proteins. To pinpoint the molecular differences between PEX11 orthologues in different kingdoms we performed further studies in cell culture with PEX11-peroxins harboring point mutations and using siRNA. Bood POZ containing gene type 2 (BPOZ-2), which contains ankyrin repeats, NLS, BTB/POZ domains and LXXLL motifs, is an adaptor protein for the E3 ubiquitin ligase scaffold protein CUL3. We isolated a cDNA encoding eEF1A1 as a BPOZ-2 binding protein by screening a human thymus cDNA library using a yeast two-hybrid system. eEF1A1 is essential for translation and is also involved in the 26S proteasome-dependent degradation of misfolded or unfolded proteins. The binding between BPOZ-2 and eEF1A1 was confirmed by pull-down and immunoprecipitation assays in vitro and in vivo, respectively. BPOZ-2 binds to eEF1A1 through the ankyrin repeats and both BTB/ POZ domains in BPOZ-2 and Domains I and III in eEF1A1. BPOZ-2 and eEF1A1 overexpressed in 293T cells co-localized as speckles within the cytoplasm. BPOZ-2 promoted eEF1A1 ubiquitylation and degradation, suggesting that eEF1A1 is a substrate of BPOZ-2. BPOZ-2 inhibited GTP binding to eEF1A1 and prevented translation in in vitro translation assay using rabbit reticulocytes.

Plant specific phi class GST, OsGSTF3: herbicide specificity and catalytic roles OsGSTF5 showed high specific activity toward chloroacetanilide herbicides used this experiment. The tau class GST enzymes displayed remarkable activity toward the diphenylether herbicide, fluorodifen. In order to elucidate the catalytic role of evolutionally conserved residues in plant GST, Tyr8, Ser13, Cys22 and Ser38 residues of the OsGSTF3 were replaced with alanine. The Ser13Ala, Cys22Ala and Ser38Ala did not affect the glutathione conjugation activity toward 1-chloro-2,4-dinitrobenzene. On the other hands, the substitution of Tyr8 with alanine resulted in a notable decrease in the specific activity toward CDNB to 11% of the wild-type value. The pK a of the thiol of GSH in the Y8A-GSH complex was estimated to be around 8.5, which was about 2.0 pK units higher than that of the wild type. Therefore, Tyr8 is essential for lowering the pK a and enhancing the nucleophilicity of the thiol of GSH in the active site of OsGSTF3, and it is major contribution of GST to catalysis. Toxoflavin produced by Burkholderia glumae causes rice grain rot and seeding rot in rice. Recently, it was found that an enzyme TflA (Toxoflavin Lyase) degrades such phytotoxin responsible for pathogenicity. Subsequently, determination of a crystal structure of TflA has been carried out using various mutants due to the difficulty in producing a crystal of the wild-type TflA. Recently, we were successful in crystallizing a mutant enzyme, and in determining its structure by using multiwavelength anomalous dispersion method. In order to clearly define the mechanism of the degradation pathway of toxoflavin, a crystal structure with the substrate bound in its active site is crucial. However, obtaining a crystal complexed with the substrate failed. Functionally active and inactive mutants were systematically produced by Ala-scanning method, and those mutants were subjected to crystallization. Among those mutants, several produced crystals complexed with a toxoflavin analog, 8-azaxanthine. By determination of 1.6Å resolution data, we found the binding site of metal in its active site, presumably manganese, and 8-azaxanthine in close vicinity, suggesting that the bound metal is critical to the catalytic activity. Crystal structure of another mutant at 1.41Å resolution was also obtained with somewhat degraded substrate, accordingly corresponding with the functional activities of the mutants. Further refinement will be carried out for better understanding of the degradation mechanism.

The essential function of TIM12 in vivo is ensured by the assembly interactions of its C-terminal domain Results: We show that Tim12 has an intrinsic affinity for inner mitochondrial membrane lipids, in contrast to the other small Tims. We find that the C-end of Tim12 is essential in vivo and its deletion crucially abolishes its assembly in complexes with the other Tims. The N-end contains targeting information and also mediates direct binding of Tim12 to its substrates. Conclusions: These results provide a molecular basis for the concept that the essential role of Tim12 relies in its unique assembly properties that allow this subunit to bridge the soluble and membrane-embedded translocases in the carrier import pathway. * E. Lionaki and C. de Marcos Lousa have contributed equally to this work. Methods: A Hidden Markov Model (HMM) approach for predicting the LPXTG-anchored cell-wall proteins of Gram-positive bacteria was developed and compared against existing methods. The HMM model is parsimonious concerning the number of freely estimated parameters, and it has been proved very sensitive and specific in the training set of 55 experimentally verified LPXTG-anchored cell-wall proteins as well as on reliable datasets of globular and transmembrane proteins.

Results: A comprehensive analysis of 94 completely sequenced genomes has been performed in order to identify such proteins in Grampositive bacteria. In total, 860 LPXTG-anchored cell-wall proteins were identified, a number that is significantly higher compared to those obtained by other available methods.

Conclusions: A total of 237 of these 860 proteins are hypothetical proteins according to genome annotations, whereas 88 of them have no homologues in the UniProt database and this might be evidence that they are members of newly identified families of CWPs. Apart from the classic LPXTG pattern, other similar patters do exist, which are responsible for the cleavage of other variants of the Sortase. We are currently working in expanding the HMM to include them in a prediction system, as well as to allow for a proper discrimination of the cleavage specificity between different types of the Sortases. The periplasmic C-terminal domain of the Escherichia coli DsbD protein (cDsbD) has a thioredoxin fold. The two cysteines in the CXXC motif serve as the reductant for the disulfide bond of the N-terminal domain which can in turn act as a reductant for various periplasmic partners. The resulting disulfide bond in cDsbD is reduced via an unknown mechanism by the transmembrane helical domain of the protein. We show by NMR analysis of 13 C, 15 N-labelled cDsbD that the protein is rigid, is stable to extremes of pH and undergoes only localised conformational changes in the vicinity of the CXXC motif, and in adjacent regions of secondary structure, upon undergoing the reduced/ oxidized transition. pK a values have been determined, using 2D NMR, for the N-terminal cysteine of the CXXC motif, Cys461, as well as for other active-site residues. It is demonstrated using site-directed mutagenesis that the negative charges of the side chains of Asp455 and Glu468 in the active site contribute to the unusually high pK a , 10.5, of Cys461. This value is higher than expected from knowledge of the reduction potential of cDsbD. In a double mutant of cDsbD, D455N/ E468Q, the pK a of Cys461 is lowered to 8.6, a value close to that expected for an unperturbed cysteine residue. The pK a of the second cysteine in wild-type cDsbD, Cys464, is significantly higher than the maximum pH value that was studied (pH 12.2). Unequilibrium melting of green fluorescent protein (GFP) was studied by differential scanning microcalorimetry in a wide range of pH and at different scanning rates. The data obtained were used to determine the kinetic parameters of heat denaturation of GFP (rate constants and activation energies of denaturation). Using the methods of circular dichroism spectroscopy, the kinetic parameters of GFP in the range of temperature-induced denaturation transitions were obtained in direct kinetic experiments. It was shown that the rate constants of GFP denaturation, obtained from microcalorimetry data, are well compatible with the rate constants from direct the kinetic experiments. Thus, it was demonstrated that, kinetic parameters of denaturation and some other structural characteristics of the transition state of the proteins can be obtained using differential scanning microcalorimetry. This approach is less tedious and allows calculating with a high accuracy all thermodynamic functions of the transition state. Increments of GFP heat capacity at transition from the native state to the transition state and from the native state to the denatured state were determined from the obtained data. This permitted us calculating such thermodynamic functions as enthalpy, entropy and free energy of GFP activation. The transition state of GFP was described. It was concluded that the extent of exposure of hydrophobic groups of the transition state of GFP varies slightly from that of the native state and the activation changes involve the entire structure of the GFP molecule. Methods: I will review the mechanism that regulates signalling from chemotactic receptors to the cytoskeleton, to elicit cell polarity and directed migration. To interpret the external gradient of chemoattractant, a cell must not only transmit the attractant signal from the cell surface to the polarity and migration machinery, but must also compare different intensities of signal at different parts of the cell surface and convert these into directed polarity.

Results: To prevent propagation of DNA damage to future generations of cells, biochemical checkpoint mechanisms induce cell cycle arrest (or apoptosis) and allow initiation of repair processes. Although checkpoints differ in biochemical detail in different organisms. Therefore we need to propose a model to answer an unsolved but fundamental question: how does the DNA damage actually activate the proteins involved. Many genes involved in cell division and DNA replication and their protein products have been identified in bacteria; however, little is known about the cell cycle regulation of the intracellular concentration of these proteins.

Conclusions: Intension to use chemical genetics' to understand regulation is necessary. 'Forward' chemical genetics is analogous to gene mutations: specific chemicals screened in libraries of structurally diverse compounds are used to identify regulatory proteins that play critical roles in complex cellular processes. crucial enzymes in the pharmaceutical and agricultural industry. They cata-lyze the oxidation of formate to carbon dioxide, coupled with reduction of NAD + to NADH so that they can be used to regenerate the expensive NADH cofactor which is used by all NAD + -dependent oxidoreductases. FDH from Candida methylica (cmFDH) was cloned and overproduced. One disadvantage of working with this enzyme is its poor thermal stability. Site directed mutagenesis techniques have been applied to increase the thermostability of cmFDH by optimizing electrostatic interactions on the protein surface. Homology modelling of cmFDH was carried out using the Insight II molecular modelling program based on the X-ray crystal structure of the FDHs from Pseudomonas sp. 101 which has about 50% similarity and Candida boidinii which has >90% similarity in their amino acid sequences.

Methods: All designed mutations in the literature to improve any kind of properties of cmFDH were performed by using Pseudomonas sp.101 as a crystal structure model until now [1] . In this work, we have designed some mutations based on Candida boidini after as its crystal structure was published by Schirwitz et al [2] . The designed mutant proteins are constructed by using Invitrogen Gene Tailor Results: Previously we had described the cloning and purification of a truncated form of HlyA, HlyA N601, that consists mainly of the NRD, and binds Ca 2+ with about the same affinity (K 0.5~1 0 -4 M) as the native protein. In the present contribution, we describe the effects of Ca 2+ binding on the surface hydrophobicity and stability of the NRD. A combination of fluorescence and IR spectroscopy, molecular exclusion chromatography and proteolysis reveal that Ca 2+ , at the same concentrations that make the native HlyA less stable and more hydrophobic, make NRD more thermodynamically stable, and its surface less hydrophobic. In addition, the tertiary structure of HlyA N601 is made more compact through Ca 2+ binding.

Discussion: These paradoxal observations suggest the hypothesis that the Ca 2+ -induced conformational changes in the NRD must be transmitted to the protein N-terminal region, rich in amphipathic helices, where the resulting changes in tertiary structure have the opposite sign to those in the Cterminal moiety. The UspDBD defines the architecture of the UspDBD-EcRDBD heterocomplex due to the significant deformation of the response element [1] . The aim of the investigation was the analysis of the role of conformational changes of UspDBD induced by EcRDBD, during the formation of UspDBD-EcRDBD-hsp27 complex. Usp is a homolog of human RXR protein.

Methods: Recombined UspDBD and EcRDBD proteins were labeled with an intein method at the C-ends of both proteins and purified on chitin column. 5¢ or 3¢-ends of the hsp27 were labeled with TAMRA fluorophore. The changes of the distances in solution from the respective C-terminus of EcRDBD or UspDBD and the 5¢ and/or 3¢ end of the response element were measured using FRET methodology and analyzed with the Fo¨rster equations.

Results: The distance between the UspDBD C-terminus and 5¢-end of hsp27 was determined in the absence and in the presence of EcRDBD and was to be: 65.5 ± 3.4Å and 71.8 ± 2.0Å , respectively. The distance between UspDBD C-terminus and hsp27 3¢-end was to be 66. Introduction: Subunit e of the F 0 /F 1 ATP Synthase (Su e) is a nonessential but well-conserved protein involved in the suprastructure of the F 0 /F 1 ATP Synthase. In Saccharomyces cerevisiae Su e contains 96 amino acids with a single transmembrane domain at the N-terminus which spans the inner mitochondrial membrane following a N-in C-out orientation. Su e is implicated in the dimerisation of the ATP synthase with a mechanism which is not yet clear. It has also been shown that affects the morphology of the inner membrane. The import of the precursor in mitochondria does not involve the well characterized translocases of the inner membrane TIM22 and TIM23, but it is affected by the inter membrane space small Tim complexes.

Results: Using in vitro binding assays, in organello import assays and lipid bilayer insertion assays we report the specific requirements of this unique import pathway for components of the outer-and inner-membrane translocation machineries. Additionally, we have identified specific sequence determinants of Su e that are crucial for its translocation.

The results so far indicate that Su e is imported with a yet uncharacterized import mechanism with distinct features. These features are proposed to have universal value for other single transmembrane domain proteins that are embedded in the inner membrane. Peroxisomes are highly dynamic organelles present in nearly all eukaryotic cells. They react upon external stimuli by changing their size, number and protein content. In higher plants, peroxisomes are involved in a variety of essential physiological and biochemical functions such as photorespiration, lipid catabolism and hormone biosynthesis. In the last decade a number of peroxin (PEX) genes have been identified, mediating peroxisome biogenesis and proliferation. However, details regarding the mechanism mediating peroxisome proliferation, which depends on the conserved PEX11-protein family members is still unclear. In yeast ScPEX11, ScPEX25 and ScPEX27 and in human cells PEX11a, b and c proteins were identified as PEX11 peroxisomal proliferation factors. In Arabidopsis thaliana, however, bioinformatic analysis revealed that the PEX11 protein family seems to consist of five homologues named AtPEX11-1 to -5 (or AtPEX11 A to E).

Our study aims to analyse the capacity of PEX11-protein family members found in yeast, humans and plants to induce peroxisomal proliferation in plants. Organism shuffling experiments were performed in such that we over-expressed the human, yeast and plant PEX11 proteins as YFP fusions and study their effects on peroxisomal appearance in plant leaves. Although we detect some variations such as in the rate of proliferation induced by orthologous PEX11 proteins, our data suggest that the function of the human, yeast and plant PEX11 proteins is highly conserved in plants.

Towards a better understanding of extracellular cytokine receptor biology: the initial characterization of a lectin/R complex During the continuous formation of blood cellular components, a process known as hematopoiesis, receptor tyrosine kinases (RTK) and their cytokine ligands are responsible for directing and proliferating the undifferentiated precursor cells to specific blood cell types. Some members of the RTK III family, such as the c-Kit and FLT3 receptors, are activated during the very early stages of hematopoiesis, making their ligands interesting targets for rational drug design. Besides binding its natural ligand, the FLT3-receptor is suggested to also recognize a legume lectin, the Dolichos lablab mannose/glucose binding lectin (FRIL). Presumably, this interaction explains the FRIL-mediated preserving effect on hematopoietic progenitors in suspension culture. To get a better understanding of the binding modus and the activation of the receptor upon ligand binding, we generated the tools necessary for the thermodynamic and structural analyses of both L/R and FRIL/R complexes. We here describe (i) the transient expression of a his-tagged Fc-FLT3 chimera using HEK293T technology, together with the purification of the secreted fusion protein via Ni-NTA chromatography; and (ii) the FRIL-lectin extraction and purification from Dolichos lablab seeds. The recombinant physiological ligand, as well as the non-natural lectin ligand, was shown to be biologically active using OCI-AML3 and THP-1 cells-based in vitro proliferation assays. Finally, we report on the first results of the kinetic and thermodynamic characterization of the binding events FRIL to the chimeric Fc-FLT3-receptor variant using surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) technology.

Comparative study on heme degradation Alginate is a linear unbranched biopolymer that in presence of certain metals yields the ionotropic cross-linked microspheres. Thus, in the present study, sodium-alginate was added into different ionic salt (ZnCl 2 , CaCl 2 and CuCl 2 ). In seeking for augmenting enzyme stability, we entrapped the enzyme, alkaline phosphatase as a model, inside alginate microspheres and determined its activity against several physicochemical parameters including various temperatures, pH, microsphere size and different types of metals. Unique to our study, we added the enzyme into the sodium-alginate gel directly (the additive form) and investigated its stability within this medium against the same parameters stated above. According to the results, this method proved to preserve enzyme activity with a higher extent of efficacy. Enzyme activity showed to be inversely proportional to the microsphere size. Among different metals used, Zn 2+ preserved enzyme activity to a higher degree (47% and 29% of the native enzyme activity) against pH of 4 and temperature of 65°C respectively as compared with Ca 2+ (28% and 26%) and Cu 2+ (24% and 17%) while the native enzyme lost its activity entirely. In the same conditions, the additive form preserved enzyme activity more efficiently (65% and 89%). Protecting the enzyme in the additive form when compared with one entrapped in microspheres thus offers an easier, faster, cheaper and above all more efficient method toward restoring enzyme stability. Introduction: CK2 is an antiapoptotic protein kinase, which regulates different survival pathways. One of these is rapresented by Akt (PKB), a kinase whose activation requires the phosphorylation of two key residues: Thr308 and Ser473. We have previously found that CK2 enhances Akt activity by directly phosphorylating Ser129; here we show that it also positively regulates the phosphorylation state of Thr308, which is not a direct target of CK2. Methods: To assess if CK2 modulates the phosphorylation of Akt Thr308 by facilitating the accessibility to Thr308, we measured the in vitro activity of the Thr308 kinase PDK1 toward different forms of Akt, previously phosphorylated or not by CK2 at Ser129. To check if phosphorylation or dephosphorylation processes are important in the control of Thr308 operated by CK2 in vivo, we transfected cells with wt or Ser129Ala Akt, and analyzed the Thr308 phosphorylation under cellular conditions where PDK1 is activated or phosphatase activity is blocked. The formation of the protective complex Akt/Hsp90 under different conditions of CK2-dependent Akt phosphorylation was also analyzed.

Results: We found that PDK1 does not discriminate between Akt phosphorylated or not at the CK2 site; on the contrary, Ser129 phosphorylation promotes a lower degree of Thr308 dephosphorylation. We also found that Hsp90, involved in protecting phospho-Thr308, is less tightly associated to Akt if Ser129 is mutated to alanine.

Conclusions: The upregulation of Thr308 phosphorylation by CK2 is mediated, at least in part, by protection against dephosphorylation, possibly involving other protein partners, such as the Hsp90 complex.

Characterization of a small GTP binding protein Gtr1, and its interaction with the high-affinity phosphate transporter/sensor Pho84 The Na + -coupled, high-affinity Pho89 plasma membrane phosphate transporter in Saccharomyces cerevisiae has so far been difficult to study because of its low activity and unusual properties. In this study we have used a pho84D pho87D pho90D pho91D quadruple deletion strain of Saccharomyces cerevisiae devoid of all inorganic phosphate transporter genes except for PHO89 to functionally characterize Pho89 under conditions where its expression is hyper stimulated. Indeed, under these conditions the Pho89 protein is strongly up-regulated and is the sole high-capacity phosphate transporter sustaining cellular acquisition of inorganic phosphate. In this quadruple deletion strain PHO-pathway regulation is lost. Even if Pho89 is synthesized in cells grown at pH 4.5-8.0 the transporter is functionally active at alkaline conditions only, with a Km value reflecting the high-affinity properties of the transporter and with a transport rate about 100-fold higher than that of the protein in a wild-type strain. Even at these hyper-expressive conditions the Pho89 is unable to sense and signal extracellular phosphate levels across the plasma membrane. In cells grown at pH 8.0 Pho89-mediated phosphate uptake at alkaline pH is cation-dependent with a strong activation by Na + ions and sensitivity to the protonophore CCCP (carbonyl cyanide m-chlorophenylhydrazone). The contribution of H + -and Na + -coupled phosphate transport systems in wild-type cells grown at different pH values was quantified. The contribution of the Na + -coupled transport system to the total cellular phosphate uptake activity increases progressively with increasing pH. were produced and compared to each other. Kinase activities of these C-terminal truncation mutants increased gradually as C-terminal sequences were removed, and zDCLK(378-677) showed 20fold higher activity than zDCLK(378-810). However, further truncation of C-terminal sequence, as in case of zDCLK(378-652), resulted in complete loss of the kinase activity, probably due to deletion of the subdomain sequence essential for the catalytic activity. Furthermore, zDCLK became activated by autophosphorylation and by amino acid substitution of Thr576 in the activation loop to Asp that mimics phosphorylated kinase. These results suggest that zDCLK is negatively regulated by C-terminal domain and positively regulated by phosphorylation.

Biochemical and structural studies of fragile X-retardation related proteins We are studying fragile X mental retardation protein (FMRP) and its paralogue Fragile X related protein -1 (FXR1P), both involved in regulation of mRNA translation and in RNA interference pathway [1] ; FMRP binds also the non coding brain-specific RNA bc1 [2] . FMRP and homologs are multi-domain proteins containing Tudor and KH domains as well as RGG motifs. These three motifs are found in a variety of nucleic acids-binding proteins, and their co-presence in FMR related proteins states their relevance in RNA metabolism. The structures of isolated Tudor and KH domains of FMRP have been reported, but nothing is yet known about the spatial arrangement of the different modules. Methods and Results: We produced different recombinant fragments of human FMRP and FXR1P and characterized them by analytical ultracentrifugation and small angle X-ray scattering. This allowed us to understand the oligomerization properties of these proteins and determine their overall 3D shape. The 31-nucleotide-long RNA fragment of bc1 has been over-produced using the two rybozyme method and characterized by NMR.

Conclusions: We determined the overall shape of FMRP and FXR1P and showed that dimerization of FMR related proteins occurs via its KH2 domain. We are using this information to characterise the interactions between FMR related molecules with nucleic acids, using a 31nucleotide-long RNA fragment of bc1 and short AU-rich sequences as model systems. PP3-74 siRNA as a tool to study S100A6 function in human epithelial HEp-2 cells L. P. Slomnicki and W. Lesniak The Nencki Institute of Expreimental Biology, Warsaw, POLAND S100A6 (calcyclin) is an 11 kDa cell-specific calcium binding protein, a member of the S100 family. So far, there are more than twenty S100 proteins identified. They are widely distributed in human tissues and implicated in many regulatory processes. S100A6, a calcium sensor, is involved in cell cycle, exocytosis, cytoskeleton rearrangement and dif- ). The aim of our work is to elucidate the S100A6 role in cellular processes. For that we prepared human epithelial HEp-2 cells stably transfected with siRNA against S100A6. Such cells have dramatically reduced S100A6 protein content confirmed by Western blot analysis. Using MTT viability assay we showed that these cells reveal increased resistance to apoptotic stimuli (induced by hydrogen peroxide) in comparison to control cells. We investigated S100A6 interactions involving p53 which might explain why the lack of S100A6 increases viability of cells exposed to proapoptotic factors. For that we applied co-immunoprecipitation, affinity chromatography, Western blot analysis, luciferase assay etc. Our results suggest that S100A6 may be involved in apoptosis via its interaction with p53. To reveal specific metal-binding properties of sMT-3 we studied binding of Zn(II), Cd(II) and Cu(I) ions to sMT-3 in comparison with human MT-3 (hMT-3), which provided detailed information about metal-binding stoichiometry of sMT-3, composition of metal-thiolate clusters and their stability against pH. Methods: Electrospray ionization mass spectrometry was used for metal binding experiments and pH studies of MT-3 metalloforms. Bathocuproine disulfonate was used for comparision of Cu(I)-binding affinities of sMT-3 and hMT-3.

Results: sMT-3 has a reduced capacity and affinity to bind zinc, copper and cadmium ions as compared to hMT-3. pH studies demonstrate that there are two 4-copper clusters in Cu 8 sMT-3 and 4-and 6-copper clusters in Cu 10 hMT-3. sMT-3 has approximately 3 times lower affinity for Cu(I) ions as compared to hMT-3.

Conclusions: Reduced metal-binding ability of sMT-3 might have physiological outcomes in extreme conditions. Both high and low levels of copper in the sheep diet lead eventually to neurological outcomes, which might be exacerbate by the reduced ability of sMT-3 to bind this ion.

Structural analysis of Lia1 from S. cerevisiae: evidence for conformational changes induced by iron displacement from its active site

The unique amino acid hypusine [N e -(4-amino-2-hydroxybutyl)lysine] occurs exclusively in one major cellular protein, eIF5A, and is formed as a result of the successive action of two enzymes, deoxyhypusine synthase (human DHS, yeast Dys1) and deoxyhypusine hydroxylase (human DOHH, yeast Lia1). Although the first enzyme of the hypusine pathway has been extensively studied, the structure and mechanism of Lia1 remain to be characterized. Hence, combined techniques were used to evaluate Lia1 catalysis, metal/substrate binding and structural conformation. Like human DOHH, recombinant Lia1 is an iron metalloenzyme. Iron is essential for enzyme activity since its loss renders the enzyme totally inactive. The separation of iron-free and iron-bound forms by gel filtration and native gel electrophoresis suggests structural differences in Lia1 tertiary structure. The ability of Lia1 to undergo conformational changes prompted us to use a set of complementary spectroscopic approaches (CD and quenching of tryptophan fluorescence) and SAXS to obtain detailed information on the processes underlying dissociation of iron at different levels of the protein organization. The additive effect of weak interactions, especially the ones established within the metal center, resulted in an active enzyme in a stabilized and compact three-dimensional fold. Loss of tertiary contacts upon iron displacement led to an elongated conformation of Lia1, in which both N-and C-terminal domains are no longer in close proximity to guarantee the proper orientation of the active groups within the active site pocket. Thus, our results demonstrate an essential structural role for iron-binding in addition to its contribution to the catalytic reaction. SUMOylation is a dynamic process of post-translational modification of proteins. Similar to the ubiquitinylation, SUMO (small ubiquitin related modifyer) is covalently attached to lysine residues of its target proteins in a three step catalytic mechanism. This SUMOylation can be reversed by specific proteases. A large number of SUMO-targets have been discovered, which underlines the function of SUMO as regulator of various cellular processes, although its physiological consequences are not precisely understood. Under hypoxia Hypoxia inducible factor (HIF)-1a has been shown to undergo modification by the three SUMO isoforms SUMO-1, -2 and -3 [1] . SUMOylation occurs within and adjacent to the oxygen dependent degradation domain of HIF-1a whereby lysine 391 and lysine 477 have been determined as specific SUMO acceptor sites [2] . This attachment of SUMO seems to influence HIF-1a stability and its transcriptional activity. 

Allosteric ligand binding at G-protein coupled receptors V. Stepanov, A. Uustare and J. Ja¨rv Tartu University, Institute of Chemistry, Jakobi, ESTONIA

In this work evidence for non-exclusive binding of agonist and antagonist with muscarinic acetylcholine receptor (mAChR m2) was obtained though kinetic analysis. Receptors expressed in Sf9 cells without coexpression of G-proteins were used. In this analysis classical tritiumlabeled mAChR m2 antagonist [ 3 H]NMS (K D = 0.43 ± 0.09 nM) was employed as the reporter ligand and the binding process was described by a two-step reaction scheme involving a fast reversible binding step with K A = 6.2 ± 1.7 nM, followed by slow isomerization step, with on-rate constant k 2 = 0.23 ± 0.03 per second and off-rate constant k -2 = 0.0076 ± 0.00074 per second. We have found that classical muscarinic agonist carbamylcholine, binding process of which with mAChR was described as fast equilibrium process, had no significant influence on mAChR m2 receptor affinity for [ 3 Two topologically distinct binding sites should exist on the receptor for this non-exclusive binding mode of the agonist and antagonist molecules to occur. While both agonist and antagonist bind to the receptor, the binding of agonist to the appropriate site interferes through receptor ternary structure changes with receptor-antagonist complex formation without the involvement of the G-proteins.

Sol g II, an allergen from Thai red fire ant, Solenopsis geminata: characterization and antibody production Phosphagen kinases catalyze the reversible transfer of the high-energy phosphoryl group of ATP to naturally occurring guanidine compounds. Arginine kinase (AK) is the only phosphagen kinase that uses an amino acid, L-arginine, as a substrate and is distributed widely in invertebrates. We determined the cDNA-derived amino acid sequences of two arginine kinases (AK1 and AK2) from the annelid Sabellastarte indica, cloned the cDNA into pMAL plasmid and expressed them in E. coli. The recombinant Sabellastarte AK2 mainly showed stronger activity for both D-and L-arginine, a very unique substrate specificity not seen before in usual AKs. In this work, fifty independent mutations were introduced around the putative substrate-binding region of Sabellastarte AK2. The L64I and L64V mutants exhibited a significant increase in enzyme activity for both D-and L-arginine than the wildtype. On the other hand, L64A, L64S and L64P caused a remarkable decrease in enzyme activity. This indicates that the 64th residue is one of the key residues in enzyme catalysis. On the other hand, the Y89T and N320E mutants displayed significant change in the ratio of (kcat/ Km for D-Arg)/(kcat/Km for L-Arg). These results, together with the prediction of three-dimensional structures of Sabellastarte AK2, suggest that 89th and 320th residues are the candidates to distinguish L-or D-arginine.

Preliminary structural characterization of DEFL, a good candidate for anticancer research The Drosophila gene deflated and its protein product, DEFL, is conserved in multicellular eukaryotes. Although DEFL is genetically implicated in the regulation of cell proliferation making it a good candidate for anticancer research, its precise function and structure are currently unknown. In this study, we first performed a bioinformatics analysis on the DEFL amino acid sequence including a combination of disorder/order and 3D-structure predictions. Our findings suggest that at least the N-terminal part of DEFL contains sequence repeats reminiscent of repeats of the HEAT-family that mediate protein-protein interactions. Their presence indicates that DEFL likely acts in a complex with other proteins and suggests a mainly a-helical structure for at least the N-terminal region. In parallel, we performed over-expression experiments of DEFL in E.coli aiming at production and purification of the protein for experimental structural analysis. The full-length (3003 bp) and the N-terminal (990 bp) coding regions of defl from D. melonogaster were cloned into the expression vector pET15b for this purpose. Both the full-length protein and the N-terminal fragment were successfully over-expressed as 6xHis-tagged proteins in BL21 plysS and BL21 cells, respectively. Expression was achieved only by tight control of cell growth combined with good aeration of the culture. Both overexpressed proteins were localized exclusively to insoluble inclusion bodies and their complete solubilization was achieved by addition of the detergent sarcosyl. The purified proteins were subsequently studied by CD spectroscopy. The far-UV CD spectrum of the N-terminal fragment showed a relatively high a-helical content in accord with our structure predictions. Introduction: Two electrophoretic isoforms of a histone H1.c subtype, H1c1 and H1c2, were identified within Guinea fowl population. These proteins form three phenotypes, c1, c2, c1c2, with two alleles c 1 and c 2 at a locus. To identify a region of H1c molecule responsible for differences between the allelic isoforms, the H1c bands from individuals with different phenotypes were cut out of the gel and treated with agents that specifically cleave peptide bonds.

Methods: The products of limited proteolysis of H1.c isoforms with N-bromosuccinimide (NBS), a-chymotrypsin or protease V8 from Staphylococcus aureus were resolved in a 13.5% polyacrylamide gel containing sodium dodecylsulphate.

Results: The NBS cleaved a peptide bond at Tyr72 moiety in the histone H1c. Only a C-terminal peptide could be detected in the polyacrylamide gel, while an N-teminal one was not visible following staining with Coomassie Blue because of its smaller molecular mass. The C-terminal peptides from the two H1c allelic isoforms differed in their electrophoretic mobilities. Similar differences in the C-peptide migrations were observed after treatment the H1c isoforms with a-chymotrypsin (which predominantly cleaved a peptide bond at Phe106) and protease V8 (which cleaved among others the peptide bond at Glu114).

Conclusion: The expected structural differences between histone H1c allelic isoforms from Guinea fowl erythrocytes seem to be located in the C-terminal part, between amino acid residue 73 and the very C-terminus.

Structural and functional characterization of aminoacylhistidine dipeptidase from Vibrio alginolyticus, a metallopeptidase with dinuclear metal center Proteins of the aminoacylase-1/metallopeptidase 20 (Acyl/M20) families were characterized to contain a mono or dinuclear zinc-binding domain at their active site. Aminoacylhistidine dipeptidase (EC 3.4.13.3, also Xaa-His dipeptidase, carnosinase, and PepD) catalyzes the cleavage and release of an N-terminal amino acid, usually neutral or hydrophobic residue, from Xaa-His dipeptide or degraded peptide fragments for amino acid utilization. We have cloned, over-expressed, and purified the wild-type protein and characterized its biochemical properties including substrate specificity, pH and temperature optima, and effects of metal ions substitution. Multiple sequences alignment, homology modeling, site-directed mutagenesis experiments, and preliminary X-ray structure determination identified putative active site residues involved in metal binding and catalysis. The functional role of these residues on enzyme catalysis and kinetics will be discussed.

A study of the energies produced on the binding of metals to iron-transport protein, transferrin, using density functional theory Introduction: Ability of mammals to distinguish bitter taste is believed to be evolved from the need to detect poisonous substances. It is logic to believe that poisonous substances interfere with the normal biological functions in mammals. Searching for bitter herbs as therapeutics has long been used in traditional medicines. In this study, we classified most drugs according to their pharmacological effects and taste, constructed the structural models of bitter taste receptors T2R5, T2R14, T2R16, T2R43 and T2R61, and identified the structural basis between bitter taste and pharmacological properties using H1 antihistaminics as an example. Methods: Therapeutic agents were grouped according to their pharmacological effect and taste. Structural models of bitter taste receptors T2R5, T2R14, T2R16, T2R34 and T2R61 and histamine H1-receptor were constructed by homology modeling. Selected H1 antihistaminics were docked into the active sites of the receptors. The active site was compared between the bitter taste receptors and the histamine H1receptor. Introduction: Juvenile hormone (JH) is essential for multiple physiological processes. It controls larval development, metamorphosis and adult reproduction. In the Lepidopterian hemolymph, 99% of JH is bound to juvenile hormone binding protein (JHBP), which protects the JH molecule from nonspecific degradation and serves as a carrier supplying the hormone to the target tissues. In G. mellonella JHBP is produced in the fat body cells. This molecule consists of 225 amino acid residues and is basic glycoprotein (pI 8.1). Until now it has not been studied whether JHBP molecules may have a membrane binding protein as a potential receptor. Methods: To test this hypothesis, fat body membrane proteins from G. mellonella larvae were isolated and tested for JHBP binding activity using surface plasmon resonance (SPR).

Results: An interaction of JHBP with fat body membrane proteins has been demonstrated. Direct capture of proteins, from tissue extracts on SPR chip surfaces followed by tandem mass spectrometry, permitted identification of ATP synthase as a JHBP binding partner. It has also been shown that this binding is inhibited by JH. In experi-ments performed with bovine F1 ATP synthase subunit, a direct binding of ATP synthase with JHBP provided a dose dependent response, consistent with a specific interaction with JHBP. The dissociation constant (K D ) revealed high binding affinity of F1 ATP synthase to JHBP with K D = 0.86 · 10 -9 M. Conclusions: The above results indicate that ATP synthase may take part in the regulation of JH signaling via JHBP.

Probing the interaction of silver nanoparticle and hemoglobin at different temperature Methods: Tissue samples were obtained from patients subjected to surgical operation due to colorectal carcinoma. Sera were obtained from patients preoperatively and postoperatively during a 6-month follow-up. Hyals were detected using HA-zymography and HASs expression was detected by RT-PCR analysis.

Results: Increased levels of Hyals were observed in cancerous samples compared with the macroscopically normal ones, in all anatomic sites examined. Hyals levels in sera were decreased perceptibly 7 days postoperatively, while 1, 3 and 6 months afterwards gradually increased to reach the amount preoperatively. HASs expression was increased in cancerous samples compared to macroscopically normal ones, being related to cancer stage. Proteases comprise the second largest group of enzymes encoded by mammalian genomes. Thus, at least 569 proteolytic enzymes have been catalogued in the human degradome. This number reflects the fundamental roles of proteases in normal and pathological processes, including cancer. In this regard, proteases facilitate the spreading of tumor cells and formation of distant metastasis through degradation of extracellular matrix components. This fact has made that these enzymes have long been associated with cancer progression. However, recent studies have revealed that different proteases can also act as tumor-protective enzymes (1) . Most of these enzymes are metalloproteases and five of them, ADAMTS-1, -8, -9, -15 and -18 belong to the ADAMTS family. We have recently reported that ADAMTS-12 also exhibits antitumoral properties in a cell-based model through modulation of Rasdependent ERK signalling pathway (2) . Now, we have found that this gene is epigenetically silenced in several types of cancer, including colon carcinomas, suggesting that this mechanism can be used to abrogate the tumor-suppressive actions of this enzyme. Likewise, we also show that ADAMTS-15 confers antitumoral properties to colon cancer cells expressing this metalloprotease, but its inactivation in the tumors occurs by mutation and not for epigenetic silencing. These findings contribute to expand the functional complexity of proteases in tumor processes.

( Nitric oxide (NO) plays a critical role in the endothelial homeostasis. It is involved in the regulation of arterial blood pressure and also shows antiatherogenic properties. Oxidative stress has known repercussions on bioavailability of NO which, among other actions, is able to induce ecSOD expression. Therefore, antioxidant substances capable of counteracting NO inactivation may improve endothelial dysfunction through an increase in ecSOD activity. This randomized controlled study was designed. to determine whether oral CoQ10 supplementation (100 and 200 mg t.i.d.) was able to improve ecSOD activity and endothelium-dependent vasodilation in patients with coronary artery disease (CAD). Twenty CAD patients (31 M/4 F, mean age 55 + 4 years, ejection fraction 57.5 + 8%) were randomized into two groups. A group (n = 10) received CoQ10 orally at doses of 300 mg/day for 1 month, while remaining 10 patients received 600 mg/day. On entry and at 4 weeks, all patients underwent brachial artery endothelium dependent vasorelaxation assessment (FMD) and measurement of endothelium-bound ecSOD activity. CoQ10 supplementation resulted in a dose dependent increase at plasma and cellular levels as determined by platelet CoQ10 quantification. Also ec-SOD and FMD increased unpon CoQ10 treatment and remarkably A correlation was found between increases in ecSOD and percent change in brachial artery diameter (r = 0.77, P < 0.0001). Both effects could be related to the antioxidant properties of CoQ10, which could protect nitric oxide from oxidative inactivation. However, a dose-dependent response could be implicated. In fact, both effects were obtained by raising plasma CoQ10 levels well above 2.5 lg/ml, which might be considered a threshold for obtaining cardiovascular benefits.

PP4A-5 Role of gelatinases (MMP-2 and MMP-9), vascular endothelial growth factor and endostatin on the clinicopathological behavior of colon and rectal cancer Introduction: Tumor metastasis and progression, which depend on angiogenic process, include the proteolytic degradation of the extracellular matrix (ECM). Matrix metalloproteinases (MMPs) are a family of ECM degrading proteinases and MMP-2 and MMP-9 are known as 'gelatinases'. We focused on the levels of MMP-2, MMP-9, TIMP-2, MT-1 MMP, vascular endothelial growth factor (VEGF) and endostatin between tumor areas compared with normal tissue specimens and investigated potential relationships between these parameters and clinicopathological variables in colon and rectal cancers. Materials and Methods: Tumor and normal tissue specimens were obtained from colon (n = 50) and rectal carcinoma (n = 34). MMP-2 and MMP-9 activity levels were examined using gelatin zymography. MT-1 MMP activity level was analysed by activity assay. TIMP-2, VEGF and endostatin levels were examined using ELISA.

Results: Significant relationships were found between colon tumor active MMP-9 and tumor invasion depth 'pT1-2 vs pT3-4' (p = 0.011) and distant metastasis. (p = 0.047). In rectal tumors, active MMP-9 level showed statistically significant relationships with metastatic disease during the diagnosis and perineural invasion (p = 0.002 and p = 0.042, respectively). Also we found statistically significant relationship between tumor proMMP-9, proMMP-2 activity levels and lymph node metastasis (p = 0.012, p = 0.021, respectively). Tumor TIMP-2 levels showed a significant relationship with recurrence (p = 0.011). The significant relation was found between tumor VEGF level and perineural invasion (p = 0.044). Also there was a statistically significant correlation between tumor VEGF and tumor size (p = 0.009 r = 0.454). Hyaluronan (HA) influences cell behavior in different ways. Many of these effects depend on HA size modification and endocytosis. However, the factors controlling endocytosis of HA remain poorly clarified. We hypothesized that the hyaluronidase Hyal2 may be one of the HA regulatory factors. Experiments were performed in the rat transformed fibroblastic cell line BB16. As the rate of endogenous expression of Hyal2 in these cell lines is low, stable transfectants of Hyal2 (BB16Hy2+) were established and compared with mock-transfected cells (BB16mock). Hyal2 overexpression markedly reduced the strong HA endocytosis displayed by BB16 cells. A portion of exogenous HA was taken up in a receptor dependent manner but the largest part was linked to a macropinocytic process. Therefore, the effects of Hyal2 on various endocytic processes were explored using specific markers. Hyal2 did not diminish receptormediated endocytosis but markedly reduced fluid-phase endocytosis. In addition, observation of BB16Hy2+ cells through transmission electron microscopy showed reduced numbers of macropinosomes, suggesting a strong inhibition of macropinocytosis. Actin filaments are a main component of macropinosome formation; Hyal2 expression disrupted the bundles of actin filaments which are a hallmark of cell protrusions.

In conclusion, Hyal2 overexpression decreases macropinocytosis through a modification of actin architecture and thereby reduces HA endocytosis. Moreover, since the macropinocytic process is used by cancer cells and antigen-presenting cells, the mechanisms and fallout of Hyal2 influence on macropinocytosis in neoplastic and antigen-presenting cells will be examined. Introduction: The number of Kilis goats bred in Turkey is about 11 million which corresponds to 25% of the goats bred in the country. These goats have adopted to the climatic conditions of the Southeastern Anatolia and give 300-400 kg of milk at every lactation. Selenium (Se) has long been considered as a highly toxic and carcinogenic element. The Se in the atmosphere is taken by plants and animals and passes to humans through food chain. The Se need of animals is mainly supplied with feed. The Se atom shows sinergical impact in animals with vitamin E and at the same time adds to the structure of Glutatyon Peroksidaz enzyme which works as a strong antioxidant. In addition, type I Iodotyreonin Deiodinaz enzyme is defined as another enxyme containing Se. The Se deficiency causes stopped the growth and decrease in efficiency in animals it results in muscular dystrophy in offsprings. This study aims to investigate Se levels in serums of blood samples obtained from 12 healthy Kilis goats. Methods: The Se levels of the serums was determined by spectrofluorometric method.

Results: The Se levels of the serums was determined by spectrofluorometric method and found to be 53,55 +-7,9 ng/ml which suggest a lower Se level compared with other animal groups.

Conclusions: It is considered that the results of this study can be a reference to other domestic animals in this region as well as serves as a basis to other studies of Se levels of other goat groups. Introduction: Atherosclerosis has been associated with increased oxidative stress and monocyte recruitment by endothelial cells. Subendothelial basement membrane proteins, such as laminins that play a central role in cell adhesion, are exposed to reactive oxygen species. Methods: We used a myeloperoxidase assay to study monocyte attachment on human umbilical cord vein endothelial cells (HUVEC) that were attached to oxidized or native laminin, in the presence or absence of anti-alphaM and anti-beta2 integrin subunit antibodies. ICAM expression by HUVEC was estimated by an ELISA assay. HUVEC attachment to oxidized or native laminin was examined using the Hemacolor kit.

Results: HUVEC attached to oxidized laminin at a higher degree as compared to native laminin, while they expressed higher levels of ICAM after their attachment to oxidized laminin. Moreover, monocytes attached at a higher degree to HUVEC that were attached to oxidized laminin. Incubation of monocytes with the antibody against the aM and b2 integrin subunits equalized the previously observed differences.

Our results indicate a modified interaction between HUVEC and the basement membranes in cases where laminin is oxidatively modified, as well as increased monocyte recruitment capacity attributed to increased ICAM-1 expression by endothelial cells that are attached to oxidized laminin with the involvement of the aM and b2 integrin subunits. This observation may represent a novel mechanism for the initiation and progress of atherosclerotic lesions. Introduction: Biglycan is absent in the normal cornea, but UVR exposure leads to a significant expression of the biglycan gene in the rabbit cornea, an effect that decreases after healing is completed, indicating the envolvement of biglycan in the corneal repair process. In the present study, we have investigated possible involvement of biglycan in the modulation of the survival of keratocytes. Methods: Keratocytes were grown either under serum free conditions to obtain quiescent keratocyte cell culture or in the presence of 10% fetal bovine serum to induce keratocyte transition into myofibroblasts (activated keratocyte phenotype). Myofibroblastic phenotype was confirmed by immunocytochemistry with anti-alpha-smooth muscle actin antibodies. Cell death was induced in both cell cultures by interleukin-1 in the presence or absence of biglycan. Histone-associated DNA fragments were assayed by using a cell death detection ELISA.

Results: Quantification of histone-associated DNA fragments by the cell death detection ELISA showed that biglycan strongly protected quiescent keratocytes from dying whereas it enhanced the death rate of activated keratocytes. Apoptotic death rate was elevated after the addition of IL-1 in both keratocyte and myofibroblast cell cultures. Coincubation with biglycan markedly reduced the number of apoptotic keratocytes but markedly increased the number of apoptotic myofibroblasts.

Conclusions: IL-1-induces apoptosis of both quiescent and activated keratocytes. However, biglycan has differential effect on apoptosis of these two cell types.

Difference in neutrophil dynamics induced by a novel superstructure of collagen Background: Recently, it was shown immune cells to interact with type I collagen using leukocyte-associated Ig-like receptor-1 (LAIR-1). Neutrophil, which is one of those cells, transmigrates through blood vessel wall and migrates into an extracellular matrix toward an inflammatory site, as a vital first immune response. Rho family protein generally plays a crucial role in regulating the cell motility and function. However, the intracellular signaling pathway of neutrophil induced by the collagen in the extracellular matrix remains unclear. Until now, we have shown that type I collagen hydrolyzed by actinidain (AP-collagen) changes the conformation by itself and the motility of neutrophil. In this study, we investigated expression of actin, Cdc42, RhoA and Rac1 in neutrophil cultured on AP-collagen assembly. Methods: We prepared pepsin-hydrolyzed type I collagen (atelocollagen) and AP-collagen from chicken skin. Neutrophil was collected from a mouse and subsequently it was cultured on a plate coated with the atelocollagen or the AP-collagen. To investigate neutrophil dynamics on the collagen assembly, we observed F-actin labeled with MFP-555-Phalloidin by using a fluorescence microscopy. To find further evidence of the actin activation, Rho family proteins were visualized by staining with specific antibodies. Results: The fluorescence observations revealed that the expression of Rho family proteins of neutrophil on the AP-collagen and the atelocollagen assembly is clearly different. Therefore, neutrophil would recognize the conformational difference between the AP-collagen and the atelocollagen as a scaffold. In conclusion, we found that the AP-collagen alters intracellular signaling pathways of neutrophil and induces its specific function. The expression of the abnormal protein Bcr-Abl is the primary cause of the chronic myelogenous leukemia (CML). Its kinase activity confers proliferative and survival advantage to CML cells. The cells containing Bcr-Abl also display altered adhesion properties. We studied the effects of Bcr-Abl inhibition on the adhesivity of CMLderived cell lines (JURL-MK1, K562) to fibronectin and on the activity of Rho/ROCK/LIM signaling pathway. This pathway links the integrins, the membrane mediators of cell-matrix adhesion, to the proteins which regulate the cytoskeleton structure. Using a quantitative and sensitive assay, we showed that Imatinib mesylate, a specific inhibitor of Bcr-Abl, induces a large decrease in the extent of cell adhesion to fibronectin-coated surfaces. Similar effect was observed after treatment with 17-AAG, an inhibitor of the chaperone HSP90 which is known to stabilize Bcr-Abl among others. Introduction: Testicular germ cell tumor is the most frequent solid tumor among young men and in most cases it can be cured. The scientific interest has been focused on the factors which contribute to the development and prognosis of the disease, such as proteolytic enzymes.

Metalloproteinase-2 and -9 belong in this category and are implicated in the degradation of extracellular matrix and consequently in cancer cell spread and metastasis. The aim of this study was to examine the presence of MMP-2 and -9 in testicular tumors. Methods: Samples from patients and healthy testes from biopsies were used. The gelatinolytic activity of MMP-2/-9 was found by gelatin zymography and the results were certified through Western blot analysis. Immunohistochemistry for MMP-2/-9 was also performed in paraffin embedded tissues.

Results: Gelatin zymography revealed that increased levels of proMMP-2/-9 and active MMP-2/-9 existed at all cancer samples compared with normal subjects. The latest were expressing only the proMMP-2/-9. The results were confirmed by Western blot analysis. Immunohistochemistry revealed that MMP-2 and MMP-9 are expressed in normal epithelial cells and secreted to the extracellular matrix in low amounts in normal testes. Increased expression of both MMPs was observed in testicular cancer. MMPs are located intracellularly in cancer cells and are also deposited in the tumor stroma.

Conclusions: MMP-2 and MMP-9 are overexpressed in both inactive and active form in testicular cancer and may contribute in the metastatic potential of the cells.

Actin-sequestering protein, thymosin beta-4 (TB4)mediated hypoxia-inducible transcription factor (HIF)-1 a stabilization in HeLa cervical tumor cells Thymosin beta-4 (TB4) is an actin-sequestering protein to control cytoskeletal reorganization. Here, we investigated whether TB4 proteins (TB4P) affect tumor microenvironment by measuring reactive oxygen species (ROS) production and hypoxia-inducible transcription factor (HIF)-1a stabilization in cervical tumor cells, since TB4P reduced paclitaxel-induced cell death rate. TB4P increased HIF-1a stabilization and transactivation, which is measured by the increase of hypoxia response element (HRE)-luciferase activity and target gene, vascular endothelial growth factor (VEGF) transcription. TB4P also elevated ROS production and ERK phosphorylation. H2O2 treatment elevated HIF-1a stabilization and ERK phosphorylation. PD98059, ERK inhibitor reduced HIF-1a increased by TB4P. Paclitaxel-induced cell death was inhibited by hypoxia conditioning that increased HIF-1a stabilization, ERK phosphorylation and ROS production. PD98059 and Nacetylcysteine (NAC) reversed paclitaxel-induced cell death which was attenuated by hypoxia. Collectively, TB4P could lead tumor cell microenvironment to hypoxia condition, which might be resulted in antitumor drug-resistance induction. It suggests that soluble TB4P could be a novel target to control tumor cell death by regulating tumor cell microenvironment.

Functional genomic approaches to uncover novel essential genes for muscle development and maintenance in Drosophila In the post-genomic era determining the function of novel proteins is becoming increasingly important to understand at the molecular level, essential processes such as tissue morphogenesis and homeostasis in the living organism. Our specific interests focused on the discovery of novel genes essential for the maintenance of muscle function in Drosophila. We took advantage of the availability of a large set of genome scale data in various model organisms including Drosophila to integrate the large amount of information in a common format and perform multiple queries regarding gene expression, protein-protein interactions, domain composition and functional requirements of fly genes in cell culture models. By applying such criteria we compiled a list of candidate 'myogenic' genes of unknown function in the fly.

We have already tested approximately 400 genes and identified several that are essential for the proper muscle function. Currently, we focus on a subset of the identified genes and examine their putative functions in additional morphogenetic processes including wing development.

The molecular basis of human Fraser syndrome Lumican -the small leucine-rich proteoglycan -has attracted attention as the molecule of extracellular matrix possibly involved in signaling pathways affecting cancer cells behavior. Its expression was reported in many types of cancer cells and its effect on tumor growth has been suggested.

Remodeling of actin cytoskeleton, induced in the response to the external stimuli is crucial for cell motility and intracellular signal transduction. The changes in actin level and cytoskeleton organization are known to accompany malignant transformation of many cell types.

We have focused on the effect of lumican on the state of actin polymerization, cytoplasmic beta and gamma isoforms expression and their subcellular distribution in human melanoma cell line A375. Statistically significant increase in monomeric actin pool was observed in A375 cells growing on lumican to distinguish from the control cells growing on glass. Western blotting analysis of the same samples showed the increase in gamma actin level in the presence of lumican. Microscopic observations of actin isoforms distribution in A375 cells revealed that monomeric beta actin was present at the cell periphery, whereas gamma actin was mostly localized in the stress fibers and thick filaments. In the presence of lumican both isoforms concentrate under cellular membrane in polymerized form.

In conclusion, we have shown that lumican induces the increase in actin level and the changes in isoforms distribution in A375 cells. These results suggest that lumican may affect melanoma cells motility by decreasing the ability to stress fibers formation and the state of actin polymerization. Introduction: Thrombospondin-1 (TSP-1) is a member of a family of five structurally related extracellular glycoproteins that plays a major role in cell-matrix and cell-cell interactions. Due to its multifunctional nature and its ability to bind to a panoply of cell surface receptors and matrix proteins, TSP-1 has been identified as a regulator of angiogenesis and tumor progression. However, the overall involvement of TSP-1 remains complex and controversial as both stimulatory and inhibitory effects in tumorigenesis have been reported. Here, we addressed the regulation of TSP-1 by growth factors such as EGF and HGF/SF and analysed its effects on human follicular (FTC-133) and papillary (TPC-1) thyroid cancer cells invasion. Results: We found that both EGF and HGF/SF increased the invasive potential of respectively FTC-133 and TPC-1 carcinoma cells. In the former cell type, EGF up-regulated TSP-1 expression in a MAPK/ ERK and PI3-kinase-dependent manner whereas in the latter cells, HGF/SF repressed TSP-1 expression through a pathway that involved activating transcription factor-1 (ATF-1). In addition, using purified protein and neutralizing antibodies, we proved that TSP-1 was, at least in part, responsible for the regulation of cell invasion but with opposing outcomes : stimulatory in follicular and inhibitory in papillary cancer cells.

Conclusions: Thrombospondin-1 can have opposite effects on cell invasion depending upon the type of differentiated thyroid carcinoma studied. These paradoxical results suggest that further investigations are necessary to understand the role played by TSP-1 in the regulation of tumor growth and metastasis in the thyroid.

Expression of MMP-2 and MMP-9 in patients with multiple myeloma Methods: MMP-2 and -9 were analyzed by substrate gel electrophoresis and Western blotting using specific monoclonal antibodies.

Results: The proforms of gelatinases were identified in the sera of most patients, but their presence was depended to the stage of the cancer. Late staged samples contained also the active forms. None differences were observed in sera obtained from the same patient at different period postoperatively. Significantly decreased amounts of both proMMP-2 and -9 were observed in sera of patients subjected to radiotherapy.

Conclusions: Gelatinases are proposed as a useful biochemical marker during cancer. The results presented here suggested that their amounts were affected in the sera of patients by the stage of cancer and radiotherapy. These observations correlated with previous findings indicating that, during laryngeal carcinoma, gelatinases in the tissue were increased in a stage related order, but substantially decreased after radiotherapy of the patients. Research question: What is the structural background of the thromboresistance of the media layer of arteries and which physiological factors modify it? Methods: Cryosections from human renal artery were perfused with citrate anticoagulated whole blood in a parallel-plate flow chamber and adhered platelets were detected by indirect immunfluorescence microscopy. Collagen fibers were detected with Masson's Trichrome (MT) staining. The structure of the vascular wall was examined with atomic force microscopy (AFM).

Results: The adhesive properties of the arterial tissues were evaluated following treatment with proteases relevant to thrombi (plasmin, thrombin) or human neutrophil granulocytes activated by formylmethionylleucylphenylalanine as a trigger of serine protease and matrix metalloproteinase release. Preincubation led to an average ten-fold increase in platelet adhesion to the media over the control level. The treatments mentioned above exposed collagen fibrils for staining with MT in the media, while in non-treated sections only the adventitia was MT positive. AFM also indicated that pretreatment with proteases removed a thin layer of extracellular matrix components, probably proteoglycans from the surface of the collagen fibers revealing their characteristic striped morphology. Conclusion: Our results suggest that neutrophil-derived and hemostatic enzymes play a role in altering the thrombogenic property of the vessel wall, which could be related to the different proteoglycan distribution in the media and adventitia.

Integrin signaling modulates cell sensitivity to apoptosis Integrin signaling controls cell physiology by specific pathways, and by cross-talking with other receptor signaling. We report by this study evidences that integrins act in modulating cell sensitivity to apoptosis induced by either serum starvation or staurosporin. Various cells (HS27, HeLa, Hep2, and DOK) were plated on collagen or fibronectin. After 2 h for adherence and spreading, apoptosis was induced by either serum starvation or staurosporin treatment. Cells were quantified for apoptosis 24 h later. In control experiments, cells plated in similar conditions on matrix protein coated surfaces, were counted for apoptosis after 24 h maintenance in medium supplemented with serum, and without staurosporin treatment. Results showed that the cells plated on collagen proved a higher sensitivity to apoptosis induced by either serum starvation or staurosporin as compared with the cells plated on fibronectin. A differential behavior was noted for various cells as a function of matrix protein used for plating. On collagen HS27 cells showed the highest sensitivity to apoptosis, while on fibronectin Hep2 cells were mostly affected. On the other hand, the sensitivity to apoptosis induced by serum starvation was lower for the cells plated on fibronectin, as compared with cells plated on collagen. Therefore, our results suggested an ability of integrins to modulate cell sensitivity to apoptosis either induced by serum starvation or staurosporin. Abstract: Carbohydrate moieties of salivary mucins play various roles in life processes, especially as a microbial trapping agent. While structural details of the salivary O-glycans from several mammalian sources are well studied, very few information is currently available for the corresponding N-glycans. The existence of N-glycans alongside the O-glycans on mucin isolated from rat sublingual gland has previously been implicated by total glycosyl compositional analysis but the respective structural data are both lacking. The advent of facile glycomic mapping and sequencing methods by mass spectrometry (MS) has enabled a structural reinvestigation into many previously unsolved issues. For the first time, high energy collision induced dissociation (CID) MALDI-MS/MS as implemented on a TOF/TOF instrument was applied to permethyl derivatives of mucin type O-glycans and N-glycans, from which the linkage specific fragmentation pattern could be established. The predominant O-glycans carried on the rat sublingual mucin were defined as sialylated core three and four types whereas the N-glycans were determined to be non-bisected hybrid types similarly carrying a sialylated type II chain. The masking effect of terminal sialylation on the tight binding of rat sublingual mucin to Galb1 fi 4Glc-NAc specific lectins and three oligomannose specific lectins were critically demonstrated in this study. In order to elucidate the immunological role of this novel protein found in crustaceans we studied by light and confocal microscopy the lectin hemocyte recognition; by spectrophotometric NBT reduction assay using elicitors and inhibitors we identified the effect of LsL on the modulation of oxidative burst mechanism on its own hemocytes. Usign western blot analyses we identified the glycoprotein attached to membrane that binds lectin on adherent cells. LsL binds 4% of total fixed circulant hemocytes (non-activated), but is able recognize 85% of adhered granular hemocytes showing filipodia and pseudopodia (activated), inducing the reactive oxygen intermediates (ROIs) production on the recognized cells. This effect was dose and time dependent (optimal: 100 ng/1 · 10 5 cells/20 minutes), is specifically inhibited by N-acetylated sugars residues (NeuAc, GalNAc or GlcNAc), superoxide dismutase ( Conclusions: These results further support the hypothesis that circulating GAGs may function as immunomodulators and its increase in release and degradation could be a biological response to modulate inflammation during disease.

Involvement of circulating hyaluronic acid in osteoclastic bone resorption of breast cancer metastatic bone disease Introduction: Syndecan-1, which is a transmembrane heparan sulfate proteoglycan (HSPG), is upregulated in breast cancer and related to poor prognosis. It may promote tumorigenesis by regulating tumor cell spreading, adhesion, proliferation and angiogenesis. Glypican-1, is a HSPG attached to the plasma membrane via GPI anchor, it is overexpressed in human breast cancer and may act to enhance the growth promoting effects of heparin-binding growth factors in breast cancer.

Zoledronate is a bisphosphonate that effectively represses osteoclastic activity by inhibiting the prenylation of small GTPases. The aim of this study was to evaluate the effect of zoledronate, on cancer cell proliferation, on the expression of syndecan-1 and glypican-1, and therefore assess its anti-tumor activity. Results: We observed a statistically significant increase of HEX activity in GDM serum compared to control groups of pregnant (p = 0.000046) and non-pregnant (p = 0.000000) healthy women and a statistically significant decrease after 3 months after GDM (p = 0.000000). A statistically significant increase of HEX activity was also found in GDM placenta tissue (p = 0.00547) in comparison to control group.

Conclusions: Our preliminary study might suggest potential role of HEX in GDM etiopathogenesis. It may be suggested that GDM causes increase in degradation of glycoconjugates.

Role of sugars on lipoprotein (a) in its interaction with endothelial cells Our hypothesis is that sugar on Lp(a) may play important role in its interaction with endothelial cell (EC) surface.

Methods: Lp(a) and Apo(a) were isolated from blood of consented donor by differential centrifugation and reduction/precipitation. The purified lipoproteins were treated with trifluoroacetic acid and analyzed on capillary high performance liquid chromatography. Enzyme-linked immunosorbent assay was used for lectin binding experiment to analyze surface sugar moiety, Lp(a) binding to EC and sugar competition assays. Introduction: Hyaluronic Acid (HA) is the only non-sulphated glycosamminoglycan (GAG) of extracellular matrix involved in inflammation, angiogenesis and tumorigenesis. HA is synthesized by HA synthases that polymerize HA using UDP-glucuronic acid and UDP-N-Acetyl glucosamine.

Aim: As HA synthesis is finely regulated, we set up a non-radioactive assay to quantify HAS activity on membrane inside the cells.

We performed experiments on primary endothelial cells (EC), HUVEC (Human Umbilical Vein EC) and HAEC (Human Aortic EC), and on immortalized cell line with endothelial origin, ECV304, that produced HA. The cells were ruptured; nuclei, cytoplasm and plasma membrane (PM) were isolated and their purity was checked by western blot. Each fraction was incubated at 37°C with non-radiolabelled UDP-sugars, digested by hyaluronidase-chondroitinase and derivatised by 2-aminoacridone. Results: Although HUVEC and HAEC did not synthesise HA, their PM and cytoplasm were able to synthesize HA after precursor incubation. PAGEFS and HPLC allowed the quantification of new synthesised HA without contaminations of HA attached to membrane in ECV304 and other GAGs. This method assed HAS activity evaluating effects of IL-1b, PolyI:C, PDGF-BB, PMA and 4-methylumbelliferon. We pointed out that phosphorylation increased HAS activity in EC. Moreover, cellular stress induced by tunicamycin increased HA synthesis in each fraction. Furthermore, HUVEC and HAEC induced HA synthesis to capture cells in blood, such as monocytes or platelets.

The described assay is a sensitive non radioactive method to asses HAS activity. Phosphorylation is the main regulator of HA synthesis. New synthesised HA chains capture monocytes by CD44-interaction with relevance for inflammation.

An adaptive role of the Ashwell receptor in mitigating the lethal coagulopathy of sepsis Introduction: The Ashwell receptor is the major lectin of hepatocytes and rapidly clears from blood circulation glycoproteins bearing glycan ligands that include galactose and N-acetylgalactosamine. This asialoglycoprotein receptor (ASGPR) activity is highly conserved throughout vertebrate phylogeny and remains a significant factor in the preparation and delivery of pharmaceuticals, yet a biological purpose of the Ashwell receptor has remained elusive. Results: We have identified endogenous ligands of the Ashwell receptor as glycoproteins and regulatory components in blood coagulation and thrombosis that include von Willebrand factor (VWF) and platelets. Properties of the Ashwell receptor including its post-natal onset of expression, high capacity, and rapid kinetics of clearance are consistent with anticipation of a sudden widespread alteration of glycoprotein structure within the circulatory system, perhaps following infection by pathogens encoding neuraminidase activity. We show that the thrombocytopenia associated with Streptococcus pneumoniae (SPN) infection, which has been attributed to the consumptive coagulopathy of sepsis, is in fact due to the host's Ashwell receptor activity. The NanA sialidase of SPN is responsible for de-sialylating pro-thrombotic components during sepsis, resulting in the formation of asialo-platelets and asialo-VWF that are rapidly cleared from circulation by the Ashwell receptor. 

Role of hyaluronan-CD44 complexes in regulation of growth factor receptor activity and in tumor progression Glycoproteomics as special branch of proteomics focuses on comprehensive study of glycoproteins and use lectin-affinity chromatography and several proteomic techniques. Glycoproteomic approach is very perspectives to search in blood serum of secreted glycoproteins as potential biomarkers of different diseases, in particular of prostate cancer (PCa). The serum level of PSA is related to benign prostatic hyperplasia (BPH) and poorly correlated with grade and stage of PCa. The aim of this research is comparative study of glycoprotein profiles in blood serum from patients with PCa and BPH. Specimens of blood serum from patients with PCa (Gleason grade 6-7, n = 33) and BPH (n = 12) were analyzed. Glycoproteins were collected on Con A Sepharose 4B, separated by 2D gel electrophoresis, stained using Coomassie Brilliant Blue R-250 and silver. Some proteins were identified by peptide mass fingerprinting based on MALDI-TOF-mass spectrometry. After affinity chromatography 52 proteins revealed on 2DE, and three of them (Mm/pI -30.0/4.8, 17.0/5.5, 14.0/6.7) were overexpressed in main part of PCa specimens, and practically undetectable in BHP. Further work will establish the significance of relationship between glycoprotein profiles in blood serum at PCa and BPH and establish the clinical utility of glycoproteomic serum assays for diagnostics.

Serglycin proteoglycan expressed by cancer cells Introduction: Molecular imprinting allows the creation of artificial recognition sites in synthetic polymers. These sites are tailor-made in situ by copolymerization of a cross-linker and functional monomers around the target molecule. After removal of the imprinted molecule, the polymeric network contains complementary binding sites with specific recognition properties. Molecularly imprinted polymers have been successfully obtained for small molecules, but proteins are more difficult to imprint due to their size, thermal instability and tendency to denature. Methods: We describe here a new strategy for the synthesis of imprinted microgels by polymerization in aqueous media with glycosidases as template-biomacromolecule. Highly specific synthetic 'antibodies' result from the strong interaction between the enzyme and an inhibitor, able to conduct a radical directly from the enzyme active site.

Results: The synthesis of glucosidase inhibitors of azasugar-type coupled to a polymerisable functionality has been performed in seven steps from methyl a-D-glucopyranoside. Inhibition constants (Ki) have shown high affinities (lM range) for the target enzymes and competitive inhibition.

The polymerization has been initiated by UV irradiation of the enzymeinhibitor complex in aqueous solution containing an acrylate-type monomer and a cross-linking agent. Control polymer has been prepared in the absence of the enzyme. The new polymers have been tested for their binding affinity and the inhibitory effect for the native enzyme.

Conclusions: Optimized conditions to obtain nanoscaled soft polymers in aqueous media have been developed. A strong anchoring point inside the cavity is beneficial for the recognition of the template, allowing to obtain high specific glycosidase inhibitors. Introduction: Frutalin is an allb homotetrameric 66 kDa D-Galactose binding lectin that stimulates natural killer cell in vitro, leukocyte migration in vivo and binding to histologic breast cancer samples. Since these findings are related to its specificity to sugars on cell surface, this study aimed at investigating frutalin interaction with biomembrane models. Methods: Langmuir monolayers were employed as biomembrane mimetic system, since they present the advantage of controlling lipid packing and the architecture of the lipid system. Furthermore, using large unilamellar vesicles (LUVs), CD, fluorescence emission and isothermal titration calorimetric (ITC) measurements were performed. Results: Frutalin strongly interacts with mixed glyco-phospho lipid systems whereas no significant alterations for pure phospholipid monolayers were observed. Among the galacto-glycolipids, the interaction is more facilitated for the galactose polar head lipid than sulfated-galactose, as observed by adsorption kinetics and ITC. Besides, surface elasticity (E) values indicate that the monolayer structure becomes more rigid in presence of frutalin. For GM1 ganglioside (Gal 1-4GalNAc 1-4Gal(3-2aNeuAc) 1-4Glc 1-1'Cer), slightly quantities of the protein adsorb, probably due to the most apart position from the interface of the D-galactose in polar head group. Results show that the native protein conformational structure was maintained even for glyco-content vesicles, and a blue shift is observed.

The results attest the protein specificity to the carbohydrate in biomembrane, and indicate a dependence of charged groups and position of the galactose group on the glycolipid polar head. In this sense, we can infer the frutalin biological activity is related to its interaction with galacto-conjugates on cell surface.

Adenosine monophosphate protein kinase regulates the synthesis of hyaluronan in human aortic smooth muscle cells Although the pathway to synthesize HA is known, little information are available on the mechanisms regulating HA synthesis. Our data showed that the cytoplasm availability of UDP-sugars regulate HA synthesis. As UDP-sugar biosynthesis requires ATP, we hypothesized that the cellular energy charge (i.e. ATP:AMP ratio) could control HA production. Methods: Cultures of SMC were used as model for Hyaluronan synthesis and cell migration, transfection and siRNA experiments were carried out in order to clarify the role of the key enzymes in the pathway of Hyaluronan synthesis. Results: In human cells the main sensor of ATP:AMP ratio is AMP activated protein kinase (AMPK). At physiological ATP concentration AMPK is not phosphorylated and not active, but at low ATP:AMP ratio AMPK became phosphorylated modifying several metabolic pathways. Treating SMC with AICAR (a chemical activator of AMPK) the HA synthesis decreased whereas the synthesis of other glycosaminoglycans remained unchanged. Using constitutive active constructs and dominant negative AMPK cells data were confirmed. AMPK knockout mice treated with AICAR did not decrease HA production. The mechanism of HA synthesis inhibition mediated by AMPK did not involved a change in gene expression. mRNA coding for HA metabolizing enzymes (i.e. UDP glucose pyrophosphorylase, UDP glucose dehydrogenase, HAS1, 2 and 3 as well as hyaluronidases) in control and AICAR treated SMC did not changed. Conclusion: AMPK activity affects the HA synthesis without changing gene expression, therefore as AMPK has a kinase activity and several enzymes belonging to HA synthetic pathway possess an AMPK phosphorylation consensus sequence, we hypothesize that AMPK, through phoshorylations, could modify the activity of some proteins involved directly in HA or in UDP-sugar precursors synthesis. Introduction: The Tim9-Tim10 complex is located in the mitochondrial intermembrane space where it chaperones a set of hydrophobic precursor proteins during their import into mitochondrial membranes. How this complex functions at a molecular level is poorly understood. Methods: We solved the structure of yeast Tim9-Tim10 and used the information to perform mutagenesis and truncation analysis in yeast. Phenotypes such as reduced growth and defects in mitochondrial protein import were then assessed.

Results: The yeast Tim9-Tim10 complex forms a hexameric assembly of alternating subunits. The core of the complex is stabilised by conserved salt-bridges which are essential for function. A Tim9 salt-bridge mutant is lethal while a Tim10 salt-bridge mutant is temperature sensitive for growth. Truncations in regions that are not involved in corecontacts do not necessarily disrupt the complex assembly. Import analysis using mitochondria isolated from a number of mutants show defects in the biogenesis of model precursors that utilise the Tim9-Tim10 complex.

Conclusion: The soluble Tim9-Tim10 complex is required for the efficient import of a set of precursor proteins. Core contacts are essential for general protein assembly while other specific regions appear important for substrate binding. Introduction: Intraneuronal accumulation of amyloid beta peptides (Abeta) and their binding to mitochondrial enzyme 17beta-hydroxysteroid dehydrogenase type 10 (17beta-HSD10) can play a key role in pathogenesis of Alzheimer disease (AD). Since cerebrospinal fluid (CSF) is in direct contact with the environment of the central nervous system, any changes in biochemical composition of the brain are predominantly reflected here. We suggest that the complex between Abeta and 17beta-HSD10 estimated in CSF could be a biomarker of AD in future, however, highly sensitive methods are needed for analysis. Methods: ELISA and optical biosensor (surface plasmon resonance method) were applied.

Results: The complex can be detected in CSF, however, its levels are very low when compared to these of free enzyme. Nevertheless, the highest amount has been detected in patients with AD when compared to nondemented controls and people with multiple sclerosis. Analysis of binding interactions in vitro supported a gradual formation of the complex and suggested possible complications to evaluate the changes occurring in vivo.

Conclusions: ELISA and surface plasmon resonance methods can be applied to analyse CSF, nevertheless, a specificity of changes in relation to AD is still under debate. Introduction: Mitochondrial complex I is an 980 kDa multimeric enzyme composed of 45 subunits. Although human complex I is associated with disease, its assembly is poorly understood, complicated by its large size and its regulation by two genomes Methods: We analyzed complex I assembly by monitoring the incorporation of either radiolabeled mtDNA-encoded translation products or newly-imported nuclear-gene encoded subunits using blue native-PAGE. We also analyzed a group of patient cell fibroblasts for defects in complex I and loss of the assembly factor CIA30. Results: We found that mtDNA-encoded subunits first assemble into intermediate complexes and require significant chase times for integration into the holoenzyme. In contrast, some imported nuclear-encoded subunits associate with pre-existing complex I subunits to form intermediates and/or the fully assembled holoenzyme. We also found that CIA30 associates with early complex I assembly intermediates. A patient presenting with encephalomyopathy was found to contain only 10% steady-state CIA30 with reduced levels of mature complex I due to defects in assembly. Genetic analysis revealed that the patient had mutations in the gene encoding CIA30. Complementing the deficiency in patient fibroblasts with normal CIA30 restored complex I levels.

Conclusions: Complex I biogenesis involves two complementary processes -(i) de novo assembly and (ii) dynamic exchange of pre-existing subunits with newly imported ones. We also found that CIA30 is involved in the early assembly of complex I and mutations in its gene can cause disease. Introduction: Cristae of the mitochondrial inner membrane were once viewed simply as a barrier that separated the mitochondria into the inner matrix and intermembrane (IMS). In many types of eukaryotic cells, crisate of mitochondria has a tubular shape. However it is not clear that the mechanism by which cristae morphology is established and maintained. The dynamic structure of cristae plays a role in mitochondrial functions.

Methods: In the last 10 years, the technique of electron tomography (ET) employing high voltage electron microscopes and rapid freezing fixation methods has revealed new information about mitochondrial membrane structure. To study the mechanism about morphological change of mitochondrial membrane, we used the knockdown and knockout system of a C. elegans mitochondrial protein and the ET technique.

Results: We measured physiological propertied related to mitochondria, such as oxygen consumption rate and reactive oxygen species level and reconstructed 3 dimensional structure of mitochondria using electron tomography. C. elegans mitochondrial protein like DIC-1 knockdown mitochondria contained numerous vesicles and the ROS level was increased. Overexpression of this protein decreased ROS level and enhanced cristae formation.

Conclusions: These results suggest that C. elegans mitochondrial protein like DIC-1 controls mitochondrial cristae/inner membrane structure, and this has a relation with the changes of ROS level. This work was supported by from the Seoul Development Institute. Introduction: Oxidative folding in the mitochondrial intermembrane space (IMS) is crucial for the import of certain cysteine-rich IMS proteins. The essential proteins Mia40 and Erv1 are key components for this mechanism functioning as a disulphide protein cascade that is functionally linked to the respiratory chain by shuttling electrons onto CytC. The subunits of the chaperone complex Tim9-Tim10 require Mia40 for their biogenesis.

Results: Here we show that interaction with Mia40 is a site-specific event: (i) the N-terminal first cysteine of the precursor is crucial for docking onto Mia40 via a mixed disulphide; (ii) release is triggered by disulphide pairing of the C-terminal cysteine onto the N-terminal one; and (iii) formation of the inner disulphide between the second and third cysteines apparently precedes the release reaction and is critical for assembly with Tim9. Discussion: The data presented here point to substrate recognition by Mia40 in a very precise and site-specific manner which supports the concept that Mia40 is not a general oxidase but rather acts specifically. Additionally, the co-ordinated steps that are proposed to occur between the first cysteine docking and the release reaction argue for a processive character of Mia40 which is strikingly different from the other known cellular protein oxidases, such as DsbA and PDI. This work provides an important framework for a mechanistic understanding of this fundamental biological process. Introduction: The high mitochondrial membrane potential (inside negative) is a powerful driving force for electrophoretic influx of cations, of which K + is the most abundant one. Because K + influx is always accompanied by influx of water, mitochondria tend to swell and the outer membrane may rupture. The K + /H + exchanger, allowing electroneutral exchange of K + against H + , is assumed to counteract this process. Recently we have identified a novel eukaryotic gene family (Y0l027 and Mrs7 in yeast, LETM1 in humans) involved in mitochondrial K+ homeostasis and volume control. Genetic screen for yol027D multi-copy suppressors led to the identification of Ydl183. Methods: We have measured mitochondrial membrane potential, mitochondrial potassium-acetate induced swelling and K+/H+ exchange in submitochondrial particles. Results: Yeast yol027D cells have strongly reduced K+/H+ exchange activities, low membrane potential and reduced respiratory growth. While double disruption yol027D mrs7D and yol027D ydl183D did not significantly change these phenotypes, triple disruption yol027D mrs7D ydl183D caused much more pronounced phenotype: K+/H+ exchange activity was below detection level, membrane potential was low and cell viability was considerably reduced. Overexpression of Yol027, Mrs7, Ydl183 or human LetM1 in yol027D mrs7D ydl183D cells rescued growth and re-established K+/H+ exchange activity. Conclusion: Yol027p, Mrs7p and Ydl183p all are likely to be involved in formation of an active K+/H+ exchange system. Lack of this activity not only affects mitochondrial functions, but lowers cell vitality. Introduction: Previous studies have established that peripheral monocytes from atherosclerotic patients display specific differences in their expression profile as compared to the normal population. Scavenger receptors (SRBI-II) and the ATP binding cassette transporter A1 (ABCA1) are essential molecules for the maintenance of cholesterol homeostasis. The aim of the present study was to correlate the phenotype of atherogenic plaques as regards to the expression of SRBI, SRBIII and ABCA1, with the corresponding phenotype of autologous circulating peripheral monocytes. Materials and Methods: Autologous plaque, control vascular tissue and blood samples were collected from 29 human donors undergoing bypass surgery of coronary arteries. Control blood samples were collected from 17 atherosclerosis negative patients, shown by coronary angiography. Using Real-time PCR the expression levels of scavenger receptors (SRBtotal, SRBI, SRBIII) and ABCA1 were quantified. Results: Real time qPCR semi-quantification analysis of the samples in bulk demonstrated that in plaques of human coronary arteries the balance of SRBI/SRB-total (p < 0.0001; unpaired t-test) and SRBIII/ SRB-total (p = 0.0449) were significantly reduced. The same reduction in SRBI/SRB-total (p = 0.0055) was observed in blood monocytes.

The above probably resulted from the increased presence of the SRBII variant, as SRBI and SRBIII expression did not differ in the sample groups. Most importantly, ABCA1 expression decreased in both plaque tissue (p = 0.0266) and in monocytes isolated from patients with atherosclerosis (p = 0.002) compared to the appropriate controls. Conclusion: The coincidence of specific events in both plaque tissue and peripheral blood monocytes indicates that atherogenesis is not restricted to the region of the vascular lesion but involves system wide changes in the monocytic cell population. Introduction: Fibrosis is an important pathological process underlying many diseases in many organs; however, the molecular mechanisms involved and the macromolecules participating during the development of fibrosis are not well understood yet. Methods and Results: We have performed proteomic analysis on a renal model of fibrosis, the unilateral ureteric obstruction, in rats. Based on the analysis of our data, we have focused on a particular protein, calreticulin, an endoplasmic reticulum resident protein not previously implicated in fibrosis, which was induced in early and late intervals during the development of fibrosis. We have confirmed proteomic data by Western blotting, immunocytochemistry and RT-PCR, where in all cases calreticulin expression was up-regulated upon ureteric obstruction both at the protein and the mRNA level from early time intervals. We have further tested the involvement of calreticulin in bleomycin-induced lung fibrosis in mice, where it was also found to be up-regulated. In cell culture experiments, renal proximal tubule epithelial cells and lung alveolar epithelial cells demonstrated up-regulation of calreticulin, upon stimulation with TGFb, a well known macromolecule involved in the fibrotic process. Introduction: Parvin is a highly conserved protein that contains two calponin homology (CH) domains in tandem. Previous studies suggest that parvin may function as an F-actin-binding protein. Our genetic study in Drosophila strongly suggest that parvin together with its binding partner Integrin-Linked Kinase (ILK) tether the actin cytoskeleton at sites of integrin adhesion in the embryonic muscle attachment sites. However it is not yet known the mechanistic aspects of parvin function in vivo. Methods: We have engineered several transgenic flies carrying full length and truncated forms of parvin fused to GFP that are expressed either by the endogenous regulatory elements of the gene or using the UAS/GAL4 system. Results/Conclusions: Our work suggested that both CH-domains as well the N-terminus region are required for parvin function in vivo. The main localization signal that targets parvin at sites of integrin adhesion resides within the CH2 domain. Overexpression of parvin in several tissues resulted in developmental defects. In particular high levels of parvin in the mesoderm caused lethality that was completely rescued upon co-expression of ILK. This result strongly indicates that ILK controls parvin function in vivo. In addition overexpression of parvin in several epithelia resulted in the missorganisation of the actin cytoskeleton and induction of apoptosis. These effects could also be rescued completely by coexpression of ILK and the inhibitor of apoptosis DIAP1. Collectively our results suggest that parvin integrates multiple signals that play pivotal role in the completion of tissue morphogenesis. Introduction: Monocytes/macrophages represent a part of mononuclear phagocyte system involved in the host response against cancer. They reveal some cytostatic/cytotoxic anticancer activities but were also suggested to promote neoangiogenesis and cancer metastasis. Since an induction of pro-inflammatory cytokine expression (for instance TNFalpha; TNF) was observed in monocytes/macrophages confronted with cancer cells both in vivo and in vitro, we focused on the influence of monocytes and monocyte-produced TNF on the invasiveness of cancer cells.

Methods: Co-cultures of elutriated human peripheral blood monocytes and human pancreatic cancer (HPC-4) cells were used to elucidate the mechanisms of the influence of monocytes on the basic HPC-4 cell invasive parameters by time-lapse microscopy and immunofluorescence techniques, followed by the analyses of the effect of exogenous TNF on HPC-4 motility.

Results: HPC-4 cells reacted to the prolonged presence of monocytes with an increased motility correlated with a transition from epithelial to mesenchymal morphology. Furthermore, an exogenous TNF elicited a corresponding induction of HPC-4 invasion. Interestingly, the induction of cell motility by monocytes was abrogated by the inhibitors of TNF production by monocytes, including pentoxyphillin, confirming that monocyte-derived TNF affects the morphology and induces motile activity of HPC-4 cells.

Conclusion: These data demonstrate an existence of the paracrine intercellular loop between HPC-4 cells and tumour infiltrating monocytes which includes TNF. Thus, the inflammatory response of the immune system to cancer, resulting from the interaction between cancer and immune cells, may be involved in the induction of cancer invasion and hence, play a crucial role during cancer progression.

Isolation of lipid rafts from atlantic cod (Gadus morhua) intestinal brush border membrane G. A. Gylfason and B. Asgeirsson Department of Biochemistry, Science Institute, University of Iceland, Reykjavik, ICELAND Abstract: Membrane lipid rafts are glycosphingolipid/cholesterolenriched membrane micro-domains that have been extensively studied during the past two decades. However, to the best of our knowledge, no studies have yet been performed on lipid rafts from the intestinal brush border membrane (BBM) of ray-finned fishes (Actionoerygii).

Our aim was to isolate and characterize lipid rafts from the BBM of Atlantic cod (Gadus morhua) intestinal enterocytes to confirm their existence and if they showed similarity to lipid rafts from other species in terms of lipid and protein content. To validate the isolation process, we assayed marker enzymes for sub-cellular organelles, including alkaline phosphatase (AP), a well-know marker enzyme for BBM and lipid rafts. AP was mapped by immunological confocal microscopy, enzyme activity, and Western blotting. We also performed lipid analysis on BBM and lipid rafts by thin-layer chromatography and 31 P-NMR. Cholesterol was measured by a conjugated enzyme assay and proteomics studies where performed by MALDI (Matrix-assisted laser desorption/ionization) and LC-ESI (Liquid-chromatography electrospray ionization) mass spectrometry from trypsin digested SDS-PAGE samples. All methods showed enrichment of AP in both BBM and lipid rafts fraction, 31 P-NMR gave high content of phosphatidylcholine in BBM as expected, but sphingomyelin was more dominant in the lipid rafts together with cholesterol. Various proteins have been associated with our lipid raft preparation such as aminopeptidase-N, prohibitin, beta-actin, and villin 2. The existence of lipid rafts containing cytoskeletal proteins has, therefore, been confirmed here for the first time in a ray-finned fish.

The non-classical export of syntaxins and their possible extracellular functions as integrin ligands Y. Hirai and K. Yamazaki Institute for Frontier Medical Sciences, Kyoto University, Kyoto, JAPAN Introduction: Elucidation of the processes involved in organogenesis requires a detailed understanding of the complex tissue interactions. The potential regulators for this process include t-SNARE molecules, which have no signal peptides and usually exist on the cytoplasmic surface of the cell membrane to mediate cytoplasmic vesicular fusions, but are temporally localized and secreted extracellularly to activate morphogenic programs in the adjacent cells. In this study, we determined key molecular elements for their extracellular presentation and the subsequent morphogenic function. Methods and Results: The cDNAs encoding mouse syntaxin 1, 2 (epimorphin), 3, 4, 5 and 6 were isolated and tagged with T7 peptide. The analyses of the transgene products revealed that the subpopulations of certain members of t-SNAREs (syntaxin2, 3 are 4) are secreted into medium with phosphatidilserine-bound synaptotagmin and annexin II, in response to the cell stress. The similar mechanisms of the non-classical export have been shown for other leaderless proteins FGF1 and IL-1. We further show that secreted syntaxin 2 (epimorphin) exerts a morphogenic function through binding to alphaV-integrin receptors and activating the FAK-MAPK signaling pathway in the target cells.

Conclusions: This study clarified the molecular elements for the nonclassical export and the action of t-SNARE molecules in the extracellular milieu, which shed light on the molecular mechanism of spatio-temporal regulation in tissue morphogenesis by proteins of double life.

Interphase-specific phosphorylation-mediated regulation of tubulin dimer partitioning in human cells P. Holmfeldt, S. Stenmark and M. Gullberg Department of Molecular Biology, Umea University, Umea, SWEDEN

The microtubule cytoskeleton is differentially regulated by a diverse array of proteins during interphase and mitosis. Op18/stathmin (Op18) and microtubule-associated protein (MAP)4 have been ascribed opposite general microtubule-directed activities, namely, microtubule destabilization and stabilization, respectively, both of which can be inhibited by phosphorylation. Here, using three human cell models, we depleted cells of Op18 and/or MAP4 by expression of interfering hairpin RNAs and we analyzed the resulting phenotypes. We found that the endogenous levels of Op18 and MAP4 have opposite and counteractive activities that largely govern the partitioning of tubulin dimers in the microtubule array at interphase. Op18 and MAP4 were also found to be the downstream targets of Ca 2 + and calmodulin-dependent protein kinase IV and PAR-1/MARK2 kinase, respectively, that control the demonstrated counteractive phosphorylation-mediated regulation of tubulin dimer partitioning. Furthermore, to address mechanisms regulating microtubule polymerization in response to cell signals, we developed a system for inducible gene product replacement. This approach revealed that site-specific phosphorylation of Op18 is both necessary and sufficient for polymerization of microtubules in response to the multifaceted signaling event of stimulation of the T cell antigen receptor complex, which activates several signal transduction pathways.

4D. Functional Cytoskeleton Dynamics To study the effects of the phosphorylation at the identified sites, we mutated them to alanine (non-phosphorylated) and aspartic acid (mimicking phosphorylation) residues, respectively.

Conclusions: Preliminary overexpression studies with these Myo9b constructs suggest that cell morphology and Rho-activity are differentially regulated by the phosphorylation of Myo9b. 

The distribution of RelA/p65 in the cytoplasm is actin cytoskeleton-dependent but not its nuclear translocation in A431 cells stimulated with EGF The actin cytoskeleton is involved in a number of processes in nonmuscle cells including response to extracellular signals, but its role in NF-jappa B translocation into the nucleus and signal regulation is not clear. We therefore checked whether the actin cytoskeleton participates in intracellular relocation and nuclear translocation of the p65 subunit of NF-kappa B in epithelial A431 cells. The p65 subunit distribution was investigated in cells spread on fibronectin after EGF treatment using confocal microscopy. Cells with an intact cytoskeleton and cells treated with cytochalasin D were analyzed. First, immunofluorescent staining showed that A431 cell adhesion to fibronectin induced partial nuclear translocation of RelA/p65, as well as localization on stress fibers and diffusely in the cytosol. Second, after EGF stimulation RelA/p65 was found in ruffles, on reorganized stress fibers, diffusely in cytosol and in discrete actin-rich cytoplasm-membrane patches. Third, further staining for actinin 1, actinin 4, vinculin and paxillin showed that the discrete p65 regions contained all these adhesion proteins. Fourth, cytochalasin D treatment caused p65 redistribution to actincontaining aggregates, and EGF further increased the number of p65containing clusters in cytoplasm. The typical adhesion proteins were also found in these aggregates. Finally, western blot confirmed that EGF caused nuclear translocation of p65 in cells with a cytochalasin D-disrupted cytoskeleton. The results suggest that an intact actin cytoskeleton is not obligatory for nuclear transport of p65 in A431 cells although there is a very tight association between of p65 and actin-rich structures in the cytoplasm. Integrin-linked Kinase (ILK) and the associated proteins Parvin and PINCH assemble a highly conserved protein complex that mediate a link between integrins and actin cytoskeleton. In order to dissect further the ILK-complex function we currently investigate how this complex form at the muscle attachment sites of the Drosophila embryo using three complementary approaches: First, we are examining the hierarchy of molecular interactions that are required for the subcellular localization of the integrin-actin linker complex components. Second, we are monitoring the dynamics of the ILK-complex in vivo using an array of transgenic flies expressing components of the integrin-actin linker complex fused to GFP. Third we are engineering a chimeric ILK form that is constitutively tethered to the plasma membrane and explore the ability to initiate the assembly of the integrin-actin linker complex independent of signals received by integrins. Meanwhile we are using an RNAi-approach and genetic mosaics to identify new roles of ILK in additional morphogenetic processes during Drosophila development. Finally we are exploring the potential functional conservation between mammalian and fly ILK using the UAS/Gal4 expression system. We describe an inflammatory response, characterized by both innate and adaptive immune cells, initiating in the des-/-hearts by day 16 post-partum. The persistence of inflammation in the absence of adaptive immunity (RAG1-/-deficiency) suggests that it is sustained by innate immune mechanisms. Anti-inflammatory molecules, such as IL1-ra and TIMP-1, produced by the injured hearts, as well as apoptosis of the infiltrating cells apparently orchestrate the gradual regression of the granulation-like tissue, leading to scar formation. Microrarray analysis conducted in 3 month-old animals revealed that the majority of the transcripts significantly up-regulated in the desminnull hearts encode for inflammation/immunity-related molecules. Overexpression of the most highly up-regulated (osteopontin, osteoactivin/ Gpnmb, MMP-12 and galectin-3) was confirmed by real-time PCR and immunohistochemical methods. To explore the role of osteopontin, found to be expressed by the inflammatory infiltrates in the desmin-null hearts, we generated mice lacking both desmin and osteopontin (des-/-OPN-/-) and estimated that they exhibit significantly reduced heart fibrosis, compared to the des-/-. Furthermore, we found considerably less MMP-12, osteoactivin and galectin-3 up-regulation in the des-/-OPN-/-hearts. Previously, we have showed that an intramolecular interaction between the amino-terminus and the kinase domain of FAK plays a regulatory role on its activity. To further reveal the molecular mechanistic insight of this regulatory event, we compared the FAK's FERM-like domain with other FERM domains. In particular, several conserved residues which are crucial for interaction with phosphoinositides found in other FERM domains were recognized and mutations of them resulted in the reduction of phosphorylation on Y397 and/or Y576. Indeed, we revealed that FAK's FERM domain possesses a high affinity toward D-3 and D-4 phosphoinositides whereas the aforementioned mutants lost the capacity binding to phosphoinositides. These results were consistent with the decreased tyrosine phosphorylation and kinase activity of FAK while the depletion of phosphate supply or blockage of phosphoinositides in the presence of neomycin in the cell culture conditions. Furthermore, we also found in the absence of phosphoinositide binding led to a significant decrease in cell motility. In contrast, overexpression of wild type FAK or an activated form of FAK (N16-FAK) could rescue this phenotype. Our findings provide a new regulatory mechanism on FAK's activity and cellular function via phosphoinositides. 

Lipocalin-2 mediates a phenotypic change similar to reactive astrocytosis Astrocytes, the most abundant glial cell type in the brain, provide metabolic and trophic support to neurons and modulate synaptic activity. In response to brain injury, astrocytes proliferate and become hypertrophic with an increased expression of intermediate filament proteins. This process is collectively referred to as reactive astrocytosis. Lipocalin 2 (LCN2) is a member of the lipocalin family that binds to small hydrophobic molecules. LCN2 can be highly induced in both inflammatory and neoplastic diseases. It is implicated in diverse physiological processes, including apoptosis. In the present study, we investigated the role of LCN2 in the cell death, morphology, and migration of astrocytes. Lcn2 expression and secretion was increased by inflammatory stimuli in astrocytes. Forced expression of LCN2 or treatment with recombinant LCN2 protein increased the sensitivity of astrocytes to cytotoxic stimuli. Iron and Bim appeared to be involved in the cytotoxic sensitization. Additionally, treatment of astrocytes with the recombinant LCN2 protein induced upregulation of glial fibrillary acidic protein (GFAP), cell migration, and the morphological change of astrocytes that was similar to characteristic phenotypic change termed reactive astrocytosis. The enhanced cytotoxic vulnerability of astrocytes correlated with the morphological change. The lcn2-induced phenotypic change of astrocytes occurred through Rho-Rho kinase-GFAP pathway, which was positively regulated by nitric oxide and cGMP. Taken together, our results indicate that LCN2 acts in an autocrine fashion to induce cell death sensitization and morphological changes in astrocytes under inflammatory condition, and these phenotypic changes may be the basis of reactive astrocytosis in vivo. Introduction: Integrins are the main family of transmembrane heterodimeric receptors that mediate cell-matrix adhesion in multicellular organisms. The main function of integrins is to trigger, upon activation, the formation of a complex network of cytoskeletal and signaling proteins such as talin, ILK, paxillin, in order to mediate the link between extracellular matrix and the actin cytoskeleton. The Calponin Homology domain-containing protein parvin is one of the proteins so far identified as a binding partner of ILK. In Drosophila there is only one parvin locus coding for a protein that displays 60% identity with the mammalian parvins. In this study we are using the power of Drosophila genetics to assess the functional requirement of parvin in the whole organism.

We have largely concentrated on the role of parvin function at the embryonic muscle attachment sites, a well characterized in vivo model system of integrin-mediated stable adhesion. We have generated mutant alleles for parvin as well as several molecular and genetic tools that enabled us to characterize the function of the protein. Allelic lines associated with loss of parvin expression are embryonic lethal, suggesting that parvin has an essential role in the completion of embryonic development. Phenotypic analysis of hemizygous embryos reveals that loss of parvin function results in defective adhesion of embryonic muscles to the epidermis. Similarities with the phenotypic defects observed in integrin and ilk mutants, strongly suggest that parvin is part of the essential molecular complex that mediates the coupling of integrins to actin cytoskeleton.

The TFIID-member TAF12 is implicated in the reduction of E-cadherin expression, whereas the AP1-factor FRA-1 in the increase of vimentin expression during epithelial-to-mesenchymal transition (EMT) in cancer Cell biological assays including morphological changes, cell growth rate, GEF activity and re-organisation of cytoskeleton were performed. Results: Wild type FGD1 had no effect on cell growth. Formation of lamellipodial structure and co-localisation with actin filament were induced in wild type FGD1 transfected cells. Growth inhibition was not observed in cells with the R636W mutation but in cells with the R443L mutation. Both mutants reduced cell migration, lamellipodial structure and co-localisation with actin filament. Conclusion: Each mutant proteins displayed distinct dysfunction in vitro that might be able to explain differences of symptoms in AAS.

Genetic analysis of the molecular machinery that couples integrins to actin in Drosophila In multicellular organisms cells change shape due to mechanical forces generated primarily intracellularly by the cytoskeleton at cell-matrix or cell-cell adhesion sites. Integrins, a main family of mechanotrasducer adhesion molecules that link the extracellular matrix to actin cytoskeleton are essential for the maintenance of cell morphology and tissue integrity in the developing organism. A well established protein complex that links integrins to actin cytoskeleton and regulates the dynamic changes in cell shape is assembled around integrin-linked kinase (ILK). Recent evidence underlines the functional importance of the ILK-complex in human diseases including cardiomyopathy and cancer. Our aim is to use the powerful genetic tools of Drosophila and dissect the in vivo role of the conserved molecular machinery that functions in concert with integrins. We are using a combination of experimental approaches including forward and reverse genetics, high resolution imaging and biochemistry, in order to analyse the spatiotemporal molecular organization and function of the integrin-actin linker complex in muscles and developing epithelia. Introduction: In circulation, platelets adhere to leukocytes forming stable complexes, a process that is mainly mediated via interactions of platelet P-selectin with its leukocyte counter-receptor, P-selectin glycoprotein ligand-1 (PSGL-1). Several chronic inflammatory diseases and acute coronary syndromes are characterised by an increase in such heterotypic interactions, raising the possibility that pro-inflammatory processes might be initiated in leukocytes upon platelet adhesion. Based on the ability of PSGL-1 to transduce intrinsic intracellular signals following engagement, we aimed to investigate any associated functional consequences of platelet adhesion on monocytes.

Methods and Results: We demonstrated increased stability of PSGL-1-mediated interaction of platelets with monocytes compared to polymorphonuclear leukocytes as well as association of monocyte PSGL-1 with Syk tyrosine kinase and ERM (ezrin, radixin, moesin) proteins. We also report differential effects of platelet binding on monocyte functional responses between non-activated and thrombinactivated platelets. In particular, monocyte CD11b expression and release of pro-inflammatory IL-1b and TNF-a were significantly upregulated upon adhesion of activated platelets, whereas non-activated platelets had negligible effects. Similarly, binding of thrombin-activated platelets but not of non-activated platelets to monocytes induced IjBa degradation, nuclear translocation of NF-jB p65 and inhibition of monocyte apoptosis in the absence of survival factors. Conclusion: Our data suggest that platelet activation status determines whether monocytes become activated following platelet adhesion. Therefore, in the absence of overt platelet activation, PSGL-1 -Pselectin-dependent platelet binding to monocytes may represent a normal physiological phenomenon with little impact on monocyte functional responses. Introduction: The aim of our analysis was to model and evaluate the possibility of interaction between protein products of the different HLA alleles and the natural killer and T-lymphocyte cells receptors from the graft recipient immunological system. This type of reactions leads often to graft versus host disease manifestation. HLA matching procedures are highly time consuming and there is a vide evidence of the significance of early donor acceptation for HSCT outcome. In cases when the donor is not perfectly matched, immunologists have a serious problem with the decision which mismatch is less harmful and hence acceptable.

Methods: First of all we performed the statistical analysis of the HLA data and after HSCT survival data collected in the Polish Central Bone Marrow Donor Registry to choose the frequent HLA mismatches between the unrelated donor and the graft recipient and afterwards apply homology structure modelling of the given alleles and ligand docking methods. All the docking studies were performed using MACROMODEL 9.5. Results and Conclusions: As the first candidate for docking we used the X-ray structures of the complexes between HLA B*08-EBV peptide and TCR LC13 receptor. We have exchanged the HLA allele in the complex to observe the influence of its substitution on the total energy of the immunological synapse. We observed that the conformation of presented peptide is the most significant factor for the recognition strength.

The release of HspBP1 from human carcinoma cells In our earlier work we have shown that keratinocytes-derived human squamous carcinoma A431 cells dynamically export Hsp70 under cell culture conditions. We have found that extracellular-Hsp70 (e-Hsp70) appears in the culture medium during short treatment of cells with either mild heat shock or pharmacological inhibitor of phospholipase C activity U73122 The purpose of the present study was to examine whether HspBP1, a known Hsp70 co-chaperone, is released from A431 cells under the same conditions and whether extracellular HspBP1 (e-HspBP1) might modulate a signaling function of e-Hsp70. We determined that the intracellular level of HspBP1 decreased during the initial phase of heat shock or treatment with U73122 and simultaneously appeared in cell culture medium. To elucidate the mechanism of HspBP1 release from A431 cells, we studied changes in intracellular distribution of the protein. We demonstrated the presence of HspBP1 in granules located at the cell periphery both in heat-shocked and U73122-treated cells. The co-localization of HspBP1 with both Hsp70 and a marker of secretory granules, chromogranin A, in the granules provides evidence that HspBP1 and Hsp70 release are mediated by secretory-like granules. These data identify a secretory pathway by which HspBP1 can be released from carcinoma cells together with Hsp70. We also demonstrate that though e-HspBP1 has no effect on EGF receptor transactivation, the mixture of e-Hsp70 and e-HspBP1 amplified EGF receptor transactivation induced by exogenously added pure Hsp70. Taken together our data suggest that e-HspBP1 is a modulator of e-Hsp70 signaling activity.

Low level long wavelength laser irradiation effects on signalling and crosstalk in stress exposed human mononuclear cell populations Introduction: Beneficial effects in treatment of hypoxic, ischemic, infected wounds, and chronic inflammatory conditions of low power laser irradiation within the tissue transparency window of 650-1000 nm are yet far from being explained, as there are many aspects of intercellular communication in various pathological conditions. The aim of this study was to supply new data concerning soft laser irradiation effects on crosstalk of adherent and non-adherent human mononuclear cells in stress conditions. Methods: Peripheral blood derived lympocytes and monocytes/monocyte-derived dendritic cells were cultured in standard conditions, in presence/absence of growth factors/cytokines, virtually alone and in coculture. We exposed various mononuclear cell populations to energy/ nutrient deprivation caused stress and/or to different therapeutic agents (low level laser irradiation, natural antioxidants) separately and/or in coculture. Cells were further cultured virtually alone and/or in coculture. Using appropriate molecular reporters, we followed up changes occurring in various cell populations survival/proliferation rates, and death-style choices by fluorescence microscopy and flow cytometry. Endochondral skeletal development begins with the formation of a cartilaginous template, where mesenchymal cells aggregate and increase in density prior to their overt differentiation into chondrocytes. A process of prechondrogenic condensation whereby mesenchymal cells aggregate occurs through cell migration and proliferation. However, the exact mechanism behind this aggregation has yet to be elucidated, although cell collision has been implicated. The protein modulation by protein kinase C (PKC)-dependent phosphorylation is critically involved in the regulation of migratory phenomena involving receptor-mediated adhesion, membrane protrusion, and the formation of discrete cell-matrix adhesion sites linked to a reorganization of the actin cytoskeleton. Investigation of the potential involvement of PKC isoforms i n the control of cell motility as analyzed by a wound-healing assay showed that PKCd inhibition was correlated with suppressions of wing bud mesenchymal cell migration and integrin b1 expression in a densityindependent force. Perturbation of integrin b1 function using its functional blocking antibody suppressed the migration of wing bud mesenchymal cells. Furthermore, the phosphorylation levels of Src and focal adhesion kinase (FAK) were decreased by PKCd inhibition. Wound-healing assay after either treatment of PP2, an inhibitor of Src family kinase, or electroporation of FAK specific siRNA, showed suppressed cell migration. Taken together, our data pointing to novel founding that PKCd may act as a positive regulator for cell migration, an essential step for processing prechondrogenic condensation, through regulation of integrin b1 signaling at focal adhesion complex.

Production of soluble CD69 receptor in Escherichia coli, and its use in animal tumor therapies Introduction: Connexins (Cx) are gap junction proteins that assemble into channels, which mediate intercellular communication. Cx43 has been shown to interact with caveolin-1 (Cav-1), but the role of this association and whether other Cavs bind Cx43 had yet to be established. Methods: The role of the endogenous interaction of Cx43 with Cav-1, and possibly with Cav-2, was mainly investigated in rat epidermal keratinocytes (REKs). The Cx43/Cavs interaction was assessed using coimmunoprecipitation, colocalization, and Far Western assays. A mutational analysis was used to determine which portion of the Cx43 molecule is involved in its interaction with Cavs. The role of Cavs in regulating Cx43 was studied using both over-expression and RNAi knockdown approaches and gap junctional intercellular communication (GJIC) was measured by dye transfer assays. The presence of Cx43 in lipid rafts was determined by Triton X-100 fractionation and sucrose gradient centrifugation. Results: Cx43 coimmunoprecipitates and colocalizes with Cav-1 and Cav-2 in REKs. Our mutation and Far Western analyses revealed that the C-terminal tail of Cx43 is required for its association with Cavs and that the Cx43/Cav-1 interaction is direct. Our results indicate that newly synthesized Cx43 interacts with Cavs in the Golgi apparatus and that the Cx43/Cavs complex also exists at the plasma membrane in lipid rafts. We demonstrate that caveolins regulate GJIC and that the presence of Cx43 in rafts may contribute to the mechanism modulating GJIC. Conclusion: Our results suggest that the Cx43/Cavs association occurs during exocytic transport and clearly indicate that caveolin regulates GJIC.

Prolonged Thy-1 stimulation of astrocytes induces migration but not proliferation Introduction: Astrocytes, a type of macroglial cells of the central nervous system convert to a fibroblast-like morphology upon brain injury. These so called 'reactive astrocytes' also proliferate and migrate to the wounded area to form the glial scar, which constitutes a major impediment to neuronal regeneration. Such morphological changes observed in astrocytes in vivo may be related to those triggered in vitro by Thy-1, an abundant neuronal glycoprotein that binds to avb3integrin in astrocytes provoking dramatic changes in cell shape and increased cell adhesion.

Methods: Thus here, we tested the hypothesis that sustained Thy-1 engagement of avb3integrin stimulates astrocyte migration and/or proliferation by using both wound healing and proliferation assays.

Results: An astrocyte monolayer was scratched with a pipette tip and migration into the cell-free zone was monitored after treating the cells for different times with soluble Thy-1-Fc-protein A complexes. Thy-1 induced the formation of filopodia, lamellipodia and also migration of astrocytes into the wounded area only when it was present for prolonged periods of time. Such migration was inhibited when adding anti-av or anti-b3integrin antibodies. Moreover, according to results obtained with MTS proliferation and Trypan blue exclusion assays, this effect was not due to cell proliferation.

Conclusions: Our data indicate that binding of Thy-1 to its receptor (avb3integrin) initially stimulates cell adhesion and changes in cell shape, followed by cell migration in a sequence similar to that described for astrogliosis. Introduction: Epimorphin (also known as syntaxin2) is a stromal signaling factor that is temporally secreted via a non-classical route to regulate the morphogenesis of various epithelia including skin epidermis. Physiological stimuli lead to its membrane translocation and subsequent extracellular secretion, which generates an epimorphin gradient in the epidermis. In the present study, we addressed how epimorphin signaling impacts the behaviors of epidermal keratinocytes. Methods: We first engineered HaCaT cells overexpressing cell-surface epimorphin, as the result of which efficiently receive an excessive epimorphin signal from adjacent cells via juxtacrine pathway, and analyzed the effects on the cellular growth and differentiation. Next, two aspects of terminal differentiation of epidermal keratinocytes have been studied in cell culture, calcium-induced cornified cell envelope formation and cell detachment-induced anoikis. In addition, to better define on epidermal morphogenesis and differentiation, we developed threedimensional (3D) organotypic cell culture model using gene-transfected cells.

Results: Artificial stimulation of HaCaT cells with extracellular epimorphin by a juxtacrine pathway triggered initiation of their early differentiation program. Intriguingly, however, sustained epimorphin signaling appeared to severely attenuate the terminal cornification. In the organotypic culture of HaCaT cells, overexpression of epimorphin impaired the successive differentiation program in the stratified epidermis-like structures; the cells underwent aberrant multicellular arrangement with a presentation of mid-differentiation markers throughout all the cell layers.

Conclusions: These results demonstrate that inadequate epimorphin elicits an abnormal differentiation response in keratinocytes and indicate a causal function of the epimorphin signaling gradient for the establishment of differentiated epidermal structure in the skin.

Quorum sensing in Zymomonas mobilis E. Stavrou and K. Pappas Department of Genetics and Biotechnology, Faculty of Biology, University of Athens, Athens, GREECE Introduction: Zymomonas mobilis is a bioethanol producing a-proteobacterium. Quorum sensing (QS) is directly related to high-cell density behavior of organisms and was thus investigated for Z. mobilis, as immediately relevant to its biotechnological nature. Methods: AHL quormone production in Z. mobilis CP4 was assayed using A. tumefaciens, C. violaceum and E. coli biosensors, by TLC and by HPLC/GC-MS (in collaboration with Dr A. Eberhard, Cornell University). Growth-dependent AHL production was tested by reporter strain Miller assays. AI-2 signal production was assayed by a V. harveyi lux sensor. Presence of QS-related genes in the Z. mobilis ZM4 genome was queried by in silico search for conserved-motif harbouring proteins and by BlastP-ing with 60 characterized QS-related proteins. Results: Z. mobilis does not likely engage in AI-2 signalling, as it failed to activate the relevant sensor, lacks a luxS homolog and possesses an SAH hydrolase homolog, indicating AI-2 precursor breakdown. Contrarily, Z. mobilis might produce AHL-type pheromones, since it activated agonistic sensors, inactivated antagonistic ones and tested positive for two putative AHLs in TLC experiments. HPLC/GC-MS supernatant extract analysis also revealed presence of 3-oxo-HSL species. Peak culture bioactivity was detected in early stationary phase and in rich medium growth, and was not affected by culture aeration.

In the CP4 genome, putative AHL synthase (HdtS) and AHL lactonase (AttM, AiiA) homologs were detected, amongst other less significant hits. Conclusions: Z. mobilis seems to engage in cell-cell signalling through release of AHL-putative compounds, whereas it does not appear to possess the AI-2 interbacterial signalling system. Introduction: Toposome, an abundant protein in the sea urchin egg and embryo, has been shown to mediate membrane-membrane interactions in a calcium-dependent manner. The effect of calcium is mediated through secondary and tertiary structural changes induced in toposome (1) . To further investigate toposome function, we have determined the subcellular localization of this protein in the egg and embryo.

Results: A polyclonal antibody was prepared against purified toposome and shown to be monospecific. When eggs were analysed by immunogold labelling, toposome was localized to the cortical and yolk granules as well as the plasma membrane. One-hour post fertilization, label was detected in the apical ECM, the hyaline layer, as well as the yolk granules and the plasma membrane. By 24 hours post fertilization, the hyaline layer and basal lamina ECMs were labelled as well as the apical and basal plasma membranes and the yolk granules. Similar labelling patterns were seen in gastrula and pluteus stage embryos: toposome was detected in both the hyaline layer and basal lamina, the yolk granules and the apical and basal plasma membranes.

Conclusions: These results clearly identify toposome as a protein appropriately distributed to engage in membrane-membrane interactions in the egg and developing embryo. While much progress has been made in learning how several junction proteins contribute to cell polarity, little is known about the contribution of lipids to cell polarization and cell adhesion. We use Drosophila as a system to study the role of lipids ((glyco)-sphingolipids and phospholipids) in the establishment of cell polarity and cell adhesion. We concentrate on the formation of septate junctions (SJ), which sustain the permeability diffusion barriers, and are found among epithelial cells and among glial cells that enwrap the nervous system. As a handle to the genetic study of lipids in this context, we investigate the contribution of lipid metabolism genes to the process of SJ formation. Methods: We are screening mutants in sphingolipid, glycosphingolipid, and phospholipid metabolism genes using an established dye penetration assay, to probe the 'tightness' of the epithelial permeability barrier. Moreover, we try to develop reagents to visualize different lipid subtypes, to characterize the subcellular distribution of different lipids in the polarised cells of Drosophila epithelia.

Results: We have compiled a list of mutants with disrupted barriers.

Conclusions: Several mutations in lipid metabolism genes present disrupted epithelial barriers. We assume that this disruption is caused by abnormal formation of the SJ. Further analysis of the molecular, cellular and ultrastructural defects of the mutants is required. Introduction: Hemodialysis patients (HD) are at the high risk of cardiovascular diseases (CVD) and oxidative stress is considered as a significant risk factor and the unifying mechanism for other cardiovascular factors. In this study, the prooxidant-antioxidant balance (PAB) before and after dialysis in HD patients and its correlation with other parameters were determined.

Methods: Sera of patients (n = 21), under regular HD, were collected before and after a HD session, and of a control group (n = 22). Prooxidant-antioxidant balance, along with clinical and biological parameters were measured.

Results: A significant increase of PAB value was observed in HD patients (before HD) as compared with healthy subjects. A significant increase of PAB value was noted in HD patients after dialysis. Also a significant correlation was observed for PAB values before and after dialysis. Before HD, a significant correlation was established between PAB value and fasting blood sugar, calcium and CRP. Also, a significant inverse correlation was established between PAB value and uric acid, serum creatinine, albumin and albumin/globulin ratio. This correlation was not obvious after HD. Prooxidant-antioxidant balance value, after dialysis, correlated significantly with serum iron concentration and inversely with alanine transaminase.

Conclusions: This study shows that the increased oxidative stress in HD patients, is not ameliorated by dialysis, and even more, it exposes HD patients to the risk of CVD. Prooxidant-antioxidant balance could be applied for devising strategies to reduce oxidative stress, to lessen or delay the progression and the complications of CVD in HD patients. Conclusions: SRIF and the sst 2 analogs, administered intravitreally, protect the retina from excitotoxicity. Further studies are essential to ascertain the therapeutic relevance of these results. Acknowledgements: Funded by GSRT (PENED) and Pythagoras.

Correlation of proxidant-antioxidant balance with platelet parameters in patients with diabetes type 2 Introduction: Altered platelet morphology and function have been reported in patients with diabetes mellitus. They are likely to be associated with the pathological processes, and increased risk of vascular disease (such as atherosclerosis) seen in these patients. It has been reported that platelets and prooxidants interact with each other in a positive feedback manner. However, according to the best of our knowledge; a direct correlation between oxidation stress and platelet parameters has hitherto not been established.

Methods: The aim of this study was to examine the correlation between prooxidant-antioxidant balance (PAB) and platelet parameters in patients with type 2 diabetes without vascular diseases. Prooxidant-antioxidant balance and platelet parameters such as platelet count (PC), mean platelet volume (MPV), plateletcrit (PCT) and platelet density width (PDW), along with other clinical and biological parameters were estimated in 61 consecutive type 2 diabetic patients and 62 age-matched healthy volunteers.

Results: The results were compared between the two groups and their correlations were determined. A significant positive correlation of PAB with PC, and PCT was observed. The mean PCs and PCT in the diabetic patients were significantly higher than the control group. However, both groups remained within the normal ranges. MPV and PWD did not significantly differ between the two groups.

Conclusions: The data from this study demonstrate a positive correlation between oxidative stress and PC for the first time. The implications of this observation in higher risk for thrombosis among diabetics and other patients with high oxidative stress merits further investigation.

Oxidative stress is significantly correlated to minor changes in hemoglobin concentration Results: A significant negative correlation of PAB with Hb was found in the diabetic patients and also in dialysis patients. A marginally significant negative correlation was found between PAB and Hb in healthy volunteers. PAB was significantly higher in diabetic and hemodialysis patients in comparison with healthy volunteers. Also, PAB was significantly higher in hemodialysis patients in comparison with diabetic patients.

The decreased Hb is associated with increased OS. Oxidative stress may be the third mechanism of anemia injury, which worsen the myocardial ischemic insult. This study suggests in anemia, in addition to Hb correction, the correction of OS should be taken into consideration that further research could be clarify the benefit of OS correction. Both the synthetic and catabolic pathways of polyamines are under restricted control to maintain the homeostasis. When cellular polyamine decreased, the synthetic mechanism will be stimulated. In case a specific type of polyamine exceeded or deficient, the interconversion reactions will be activated to interchange them from each other that provides another route to maintain the homeostasis. Despite of the ubiquitous polyamine synthetic and interconversion reaction, diamine oxidase (DAO), found abundantly in pregnant uterus and placenta, is also a possible pathway to catabolize polyamine.

Methods: We used real-time PCR to monitor the expression profiles of polyamine synthetic enzymes, such as onithine decarboxylase and S-AdoMet decarboxylase, metabolic regulator, such as antizyme, and catabolic enzymes, such as spermidine/spermine acetyltransferase (SSAT), polyamine oxidase (PAO), spermine oxidase (SMO) and DAO in mouse uterus, placenta and embryo at different pregnant stages. Results: Our data indicated that the expression-level of synthetic enzymes were high in embryo, placenta and uterus during E9.5E11.5. The expression of SSAT was also upregulated in this stages. On the other hand, the catabolic oxidases, PAO and SMO, were not induced. The expression of DAO was high in the early implantation but declined after the mid-stage of pregnancy in uterus and placenta.

Conclusions: During E9.5E11.5, the polyamine synthetic enzymes and SSAT both upregulated that suggest polyamines might be converted to acetyl polyamines, the export form. Since DAO is also a secreted enzyme, acetyl polyamine might be the substrates of DAO and their metabolites may play roles in pregnancy.

PP4-9 Apoptosis induced by an acidic deoxyribonuclease (DNase II) from Bothrops alternatus snake venom in MDCK cells Introduction: Snake venoms contain enzymes that degrade nucleic acids and their constituents. Acidic deoxyribonucleases (DNase II) have been implicated in DNA fragmentation during apoptosis in mammals. In this work, we used MDCK cells to investigate the apoptotic activity of DNase II purified from Bothrops alternatus snake venom.

Methods: DNase II was purified in five chromatographic steps. Cultured MDCK cells were incubated with B. alternatus DNase (>100 U/ ml) and cytotoxicity was assessed by neutral red uptake and lactate dehydrogenase release. Cellular morphology was assessed by the Feulgen reaction, toluidine blue staining and phase contrast microscopy. Cell death was quantified by flow cytometry using annexin V-FITC and propidium iodide. Caspase three, eight and nine activities were assayed spectrophotometrically, and caspase three, eight and nine, Bcl-2 and PARP protein expression was analyzed by western blotting.

Results: DNase II was cytotoxic to MDCK cells at concentrations >400 U/mL (>20 lg/mL) and produced morphological alterations that included cell shrinkage, swelling and detachment from the monolayer, nuclear condensation and/or fragmentation, and cytoplasmic vacuolization. There was also DNA fragmentation and an increase in the number of apoptotic cells and, to a lesser extent, necrotic cells. Caspases three, eight and nine were activated and PARP showed concentration-and time-dependent cleavage following incubation with DNase II. There was also a decrease in Bcl-2 expression. Introduction: Batten disease is a fatal neurodegenerative disorder caused by mutations in Cln3. The yeast model for Batten disease revealed that the disruption of Cln3 function induced a limitation in nitric oxide (NO) production. We proposed that a similar alteration may also be present in cells from patients possibly contributing to neuronal dysfunction 1 . To further investigate this hypothesis, we studied the Cln3 ex7/8 knock-in mouse, which harbour the most common disease-causing CLN3 mutation. Methods: Behavioural phenotyping protocols were applied in developing pups and young adult mice to determine whether Cln3 ex7/8 knock-in mouse recapitulate the major clinical features of the disease. Subsequently, the in vivo kinetics of NO production in the brain of these animals was quantified by means of electrochemical microsensors. Results: We detected behaviour anomalies early in the development. Specifically, Cln3 ex7/8 pre-weaning pups show delayed acquisition of neuronal reflexes. Furthermore, young adult animals present decreased levels of exploratory activity and marked motor deficits as early as 8 weeks of age. We measured the concentration dynamics of endogenous NO production in selected brain regions in response to NMDA stimulation and, apparently, the results point to altered production. Methods: Cells were cultured at 37°C (5% CO 2 ) in RPMI1640. Proteins were identified by immunoblotting.

Results: In wild type cells, proliferation proceeds for approximately 6-7 days, than progressively decreases becoming undetectable after 14 days. This growth arrest is accompanied by proteolytic degradation of several proteins directly involved in lymphocyte functions such as c and h PKC isozymes together with talin and Focal Adhesion Kinase (FAK), all recognized as calpain substrates. In cells overexpressing calpastatin, the rate of proliferation during the first 7 days is 40-50% reduced, and then proliferation proceeds without the enter into the G 0 stage. After 15 days, the rate of cell accumulation was 30% reduced. This loss of growth arrest was accompanied by an almost complete protection of PKC, talin and FAK. The requirement of calpain digestion in the entry of cells into the G 0 stage was further supported by similar effects on both cell growth and proteolytic degradation obtained following cell exposure to a synthetic calpain inhibitor. Conclusion: These results provide a direct demonstration that activation of calpain is required to exit from cell cycle and to enter a G 0 stage. Thus a balanced calpain-calpastatin ratio is required for the maintenance of a normal rate of cell accumulation. All the large primates encounter numerous DNA and RNA viruses but rarely suffer from any serious illness. Viruses also interact with each other directly. Viruses from the same species sometimes compete with each other, causing one to eventually succeed in the host cell and block infection by others. Viruses from heterologous species, can co-infect cells cooperatively. Viruses also interact with each other directly. In recent years, it has become evident that HIV-1 infected individuals when co-infected with either Herpesvirus 6 (HHV-6), HHV-7 or Hepatitis GB-C (GBV-C) may live three times longer as compared to those without these co-infections. Recent studies have proposed the interesting perspective that viral gene expression is downregulated by host micro-RNAs (miRNAs), small non-coding RNAs well known as post-transcriptional gene silencer. Recently, we have shown an inverse correlation between the numbers and relative degree of homology of miRNAs to the relative replication capacity of a specific retroviruses and various lentiviruses. In order to evaluate that miRNA-based silencing may be involved in HHV-6, -7 and GBV-C co-infections in HIV-1 infected individuals we computationally analyzed the miRNAs that have significant homologies to both HIV-1 and the co-infecting viruses. We discovered that miRNAs with over 80% homology to various targets in HHV-6, -7 and GBV-C genomes also mutually target HIV-1. We postulate that mutually targeting miRNAs serve to inhibit HIV-1 replication in individuals co-infected with these viruses. These results also suggest that miRNAs extensively mediate antiviral defenses in humans.

Wolbachia ankyrins and their potential role on Drosophila-Wolbachia symbiosis Wolbachia-host interactions range from many forms of reproductive parasitism to mutualistic symbioses. Despite their common occurrence and major effects on host biology, little is known on the molecular mechanisms that mediate Wolbachia-host interactions. The sequencing of the genomes of different Wolbachia strains (wMel, wRi) that infect insect hosts revealed an unusually large number of genes encoding proteins with ankyrin repeat domains. We are investigating the role of these proteins. Methods: The distribution of ankyrin genes in different Wolbachia strains was determined by PCR and Southern analysis. Semi-quantitative RT-PCR analysis was also performed on RNA extracts from different developmental stages and tissues. Selected ank-proteins were expressed transiently in insect cells as GFP chimeras and their subcellular localization was analysed by confocal microscopy. Introduction: The most distinctive feature of the dimorphic pathogenic fungus Paracoccidioides brasiliensis is the multiple budding phenotype of its yeast form, a phenomenon that still remains unclear. This work focuses on the P. brasiliensis homolog to the Rho-like GTPase Cdc42p (Pbcdc42p), a protein implicated in the development of multiple buds in Saccharomyces cerevisiae, and its relevance in controlling cellular morphology and virulence. Methods: We have isolated PbCDC42, functionally complemented S. cerevisiae Dcdc42 cells, and evaluated cellular morphology and bud scar patterns. PbCDC42's expression was down-regulated in P. brasiliensis yeast cells using antisense-RNA (aRNA) and several parameters were evaluated: cell size, viability, and morphological transition. In vitro and in vivo assays were performed using either macrophages to analyze phagocytosis or C57BL/6 mice to compare the virulence of the wild-type and mutant strains upon intravenous infection. Results: Expression of PbCDC42's expression in S. cerevisiae Dcdc42 cells induced altered cellular morphology and deregulation of the temporal/spatial control of bud emergence. In P. brasiliensis, PbCDC42-aRNA cells were smaller with an increased homogeneity of cell size and viability. Moreover, transition from yeast-to-mycelia was delayed without alterations in mycelial growth. Regarding in vitro and in vivo infection, knock-down of PbCDC42 led to increased phagocytosis and survival. Conclusions: Overall, our data indicates that Pbcdc42p has an important role in controlling cellular morphology and seems to present itself as a relevant virulence factor, suggesting that P. brasiliensis wild-type morphology is important for pathogenesis. The etiological role of high-risk human papillomaviruses (HPV) in preinvasive and invasive cervical cancer has been demonstrated by epidemiological and molecular studies. There is compelling evidence that during carcinogenesis viral oncogenes altered cell cycle progression. On the other hands, cell cycle depends on the sequential activation of a series of cyclins. The aim of this study is to investigate the expression of three cyclins in swabs harvested from patients presenting cervical preneoplasic lesions. Methods: From a 268 cohort of HPV positive women, we selected 52 patients (24-48 years) with mild and severe dysplasia and presenting only HPV16 type (in InnoLipa) and eight patients without HPV infection and normal cytology were used as negative control. The liquidbased cervical specimens of the selected patients were subjected to immunocytochemical and molecular techniques for cyclin A, D and E expression and for viral E6/E7oncogene mRNA expression. Results: Marked cyclin D1 immunoreactivity was found in 16% of HGSIL cases as compared with LGSIL lesions (9%) while cyclin E immunoreactivity presented a significant increase from normal to HGSIL lesions. Increased cyclin E expression induces a proliferative effect and might be correlated to progression through the cell cycle. Cyclin A expression was also increasing with the lesions' severity but also with the viral mRNAs levels.

Conclusions: Cyclin A expression and cyclin E immunoreactivity correlate strongly with viral mRNA expression and lesions' severity. Cyclin D1 immunoreactivity was moderately elevated and no significant correlation with morphologic features in liquid-based specimens and viral mRNA was noted. They annually cause clinical illness in 300-500 million people with 1.5-2.7 million deaths, mainly caused by Plasmodium falciparum. The rapid multiplication of P. falciparum within human erythrocytes is directly associated with its pathogenesis. We have identified a new metabolic pathway (SDPM) in P. falciparum that links hemoglobin catabolism and amino acid transport to membrane biogenesis and parasite cellularization 1-6 . A key step in this pathway is controlled by a phosphoethanolamine methyltransferase, PfPMT, a member of a family of methyl-transferases only found in plants, worms and protozoa. Results: Our studies demonstrated that PfPMT plays an essential function in parasite's replication, cellularization and sexual differentiation.

We have developed an enzyme-coupled colorimetric assay to screen for compounds that inhibit PfPMT activity and block parasite infection and transmission. In addition to the phosphocholine analog miltefosine, two new compounds amodiaquin and hexadecyltrimethylammonium known for their antimalarial activity were found to inhibit PfPMT activity. The trophoblastic cell represents the main functional unit of the placenta. It proliferates, migrates, and invades the maternal tissue in a way that is similar to malignant tumors. Nevertheless, these processes are tightly controlled by stringent spatial and temporal confines. Therefore, the trophoblastic cell, as 'a well-behaved tumor', represents an ideal model system to investigate several cellular processes. Infection with genital human papillomaviruses (HPV) represents the most common sexual transmitted infection in fertile women. HPV are small circular double stranded DNA viruses, which comprise open reading frames gathered in a regulatory early (E) and structural late (L) regions. Infection with HPV-16 is the necessary cause of over 99% of human cervical cancers. However, several studies report that HPV viruses could infect trophoblasts during pregnancies. Surprisingly, HPV can replicate in vitro in trophoblasts. Higher HPV infection frequency has been reported to be associated with some spontaneous abortion and gestational trophoblastic diseases. In this report, we have studied the impacts of HPV-16 early proteins on the growth, cytotoxicity, adhesiveness, differentiation and migration of trophoblastic cells. Our results showed that hydrophobic E5 protein is localized in many interne membranes compartments of the trophoblast. E5 could increase cell growth, meanwhile, it seemed cytotoxic. However, the presence of E5, E6 and E7 could reverse the cytotoxic effects of E5. In addition, E5 decreased the ability of the trophoblastic cells to attach to the support and to the endometrial cells. These observations suggest that E5 could modify the adhesiveness of the trophoblastic cells. Introduction: Phosphothreonine lyases are a recently described class of enzyme that use an elimination reaction to cleave phosphate from phosphothreonine of the pThr-X-pTyr motif of MAP kinases such as ERK1/2, p38 and JNK, leading to a dehydrobutyrine (Dhb) residue. Therefore, the post-translational modification that results from phosphothreonine lyase activity, unlike phosphatase activity, does not allow rephosphorylation on the same residue. Several pathogenic bacteria, including Salmonella and Shigella, inject phosphothreonine lyases into the host cell through a type III secretion system. This disrupts immune system signaling and allows bacterial virulence. Results: A 1.4 Å structure of the Chromobacterium violaceum VirA phosphothreonine lyase was determined by X-ray crystallography using a selenomethionine derivative by MAD (Multiwavelength Anomalous Diffraction). It adopts a fold that is similar to the recently published Salmonella SpvC structures. The VirA phosphothreonine lyase is catalytically activity against phosphorylated ERK2 protein and phosphopeptide as demonstrated by both western blot and phosphate release assay. Conclusion: A phospho-mimetic sulphate reveals the active site. Lys136 is positioned to act as a catalytic base in the b-elimination reaction. A catalytic diad of Asp201 and His106, would act as a general acid to protonate the leaving phosphate. Catalytically active VirA is consistent with a functional Chromobacterium violaceum type III secretion system. The lack of Arg90 suggests that VirA, unlike SpvC, may not be specific for dual phosphorylated MAPK. Results: Soil application of R. solani alone and B. licheniformis in combination with R. solani induced a significant increase in the activities of b-1,3-glucanase and phenylalanine ammonia-lyase (PAL). Peroxidase activity in both fungal and bacterial treatments was also significantly stimulated. In contrast, peroxidase activity was reduced upon inoculation of plants with a mixture of B. licheniformis-R. solani. Conclusions: Results indicated that generally transcripts of defencerelated genes as well as some enzymatic activities were induced in all treatments, including that with Bacillus spp. alone.

Detection of humoral responses to the hepatitis C virus core+1 protein in patients with HCVassociated hepatocellular carcinoma G. Dalagiorgou 1 , N. Vasilaki 1 , A. Kakkanas 1 , E. Aslanoglou 1 , D. Vassilopoulos 2 , S. Hadziyiannis 2 and P. Mavromara 1 1 Molecular Virology Laboratory, Hellenic Pasteur Institute, Athens, GREECE, 2 Academic Department of Internal Medicine, Hippocration General Hospital Athens, Athens, GREECE Introduction: The hepatitis C virus (HCV) is a major cause of acute and chronic liver diseases which frequently lead to liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC). The viral genome possesses a reading frame overlapping the core gene at +1 nucleotide (core+1 ORF) that recently was reported to be translated by alternative mechanisms into proteins designated core+1/ARFP/F. Converging data from several laboratories support that the core+1 ORF stimulates specific B-cell and T-cell immune responses in HCV-infected patients, suggesting that it is expressed during natural infection. The aim of this study is to characterize the prevalence of circulating antibodies against core+1 protein in HCV infected patients during different stages of disease. Methods: The HCV core+1 ORF of genotypes 1a and 1b, were cloned and expressed in pET 20(+) prokaryotic expression vector, which allows fusion of a foreign protein with a 6X His cassette at the carboxy-terminus. Also, peptides corresponding to different regions of the core+1 protein were synthesized. Both purified recombinant proteins and peptides were used for the development of home-made enzyme-linked immunosorbent assays. Results-Conclusion: Firstly a sera panel of patients with HCV-associated hepatocellular carcinoma (HCC). Sera from healthy individuals were used as negative control. Our results demonstrate a statistically important higher prevalence of anti-core+1 antibodies in sera from HCV-HCC patients as compared to chronically infected HCV patients suggesting a role of core+1 in advanced liver disease and cancer. Methods: To clarify the role of HuR in innate responses we generated a system for its innate ablation based on gene-targeting and Cre/loxP mediated recombination. Using this system we study: (a) HuR dependent in vivo cellular responses in myeloid ontogeny, (b) responses to innate sensitizers in vivo and (c) gene expression assays in ex-vivo cultures. Results: We demonstrate that mutant animals show partial defects in myelopoiesis due to defects in CSF-signals driving myeloid differentiation. Furthermore, we demonstrate that HuR deficient macrophages display exacerbated cytokine production profiles in response to TLR ligands relating to altered translational outputs. Similarly, mutant mice appear more sensitive to models of systemic and organ specific acute inflammatory assault. In addition, we reveal interfere with the SAPKmediated intracellular pathways modulating cytokine biosynthesis. Currently, we are analysing the potential interplays between HuR and other ARE-binding proteins of relevance to innate activation. Conclusion: Our data suggest that HuR acts in a complex yet tissue specific fashion to modulate differentiation signals and inflammatory gene expression towards myelopoiesis and inflammation. Dermatophytes are a group of morphologically and physiologically similar filamentous fungi that have the capacity of invading keratinized human and animal tissues. Cysteine proteinases are widespread in nature and have different physiologic functions. Their activity is regulated by different mechanisms of which protein inhibitors are of great importance. To our knowledge, no protein protease inhibitor from dermatophyte fungy has been reported so far. The inhibitor was isolated from mechanically and ultrasonically disintegrated fungal mycelia using the affinity chromatography, ion exchange chromatography and reverse phase HPLC. The isolated inhibitor was analyzed by SDS-PAGE and Western blot analysis. The inhibitory properties against different proteinases have been determined. The enzymatically prepared peptide fragments were separated by HPLC and sequenced, using automated Edman degradation method. The obtained sequences were compared to non-redundant protein sequence databases using blastp algorithm at NCBI. It has been proven that the isolated protein inhibits proteolytic activity of papain, cathepsin B, but not cathepsin H. SDS-PAGE analysis of inhibitor preparation showed bands at 12, 24 and 48 kDa, suggesting multimeric composition of the protein. So far no sequence homology has been found between the partial protein sequence of T. mentagrophytes inhibitor and any known cysteine protease inhibitor, implying its structural uniqueness.

The neutrophil-activating protein of Helicobacter pylori promotes myeloperoxidase release from human netruophils Helicobacter pylori (H. pylori) is closely associated with chronic gastritis, peptic ulcer disease, and gastric adenocarcinoma and lymphoma.

Helicobacter pylori neutrophil-activating protein (HP-NAP) is an important virulence factor of H. pylori for its pathogenesis. To investigate how HP-NAP activates human neutrophils, HP-NAP was expressed in Escherichia coli (E.coli), purified by the two sequential gel filtration chromatographies, and then subjected to filtration for the removal of endotoxin. The recombinant HP-NAP was able to stimulate human neutrophils to produce reactive oxygen species (ROS) as examined by luminol-enhanced chemiluminescent assay. HP-NAP can also promote the secretion of myeloperoxidase (MPO) in human neutrophils. The finding of this new function of HP-NAP in human neutrophils suggests that there should be more host immune responses of HP-NAP awaited to be explored.

The obligate intracellular parasite Toxoplasma gondii secretes a soluble phosphatidylserine decarboxylase Introduction: Leishmaniasis is a group of diseases caused by protozoan parasite Leishmania. The disease affects over 12 million people in 88 countries. DNA fragmentation was observed in the parasite or the host cells or both. The aim of the present work was to assess DNA damage induced in mice skin cells.

Methods: BALB/c mice were randomly divided into two groups as followed: control and experimental groups that were infected with 3 · 10 6 stationary phase L. major promastigotes. Seven mice in each group were killed after 20, 40, 60, 90 and 120 days of injection. Total DNA was extracted from skin cells and compared with DNA from control cells using agarose gel electrophoresis and diphenylamine reaction.

The results show that until 40 days of injection, the cells infected with Leishmania did not induce any DNA damage. The presence of denatured DNA was revealed, as a prominent smear DNA fragmentation after 60, 90 and 120 days of injection. Quantitative DNA fragmentation analysis of cells showed produce only 12-15% of DNA fragmentation in compared to the control (9%).

The data suggest that DNA damage in Leishmania infected cells is time-dependent and provide possibilities of existence of apoptotic pathway in Leishmania infected cells. However, the present data does not stand to clearly confirm to situation and further investigation need be carried out. Methods: BALB/c mice were randomly divided into two groups as followed: control and experimental groups that were infected with 3 · 10 6 stationary phase L. major promastigotes. Seven mice in each group were killed after 20, 40, 60, 90 and 120 days of injection. The spleen of each groups were removed and the enzyme activities of SOD and CAT as well as GSH and MDA levels were determined by spectrophotometric assays.

Results: The results of show that the increased SOD activity was not accompanied any change in CAT activity after 60, 90 and 120 days of injection. The depletion of GSH content and an increased MDA level were observed after 90 days of injection.

Conclusions: The data suggest that the antioxidant defense systems in Leishmania infected spleen cells are significantly altered. The depletion of GSH level and the increased lipid peroxidation induce oxidative stress and cell death.

Translation mediated by the internal ribosome entry site (IRES), of the hepatitis C virus (HCV) genomic RNA, is regulated positively by the phosphorylation of the eukaryotic translation initiation factor 2a (eIF2a)

Introduction: The single-stranded positive-sense RNA genome of HCV, is translated by a cap-independent mechanism, that evolves an IRES, that apart of the eIF2-GTP-Met-tRNA i complex and eIF3 does not require other canonical eIFs to initiate translation. This study investigated the effect of the eIF2a phosphorylation status on HCV IRES.

Methods: The effect of the over-expression of eIF2a or a non-phosphorylatable eIF2a (muteIF2a) on HCV IRES, EMCV IRES and capdependent translation was studied in co-transfection experiments using a bi-cistronic expression vector or a replication-defective HCV-replicon expressing plasmid. In addition endoplasmic reticulum (ER) stress conditions were applied, to investigate the effect of the elevated levels of eIF2a phosphorylation.

The over-expression of eIF2a or muteIF2a, down-regulated HCV IRES activity whereas, cap-and EMCV IRES-dependent translation were stimulated. However, ER stress resulted in a strong activation of HCV IRES while no or negative effect was observed on cap-and EMCV IRES-dependent translation, respectively. Moreover, under stress conditions the ratio of HCV IRES-dependent to cap-or EMCV IRES-dependent translation increased by eIF2a over-expression as compared to muteIF2a overexpression. These results suggest that elevated levels of un-phosphorylated eIF2a inhibit HCV IRES function and indicate that HCV IRES requires the phosphorylation of eIF2a subunit. This is in contrast to cap-and EMCV IRES-dependent translation initiation.

Bactericidal activity of synthetic human betadefensin 1 against different bacterial species Introduction: Human beta-defensin 1 (hBD-1) is a 36-amino acid cationic peptide of the innate immune system that serves as anti-microbial molecule. The objective of the study was to determine the bactericidal activity of synthetic hBD-1 against different bacterial species with regard to their anti-microbial resistance phenotypes.

Methods: Ninety-one nosocomial pathogens belonging to eight different bacterial species were exposed to different concentrations of HBD-1 that was prepared synthetically, purified and in vitro oxidized. Results: The native disulfide connectivity was found essential for the bactericidal activity of hBD-1, while sodium chloride concentration was reversely associated with its potency. hBD-1 exhibited high bactericidal activity against Acinetobacter baumannii, Pseudomonas aeruginosa, Enterococcus faecalis, Enterococcus faecium and Staphylococcus aureus clinical isolates (the concentration of 10 lg/ml of hBD-1 was lethal for > 99% of the bacterial cells). Characteristically, A. baumannii isolates that exhibited multidrug-resistant phenotypes were susceptible to the lowest concentrations of hBD-1 [demonstrarting a virtual 90% lethal dose (vLD 90 ) < 4.5 lg/ml]. Bactericidal activity of hBD-1 was less pronounced against E. coli, K. pneumoniae and P. mirabils isolates (the vLD 90 varied from 5 to 50 lg/ml of hBD-1) but was significantly enhanced against isolates of these species that exhibited resistance to amoxicillin/clavulanate, aztreonam and cephalosporins (vLD 90 £ 10 lg/ ml of HBD-1).

Conclusions: These observations suggest that the natural hBD-1 has a potential therapeutic role against bacterial pathogens and particularly against those exhibiting multidrug-resistant phenotypes. 

The binding and activation of the host kiningenerating system on the cell walls of pathogenic Methods: The binding of proteins comprising the host kinin-generation system, including factor XII, prekalikrein and high molecular weight kininogen (HK), to the surface of Candida spp. in both the yeast and filamentous forms was analyzed by the ligand-binding microplate assay and ligand-blotting. A release of kinin-like peptides was detected with a specific immunochemical kit.

Results: All components of the kinin-generating system were shown to adhere to the Candida cell walls, with the following order of binding strength: factor XII > HK > prekalikrein. The pathogen surfacemediated activation of factor XII triggered the kinin release. Additionally, the fungal secretory proteinases were shown to posses the kininogenase activity, i.e. they were able to release kinin-like peptides from HK.

Conclusions: Candida fungi can trigger the kinin production at the site of infection by the activation of host's own kinin-generating cascade or by direct action of fungal proteolytic enzymes on host kininogen.

Antimicrobial properties and novel designs of analogs of pleurocidin isolated from winter flounder J. Lee, W. S. Sung and D. G. Lee School of Life Sciences and Biotechnology, College of Natural Sciences, Kyungpook National University, Daegu, REPUBLIC OF KOREA Pleurocidin (Ple) is a 25-residue peptide derived from the skin mucous secretion of the winter flounder, Pleuronectes americanus. In this study, we investigated antimicrobial properties of Ple and its enantiomers. Ple showed potent antimicrobial activities by disrupting membranes without high hemolysis. To develop this peptide as novel therapeutic agents, we designed analogs of which a net hydrophobicity was decreased. The analogs were synthesized by arginine or serine substitution at positions 1, 3, 12, 19 or 21 at the hydrophobic face of Ple without changing its amphipathic structure. The result exhibited that the analogs maintained antimicrobial activities and abilities for permeabilizing phospholipids membranes without hemolysis. In succession we designed a synthetic enantiomeric peptide composed of all-D-amino acids to enhance a proteolytic resistance. The proteolytic resistance was confirmed by HPLC with the digestion of proteases. To investigate the antibiotic effect of L-and D-Ple, an antibacterial activity and hemolytic effect were tested. D-Ple showed a decreased antibacterial activity and a dramatically decreased hemolytic activity compared with L-Ple. The hemolytic effect of analog was further confirmed by using calcein leakage measurement with liposome. To elucidate these results, the secondary structure of the peptides was investigated by circular dichroism (CD) spectroscopy. The results revealed that D-Ple, as well as L-Ple, had typical alpha helical structures which were mirror images, with a different helicity. These results suggested that the discrepancy of the structure between the two peptides made their antibacterial activity distinct. Rev is a small regulatory protein of HIV-1 essential for virus replication. Above all, Rev (35-50) is known for a nuclear localization signal (NLS) and requisite for shuttling between the nucleus and the cytoplasm in the host cell. In this study, we investigated the antimicrobial effects of Rev-NLS toward apoptosis of fungal cells and suggested the possibility as a therapeutic agent by synthesizing D-Rev-NLS. First of all, Rev-NLS had potent antimicrobial activities in energy-and saltindependent manners without hemolytic effect and accumulated in the nucleus of C. albicans without membrane damage after binding to microbial cell wall. Also we found out that Rev-NLS influenced on the cell cycle arrest at S phase as a result that it bound to chromosomal DNA. The result of fluorescence study indicated that tryptophan is the key for interacting with cell membrane to penetrate into the nucleus. Subsequently we investigated phosphatidylserine (PS) translocation caused by apoptosis followed above results. The result exhibited that Rev-NLS induced an early apoptosis of C. albicans. Finally to aim for which was the application of therapeutic agents, we designed a synthetic enantiomeric analog peptide composed of all-D-amino acids to enhance a resistance to proteases. D-Rev-NLS displayed 2-fold more potent antimicrobial activity than that of L-Rev-NLS, and it also showed a proteolytic resistance against various proteases.

Interactions between proteins of the symbiotic polydnavirus CcBV of the endoparasitic wasp Cotesia congregata and proteins of the NF-jB pathway in insect and mammalian cell lines The aim of this study was to investigate the virulence and pathogenicity hallmarks of Escherichia coli isolated from hospital surfaces. Material and methods: One hundred Enterobacteriaceae strains belonging in majority to E. coli, followed by Klebsiella oxytoca, K. pneumoniae) and Enterobacter cloacae species isolated from surfaces in the hospital environment were identified both by biochemical and serological tests. The virulence tested features were: adherence and invasion capacity on HeLa cells by Cravioto adapted method, adherence on inert substrata quantified by slime test, production of extracellular enzymes and exotoxins (haemolysins and other pore-forming toxins, amylase, mucinase, gelatinase, caseinase, aesculin hydrolysis).

Results and dicussion: The tested strains exhibited different virulence patterns varying with the source of isolation, but also with the taxonomic affiliation. 45% of the enterobacterial strains isolated from hospital surfaces exhibited adherence ability to the cellular substrate, with aggregative (24%) and respectively, diffuse (21%) patterns and 87% of the same strains colonized the inert substrate, as demonstrated by the positiv slime test. 82% of the tested strains produced alpha-heamolysis and 8% bet-haemolysis. As concerning the other soluble enzymatic virulence factors, 66% produced mucinase, 13% DN-ase, 39% caseinase, a reduced number of strains produced lipase (8%), gelatinase (7%) and lecithinase (4%).

The search for coding sequence of glycine-rich proteins (GRP) identified in Chelidonium majus L. . The goal of present study was to determine the coding sequence of glycine-rich proteins (GRPs), which possess RNA-binding properties, are accumulated in the vascular tissues and act in defence mechanisms of the plant. Methods: Genomic DNA was isolated using DNeasy Plant Mini Kit (Qiagen) from 14-day old Ch. majus seedlings. To obtain portion of GRP gene, PCR was carried out using degenerade primers GRP1 (5¢-TGYTTYGTNGGNGGNCTNG-3¢) and GRP4 (5¢-CCNCCNCCRTANCC-3¢), which were designed based on highly conserved regions of GRP [2] . PCR products of about 800 bp were cloned using pGEM-T Easy vector (Promega) and sequenced. Results: Obtained sequences were analysed using NCBI VecScreen, BLASTn and BLASTp tools (www.ncbi.nlm.nih.gov). DNA fragment of 375 bp showed high sequence identity with glycine-rich RNA-binding protein (GRP) mRNA sequence from Glycine max for nucleotide alignment, with 73% max. identity, 154 total score and E-value of 2e-34, and putative glycine rich protein from Rumex obtusifolius for protein alignment, with 125 total score and E-value of 1e-27. Moreover, the sequence covers highly conserved region named RNA recognition motif, which is probably diagnostic of an RNA binding protein.

Conclusions: Results of this study show, that it was possible to determine a highly conserved part of GRP protein's gene from Ch. majus. Results and Discussion: We show here that soluble ACT inserts into plasma membrane microdomains, with independence of the presence or not of its integrin receptor, and induces a calcium influx, through the activation of several types of calcium-channels, that activates integrins and form unadhesive ACT-integrin complexes. The impairment of the adhesive properties of immune cells seems an intelligent way to be selected by any pathogen to modulate the host immune response in its own profit.

Helicobacter pylori CagA protein phosphorylation in early infection stages of gastric epithelial cells coincides with FAK dephosphorylation and is compensated by increased Pyk2 activity Introduction: Helicobacter pylori (Hp) CagA protein has been proposed to deregulate key signaling elements in gastric epithelial cells following its phosphorylation on repeating EPIYA motifs (A:EPIYAKVNK, B:EPIYAQVAKK, C:EPIYATIDDLG). We describe herein, the effect of CagA phosphorylation on focal adhesion kinase (FAK) and Pyk2 activation following infection of gastric epithelial cells with Hp clinical strains isolated from gastric biopsies. Methods: CagA, FAK, Pyk2 expression was detected in total lysates of AGS cells infected over 36 hours with pairs of naturally occurring isogenic Hp strains expressing CagA protein with two or three EPIYA motifs (AB-ABC), three or five (ABC-ABCCC) and two pairs with three or four motifs (ABC-ABCC). Tyrosine phosphorylated proteins were detected by western blotting following immunoprecipitation with antiphosphotyrosine antibody. Levels of activated pFAK were also evaluated by a specific antibody against pTyr397-FAK autophosphorylation site. Results: Following Hp infection of AGS cells in vitro, we observed that CagA species harbouring more EPIYA-C motifs exhibited higher levels and rates of tyrosine phosphorylation. Infection with all Hp strains induced higher levels of Y-397 FAK dephosphorylation with total phosphotyrosine levels of FAK remaining unaffected. Concomitantly, upon Hp infection we observed a time-dependent increase in phosphotyrosine Pyk2 levels and an increased rate of Pyk2 degradation products. Conclusions: Hp infection during its initial stages seems to deregulate signalling events linked to the integrin pathway, such as FAK activation. We have observed that CagA EPIYA-C time-dependent phosphorylation is accompanied by reduced pY397-FAK autophosphorylation and concomitant increase in Pyk2 activation, probably due to reduced FAK activation.

Modulation of liver specific gene promoters by the HCV core and core+1/s proteins G. Pissas, P. N. Foka, N. Vassilaki, A. Kakkanas and P. Mavromara Molecular Virology Laboratory, Hellenic Pasteur Institute, Athens, GREECE Introduction: Worldwide, about 300 million individuals are infected by the hepatitis C virus (HCV) and three quarters are suffering from persistent HCV infection, often leading to development of liver cirrhosis and hepatocellular carcinoma. However, the mechanism underlying this process is still unclear. HCV is a positive strand RNA virus within the Flaviviridae family, its genome spanning approximately 9600 nucleotides and encodes a single polyprotein of about 3thinsp;000 amino acids, processed via host cell and viral proteases into at least 10 structural and non-structural proteins. Recent studies from our laboratory and others have accumulated strong evidence for the existence of a novel protein of hepatitis C virus encoded by an ORF overlapping the core gene in the +1 frame (core+1 ORF). However, today the biological importance of this protein remains unknown. Methods: In this study, we examined the effect of core+1/S on chimeric (EalbpAT-1a, EIIpAT-1a) and non-chimeric (pAlb, pAT-1a) human liver specific promoters, given that both proteins, albumin and anti-trypsin, are produced exclusively in the liver and chronic liver disease results to a significant decrease in their expression levels. CMV promoter was used as control. Series of transient co-transfections using the annotated constructs, along with HCV core and HCV core+1 expressing constructs, were carried out into cultured cells of hepatic and non-hepatic origin. Results and conclusions: The presence of core+1/S exhibited a significant reduction in the levels of expression of the luciferase reporter gene, under EalbpAT-1a, EIIpAT-1a, pAT-1a and pAlb promoters, suggesting a putative direct, or indirect interaction of core+1/S with one, or more transcription factors associated with the albumin and anti-trypsin promoters. Results: Regarding the cytological diagnosis, HPV was detected in 75.5% from normal/inflammatory samples, in 87.5% ASCUS, 70.37% LSIL and 90.9% HSIL, which demonstrated that HPV infections are frequent. We noted that the predominant genotypes in patients were HPV16 (56%) in association with other high risk (HPV18, 51, 31). P16 immunoreactivity was markedly noted in all integrated form (38/52 HPV16 cases) LSIL or HSIL but only in two cases presenting ASCUS/ inflammatory cytology. P16 mRNA levels correlates with protein expression determined on cells isolated from swabs. Conclusions: Integration of human papillomavirus DNA into the host genome is mainly found in high-grade dysplastic lesions and points to an increased progression potential. Significant pl6 overexpression was observed in the HSIL group, over-expression of p16 acting as potential biomarker for cervical cancer progression from premalign lesions.

Influence of antagonistic microorganisms treatment on the antioxidant system of plant cells The present study focuses on the evaluation of the effects induced by some nonpathogenic bacterial strains on the antioxidant system of cucumber plants. Methods: Cucumber seeds were sterilized by exposure to 1% sodium hypocloride. Seeds were immersed for 12 hours in bacterial cultures of Bacillus licheniformis, strains B40, mB40, tB40 and Pseudomonas spp strains P7, P14, P18. Subsequently, seeds were germinated and grown in sterile conditions. Five days after the infection of seedlings with mycelium of Pythium debaryanum, the treated plants were harvested for total protein extraction. Enzymatic activity of superoxide dismutase (SOD), peroxidase (POX), catalase (CAT), total proteins and electrophoretic patterns of the isoenzymes were performed. Results: Both bacterial and fungal exposure induced an increase of SOD activity. Electrophoretic pattern does not emphasize changes in SOD isoforms. POX activity increases in all experimental pathogen infected trials. P14 and P18 colonization presented a similar effect on POX as fungal pathogen. Pathogen and bacteria induced changes in the peroxidase isoforms expression. Fungal infection determined a mild increase of CAT, although in P7 colonized plants CAT presented a higher activity and different isoforms. Conclusions: Plants colonization with beneficial bacterial strains induced in the first stage changes of the antioxidant system similar to those produced by fungal attack, and allowed a good protection against plant fungal pathogens. Methods: Five lectin preparations obtained by affinity chromatography interacted with liquid cultures of Salmonella typhymurium. The positive results of interaction were evaluated by the degree of bacteria agglutination. The most effective lectin was that isolated from potato shoots. Animal experiment consisted in four trials: control, lectin treated but not infected, infected with Salmonella, infected and lectin treated. Lectin was administered to three-days-old chicken, in the drinking water.

Results: Lectin administration induced an increase of the reactivity of the gut associated lymphoid tissue both in duodenum and caeca in non infected chickens, as compared to control group. Salmonella infected and lectin treated animals presented a remarkable epithelial integrity as compared with infected but not treated ones, that presented large tissue necrosis area. The number of heterophiles increased in all lectin administered chickens.

Conclusions: Orally administered lectin induced an immune system activation and also prevented the bacterial adhesion to the intestinal mucosa. We have shown previously that two Anopheles gambiae C-type lectins (CTLs), CTL4 and CTLMA2, block the melanization of Plasmodium berghei ookinetes in the mosquito midgut, hence promoting successful parasite development (1) . Here, we show that these lectins also play an important role in mosquito immunity against bacteria. Methods and Results: Functional genetic analysis of CTL4 and CTLMA2 was performed using in vivo RNAi. Adult mosquitoes silenced for CTL4 or CTLMA2 were more susceptible to infections by Escherichia coli but not Staphylococcus aureus. These mosquitoes exhibited a dramatic proliferation of E. coli relative to wild-type controls suggesting indeed a role for these lectins in controlling proliferation of gram-negative bacteria. Western blot analysis revealed that CTL4 and CTLMA2 are secreted into the mosquito's hemolymph and exist predominantly as a heterodimer. Interestingly, silencing either lectin triggered the loss of the other protein from the hemolymph, although no cross-silencing was observed at the mRNA level, suggesting the involvement of a potential post-transcriptional regulatory mechanism.

Conclusions: Taken together, we show that, in addition to their role in blocking Plasmodium ookinete melanisation, CTL4 and CTLMA2 are also involved in A. gambiae defence against gram-negative bacteria.

Since the role of CTLs in vertebrate immunity is well established, our data suggest that CTL-mediated defence constitute an ancient strategy in metazoan immunity. Here, we focused on evaluating the secretion role of the three motifs, which include 24 highly conserved residues. The mutant constructs were subjected to cell fractionation, and the protein secretion levels in the culture medium, outer membrane, periplasm, and on the cell surface were assessed by immunochemical techniques.

Results: When compared to the wild-type, more than half of the mutants exhibited various secretion defective phenotypes, including cell lysis, diminished secretion into the culture medium, and protein accumulation in various compartments of the cell envelope.

Conclusions: Our data suggest the function of these residues in various steps of Tsh's passenger secretion process, including barrel insertion, outer-membrane translocation, and processing. The results hence contribute to a better understanding of the auto-transporter protein secretion mechanism.

Functional annotation of ORF6, a 'pig' gene from Pseudomonas syringae B728a identified many Pss-B728a genes induced during the epiphytic phase (pigs), among them orf6 (Psyr-1182), located in the hrp/hrc pathogenicity island (PAI) encoding the T3SS. Although orf6 appears not to belong to the hrp-regulon, its location suggested a possible role in pathogenesis. We examined the ability of orf6 to elicit the plant hypersensitive response (HR) and annotated bioinformatically its putative homologues in Pseudomonas. Introduction: A major goal in plant science is the production of crops with increased and durable resistance to a wide spectrum of diseases. Engineering resistance for agricultural improvement incorporates diverse strategies to mitigate the crop losses imposed by pathogens. A promising approach to engineering disease resistance is to express a pathogen component in the plant that the plant can recognize. Recognition of these elicitor molecules then leads to the activation of a full defense response that is sufficient to restrict pathogen growth. Methods: Transgenic Arabidopsis plants were inoculated 3 hours after induction with b-estradiol. Bacterial multiplication in leaves was examined by cutting tissue from the inoculation sites, homogenization in 10 mM MgCl 2 and serial dilutions of the homogenate. Interacting Arabidopsis proteins were identified using as bait the HrpA protein in a yeast two hybrid screen against an Arabidopsis library. Results: Use of the gene encoding the Hrp pilus subunit from Pseudomonas syringae pv. tomato strain DC3000, as a transgene in Arabidopsis thaliana, showed elevated levels of resistance towards the bacterial speck disease, indicating that the signal transduction pathway leading to the expression of disease had been altered. The yeast two hybrid approach identified Arabidopsis proteins that have putative roles in defense responses.

Conclusions: In conclusion, in planta expression of the hrpA gene in A. thaliana altered the phenotypic response, after inoculation with the bacterial strain DC3000 of P. s. pv. tomato. Elevated levels of resistance appear to be specific to bacterial speck indicating a possible new role for HrpA.

Role of Burkholderia pseudomallei rpoS in regulation of kat G and dpsA genes via an oxidative regulator, oxyR Introduction: Burkholderia pseudomallei is a causative of melioidosis. Recent study showed that they survive intracellular in both non-professional phagocytes and professional phagocytes. One of the strategies intracellular bacteria use to protect an oxidative stress is to express a number of anti-oxidant enzymes and repair activities. Much of this inducible resistance depends on the oxyR gene, which governs a set of genes that constitute the oxyR regulon. To date, the possible transcriptional regulation and the mechanisms controlling the oxyR and rpoS genes in B. pseudomallei have not been extensively studied.

Methods: Here we have used the transcriptional fusion method to identify a key change in the oxyR expression of B. pseudomallei in which rpoS is absent. We also examined the expression of katG-dpsA and show that oxyR and rpoS act dependently to regulate the levels of the katG and dpsA using Northern analysis and RT-PCR. Results: Mutant strain lacking rpoS failed to increase oxyR expression during growth. The results indicate that under oxidative stress conditions the up-regulation of oxyR by rpoS enables B. pseudomallei to induce the up-regulation of dpsA and katG and also to induce the down-regulation of katG in normal growth condition. Summary: The lack of rpoS resulting in changes of oxyR transcription and also evaluated possible regulators of the oxyR gene. This study demonstrates that rpoS is a key component of the oxidative stress protection system important for the survival of the infectious pathogen B. pseudomallei.

The Anopheles REL1 signaling pathway is required for anti-fungal immunity and can reduce malaria infections F. Turlure, J. Waldock, S. Meister, F. Kafatos and G. Christophides Laboratory of Immunogenomics, Immunology and Infection, Division of Cell and Molecular Biology, Faculty of Natural Sciences, Imperial College London, London, UK Introduction: Insects rely on a basic, germ-line encoded innate immune. Two main NF-jB immune signaling pathways were identified in the malaria vector Anopheles gambiae. The Imd/REL2 pathway was shown to be active against both Gram-type bacteria and the rodent malaria parasite Plasmodium berghei, but the role of the Toll/REL1 pathway is yet unclear.

Methods: Components of the REL1 and REL2 pathways were silenced in mosquitoes. Survival rates following infections by bacteria or fungal spores were monitored, and immunity genes regulation was assessed by quantitative PCR and DNA microarrays. Knockdown mosquitoes were also infected with the P. berghei parasite, and parasite loads were recorded 7 days later. Finally, mosquitoes were co-infected with parasites and bacteria/fungi and their capacity to support development of the malaria parasite was monitored.

Results: REL1 depletion did not affect mosquito survival to bacterial infections. Nonetheless, REL1, unlike REL2, was required for wild-type survival after fungal infection, and anti-microbial peptide-encoding genes such as CEC1 were up-regulated early in infection. REL1 marginally contributed to the immune response against P. berghei but when previously fully activated by fungal challenge or depletion of its inhibitor CACT, could drastically reduce the parasite load.

Conclusions: Here we present data identifying the A. gambiae REL1 pathway as essential for the anti-fungal immune response, and highly active against P. berghei, when ectopically activated.

Hydrogen peroxide, catalase activity and response to dietary phenolics in aphids Conclusions: The phenolic pro-oxidants include mostly chemicals amongst DP and PP. The induction of CAT activity appears to be real antioxidant response against H 2 O 2 /oxidative stress derived from caffeic acid and related DP and PP, during their red-ox metabolism within the aphid tissues.

Genotyping of Helicobacter pylori strains from south Turkey Introduction: Helicobacter pylori (Hp) represent one of the most common and medically prominent infections worldwide. Hp has been asso-ciated with the development of gastric ulcer, atrophic gastritis and gastric neoplasia including mucosa associated lymphoid tissue (MALT) lymphoma. Because of wide range of clinical signs from subclinic-mild gastritis to ulcerations or neoplastic processes, depending on virulence factors of infecting bacterial strains, different aspects are present on curing patients using combined antibiotics due to troubles, expenses of therapy and development of antibiotic resistance while this infection is totally curable. Genotyping studies show that, the bacterial strains with cytotoxin-associated (cagA) gene and some subtypes of Vacuolatingcytotoxin (vacA) gene, encoding proteins with cytotoxic activity on host tissue are associated with more severe clinical outcome. The vacA gene contains s1, s2 and m1, m2 variable parts. Type s1/m1 and s1/m2 strains produce high and moderate levels of toxin, respectively, while s2/m2 strains show little or no activity. Since most vacA s1 strains are cagA positive the two markers are closely related. Methods: We have genotyped the vacA subtypes and tested the presence of cagA pathogenicity islands using multiplex PCR method in 60 biopsy specimen obtained endoscopially from stomach of dyspeptic patients from HATAY region of Turkey.

Results: Hp infection was found in 55% of cases and 63.6 of positive cases were infected with virulent strains. Furthermore, mixed infection was found in six cases. Conclusions: Our suggestion is patients with virulent strains should absolutely be cured because of probability to develop atrophic gastritis and gastric neoplasia and risks of transmission of these virulent strains to healthy people and epidemic.

Cis-acting RNA elements within the hepatitis C virus core/core+1 coding region but not core+1 proteins modulate the efficiency of virus proliferation Introduction: The HCV genome possesses a reading frame overlapping the core gene that is translated by alternative mechanisms into proteins designated core+1/ARFP/F. Moreover, in the core/core+1 coding-region four conserved RNA stem-loops have been predicted (SL47, SL87, SL248, SL443). Methods: To study the importance of the core+1 frame and core-RNA structures on replication and virus production, five non-sense mutations (stops 1-5) predicted to abolish core+1 translation without changing core aminoacids but affecting the first three core-RNA stem-loops, as well as extensive substitutions designed to disrupt the four stem-loops, were introduced into JFH1 and chimeric H77/JFH1 genomes. Introduction: The Gram-negative, human opportunistic pathogen, Haemophilus influenzae, is a cysteine as well as a glutathione auxotroph. Chemically defined minimal medium (MIc medium) sustains growth only when supplemented with an organic cysteine source, such as cystine or glutathione disulfide. This property was exploited in a biological assay to screen a H.

influenzae Rd transposon insertion mutant library for gene(s) involved in glutathione disulfide transport.

Results: Approximately 12 000 kanamycin-resistant insertion mutants were screened for loss of the ability to grow on MIc-plates supplemented with glutathione disulfide as an organic source of cysteine. These screening experiments and subsequent PCR-based identifications led to the selection of only one kanamycin-resistant mutant that was consistently abrogated in its ability to grow on glutathione-supplemented MIc-plates, while being indistinguishable from the wild-type on cystine-supplemented plates. for which then single knockouts were constructed using a homology-based insertion mutagenesis strategy and H. influenzae Rd as the parent strain. Only the HI0853 knockout strain was abrogated in its ability to grow on glutathione disulfide MIc-plates. Conclusions: Our results suggest that the HI0853 gene product is exclusively responsible for binding glutathione disulfide in the periplasm of H. influenzae and is recognized by the HI1187 gene product containing Dpplike ABC-transporter for import. Finally, using wild-type, knock-out and complemented strains, we will here present some of the phenotypic alterations accompanying the loss of HI0853 expression.

Biosynthesis of D-arabinose in mycobacteria: a novel bacterial pathway with implications for antimycobacterial therapy

Institute of Public Health, Brussels, BELGIUM Introduction: Decaprenyl-phospho-arabinose (b-D-arabinofuranosyl-1-Omonophosphodecaprenol), the only known donor of D-arabinose in bacteria, and its precursor: decaprenyl-phospho-ribose (b-D-ribofuranosyl-1-O-monophosphodecaprenol), were discovered in 1990 and first described in 1992. In the route to D-arabinofuranose, the decaprenyl-phospho-ribose 2¢-epimerase converts decaprenyl-phospho-ribose to decaprenyl-phosphoarabinose which is a substrate for arabinosyltransferases in the synthesis of the cell-wall arabinogalactan and (lipo)arabinomannan polysaccharides of mycobacteria.

Results: The first step of the proposed decaprenyl-P-arabinose biosynthesis pathway in M. tuberculosis and related actinobacteria, is the formation of D-ribose 5-phosphate from sedoheptulose 7-phosphate catalyzed by the Rv1449 transketolase, and/or by the isomerization of D-ribulose 5-phosphate catalyzed by the Rv2465 D-ribose 5-phosphate isomerase. D-ribose 5phosphate is a substrate for the Rv1017 phosphoribosyl pyrophosphate synthetase which forms 5-phosphoribosyl pyrophosphate (PRPP). The activated 5-phosphoribofuranosyl residue of PRPP is transferred by the Rv3806 5-phosphoribosyltransferase to decaprenyl phosphate, thus forming 5'-phosphoribosyl-monophospho-decaprenol. The dephosphorylation of 5'-phosphoribosyl-monophospho-decaprenol to decaprenyl-P-ribose by the putative Rv3807 phospholipid phosphatase is proposed to be the committed step of the pathway. A subsequent 2'-epimerization of decaprenyl-P-ribose by the heteromeric Rv3790/Rv3791 2'-epimerase leads to the formation of the decaprenyl-P-arabinose precursor for the synthesis of the cell-wall arabinans in Actinomycetales. The mycobacterial 2'-epimerase Rv3790 subunit is similar to the fungal D-arabinono-1,4-lactone oxidase, the last enzyme in the biosynthesis of D-erythroascorbic acid, thus pointing to evolutionary links between the D-arabinofuranose and L-ascorbic acid-related pathways.

Conclusions: Decaprenyl-phospho-arabinose and decaprenyl-phosphoribose have served as model compounds in the development of new inhibitors and potential anti-tuberculosis drugs. Other potential targets of the D-arabinose pathway will be discussed. TLR4 is expressed in different cell types including myeloid cells, the key effectors of innate immune reactions. By now many aspects of TLR4 downstream signalling remain unclear.

Methods: THP-1 human leukaemia myeloid macrophages were used. Western blot analysis, ELISA set-ups, reverse transcription (RT)-PCR and DOTAP transfection were used mainly to conduct the study reported.

In this study we have found that LPSinduced TLR4 signalling triggers cross-talk of hypoxia inducible factor 1 alpha (HIF-1a) and ASK1 downstream pathway in THP-1 human myeloid monocytic leukaemia cells. Both pathways are activated via redox-dependent mechanism associated with tyrosine kinase/phospholipase C-1c-mediated activation of protein kinase C a/b that activates NADPH oxidase and therefore production of reactive oxygen species that activate both HIF-1a and ASK1. TLR4-dependent activation of PI3-kinase and direct S-nitrosation were found to downregulate ASK1 during TLR4 downstream signalling protecting the cells against LPSinduced, ASK1-mediated apoptosis. ASK1 contributes to stabilisation of HIF-1a protein via activation of p38 MAP kinase that directly phosphorylates HIF-1a. Knockdown of HIF-1a in THP-1 cells with siRNA suggested that this protein is critical for regulation of energy metabolism and protection of LPS-stimulated cells against ASK1induced programmed death. Furthermore, HIF-1a protein supports TLR4-dependent IL-6 production by protecting the cells against depletion of ATP. Cephalosporin C (CPC) is produced by the fungus Chephalosporium acremonium. Semisynthetic cephalosporin antibiotics are industrially synthetized from 7-aminocephalosporanic acid (7-aca) obtained by chemical deacylation of CPC. However, this process implies several expensive steps and treatment of toxic wastes. Therefore great efforts have been made to find a bioprocess, converting CPC in 7-aca. The enzyme cephalosporin acylase (CA) catalyzes the conversion of CPC in 7-aca. Indeed several CAs isolated from genus Pseudomonas and Bacillus use glutaryl-7-aca as primary substrate and have low specificity for CPC. Thermophiles are microorganism adapted to survive at high temperature. Enzymes isolated from these sources represent useful tools for industry because of the higher stability respect to their mesophilic counterparts. Here we report the cloning, purification and characterization of an enzyme belonging to the CA family isolated from the thermophilic eubacterium Geobacillus thermodenitrificans. Genome sequence of G. thermodenitrificans has recently become available. Cell extracts of the eubacterium showed CPC bioconversion evaluated by HPLC analysis. A gene annotated as CA was amplified by PCR, inserted in pET-28b and transformed in BL21 (DE3) E. coli strain. The protein containing an His-tag was purified by Ni-affinity chromatography. The amino acid sequence showed 30-40% of identity with proteins of CA family. Analyses in SDS-PAGE showed three protein bands of 60, 20 and 40 kDa, likely corresponding to the precursor polypeptide, the a and b subunits, respectively. Biochemical characterization of CA from G. thermodenitrificans could provide new insight in the substrate specificity of the enzyme and improve the production of antibiotics. Introduction: Like multicellular organisms, the yeast Saccharomyces cerevisiae can undergo programmed cell death (PCD) induced by different environmental stress. By using an experimental system in which yeast PCD is induced by acetic acid (AA-PCD), we investigated both cell survival and a variety of processes occurring en route to death, including: production of reactive oxygen species (ROS) and its regulation, the involvement of proteasome and metacaspase YCA1, cytochrome c release. Methods: AA-PCD was induced in exponential yeast cells. Catalase (CAT) or superoxide dismutase (SOD) was over-expressed in yeast cells and YCA1 gene-knock out strain was constructed. ROS formation, enzyme activities and cytochrome c release were assayed by fluorescence microscopy, spectrophotometric and immunoblotting analysis.

5C. Life Adaptations in Extreme Environments 5C. Life Adaptations in Extreme Environments PP5C

Results: We show that en route to AA-PCD: ROS are produced in a CAT and SOD dependent-fashion; proteasome activation is needed for AA-PCD to occur; cytochrome c is released from coupled mitochondria and can work as an electron donor and a ROS scavenger; YCA1 is dispensable for AA-PCD to occur and caspase inhibitor zVAD-fmk does not prevent AA-PCD. Conclusion: The picture emerging from these results is as follows: early H 2 O 2 production occurs in AA-PCD in a manner modulated by CAT and SOD. Proteasome is transiently activated starting 60 minutes after PCD induction. Release of cytochrome c occurs starting at 60 minutes of AA-PCD and is complete at 150 minutes. YCA1 participates in AA-PCD also in a manner unrelated to its caspase-like activity. Introduction: Aquatic hyphomycetes generally dominate leaf decomposition in unpolluted habitats. Surprisingly, an amazing diversity was found in high polluted waters of former industrial sites in Central Germany [1] . Some isolated strains have evolved biochemical adaptations. Methods: Using integrated methods for separation and structural analysis the thiol peptide levels and the structure and metal binding capacity of a novel metallothionein were elucidated. Biotransformation reactions of organic pollutants were assessed with fungal cultures and isolated enzymes, using mass-spectrometric analysis.

Results: Some Heliscus lugdunensis strains isolated from high heavy metal (HM) contaminated habitats show under Cd exposure biochemical divergences in intracellular HM accumulation, and in the thiol metabolism (increase of glutathione pool, induction of phytochelatin 2 and a novel metallothionein). H. lugdunensis also convert the insecticide metabolite 1-naphthol into sulphate conjugates and 1-methoxynaphthalene, indicating the involvement of phase II enzymes [2] . Clavariopsis aquatica metabolize the xenoestrogen nonylphenol and the polycyclic musk fragrances Galaxolide and Tonalide. An extracellular laccase isolated from C. aquatica is able to oxidize such pollutants [3] . Conclusions: Studies underway will explore the potential of thiol peptides as intracellular agents for HM detoxification by aquatic fungi. Organic pollutants are converted by AQHs, involving both intracellular and extracellular enzymes. 

The cellular potassium is used predominantly for the maintenance of the intracellular turgor, therefore the influx of potassium is supposed to be the first adaptation mechanism of bacterial cells to osmotic up-shift. For such adaptation, bacteria evolved several types of potassium transport systems working under different K+ concentration. The inducible high affinity system (Kdp) which works in limiting K+ concentration was identified in E.coli. Sequence homologues of the Kdp system were identified in many bacteria, but not in Bacillus subtilis.

The Bacillus subtilis L-42 mutant strain, which displays limited growth and inability to cope with hyper-osmotic shock in a defined media with a potassium concentration below 1 mM, was isolated by non-specific transposon insertional mutagenesis followed by an enrichment selection in media with a potassium concentration below 0.5 mM. Localization of the mutation was provided, only one insertion of recombinant transposon was found in the mutant chromosome, localized into the yxkO gene, a putative ribokinase with unknown function. The mutant reveals considering pleiotropic effect, is involved in adaptation to osmotic, K+ ions limitation stress, differ in morphology of colony growth. Conclusion: Further genetic and physiological characterizations of the mutant were done and it can be assumed that the product of the yxkO gene is involved in regulatory pathway of the general stress response to cope with heat, ethanol stresses and limiting concentration of potassium and osmotic up shift when no other compatible solute are available.

The ECF sigma factor sigR is involved in Caulobacter crescentus response to cadmium R. Lourenco and S. Gomes Depto. de Bioquıḿica, Instituto de Quıḿica, Universidade de Sa˜o Paulo, Sa˜o Paulo, BRAZIL Introduction: For their protection against oxidative injuries caused by cadmium exposure, cells increase the expression of genes involved in the reduction of intracellular levels of this metal and in the repair of damaged molecules and regulatory genes. Caulobacter crescentus sigR gene, encoding an ECF sigma factor necessary for the transcriptional response to singlet oxygen, is one of the genes involved in the bacterial response to cadmium. Methods: To characterize the kinetics of induction of sigR expression by cadmium, transcription fusion assays and western blot experiments were performed with cells exposed to this metal. In addition, a genome-wide transcription profiling was used to determine the set of cadmium-induced genes under the control of SigR.

Results: A transient increase in sigR expression was observed following treatment of Caulobacter cells with cadmium in a SigR-dependent manner. A similar expression pattern was evident when cells were exposed to organic hydroperoxide but not to superoxide anion or hydrogen peroxide. The transcriptome approach revealed that SigR regulates a limited number of genes involved mostly in repair of damaged molecules, and that important defense mechanisms against cadmium, such as efflux pumps and some enzymatic antioxidants, are not under the control of this ECF sigma factor. Conclusions: Our data provide evidence that cadmium induces sigR expression in Caulobacter through an increase in cellular peroxidation, and therefore, singlet oxygen production, resulting in the upregulation of genes that allows cells to repair the oxidative damages generated by this ROS.

Effect of various starches on production of aamylase in thermophilic Bacillus licheniformis Introduction: Today, microorganisms and their enzymes are used in many biotechnological and industrial applications. Amylases are enzymes which hydrolyse starch molecules to give diverse products including dextrins and progressively smaller polymers composed of glucose units. Recent research with thermostable a-amylase has concentrated on the enzymes of thermophiles and extreme thermophiles and little is known about the properties of the enzymes produced by these organisms. These enzymes are used in the textile and paper industries, in starch liquefaction, as a food adhesive, and in sugar production. Materials and Methods: In this study, mud samples were collected from Davutlu Hot Springs (Diyadin/Ag˘rı). Biochemical and 16S rRNA analyses of thermostable bacteria were conducted and these bacteria were found to be a strain of Bacillus licheniformis. Their ability to produce a-amylase of industrial importance was studied in two different basal media (BM1 and BM2) containing different concentrations (0.5-3%) of various starches (soluble, corn, potato and wheat). The optimum pH and temperature for enzyme activity and enzyme production time for this enzyme are 7.0, 70°C and 24 hours, respectively. The enzyme activity was determined by Bernfeld method. Results: The gram positive, spore-forming, motile moderately thermophilic bacteria was found to be a strain of Bacillus licheniformis analysed by 16S rRNA comparison. It was found that the a-amylase activity in B.licheniformis grown on BM1 and BM2 without starches were 57 l/mg and 107 l/mg, respectively. The enzyme production in BM1 was increased by addition of soluble starch up to 2% (125 l/mg), while a slight increase was obtained at 1% of potato starch (82 l/mg). However, there was a great increase in BM2 containing both potato starch and soluble starch. The enzyme activity in BM2 with 2% soluble starch was 337 l/mg, while it was 435 l/mg that with 1% potato starch. Both corn and wheat starches at different concentrations tested did not have an effect on the enzyme production.

Conclusions: The present study shows that a-amylase is produced in great extent at BM2 in the presence of both soluble and potato starch which shows that a-amylase of thermopilic Bacillus licheniformis strain can be used for biotechnological and industrial applications. In extreme environment, some bacteria can form spores which are metabolically dormant and can survive million of years [1] . Usual food preservation treatments can inactivate spores, but strongly reduce organoleptic qualities. Emerging technologies like high hydrostatic pressure allow better preservation of organoleptic qualities but fail to completely inactivate spores [2] . A better comprehension of spore resistance mechanisms is needed to optimize these processes. When spores were placed in environment at different water activities, they did not have the same heat resistance [3] . These tests were done with spores grown in optimum conditions (water activity 0.993) which seldom happen in nature. In this work we studied the influence of the water activity of sporulation medium on spore resistance, in relation with core water activity. Sporulation was initiated at water activity levels ranging from 0.950 to 0.993, using different depressor solutes (NaCl or glycerol). Spore resistance was tested by heating at 97°C during 60 minutes and by pressurising from 150 MPa to 550 MPa. Compared to spores grown in optimum conditions, spores grown at lower water activity were smaller, much more heat sensitive, and had the same high pressure resistance. These spores could germinate at lower water activity level, which suggests a lower core water activity. Such modifications in the water activity level of the sporulation medium could influence core water activity and clarify the relation between core water activity and spore heat resistance. Introduction: The universal cellular stress response (CSR) is modulated, amongst others, by heat, anticancer agents and hormones. Thyroxine (T 4 ) has been shown to confer resistance to a subsequent potentially lethal insult in yeast, while doxorubicin (DOX) is known to decrease cell survival. Considering the homeostatic character of hormones, this study aimed to explore the role of T 4 in yeast survival during the CSR upon treatment with the anticancer agent DOX. Methods: The CSR was evaluated by determining microscopically the viability of Saccharomyces cerevisiae after heat shock (HS, 53°C, 30 minutes). The effect of long-term (27°C, 24 hours) treatment with 0.1 mM T 4 on cell survival upon long-or short-term (2 hours prior to HS) administration of 0.01-0.09 mM DOX was investigated (n = 3-10). Statistical analyses were performed by ANOVA and non-parametric tests. Control cultures in the absence of any agent were included in all experiments. Results: Long-term DOX administration decreased dose-dependently (p < 0.05) both cell number and viability before and after HS, shortterm treatment showing no effect. T 4 induced no alterations in growth and viability before HS (p > 0.05). However, the presence of T 4 significantly increased viability after HS upon long-or short-term DOX administration (p < 0.05). Interestingly, T 4 reversed considerably the extent of dead cell aggregates and the morphological alterations observed upon plating the cultures exposed to DOX alone. Conclusions: The protective action of T 4 against DOX toxicity in this eukaryotic experimental model provides the lead for the investigation of the interactions between hormones and anticancer agents under stress conditions and their potential implications in cancer chemotherapy.

A. Spence and B.P. Kelleher School of Chemical Sciences, Dublin City University, Dublin, IRELAND Introduction: Bacterial enumeration in soil environments estimates that the population may reach approximately 10 10 /g of soil and comprise up to 90% of total soil microbial biomass. Bacteria are present as single cells or multicell colonies and often strongly adsorb onto mineral surfaces such as sand and clay. The interactions of microbes and microbial biomolecules with these minerals have profound impacts on the physical, chemical and biological properties of soils. Method: Microbes were cultivated from a clay-loam soil from Teagasc, Ireland, and the microbial titre determined. Microbial proteins were isolated and the concentration evaluated using the Bradford assay. Quantified microbes and proteins were sorbed to montmorillonite and kaolinite and their equilibrium adsorption determined (initial concentration -equilibrium supernatant). Clay mineral-complexes were characterized by X-ray diffraction (XRD), Scanning electron microscopy (SEM) and Fourier-transform infrared (FT-IR) spectroscopy. Results: Montmorillonite and Kaolinite adsorbed 3.71x10 10 and 3.33x10 10 cells/g, respectively. Equilibrium adsorption of proteins onto montmorillonite and kaolinite was determined at 920 mg/g and 728 mg/g, respectively. XRD showed that microbes and proteins intercalated the interlamellar region of montmorillonite resulting in montmorillonite-microbial and montmorillonite-protein complexes with 001 d-spacing increases of 1.5 and 1.6 nm, respectively. SEM indicated that microbes and proteins also adsorption on montmorillonite surfaces. XRD spectra confirmed that microbes and proteins did not penetrate kaolinite, indicating that adsorption occurred primarily on external surfaces, as also suggested by SEM. FT-IR spectra were indicative of montmorillonite-microbial and -protein, and kaolinite-microbial andprotein complexes. Conclusions: Montmorillonite indicated a greater affinity for soil microbes and soil microbial proteins when compared with kaolinite.

Response of unicellular green algae to irradiance stress R. Thaipratum and K. Yokthongwattana Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, THAILAND Understanding the molecular mechanisms how microalgae adapt to short-term and long-term irradiance-stress conditions may broaden our knowledge of how plants counteract with excess light energy and maintain their photosynthetic productivity. Regardless of extensive research on physiological responses of plant to strong irradiance, many issues have not been fully addressed. For example, even though zeaxanthin (Z) is believed to play a photoprotective role via dissipation of excessive light energy as heat and prevent oxidative damage, its exact functions have not been clarified. Constitutive accumulation of Z in the zea1 strain, a mutant of Dunaliella salina, does not affect the rate of photodamage of the photosynthetic apparatus from photoinhibition compared to wild type whereas Chlamydomonas reinhardtii, npq1, resists to high light. From chlorophyll fluorescence measurement in presence of inhibitor, nigericin, we found that photoinhibition quenching was the predominant nonphotochemical energy dissipation process in Dunaliella salina wild type and a mutant constitutively accumulating zeaxanthin (zea1). However upon expose to high light zea1 has higher qE than wild type. Our data also show that growth rate of mutant is lower than wild type when grown under a photon flux density of 150l mol/m 2 /s whereas when grown under lower photon flux density (30 lmol/m 2 /s) data show no difference in rate of growth. The difference in growth rate may be come from quenching by Z in mutant. A major regulatory mechanism evolved by microorganisms to combat stress is the regulation mediated by (p) ppGpp (the stringent response molecule), metabolised by Rel proteins. These are divided into bifunctional and monofunctional proteins based on the presence or absence of the hydrolysis activity. While these proteins require Mg 2+ for (p) ppGpp synthesis, high Mg 2+ was shown to inhibit this reaction in bifunctional Rel proteins from M. tuberculosis and S. equisimilis. This is not a characteristic feature in enzymes that use a dual-metal-ion mechanism, like DNA polymerases that are known to carry out a similar pyrophosphate transfer reaction. Comparison of polymerase Polb and Rel Seq structures that share a common fold, led to the proposal that the latter would follow a single-metal-ion mechanism. Surprisingly, in contrast to bifunctional Rel, we did not find inhibition of pppGpp synthesis at higher Mg 2+ in the monofunctional RelA from E.coli. We show that a charge reversal in a conserved motif in the synthesis domains explains this contrast; an RxKD motif in the bifunctional proteins is reversed to an ExDD motif. The differential response of these proteins to Mg 2+ could also be noticed in fluorescent nucleotide binding and circular dichroism experiments. In mutants where the motifs were reversed, the differential effect could also be reversed. We infer that although a catalytic Mg 2+ is common to both bifunctional and monofunctional proteins, the latter would utilize an additional metal binding site formed by ExDD. This work, for the first time, brings out differences in (p) ppGpp synthesis by the two classes of Rel proteins. 

Microbial community shifts during the annual operation of Messolonghi solar saltern, Greece Introduction: Significant efforts are currently undertaken world-wide to unravel the microbial diversity in extreme environments including solar salterns. A combination of culture-dependent and independent (DNA microarray) approaches was employed to determine the microbial community dynamics during the annual operation of a Greek solar saltern.

Methods: Four samples, sediment, hypersaline water, saturated brine and crude salt, were taken from Messolonghi solar saltern, Western Greece, during a functional cycle in a period of a year. The structure of microbial communities of the above samples was examined by cultivation and a high density oligonucleotide microarray (Phylochip) that permits simultaneous identification of almost 9,000 taxa. Results: By the cultivation-dependent approach, 219 and 132 Bacterial and Archaeal isolates respectively, were recovered from samples coming from the four different functional stages of the saltworks. All bacterial strains, including four potential new species, were phylogenetically related to 43 members of Actinobacteria, Firmicutes and c-Proteobacteria. The archaeal isolates were placed within the family of Halobacteriaceae. The culture-independent approach, revealed a significant bacterial diversity related to 417 members of almost all bacterial phyla. At the four stages analysed, the archaeal diversity consisted of both members of Crenarchaeota and Euryarchaeota.

Conclusions: The present study indicates vast changes in the microbial community structure of Messolonghi solar saltern due to the drastic environmental changes occurring during its annual operation, denoting a rapidly adaptive dynamic ecosystem.

Antagonistic action of antioxidants during the oxidative stress response in eukaryotic cells E. Ch. Yiannakopoulou and E. Tiligada Department of Pharmacology, Medical School, University of Athens, Athens, GREECE Introduction: Yeast cells exposed to adverse conditions employ a number of defence mechanisms to respond efficiently to stress. Stress response can be modulated by a number of pharmacologically active agents, including the antioxidant compounds N-acetylcysteine (NAC) and glutathione (GSSG). Since in real life organisms are exposed to several antioxidants simultaneously, there is growing interest in the influence of antioxidants interactions on cell growth and survival. The aim of this study was to investigate the effect of NAC and GSSG coadministration on the oxidative stress response in the unicellular eukaryotic organism Saccharomyces cerevisiae. Methods: Oxidative stress was induced by incubating post-logarithmic S. cerevisiae cultures in the presence of 1.5 mM H 2 O 2 for 1 hour. NAC and GSSG were administered at 30 mM and 0.5 mM, respectively either 1 hour prior to (pretreatment) or during oxidative stress. Yeast cultures were exposed either to each compound alone or to both antioxidants. Subsequently, cell survival and proliferation after oxidative stress was evaluated by counting the colony forming units following plating of culture aliquots in agar.

Results: NAC and GSSG, when administered alone during either pretreatment or oxidative stress, increased cell survival (p < 0.001) compared to the absence of any agent. On the contrary, the presence of both NAC and GSSG either prior or during oxidative stress had no effect on cell survival (p > 0.05). Conclusion: NAC or GSSG, when administered alone, protected yeast cells from oxidative stress, while upon co-administration they failed to exert their protective action, possibly by antagonizing each other at the particular doses. Introduction: Oxygen supply is essential for survival of the chytridiomycete Blastocladiella emersonii. However, because of its saprophytic lifestyle, this fungus seems to be adapted to low aerated environments and thus can be an interestingly model of study the hypoxic stress response. In addition, comparison between hypoxia and other stresses such as cadmium exposure and heat shock may shed light on how this organism responds to extreme environments. Methods: In order to gain insight into the transcriptional program that directs the physiological and metabolic alterations to endure hypoxia stress, we analyzed the expression profile of about 3,773 genes in B. emersonii cells exposed to different concentrations of dissolved oxygen using cDNA microarrays and qRT-PCR. Results: We found that B. emersonii cells respond to oxygen deprivation mainly by altering the expression of genes required for energy production and consumption in an effort to maintain ATP levels by increasing anaerobic carbon metabolism and decreasing energy-consuming processes. The comparison of these microarray data with cadmium exposure and heat shock data recently obtained in our laboratory showed that these kinds of stresses share some similar transcriptional responses indicating that despite acting through different mechanisms, they may trigger similar adaptative and protective responses.

Conclusions: The transcriptional profile of B. emersonii in response to hypoxia and transient anoxia presented in this work reveals a key reason why this organism is able to survive in the absence of oxygen. Our study reveals crucial differences and similarities between hypoxia, cadmium and heat shock stress responses. Metallo-b-lactamases (MBLs) are considered an emerging family of Zn 2+ -dependent enzymes that significantly contribute to the resistance of many nosocomial pathogens against b-lactam antimicrobials. Since these plasmid-encoded enzymes constitute specific molecular targets for b-lactams, their exact mode of action is of great importance for deploying efficient anti-infective treatments and for the control of severe multi-resistant nosocomial infections, which becomes a global problem. A novel hybrid VIM-1/VIM-2-type b-lactamase (named VIM-12), has been recently identified in a clinical isolate of K. pneumoniae in Greece. This enzyme exhibits high sequence similarity with VIM-1 at its Nterminal region and with VIM-2 at its C-terminal region raising the question whether this sequence similarity reflects also in a similar functional role. Moreover, the possible contribution of this novel b-lactamase to the overall antibiotic resistance of this specific clinical isolate was investigated. The gene coding for VIM-12 was cloned and expressed and the recombinant enzyme was used for detailed kinetic analysis, using a variety of b-lactam antibiotics. VIM-12 was found to exhibit narrow substrate specificity, compared to other known b-lactamases, limited mainly to penicillin and to a much lesser extent to imipenen. Interestingly, meropenem was found to act as non-competitive inhibitor of the enzyme, although the active site of VIM-12 showed complete conservation of residues among VIM enzymes. We conclude that VIM-12 represents a novel and unique member among the known metallo-b-lactamases, exhibiting atypical substrate specificity. The F plasmid encodes a type four secretion system (T4SS) involved in conjugation. F TraJ is reported to be a 229 amino acid positive activator of the major transfer operon that relieves H-NS repression, although this mechanism is unknown.

Methods: BLAST analysis of TraJ from different F-like plasmids indicated clusters of highly conserved residues including within a predicted helix-turnhelix (HTH) DNA binding motif. Mutations at conserved residues were assayed for effects on conjugation, protein production, in vitro electrophoretic mobility shift assay (EMSA) and in vivo Chromatin immunoprecipitation assay (ChIP assay).

Mutations of all conserved amino acids in the putative HTH motif abolish or highly diminish conjugation. Moreover, a DNA band corresponding to the main transfer promoter region was detected from the ChIP assay, only in a strain producing TraJ. Therefore, F TraJ is a DNA binding protein that counteracts H-NS possibly by competing for binding sites in the F transfer control region. The C-terminal is crucial for conjugation as well, as deletion of the last six residues, not including the HTH site, disrupts mating ability. TraJ is a 226 amino acid protein, as elimination of the first methionine, but not the methionine on the fourth position, did not affect protein production or mating ability.

Application of TRACA to the human intestinal microbiota: insights into tetracycline resistant genes of the mobile metagenome using PCR based screen P. D. Scanlan and J. R. Marchesi

Introduction: The human distal colon represents a diverse and complex eco-system with bacterial cell numbers ranging in density from 10 11 to cell-10 12 cells per gram of faeces. The vast majority (60-80%) of bacterial species within the human GI are refractory to cultivation and to access plasmids that comprise this fraction of the community a novel culture independent method of plasmid capture was applied; TRACA (Transposon Aided Capture). Methods: The TRACA method was applied to faecal samples from different individuals and a PCR based screen of resultant TRACA plasmids was used to A) assess the diversity of Tetracycline genes from GI plasmids and B) determine the congruency of results for Tetracycline resistant genes found from PCR screens of the faecal metagenome compared to the mobile metagenome. Introduction: The presence of tellurite resistance genes has been reported in relatively wide spectrum of human pathogens, despite the fact that tellurium, as well as its soluble salts are both rare in the environment and are no longer in use as antimicrobial agents in treating numerous human disease. A driving force to intensify our studies on ter locus has been the discovery of its presence in the food-borne hemorrhagic pathogen Escherichia coli O157:H7. We have studied the previously sequenced and described ter operon found on a large conjugative plasmid pTE53 which was originally isolated from uropathogenic strain Escherichia coli KL53. Methods: We have cloned and expressed essential genes (terBCDE) of Te R in the pET28a(+) and Duet Coexpression vectors. We have prepared the fusion genetic construct of potential promoter region and gfpUV reporter gene.

Results: Ter proteins have been successfully expressed and their biological activity was proved by the two plasmid complementation system. The previously real time PCR analysis has determined that the genes terZ, terA, terB, terC, terD and terE are represented by a single RNA transcript, forming a single transcriptional unit that is expressed from a single promoter. It has been predicted also by bioinformatics analysis. We functionally confirmed the existence of such promoter by cloning of the potential promotor sequence prior gene terZ in the fusion with reporter gfpUV gene. Conclusion: However, terBCDE genes and their products are known to be essential, thus far the tellurite resistance mechanism of Escherichia coli still remains mystery. .) . MbeA in the injector was titrated onto MbeC in the calorimetric cell. The heat evolved following each 10 ll injection was obtained from the integral of the calorimetric signal. The heat due to the binding reaction was obtained as the difference between the heat of reaction and the corresponding heat of dilution. Analysis of data was performed using Microcal Origin software.

The titration experiments showed that the two proteins interacted with a 1:1 stoichiometry and a K D value of approximately 21 lM was estimated. In this study, we tried to clarify the alteration. Methods: MIC was determined by the agar dilution method, and then, double disc test and PCR were performed for phenotyping and genotyping. The whole erm(B) gene and its regulatory region were sequenced. Also, the growth rate was measured and beta-galactosidase assay was performed to observe its expression pattern. Results: Among cMLS clinical isolates having erm(B), five of thirty three Enterococcus facium (15.2%) and seven of thirty five (20.0%) E. faecalis were identified having the alteration. erm(B) gene of Tn917 was maintained and alterations were only found in the regulatory region. Thirteen nucleotides were inserted after the 7th amino acid in the leader peptide. This insertion led frame shift, and the stop codon appeared four nucleotides earlier than original Tn917. Typical Tn917 has 28aa-long leader peptide, but the mutant revealed having 31aa-long leader peptide. The altered regulatory region was similar to that of Tn5398 of Clostridium difficile. Growth curve and beta-galactosidase assay revealed that this alteration makes erm(B) gene express constitutively.

Conclusions: In this study, we identified novel alteration in the erm(B) regulatory region similar to Tn5398 of C. difficile. This alteration seemed to lead constitutive expression of erm(B) gene and typical cMLS phenotype. Conclusion: Ectoine biosynthesis in Z. mobilis recombinants is probably impaired because of interferences with its metabolic flux, as Z. mobilis is strictly dependent on glucose oxidation for energy production. This constraint limits significantly the perspective of Z. mobilis use as an engineered host for biotechnological applications derived from ectoine production. Introduction: In industrial yeast bioprocesses, yeast cells frequently encounter osmotic stress. Cellular response to hyperosmolarity in bakers' yeast as a model organism has attracted attention since principles of osmoadaptation are conserved across eukaryotes. Thus yeast strains having resistance to high osmolarity have great importance in understanding the cellular mechanism of osmoadaptation. In this study, evolutionary engineering strategy based on applying selective pressure towards desired phenotype was adopted.

Methods: Sorbitol was used as the agent to generate osmotic stress. Sorbitol stress was applied on ethyl methane sulfonate (EMS) mutagenized genetically diverse population of the yeast S. cerevisiae in batch cultures. Survivors of a stress condition were exposed to higher stress levels, thus, osmotic stress was applied increasingly and continuously for 30 mutant generations. Individual mutants were selected randomly from the final mutant population of sorbitol stress, which have resisted significantly higher osmotic stress levels than the minimum inhibitory sorbitol concentration of the wild type strain. Results: Mutant population was exposed to osmotic (sorbitol) stress starting from 1M to 3.4M sorbitol concentration. Using a highthroughput, most probable number (MPN)-based method, individuals from the final population were investigated for their resistances to sorbitol stress and to various common stresses such as freeze-thawing, heat, oxidative stress, and salt stress. Resistances were compared with those of the wild type strain in terms of percent survival. Conclusions: Osmotic stress-resistant yeast mutants were successfully selected using evolutionary engineering approach. Understanding the genetic background of osmotic stress resistance in yeast will help improve efficiency in yeast bioprocesses. Methods: Different regimes of aeration intensity (pO 2 ) were used to change cellular energy metabolism and to estimate its effect on L-valine synthesis by recombinants of C. glutamicum ATCC 13032.

Results: pO 2 in the range 32-35% from saturation was estimated as optimum aeration intensity to achieve maximum cellular growth rate, pO 2 below 20% -as optimum aeration to increase the cell specific rate of valine synthesis (q Pmax was 0.17 ± 0.02 or 0.07 ± 0.02 g valine g/ cdm/hour depending on pO 2 regimes during cultivations, e.g. 17 ± 2% or 34 ± 2%, respectively). An increase in valine synthesis under conditions of decreased aeration intensity directly related to a significant decrease in CN-sensitive respiration coupled to ATP generation, increase in intracellular pyruvate concentration and acetohydroxy acid synthase activity in cells.

Conclusions: A slight decrease in the rate of energy generation by oxidative phosphorylation, achieved by maintenance of restricted aeration intensity during cultivations, was estimated as a suitable method to increase L-valine overproduction by recombinant C. glutamicum strains. Therefore in order to maximize L-valine synthesis productivity and amino acid yield from glucose, different regimes of pO 2 during fed-batch cultivations of strains were maintained.

A monomeric aldehyde dehydrogenase as part of a pAO1 encoded pathway for sugar utilization Introduction: The 165 kb catabolic plasmid pAO1 confers to its host Arthrobacter nicotinovorans the ability to grow on nicotine. Besides the well characterized pathway for nicotine degradation, it also carries a gene cluster of a hypothetical pathway for carbohydrate utilization. This cluster consists of ORFs of a transcriptional regulator, of a sugar ABC-transport system, and of several putative dehydrogenases and oxidoreductases. The Orf39 product, a hypothetical aldehyde dehydrogenase, was produced recombinantly, purified and characterized biochemically as starting point for investigating the functional role of this pathway. Methods: Gene cloning, expression and protein purification was performed according to standard methods. Enzyme activity was determined by monitoring at 340 the formation of NAD(P)H in the presence of different substrates.

Results: The 49.5 kDa protein had aldehyde-dehydrogenase activity, preferring substrates in the following order: butylaldehyde, glyceraldehyde, glutaraldehyde, benzaldehyde, formaldehyde. Enzyme activity was lost in the absence of b-mercaptoethanol, suggesting the presence of an essential Cys-SH residue during the catalytic reaction. Unlike human, sheep or rat aldehyde-dehydrogenases, the ORF39 product run as a monomer on GPC. The enzyme prefered NADP + over NAD + , with a ratio of 4:1.

Conclusions: The product of Orf39 of pAO1 of A. nicotinovorans encodes a monomeric, NADP + -dependent aldehyde-dehydrogenase. It may be part of a plasmid-transmissible pathway allowing for sugar catabolism. Introduction: The AtoSC two-component system in E. coli activates atoDAEB operon genes to catabolize short-chain fatty acids, acetoacetate being the inducer. The AtoC response regulator was first described as the Antizyme of the polyamine biosynthetic ornithine decarboxylase. The AtoC binding site within the ato promoter was experimentally verified as an inverted 20 bp palindromic repeat. Transcriptome and phenotypic analyses however, indicated the involvement of atoSC to various other cellular activities, such as flagellar synthesis. Here we report a bioinformatic analysis for the localization of the AtoC binding motif within the E. coli genome that could indicate a putative implication, direct or indirect, of the AtoC in other operons regulation.

Methods: A genome-wide promoter analysis was performed using a program for the de novo detection of overrepresented motifs within promoters. The motif sampling was initiated using the intergenic region where the AtoC binding site is located. By applying an iterative algorithm, including TCS regulated promoters, the program resulted to promoter sets comprising high-scoring putative cis-regulatory elements. The AtoC binding ability to these elements was tested by chromatin immunoprecipitation, using anti-AtoC polyclonal antibody.

Results: MotifScanner identified occurrences of different motifs connected to the ato palindromic sequence, and those related to established AtoC functions were singled out and used as templates for the design of primers, in order to analyse the immunoprecipitated chromatin in strains lacking or comprising atoSC, with or without acetoacetate induction.

Conclusions: Experimental verification of predicted correlations could provide an insight to gene regulatory networks in bacteria, enabling us to foresee potential targets for response regulators.

Glutathione is essential to initiate development in Dictyostelium discoideum Introduction: Glutathione is the most abundant non-protein thiolcontaining compound and known as a major source of reducing equivalents in eukaryotes. In Dictyostelium discoideum glutathione is required for growth and development. Disruption of gcsA encoding cglutamylcysteine synthetase (GCS), an essential enzyme in glutathione biosynthesis, resulted in glutathione auxotrophy and failed to develop. Methods: Cellular reactive oxygen species (ROS) level was measured by using 2,7-dichlorofluorescin diacetate (DCFH-DA) and detected by a fluorescence spectrophotometer. And concentration of intracellular methylglyoxal was analyzed by quantification of quinoxaline, the derivative of methylglyoxal, with high performance liquid chromatography (HPLC). Results: The transcriptional expression of gcsA was induced as aggregation progressed. To investigate roles of glutathione in development of D. discoideum, Cellular ROS level was measured. In KAx3 cells, cellular ROS level was increased as soon as the nutrition exhausted but in a short time the level was decreased. The cellular ROS level of GCSnull mutants was higher and sustained longer than KAx3 cells. And interestingly as D. discoideum started to develop, the concentration of intracellular methylglyoxal was decreased in both KAx3 and GCS-null cells. While the level of methylglyoxal in KAx3 cells decreased gradually, the level of methylglyoxal in GCS-null mutants increased. Conclusions: According to the above results we hypothesized that there might be correlations between intracellular ROS, methylglyoxal and glutathione during early development of D. discoideum. The study of ROS and methylglyoxal in GCS-null mutants could help to understand the transition from growth to development in D. discoideum.

The key role of flavin reductases in alkane metabolism in oil-degrading bacteria Introduction: Over the past few years, a new and ecophysiologically unusual groups of marine oil-eating bacteria has been recognized. Alcanivorax borkumensis SK2 is a key marine oil-degrading bacterium that its complete genome was sequenced in late 2005.

Methods: The genes of four flavin reductases and two rubredoxins of A. borkumensis SK2 were amplified from its genomic DNA. Finally cloning of mentioned genes was performed in an expression vector (pET21a). Desired proteins were produced in BL21 cells, in LB medium (contained 100 lg/ml ampicillin). At an OD 600 of 0.8, protein expression was induced by 1 mM IPTG and continued overnight at 30°C (150 rpm). Cell cultures were centrifuged, resuspended in Tris-Hcl buffer (0.1M, pH 6.8) and sonicated.

Results: SDS-PAGE gel electrophoresis of the protein preparations confirmed considerable expression of desired proteins. Discussion: Functional genomic analysis of Alcanivorax borkumensis SK2 has provided significant insights into the genomic basis of the efficiency and versatiity of its alkane degradation. It has two systems for alkane mineralization (alkane hydroxylase and cytochrome P-450). Both enzymes are flavin-dependent monooxygenases. So far, in many reports have been shown that the enhancement of flavin reductase activity, has considerable effect on the mono-oxygenase activity. The main aim of the next step of this project is to find the key residues that have essential roles in electron transfer, and change them by site-direct mutagenesis (in order to enhancement of flavin reductase activity and finally alkane degradation efficiency). While classic models of allostery are described by transitions of oligomers of a defined number of subunits, many authors proposed more extended schemas including association-dissociation equilibria (e.g., [1] references therein). Since intracellular diffusion of molecules is strongly dependent on size, and consequently on association state, the significance of dynamic variation of mobility was examined by numerical simulations. The results show that if a relevant ligand is introduced in a non homogeneous manner, a condition that is often valid in vivo, then the macromolecule will be distributed in an uneven way. On the other hand, the examination of a considerable number of examples of ligand-modulated association reported in bibliography on energy metabolism and the basic biosynthetic pathways shows that in most cases specific association patterns are followed: In the majority of instances the substrate or activating effector induces the formation of bulkier forms. The second most frequent motif is association induced by a feedback inhibitor. It is intriguing that the latter scheme appears in almost all cases when the substrates have to fullfil other major metabolic roles. These recurring motifs, in conjunction with the numerical results, suggest that allostery may be at the base of spatial organization of the main metabolic pathways. Besides, Bioinformatics became a fundamental tool for development of new approaches as functional genomics, comparative genomics, pharmacogenomics and others. We developed the AnEnp, a tool that groups proteins based on sequence similarity of enzymatic function that allows the search of analogies between organisms. It is capable of clustering protein sequences and visualizing the results; it also carries out similarity searches via BLAST and/or HMMER and reconstructs metabolic maps. We used AnEnp to find putative analogies of enzymatic functions between Homo sapiens, Trypanosoma cruzi and Leishmania major, using as model the biochemical pathways for lipid, energetic and amino acid metabolism. We selected a set of analogous enzymes from T. cruzi and L. major to perform cloning, expression, purification and analyze their potential for drug development. Now a day we have done this strategy for two enzymes from mevalonate pathway from T. cruzi and another two enzymes, one from pentose shunt pathway and other from tricarboxylic cycle. Those enzymes were cloned, expressed, purified and were used to developed polyclonal antibody, which ones were used for identifying constitutional expression on these parasites. The primary structures of these enzymes are being making by homology modeling.

Towards the characterization of the glucose tolerance mechanism in the ethanologenic saccharophylic bacterium Zymomonas mobilis V. Hatzivasiliou, A. Christogianni, A. I. Koukkou and C. Drainas Department of Chemistry, University of Ioannina, Ioannina, GREECE Introduction: Elevated osmotic pressure is an environmental stress for microbial cells. Several mechanisms have been described for the adaptation to osmotic pressure due to high concentrations of salts or sugars. They all involve responses to signals triggering intracellular production of compatible solutes, which act as osmo-protectants. Zymomonas mobilis is exceptionally tolerant to extremely high concentrations of glucose, conditions that do not lead to production of any known compatible solute. An osmo-regulated operon (glc) involved in glucose tolerance has been identified and sequenced by our team, comprising of four putative structural genes (Christogianni, et al., J. Bacteriol. 2005; 187: 5179-5188), the function of which is yet unknown. Methods: To investigate this apparently new osmo-regulatory mechanism we studied the expression of the natural products encoded by the four genes of the glc operon under conditions of growth on high glucose concentration by western blot analysis. Results: Polyclonal antibodies for each gene product were isolated using PCR amplified and overexpressed recombinant proteins of the glc operon as antigens. The natural products of the four genes detected in Z. mobilis cell-free extracts were found to be inducible and appeared at higher molecular weights than expected. Treatment with KCl and urea recovered the expected molecular weights.

The natural products of the Z. mobilis glc operon are induced under high glucose concentrations and possibly are involved in intracellular interactions, the nature and role of which remains to be clarified.

Re-examination of the rationale for the use of bipyridylium cation radicals as site-specific electron donors for respiratory nitrate reduction A. Illichmanova, M. Janu, V. Sedlacek and I. Kucera Department of Biochemistry, Faculty of Science, Masaryk University, Brno, CZECH REPUBLIC Bacteria express two distinct nitrate reductases that are associated with the respiratory chain: the membrane-bound, cytoplasmically oriented Nar, and the periplasmic Nap. In Paracoccus denitrificans Nar is expressed under anaerobic conditions in the presence of nitrate which must be transported across the cytoplasmic membrane to reach the active site of this enzyme. The periplasmic nitrate reductase Nap is expressed during the aerobic growth on butyrate even in the absence of nitrate. The activity of these two enzymes is usually measured using colored bipyridylium cation radicals methyl viologen (MV) or benzyl viologen (BV) as electron donors. It is generally believed that the more polar MV does not enter the cell and hence donates electrons exclusively to Nap, while oxidation of hydrophobic BV reflects the action of both enzymes. This issue was further examined by a kinetic analysis of nitrate reduction in the wild-type strain and in the Nar or Nap mutant strains. The results show that (i) MV is a quite effective donor for either enzyme, (ii) BV blocks the active transport of nitrate to the active site of Nar, and (iii) BV functions as an ionophore for nitrate at higher concentrations. Acknowledgement: This work was supported by the grants from FRVS (G4/1760/2008) and the Ministry of Education, Youth and Sports (MSM 0021622413).

The up-regulation of thiamine-synthesizing, activating and utilizing enzymes in baker's yeasts under conditions of oxidative stress A. Kozik and E. Kowalska Jagiellonian University, Faculty of Biochemisty, Biophysics and Biotechnology, Krakow, POLAND Introduction: Thiamine diphosphate (TDP) is a universal coenzyme involved in the major pathways of carbohydrate and amino acid metabolism. An up-regulation of several TDP-dependent metabolic routes under conditions of oxidative stress has been observed in many organisms, including baker's yeasts which posses the ability to synthesize thiamine (vitamin B 1 ) de novo. We hypothesize that the biosynthesis of thiamine and its further activation to TDP, should also add to the yeast response to the stress conditions. In this work, we preliminarily test this hypothesis by analyzing the changes in the levels of representative thiamine-synthesizing, activating and utilizing enzymes in yeasts subjected to the oxidative stress. Methods: Baker's yeast strain BY4741, grown in the complete YPD medium or in a minimal EMM2 medium free of thiamine, was stressed by treatment with 0.5 mM hydrogen peroxide or higher temperature (37°C). Activities of antioxidant enzymes (glutathione peroxidase, superoxide dismutase etc.), thiamine phosphate synthase (TPS), thiamine pyrophosphokinase (TPK) and transketolase (TK) were determined.

Results: As expected, the hydrogen peroxide-and higher temperaturetreatment of yeasts resulted in the increase of the levels of antioxidant enzymes. In a correlation with this well known response against the oxidative stress, the activities of TPS, TPK and TK were also enhanced under all conditions studied. The highest increases, up to 240 and 285% of the control, respectively, were recorded for TPS and TPK in yeasts grown in the absence of thiamine and at the higher temperature. University of Thessaly, Larissa, GREECE Pseudomonas entomophila was recently identified to be the only pseudomonad that naturally infects and induces lethality of Drosophila melanogaster [1] . Complete sequencing of the 5.9-Mb P. entomophila genome exposed potential virulence factors aiding the pathogenic nature of this bacterium but experimental evidence is still lacking for most of them [2] . Cyanogenesis (e.g. HCN production) has been demonstrated in a small number of bacterial species. The presence of hcnABC gene cluster (encoding HCN synthase) in P. entomophila genome led us to assume that P. entomophila produces cyanide. HCN production was measured in liquid cultures using a cyanide ion-selective microelectrode and on solid media using a standard method [3] . HCN concentration was found 70-80 lM in liquid cultures and roughly 500 lM on solid media demonstrating unequivocally that P. entomophila produces HCN. In contrast to the wild type, a mutant strain (DGacA) does not produce any HCN in liquid culture but produce some HCN on solid media (roughly 100 lM). These data demonstrate that the GacS-GacA two component regulatory system affects in a positive manner HCN production in P. entomophila, though a second unknown regulator affects HCN production under certain physiological conditions. In conclusion we demonstrate for the first time cyanide production by P. entomophila, we determine genetic factors that affect HCN production and we discuss the implications of HCN production in pathogenicity exerted by P. entomophila. Laboratory of Chemistry, Department of Science, Agricultural University of Athens, Athens, GREECE Introduction: Acid tolerance response (ATR) can be induced in Streptococcus macedonicus at mid-log phase after auto-acidification, transient exposure to acidic pH or acid habituation as well as at stationary phase [1] . Methods: RNA arbitrarily primed PCR and Fourier Transform Infrared Spectroscopy (FT-IR) were employed to compare the transcriptional profiles and the whole-cell chemical composition of these epigenetic phenotypes, respectively. Results: RNA fingerprints revealed significant differences among the phenotypes indicating that gene expression during ATR is influenced not only by the growth phase but also by the treatments employed to induce the response. The genes coding for the mannose-specific IID component, the 1,2-diacylglycerol 3-glucosyltransferase, the 3-oxoacylacyl carrier protein, the large subunit of carbamoyl-phosphate synthase and a hypothetical protein were found to be induced at least under some of the acid adapting conditions. Furthermore, principal component analysis of the second derivative transformed FT-IR spectra segregated S. macedonicus phenotypes individually in all spectral regions that are characteristic for major cellular constituents like polysaccharides of the cell wall, fatty acids of the cell membrane, cellular proteins and other compounds absorbing in these regions. These findings evidence major changes in cells' chemical composition due to acid adaptation that were clearly different to some extent among the phenotypes. Conclusion: Overall, our data demonstrate the plasticity in ATR of S. macedonicus that reflects the inherent ability of the bacterium to adjust the response to the distinctiveness of the imposed stress condition probably in an attempt to maximize its adaptability. In bacterial cell cycle, timing of chromosome replication is tightly linked to the cell size. The replication is initiated at oriC by DnaA-ATP that is consequently released as inactive DnaA-ADP. Rejuvenation of DnaA-ADP to DnaA-ATP is promoted by its binding to acidic phospholipids enriched membrane. We have previously reported a phenomenon of a cooperative transition of DnaA between two functional states, with low and high nucleotide dissociation rates, which is driven by the density of the protein (crowding) on the membrane surface. Furthermore, the concept of lipid domains in bacterial membrane and their functional role got an experimental support in recent years. Combining these phenomena allowed us to suggest a new mechanism for the DNA initiation control in bacteria underlying transduction of the continuous cell growth to the discontinuous chromosome replication. Initiation of DNA replication occurs when the cell mass reaches a critical value, the initiation mass, that is determined by the cellular level of DnaA. Excess of DnaA causes decrease in initiation mass and overinitiation. We used a mutant DnaA defective in DNA binding, produced in E. coli in a controlled fashion, to create a crowding effect on the membrane without affecting initiation. Thus, delaying the native DnaA activity transition on the membrane (lowering the rejuvenation rate) should cause an increase in initiation mass, in contrast to the native DnaA expression. The initiation mass was determined in a wide range of mutant and native DnaA levels and the results are discussed in the frame of the suggested hypothesis.

EglD, an expansin-like protein with putative endonuclease activity is localized in the conidial cell wall of Aspergillus nidulans D. Bouzarelou and V. Sophianopoulou Institute of Biology, National Centre for Scientific Research ''Demokritos'' (NCSRD), Athens, GREECE Aspergillus nidulans asexual reproduction leads eventually to cell differentiation. A conidiospore germinates to form tubular hyphae that will develop into the mycelium, a network of interconnected cells that differentiate into conidiophores. Although the process of conidial germination in filamentous fungi has been extensively studied, many aspects remain to be elucidated since the asexual spore or conidium is vital in their life cycle. The fungus cell wall is a complex structure consisting of polysaccharides such as glucans and chitin, and a minority component of proteins and lipids together with galactose and mannose. Breakage and reformation of cell wall polymer bonds along with the maintenance of cell wall plasticity during conidia germination depend upon a range of hydrolytic enzymes whose activity is analogous to that of expansins, a highly conserved group of plant cell wall proteins with characteristic wall loosening activity. In the current study we identified and characterized the eglD gene in A. nidulans, an expansin-like gene the product of which shows strong similarities with bacterial and fungal endo-b1,4-glucanases. The eglD gene is constitutively expressed in all developmental stages and compartments of A. nidulans asexual life cycle. However, the EglD protein is exclusively present in conidial cell walls. The role of the EglD protein in morphogenesis, growth and germination rate of conidia was investigated. Our results show that EglD is a conidial cell wall localized expansin-like protein, which could be involved in cell wall remodeling during germination. Introduction: Hyaluronan, sulfated glycosaminoglycans and proteoglycans versican and decorin play important roles in cancer. The present study investigates their expression and chemical structure in cartilaginous and non-cartilaginous tissues in laryngeal cancer. Methods: Proteoglycans were characterized immunohistochemically and by western blotting; their expression was examined by RT-PCR. Glycosaminoglycans were purified and characterized by gel chromatography and HPLC. Results: Proteoglycans were overexpressed in cancer, versican much more than decorin and correlated with their stage-related accumulation within the tissue. Decorin expression was higher to versican in normal larynx; their disproportionate overexpression during cancer resulted in equimolar expression. In cartilaginous tissues, the amounts of glycosaminoglycans decreased in contrast to non-cartilaginous, where glycosaminoglycans increased significantly. In all cases, 4-sulfated/6-sulfated and non-sulfated/sulfated disaccharides were significantly increased. The sulfated glycosaminoglycans from both cancerous tissues were of lower M r with increased polydispersity and heterogeneity compared to normal. The high M r hyaluronan was also significantly decreased. Of particular interest was the great increase of low M r hyaluronan (330 000-890 000) in non-cartilaginous tissues.

Conclusions: The kind and the extent of the alterations observed in both proteoglycans and glycosaminoglycans, presenting an intense stage-related behaviour, depended on the tissue origin and could be associated with the malignant phenotype of laryngeal cancer. for experiments. When dissociated cells from blastula-stage medaka embryos were incubated with a conditioned medium from a medaka cell-line, they differentiated into many rhythmically contracting cells. This differentiation occurred in a monolayer of primary culture. We purified the protein factor and identified it as medaka activin A2 (homodimer of inhibin betaA subunit). Methods: We analyzed the contacting cells by immuno-staining, electron microscopy, expression of marker genes, action potential recordings and pharmacological responses. The protein factor was purified by column chromatography and analyzed by mass spectrometry.

Results: All analyses of the spontaneously contracting cells showed the characteristics of myocardial cells. We purified the cardiac cell-inducing factor from the conditioned medium by 523 100-fold with 8% recovery of the protein. Analysis of amino-acid sequence revealed the factor to be medaka activin A2. In addition, recombinant human activin A showed the same cardiac cell-inducing activity to medaka embryonic cells.

Conclusions: Our results demonstrate that activin A is a potent factor for medaka myocardial cell differentiation. This culture method should provide a novel and simple experimental system to study cardiomyocyte differentiation in vitro.

The involvement of endocannabinoid system in rat adipocyte differentiation Introduction: The endocannabinoid system (EC) has been shown to play a role in energy homeostasis mainly through feeding modulation. Anandamide [N-arachidonoylethanolamine (AEA)], one of the most-studied endocannabinoids, is effective primarily by binding to CB1R and CB2R cannabinoid receptors and is subjected to intracellular degradation to arachidonic acid by fatty acid amide hydrolase (FAAH). A recent study reports that 3T3-L1 preadipocytes possess a functional endocannabinoid system which elements are modulated during cell differentiation to adipocytes. We have already shown that AEA is an inducer of rat adipocyte differentiation. In this study we investigate the existence of CBRs and FAAH in rat preadipocytes and the possible modulation of their expression during the differentiation process induced by AEA. Embryonic stem cells (ESC) give rise to the possibility that stem cell therapy can be introduced in the treatment of incurable diseases, as their pluripotency indicates that they can be grown indefinitely without losing the capacity for multi-differentiation. However, before cell replacement therapies can be used, the problems with the tumorigenicity of undifferentiated ESC must be solved. In the present study, we attempt to find biomarkers with which to detect undifferentiated ESC that may cause the formation of tumors. We induced neuronal differentiation from R1, a mouse ESC-line, using a five-step method and simulta-neously measured the values of telomerase activity (TA). And then we set up modified method using physical removal colony, formed by undifferentiated ESC. To investigate the telomerase has relation with ESC formation in vivo, TERT-knock down cells were established by TERT-shRNA using lentiviral particle. We subcutaneously injected these cells into nude mice as immune deficient animal model for teratocarcinogenicity and measured incidence and volume of tumor formed in mice for 4 weeks. The tumor incidence and volume were significantly decreased by modification of differentiation protocol as removal colony, and volumes of formed tumor by TERT-knock down ESC showed significantly decreased against those of null ESC. In these results, we recognized that telomerase containing TERT and removal of undifferetiated ESC highly expressing telomerase activity has important roll in reduction of tumor formation on ESC teratocarcinogenicity, suggesting that telomerase appears to be a useful biomarker for the assessment of the safety of stem cell-based therapies using ESC. Introduction: The structural damage of skeletal muscle fibres may be caused by different events, such as the cumulative effects of a series of contractions that could mimic an intense excercise. Sphingosine 1-phosphate (S1P)-formed by sphingosine kinase (SphK) has been recently reported to exert a physiologically important biological activity in myoblasts, acting simultaneously as anti-proliferating and pro-differentiating agent through the activation of a specific S1P receptor [1] . However, the importance of S1P in repair and prevention of muscle damage has not been investigated yet. To this end, muscular injury has been induced in fast skeletal muscle fibres by cycles of contractures in a K+ solution alternated by recover in Ringer solution, in the presence and in absence of S1P. Tissue damage was estimated by electrophysiologial, biochemical and morphological analysis. Results: Our results showed a marked decreased resistance and a depolarization of the resting membrane potential after K + -contractures, paralleled by an higher level of SphK activity and S1P production. Interestingly, muscle damage was significantly prevented by the addition of S1P to the bath solution during the contractures. In these experimental conditions, satellite cells obtained from these muscle fibers, exhibited an increased proliferation activity, as judged by Western analysis and MTT assay and, when incubated in serum-free medium, showed an enhanced ability to accomplish myogenic differentiation.

Conclusions: Altogether these findings support the idea that SphK/ S1P act as a survival system, reducing cell damage and contributing to the activation of quiescent staminal cells and their differentiation after structural damage. Methods: Human MSC were isolated from BM samples and expanded in vitro. Cells were challenged with abscisic acid (ABA, a plant hormone) or cyclic ADP-ribose (cADPR, a calcium mobilizer) and the proliferation rate, differentiation abilities and cytokine release patterns were explored together with the underlying signal transduction pathway, through biochemical and molecular biology techniques.

Results: Extracellular cADPR and ABA stimulate the in vitro proliferation of MSC as well as the production of immunomodulatory cytokines, without affecting their differentiation properties. Both signal molecules doubled the number of colonies, increased cyclo-oxygenase-2 expression and prostaglandins production and enhanced the release of several cytokines. The transduction pathway of ABA involves a PTX-inhibitable receptor, PKA activation, CD38 ADP-ribosyl cyclase stimulation, cADPR production, intracellular free Ca ++ increase. Interestingly, in the presence of cytokines such as BMP-7, TNF-a and IL-8, MSC were able to synthesize and release ABA. Methods: Ca 2+ activity was measured with confocal microscope. RT-PCR and western blot were used to detect gene and protein expression, respectively. siRNA technique was applied to knockdown gene expression.

Results: cADPR increased the frequency of spontaneous Ca 2+ oscillations, and this effect was abolished by pre-treatment with the specific cADPR antagonist 8-Br-cADPR, but not by ryanodine, the classic targets of cADPR in other types of cells. RT-PCR showed no gene expression for ryanodine receptors in hMSCs. The cADPR-induced increase of spontaneous Ca 2+ oscillations was abolished by the TRPM2 channel inhibitors. Knockdown of TRPM2 channels showed no significant effects on spontaneous Ca 2+ oscillations; however, it abrogated the cADPR effect on spontaneous Ca 2+ oscillations. We further found that cADPR increased hMSCs proliferation, but had no significant effects on osteogeneic and adipogeneic differentiation. The effect of cADPR on hMSCs proliferation was antagonized by 8-Br-cADPR or by the selective knockdown of TRPM2 channels.

Conclusions: Our results demonstrate that cADPR regulate cell proliferation by modulating spontaneous Ca 2+ oscillations mediated by TRPM2 channels, but has no effects on osteogeneic and adipogeneic differentiation in hMSCs. There are several parameters affecting reprograming of somatic cell ino ocyte cytoplasm. The objective of this study was to examine the effect of cell type, sex of cell and embryo culture medium on somatic cell cloning (SSC). In the first experiment, five different medium combinations were compared to examine the effec to fcl on embryo development. In these condexperiment, we investigated the effect of source and sex of cells on SSC.Bovineoocytes isolated from slaughterhouse ovaries were matured in TCM199 supplemented with fetalbovineserum (FBS), sodiumpyruvate, penicillin/ streptomycin EGFbFSH, and bLH. After maturation, cumulus cells were removed and oocytes previously stained with Hoechst were nucleated by aspirating the first polar body and the metaphase II plate. A single cells derived from cartilage tissue of two different strain of cow was inserted into the perivitelline space of the enucleated oocyte. Oocyte-cell couples were fused by a DC pulse of 133V/500 (m for30 (s in the Zimmerman's medium.-After fusion, fused NT units were activated using a combination of CaI(5lM for5 min),CD(2.5 lg/ml)+cycloheximide(CHX,10lg/ml) for 1 h and CHX alone for 4 h. After activation, NT units were cultured in Sage medium for 72 h and then additonal 4-5 days in five different medium combinations (Group1:Sage with 8mg/mlBSA Group2:Sage with10%-FCS;Group3:Sage with10% Serum replacment; Group 4:Sage with 8mg/ mlBSAand 5%FCS; Group5:Sage with 4mg/mlBSAand5% FCS). The differences among groups were analyzed. In the fifth group, blastocyst rate (39,3%) was higher than the other groups.When the compared the blastocytes rates between the first (18,36%) and the fifth groups (39,3%); the significant difference was found. In the second experiment, cell sources and sex were analyzed. Donor cells from two different native bovine strains(anatolian black and grey) were used.When used cells from the Anatolian black cow; there was not found significant differences between development of female and male cloned embryos (28,5% versus 26,6%) to blastocyt stage.-However; when the Anatolian black and Grey cloned embryos from male cells were compared, the significant differences were visulalized on blastocysts (26,6% versus 38,2%, respectively) development. This results showed that blastocysts developed higher in the fifth group than the other groups.In addition source of cells were affected that SSC blastocysts rates. Stem cells undergo continuous self-renewal by a combination of proliferation and apoptosis. During embryogenesis though, many of the proteins that contribute to the execution phase of apoptosis in somatic and malignant cells are not activated in the same way. In this communication we have focused to study the apoptotic function of gammaH2AX in embryogenesis, utilizing mouse embryonic stem (mES) cells from wild type and knockout H2AX mice, and in vitro differentiated in embryoid bodies. We have previously shown in somatic and malignant cells that gammaH2AX is induced massively upon activation of apoptotic endonucleases, but it cannot be detected by immunocytochemistry, as it becomes masked in a caspase-6-dependent manner. Straurosporineinduced apoptosis in wild type and H2AX +/-mES cells resulted in gamma-H2AX formation detected by immuoblotting, indicating DNA fragmentation. In contrast to the apoptotic masking of gammaH2AX in differentiated cells, immunocytochemistry performed in apoptotic mES cells showed a foci-like pattern, resembling the gammaH2AX pattern induced by Apoptosis-Inducing Factor prior to caspase activation. In accordance, co-treatment of apoptotic cells with caspase inhibitors show no effect, indicating that the gammaH2AX apoptotic pattern in mES cells is caspase-independent. Further, apoptosis induction with staurosporine in mouse H2AX -/-ES cells resulted in massive chromatin relaxation and disorganization. This effect is unique to the doublestrand breaks generated by staurosporine in the course of apoptosis, as it is not observed in irradiated H2AX -/-mES cells. The above described distinct features of apoptotic gammaH2AX pattern in early embryogenesis faint away, as cells differentiate progressively to ectoderm, mesoderm and endoderm lineage.

Induction of gamma-globin expression and HbF production after episomal gene transfer of a synthetic zinc-finger activator, in K562 and murine progenitor cells The transgenic expression of antifreeze protein (AFP) gene holds great promise for conferring freeze resistance to commercially important plant and animal species. Four types of antifreeze proteins (AFPs) have been reported and characterized in several species of polar fish. In order to prevent serum freezing, some fish species stay in deep waters where they avoid contacting with nucleating ice crystals and can thus survive in a super cooled state. In our previous study, we have generated the transgenic mice carrying ocean pout antifreeze protein (typIII) gene (the gene from kOP5) and investigated the biological functions of AFP in a mammalian system. In this study, AFP transgenic mice's and normal mice's cells were used to show expression of AFP proteins by immunohistochemistry and western blot analyzes in cells' supernatants. In this experiment, fibroblast cells which obtained from AFP and normal mice's tails were used. Cells were frozen 2 · 10 6 number each vial. After thawing the vials, cell numbers were counted. 2 · 10 6 cells in AFP group and 1.5 · 10 6 cells in control group were alive. Cells were plated in Petri dishes and cell supernatants were collected for western blot analyzes. Immunohistochemical staining was done to show AFP expression (SP2-III for primary antibody,1:1000) and H-E staining for cells morphology. As a result; AFP cells were positive for SP2-III antibody in a limited scale but control cells were not positive for SP2-III antibody. There was not found morphological differences between AFP and control cells after thawing. In AFP cell culture, the death rates were lower than control cell culture (8.3% and 12.5% respectively). In western blot analysis, AFP cells' supernatant was positive for AFP. Thereby, AFP cells were showed the paracrine feature to releasing AFP. It is the first time to show Fish AFP expression in fibroblast cells. Also, this study was showed the freezing effects on AFP cell culture.

Introduction: The structural damage of skeletal muscle fibres may be caused by different events, such as the cumulative effects of a series of contractions that could mimic an intense excercise. Sphingosine 1-phosphate (S1P)-formed by sphingosine kinase (SphK) has been recently reported to exert a physiologically important biological activity in myoblasts, acting simultaneously as anti-proliferating and pro-differentiating agent through the activation of a specific S1P receptor [1] . However, the importance of S1P in repair and prevention of muscle damage has not been investigated yet. To this end, muscular injury has been induced in fast skeletal muscle fibres by cycles of contractures in a K + solution alternated by recover in Ringer solution, in the presence and in absence of S1P. Tissue damage was estimated by electrophysiologial, biochemical and morphological analysis. Results: Our results showed a marked decreased resistance and a depolarization of the resting membrane potential after K + -contractures paralleled by an higher level of SphK activity and S1P production. Interestingly, muscle damage was significantly prevented by the addition of S1P to the bath solution during the contractures. In these experimental conditions, satellite cells obtained from these muscle fibers, exhibited an increased proliferation activity, as judged by Western analysis and MTT assay and, when incubated in serum-free medium, showed an enhanced ability to accomplish myogenic differentiation.

Conclusions: All these findings support the idea that SphK/S1P can act as a survival system, reducing cell damage and contributing to the activation of quiescent staminal cells and their differentiation. Introduction: Rat neural stem cells (rNSCs) with self-renewal and multilineage potential are considered a good candidate for cell replacement of damaged nervous tissue. In vitro experimental conditions can differentiate these cells into neuronal phenotypes. The purpose of the present study is to characterize the effects of dibutyryl cyclic AMP (dbcAMP)-mediated differentiation, and by which molecular mechanism regulated in rNSCs. Methods: To test whether the rNSCs-derived neurons could express neuronal specific properties, the differentiated cells were characterized by (i) morphological analysis, (ii) immunostaining for neuronal specific proteins, and (iii) the expression of characteristic neuronal ion channels. Results: Using immunostaining and immunoblot analysis, we have shown that some neuronal proteins, including neurofilament, microtubule-associated protein 2 (MAP2) and TUJ1, are expressed in dbcAMP-treated cells but are almost absent in untreated cells. To further confirm the differentiated cells to be neuronal cells, functional characterization of these cells was conducted using calcium imaging analysis system. Moreover, treatment of rNSC with H89, a PKA inhibitor, partially blocked the effect of dbcAMP. On the other hand, to address the significance of signal transduction pathways involved in the differentiation induced by dbcAMP, we used several potent inhibitors, such as KN93, a CaMKII inhibitor; two types of MEK inhibitors, U0126 and PD98059 and okadaic acid, an inhibitors of PP2A/2B. All of these inhibitors could block dbcAMP-stimulated neuronal differentiation significantly. Conclusions: Taken together, the results of the present study indicate that multiple signaling pathways are involved in the differentiation of rNSCs.

Linked mechanisms of neurogliogenesis and apoptosis in adult rat brain Introduction: There is overwhelming evidence that processes of neurogliogenesis (nervous and glial cell proliferation, migration and differentiation) as well as genetically programmed cell death (apoptosis) occur throughout ontogeny up to adulthood and can be considered an important element in mechanisms underlying brain function. The aim of the study was to investigate the possibility of a structural-functional association between neurogliogenesis and apoptosis in adult rat brain. Methods: 5-brom-2-deoxyuridine (BrdU) was detected in brain structures by immunoblotting analysis. Activity of caspase 3 was determined using a spectrophotometric method and DNA internucleosomal fragmentation was analyzed by DNA electrophoresis with gel-documentation system analysis.

Results: Simultaneous detection of BrdU (a marker of neurogliogenesis) in native and fragmented DNA, plus the activity of caspase 3 (essential for apoptosis) in adult rat cerebellum, hippocampus and cerebral cortex 24 hours, 3, 14 and 30 days after BrdU administration was carried out. Differences in dynamics along with expression of neurogliogenetic and apoptotic parameters were disclosed in the brain structures studied. Additionally, correlations between proliferation, differentiation and programmed cell death in the adult brain were unveiled.

Conclusion: These studies confirm the hypothesis that processes of neurogliogenesis and apoptosis in the adult rat brain are closely associated. Acknowledgements: Support by Russian Fund of Fundamental Research. Results: The transport of hemin was found to be a temperature-and time-dependent process. Although some hemin was exported after cell washing, intracellularly accumulated hemin was found complexed with cytoplasmic and nuclear proteins. In addition, hemin was also found to bind to DNA fragments and dissociate complexes formed between nuclear transcription factors and b-or e-globin gene promoters.

Conclusion: Intracellular hemin acts as signalling and/or ligand molecule to mediate the interactions of certain transcription factors with specific gene promoter regions. Introduction: Nasal polyposis is a chronic inflammatory disease of the nasal mucosa, that characterised by inflammatory cell infiltration, modifications of epithelial differentiation, tissue remodelling, extracellular matrix accumulation, and oedema. The insuline-like growth factor binding protein-3 (IGFBP-3) belongs to a family of binding proteins, which bind the insuline-like growth factors, IGF-I and IGF-II, thus regulating their effective abilities. The aim of the study was the detection and identification of enzymatic activity able to degrade the IGFBP-3 in nasal polyps tissue extracts. Methods: Human nasal polyps specimens were extracted with 0.15M Tris-HCl pH 7.5, buffer, centrifuged at 12000g and the supernatants were collected. The serine protease activity in extracts was purified by affinity chromatography, using an agarose-p-amino-benzamidine gel. The study of IGF-I/IGFBP-3 interaction was performed by a solid phase method. Results: Extracts of nasal polyps tissue contained IGFBP-3-degrading activity, which was significantly inhibited by benzamidine hydrochloride. Affinity purified IGFBP-3-degrading activity cleaved the IGFBP-3 in two major fragments of approximately 27 and 14 kDa molecular mass, which were not able to bind IGF-I. This activity was rather specific for IGFBP-3, since it did not degrade casein, ovalbumin and gelatin. The IGFBP-3, which significantly suppressed the IGF-I-induced proliferation of nasal polyp fibroblasts, upon treatment with the affinity purified IGFBP-3-degrading activity lost its suppressive activity.

The nasal polyps IGFBP-3-degrading activity may be responsible for the in situ release of IGF-I from IGF-I/IGFBP-3 complexes, which in turn may stimulate the growth of both epithelium and blood vessels in the sinuses, contributing into polyposis pathogenesis.

The nuclear role of SWAP-70 in B cell activation in the growth of prostate and breast cancers. The over-expression of EGFR is correlated with resistance to endocrine therapy. Both tumours show an early hormone-responsive stage towards a hormone-refractory stage, which is more aggressive with a very poor prognosis. The main aim of this study was to report the crosstalk between EGFR and VIP signalling pathways. Methods: We evaluated the effect of a ligand-independent activation of EGFR in two human hormone-dependent cancer cell lines. VIPinduced EGFR trans-activation was investigated in breast (T47D) and prostate (LNCaP) cells. In our study, semiquantitative and quantitative RT-PCR, western blot and immunocytochemical assays were performed. A specific VIP antagonist, JV-1-53, was used to block VIP effects.

Results: In breast cancer cells, VIP modulates the expression of EGFR protein as well as the corresponding phosphorylated form. In prostate cancer cells, VIP regulates both mRNA and protein levels of EGFR. In addition, VIP-induced EGFR transactivation shows a different pattern as compared with breast cancer cells by means of early and late responses. The direct involvement of VIP receptor is also demonstrated by using JV-1-53 analogue. Conclusion: VIP induces a different mechanism of ligand-independent activation of EGFR in human hormone-dependent breast and prostate carcinomas. Although the stimulation pattern is different, these findings support the potential therapeutic value of available VIP receptor antagonists, as well as their usefulness in combined treatments with EGFR antibodies and tyrosine kinase inhibitors in human hormonedependent cancers. localized on X, Y and Z catalytic subunits, respectively. A relationship between its content and activity and cell senescence has been established. Osteoarthritis is a joint degenerative disease highly associated with ageing. Many studies demonstrated the beneficial effects of estrogens in osteoarthritis. The aim of the present study was to determine the effects of estrogens and phytoestrogens on proteasome expression and activity in chondrocytes from patients with osteoarthritis. Methods: Articular cartilage chondrocytes were cultured in the absence or presence of 17 b-estradiol or genistein for 24 hour. The proteasome CT-L activity was determined, using the fluorogenic substrate Suc-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin. The estrogen receptors (ERa, ERb) and proteasome subunits expression at mRNA levels was ascertained by RT-PCR and cell viability was documented by MTT method. Results: Genistein and estradiol up-regulated estrogen receptors expression, with genistein and estradiol to be more effective on ERa and ERb, respectively. They also caused a significant up-regulation and down-regulation on the expression of Z and Y subunit of proteasome, respectively. The expression of X subunit was up-regulated by genistein, while estradiol had no effect on it. The ATP-independent proteasome CT-L activity was enhanced by genistein and estradiol, mainly by genistein. An increased chondrocytes survival, in response to proteasome inhibitor MG-132, was observed in the presence of both agents, with genistein to be more effective.

Conclusions: Estrogens and phytoestrogens possibly exhibit their chondroprotective role, via up-regulation of proteasome expression and activity, thus maintaining chondrocytes homeostasis. Morbidity and mortality due to prostate cancer are mainly a result of prostate cancer metastases. After the initial neoplastic transformation of cells, the process of metastasis develops through a series of sequential steps, which involve tumor proliferation, loss of adhesion, local invasion and migration from the primary tumor. Previously, we reported that Vasoactive Intestinal Peptide (VIP) plays an important role in the initiation of prostate cancer because this peptide enhances ciclooxygenase 2 (COX-2) [1] .The aim of this study was demonstrated that VIP is implicated in progression to malignancy. We analyzed VIP effect in two human prostate cell lines: androgen-dependent (LNCaP) and independent (PC3) cancer cells. 3 H-thymidine assay showed that VIP significantly increased the cell proliferation by 70% in both cell lines compared with untreated controls. Immunoblotting of Cyclin D1 showed a time-dependent VIP increase of Cyclin D1 expression that reached the maximum level at 8 and 6 hours in LNCaP and PC3 cells, respectively. Adhesion assay showed that both cells rapidly whereas and VIP decreased significantly these adhesions. Immunoblotting of Ecadherin demonstrated that VIP decreased the E-cadherin expression in both cell lines. Finally, invasion assay showed that VIP increased the invasion process in both cancer cell lines. All these effects appeared faster at advanced cancer progression. Thus, the implication of VIP in these three steps of metastasis process (proliferation, loss of adhesion and invasion) suggests the involvement of VIP in prostate cancer progression as a pro-metastatic factor. Introduction: It has recently been reported that oxytocin (OT) is produced by some tumoral cell types, and that OT receptors (OTRs) are expressed in a variety of cell types. Among these, human umbilical vein endothelial cells (HUVECs) respond to OT with an increased proliferation, suggesting a possible role for the hormone in the regulation of angiogenesis. Methods: We employed chemotaxis and chemoinvasion assays to characterize the effect of OT on HUVEC motility, and immunoblot analysis to study its molecular mechanisms of action.

Results: We showed that OT stimulates migration and invasion in HUVECs via OTR activation. We identified the Gq coupling of OTRs and phospholipase C (PLC) as the main effectors of OT's promigratory action in HUVECs. We also found that OT stimulates the phosphorylation of endothelial nitric oxide synthase (eNOS) via the phosphatidylinositol-3-kinase (PI-3-K)/AKT pathway, and that the activation of PI-3-K and formation of nitric oxide (NO) are required for the promigratory effect of OT. Interestingly, both the AKT and eNOS phosphorylation induced by OTR activation depend on PLC activity. These findings suggest a new intracellular pathway (currently under investigation) which plays a major role in controlling the OTR-induced migration of HUVECs. Furthermore, OT induced HUVEC sprouting in a 3-D collagen matrix.

Conclusions: The ability of OT to stimulate HUVEC motility, invasion and sprouting suggests that the hormone can participate to physiopathological processes where activation of endothelial cells plays an important role, like for example angiogenesis.

On the role of the energy signaling cascade of AMPK, PPARs, leptin and adiponectin in the metabolic remodeling and cardiac function of sucrose-induced metabolic syndrome Angiogenesis the growth of new blood vessels is an important natural process occurring in the body, both in health and in disease. Angiogenesis occurs in the healthy body for healing wounds and for restoring blood flow to tissues after injury. There are at least 20 angiogenic growth factors. TNF-a is also an angiogenic factor. Tumor necrosis factor-a is a cytokine involved in systemic inflammation and is a member of a group of cytokines that all stimulate the acute phase reaction. TNF causes apoptotic cell death, cellular proliferation, differentiation, inflammation, tumorigenesis, and viral replication. Adrenomedullin (AdM) is a potent, vasodilator peptide that was originally isolated from human pheochromocytoma. AdM signaling is of particular significance in endothelial cell biology since the peptide protects cells from apoptosis, promotes angiogenesis, and affects vascular tone and permeability. In this study, we investigated the effects of adrenomedullin and methyl.-adrenomedullin treatment on TNF-a levels in some rat tissues. Eighteen male wistar rats were divided into three groups: Control group, Adrenomedullin group, methyl-adrenomedullin group. In AdM treated group and met-AdM treated groups, animals received intraperitoneal (i.p) injection of AdM and met-AdM (2000 ng/kg body weight) once a day during a week. Tnfa levels were measured using The Biosource Rat TNF-a Immunoasssay Kit. ANOVA and Duncan test was used in statistically analysis and the value expressed as mean ± SD. In conclusion, in the groups treated AdM and met-AdM, TNF-a Levels were increased compared with controls. The differences were significant (P < 0.05).

Identifying the effects of adult-onset hypothyroidism on mouse cerebral cortex using quantitative gas chromatography-mass spectrometry metabolomics Introduction: Adult mammalian brain has been considered, for long time, as metabolically non-responsive to thyroid hormones, mainly because measurements in adult rat brain slices had indicated that the oxygen consumption rate remained unaffected by their thyroidal status. However, the information about the underlying biochemical network activity that could support this perception is still obscure and fragmented. Technological developments in quantitative metabolomic profiling analysis have enabled the acquisition of the metabolic fingerprint of any biological system, providing thus an integrative view of metabolism. In this study, quantitative Gas Chromatography-Mass Spectrometry (GC-MS) metabolomics was used to determine changes in the metabolic physiology of mouse cerebral cortex induced by adult-onset hypothyroidism.

Methods: The free polar metabolomic profile of the cerebral cortex from 120-days-old control and hypothyroid (1% KClO 4 in the drinking water for 60 days) Balb-c male mice was acquired using Saturn 2200 GC-(ion trap) MS (Varian Inc.). The profiles were appropriately normalized [1] and analyzed using multivariate statistical analysis techniques (TM4 MeV, V3.1), to identify the metabolites, whose concentration was significantly changed from the control to the hypothyroid state.

Results: Analysis of the metabolic network activity based on the characteristic metabolites provided evidence that hypothyroidism does affect the metabolic physiology of the cerebral cortex. Importantly, significant changes were observed in the tricarboxylic acid cycle (TCA) activity.

Conclusions: Metabolomic profiling analysis enabled the differentiation between control and adult-onset hypothyroid mouse cortex. The metabolic pathways whose activity was mainly affected were identified. TrkA receptor activation is a pivotal event for neuronal cell differentiation and survival. However, its over-activation or removal of its ligand NGF tends to lead to cell death. Recently, we showed that TrkA overexpression can induce massive cell death, some of which via apoptosis.

Here we also show the cell death induced by TrkA is associated with autophagy via accumulation of reactive oxygen species (ROS). TrkA induced cells showed many GFP-LC3 punctate formation, development of acidic vesicular organelles (AVO) and formation of autophagosomes, which were blocked by the addition of autophagy inhibitors. In addition, expression of autophagy-related proteins (LC3-II, Beclin 1, Atg5-Atg12 conjugate) was altered during TrkA-mediated cell death.

In particular, reactive oxygen species were dramatically accumulated in TrkA-induced cells, and this ROS accumulation was closely associated with reduction of catalase and caspase 8 expression. Taken together, TrkA overexpression causes ROS accumulation via catalase reduction, leading to autophagic cell death. Introduction and results: The serine/threonine kinase PDK1 plays a central role in cellular signaling by phosphorylating members of the AGC family of kinases, including PKB/Akt. We now present evidence showing that PDK1 is essential for the motility of vascular endothelial cells and it is involved in the regulation of their chemotaxis. Endothelial cells differentiated from mouse embryonic stem cells lacking PDK1 completely lost their ability to migrate in vitro in response to VEGF-A. In addiction, PDK1 -/embryoid bodies exhibit evident developmental and vascular defects that can be attributed to a reduced cell migration. Moreover, the over-expression of PDK1 increased the endothelial cell migration induced by VEGF-A. The ability of PDK1 to regulate cell chemotaxis is dependent on its PH and catalytic domains. As cell adhesion on extra-cellular matrix is essential during cell migration, we analyzed the role of PDK1 in this process. First, we observed that shRNA-mediated PDK1 knock-down inhibited endothelial cell adhesion on vitronectin and, in a lesser way, on fibronectin. In addiction, PDK1 co-immunoprecipitated with both avb3 and a5b1 and it co-localized with avb3 during the early phases of adhesion on vitronectin. Moreover, PDK1 knock-down endothelial cells displayed paxillinstained focal adhesions thicker and longer compared to wild-type cells.

Finally, we observed that PDK1 knock-down inhibited integrin avb3 endocytosis. The serine-threonine kinase PAK1 is activated by small GTPase-dependent and -independent mechanisms. However, the role of tyrosyl phosphorylation in PAK1 regulation is unclear. We showed that prolactin (PRL)-activated tyrosine kinase JAK2 phosphorylates PAK1 in vivo and that WT but not kinase dead JAK2 directly phosphorylates PAK1 in cells and in an in vitro kinase assay. Using mass spectrometry and 2D peptide mapping, we identified tyrosines 153, 201 and 285 in PAK1 as sites of JAK2 kinase tyrosyl phosphorylation. Experiments using PAK1 with tyrosines 153, 201 and 285 mutated to phenylalanines (PAK1 Y3F) suggest that these tyrosines serve as regulatory sites in PAK1. Phosphorylation of these tyrosines by JAK2 increased PAK1 kinase activity by more than two times and decreased staurosporineinduced apoptosis. To test whether pTyr-PAK1 participates in cytokine-stimulated actin reorganization, we overexpressed WT or Y3F PAK1 in 3T3 F442A cells that have the growth hormone (GH) receptor and MCF7 cells that have the PRL receptor and assayed membrane ruffling. Overexpression of WT PAK1 stimulated membrane ruffling in response to GH and PRL compared to control cells whereas the Y3F PAK1 blocked the ruffling. We examined the effect of pTyr-PAK1 on cell motility using a gold particle motility (phagokinetic) assay. The cells overexpressing PAK1/JAK2 or PAK T423E exhibited increased cell motility compared to cells overexpressing either WT PAK1/JAK2 K882E or 3YF PAK1. Our findings indicate that JAK2 tyrosine kinase phosphorylates PAK1 and this phosphorylation plays an important role in cell survival, cell motility and cytokine-stimulated membrane ruffling.

Androgen receptor participation in nuclear b-catenin expression and prostate cancer progression J. Duliqska-Litewka and P. Laidler Chair of Medical Biochemistry, Jagiellonian University Medical College, Crakow, POLAND Prostate cancer constitutes a serious males, health problem and has a high prevalence to metastasize making it a very difficult cancer to treat if not detected early. Androgen receptor (AR) controls the expression of genes involved in cell proliferation, migration, differentiation, and cell death. Increasing cellular levels of the AR not only intensify androgen-induced cell proliferation but also increases the sensitivity of prostate cancer cells to androgens, allowing them to grow in a low androgen environment. b-catenin acts as coactivators of AR but also Akt modulates androgen signaling. We used siRNA constructs (Ambion) to observe the AR's and Akt's effect on androgen-induced transcription, cell proliferation (BrdU and crystal violet assay) and migration (Boyden chamber) in prostate cancer cell lines (LNCaP, Du145, PC-3). AR, pAkt(Ser-473), KLK3, GSK-3b-P, b-catenin, E-, N-cadherins, cyclinD1, cyclinG1, p21, p27 and c-myc expression was determined by western blot and RT-PCR analysis. Nuclear translocation of beta-catenin was studied using NuCLEAR Extraction Kit.

Silencing of AR with siRNA in LNCaP cell line significantly reduced proliferation (45-70%), expression of KLK3, cyclinD1, cyclinG1, nuclear beta-catenin but increased expression E-cadherin, p21 and p27. Akt takes over the function after longer silencing of AR, restores height proliferation and inverts expression of studied proteins. We showed the role of AR and Akt in proliferation and their reciprocal interaction in b-catenin signaling pathway which may directly contribute to cancer progression in cause of loss of androgen dependence. Introduction: 17b-Hydroxysteroid dehydrogenases (17b-HSDs) control intracellular availability of active sex steroids in gonads and peripheral tissues. So far, fourteen different 17b-HSD types have been described. All of them belong to the short-chain dehydrogenase family except 17b-HSD5, which is an aldo-keto reductase. To date, only 17(-HSD types 7 and 8 promoters have been characterized. In both cases CCAAT boxes are necessary for transcription, and C/EBPb binding to these motifs was described in the latter. Here, we analyze the role of CCAAT boxes, C/EBPb and 17b-estradiol in the transcription of human 17 (-HSD types 1 Introduction: Cycloheximide (CHX), a protein synthesis inhibitor with high toxicity, induces apoptosis in some cell types. We present a case report of a pregnant rat which developed a malignant giant breast tumor after treatment with CHX. We try to investigate the mechanism of action of CHX by determining the caspase-3 activity in liver. Methods: Pregnant rats (Wistar) were injected with CHX (3 mg/ kg.b.w.) on 10 th , 11 th gestational days, and 24 hour before sacrificing. Pregnancy on most of the rats resulted in abortions and teratomas, while on one of those, a big breast tumor developed rapidly, which in 2 months became enormous. Three months after the tumor was first observed, we anesthetized the animal, removed the tumor by a surgical operation and sacrificed it. Liver also removed and homogenized. Following subcellular fractionation according to the method of Nordlie and Lardy, the caspase-3 activity was determined in crude, nuclear, and cytosolic+ribosomal fractions by a colorimetric assay (kit, Sigma), based on the hydrolysis of the peptide substrate acetyl-Asp-Glu-Val-Asp p-Nitroaniline (pNA) moiety. Tissue samples from the breast tumor were proceeded for histological sections in order to observe its histological structure. Paraffin sections 4 lm thick were stained with Hematoxylin, Eosin and Trichrome Masson. Introduction: The local and systemic inflammatory response induced by brain hypoxia is an important target for the actual research. Our aim is to study the neuronal growth factor -neurotrophin 3 (NT3) and the inflammatory cytokines (TNFa, IL-6 and IL-10) in serum and cerebrospinal (CSF) patients with ischemic stroke, in order to establish possible correlations between their dynamic and activity. Methods: Forty-five patients aged 67.65 ± 10.11 years. were investigated after computed tomography-confirmed IS. The biological samples were represented by blood and CSF. NT-3 and cytokines were determined using ELISA kits. Results: No significant changes were obtained for TNFa and IL-10. Considering NT3 and IL-6 levels, patients were divided in three groups. Group 1 (2-5 days after IS), with significant higher levels of NT3 and IL-6 than controls. Group 1 patients presented significantly increased CSF IL-6 and NT3 levels compared with serum (p < 0.05). Group 2 (7-11 days after IS), with higher levels of IL-6 than controls (p < 0.05), but lower than group 1 (p < 0.05). NT3 serum and CSF levels were significantly increased (p < 0.05) compared to controls and group 1. Group 3 (13-26 days after IS): with higher levels of IL-6 and NT3 than controls (p < 0.05), but lower than group 1 and group 2 (p > 0.05).

Conclusions: the peak of the stroke-induced inflammatory response is between 7th and 11th day after IS, as well as the NT3 release from central nervous system. It is to be proved if NT3 high level is a proapoptotic or a pro-regenerative factor. Introduction: Coexpression of EGFR and HER-2 has been reported in a subset of colon cancers and may cooperatively promote tumor cell growth and survival. In the current study, two tyrosine kinase inhibitors gefitinib and lapatinib, were evaluated in two colon cancer cell lines, DLD-1 and Caco-2. The aim of the study was to investigate their effect on tumor cell proliferation and apoptosis. Methods: Cell proliferation was estimated using the MTT assay and apoptosis was evaluated by DNA fragmentation and the Annexin V binding assay. EGFR and HER-2 protein and mRNA levels were evaluated by immunoblotting and quantitative RT-PCR, respectively. The solution of the question about existing of the second route of ammonia assimilation in higher plants is very important for plant biochemistry. There is a number of works in which it is underlined that NADP-specific glutamate dehydrogenase (NADP-GDh) can play role in ammonia assimilation. However up to date nobody succeeded to study kinetic characteristics of NADP-GDh. We also couldn't purify the soluble form of NADP-GDh. In this reason we decided to isolate it. Using chromatography on column with nanostructured carbon sorbent we purified spherosome bound NADP-GDh from filling seeds of wheat. This spherosome bound NADP-GDh has high affinity to ammonia. Its K M is equal to 1.3 lM. We studied the activation of this enzyme on the model system on the unembryonated dry wheat seeds that were treated by fusicoccin. It was established that fusicoccin activates spherosome bound NADP-GDh by increasing of level of cytosolic calcium. It was established that mechanism of activation of NADP-GDh of spherosome includes two stages: first is binding of cytosolic Ca 2+ with GDh of spherosome, the second stage is the phosphorylation of spherosome enzymes. Thus we discovered the new route of assimilation of ammonia which is carried out by spherosome bound NADP-GDh. The activation of this enzyme is controlled by fusicoccin, Ca 2+ and phosophorylation. Introduction: TNFa is a proinflammatory cytokine, which is produced by many cell types, including adipocytes. Today, much effort has been given to clarify the role of TNFa in obesity and insulin resistance. TNFa affects adipocyte function in various ways including their implication in inflammation, which accompanies the development of obesity. The inflammatory lipid mediator PAF often mediates the effects of TNFa on other cell types. We have already shown that TNFa induces PAF biosynthesis sevenfold higher in adipocytes than in preadipocytes. In this study, we investigate the involvement of PI3K and ERK1/2 in this process. Methods: Adipocytes and preadipocytes were isolated from the epididymal fat pads of male Wistar rats. The cells were treated with TNFa in the presence or absence of wortmannin. PAF production and ERK1/2 activation were determined by using [ 3 H]-lyso-PAF as a substrate and western blotting, respectively. Results: Wortmannin enhances TNFa-inducing effect on PAF syntesis in adipocytes, while it inhibits this effect in preadipocytes. TNFa affected differentially ERK1/2 activity in the two cell types, as it enhances the activity only in preadipocytes. Finally, wortmannin potentiated TNFa effect on ERK1/2 activity in adipocytes and inhibits TNFa effect in preadipocytes.

Conclusions: TNFa is more efficient inducer of PAF synthesis in adipocytes than in preadipocytes suggesting a maturation of TNFa-PAF interrelationship during cell differentiation. PI3K and ERK1/2 are involved in this process, though the exact mechanism of ERK1/2 implication remains to be seen. 

In vitro activation of Nrf2-mediated oxidative stress response in human cerebral microvascular endothelial cells by resveratrol Introduction: It is well established that hypoxia (H) followed by reoxygenation (R) results in generation of reactive oxygen species (ROS) and subsequent tissue damage. Nuclear factor erythroid-related factor 2 (Nrf2) is a redox-sensitive transcription factor which is involved in the transcriptional regulation of many antioxidant genes. Resveratrol (RSV), a polyphenolic phytoalexin, has been reported to possess antiinflammatory, anticarcinogenic, and antioxidant activities. In this study, we examined the neuroprotective effect of RSV against H/Rinduced oxidative stress using human cerebral microvascular endothelial (HCMVE) cells in vitro. Methods: HCMVE cells were pretreated with RSV (10 lM) for one hour and the cells were exposed to oxygen glucose deprivation (OGD) to mimic H/R (6 hour OGD/24 hour reoxygenation) for the generation of ROS. Intracellular ROS production was measured flourometrically by using hydroxyphenyl fluorescein at 10 lM concentration. HCMVE cells were incubated with RSV (10 lM) and the activation of Nrf2 was analyzed by western blotting. Induction of Nrf2 target genes mRNA expression was determined by Real-time PCR.

Results: H/R induced the ROS generation and pre-treatment of cells with RSV significantly reduced H/R-induced production of ROS. RSV induced nuclear translocation of Nrf2 1 and 3 hours after RSV incubation. RSV also induced the mRNA expression of Nrf2 and its target genes such as glutamyl cysteine synthetase (GCS), heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase-1 (NQO1), 24 hours after RSV treatment.

Conclusions: These results suggest that RSV augments cellular antioxidant capacity through activation of Nr2 and induction of Nrf2 target genes, thereby protecting HCMVE cells from oxidative stress. 3-phosphoinositide-dependent protein kinase-1 (PDK1) is a master kinase that activates various intracellular serine/threonine kinases, which are involved in diverse cellular processes including cell survival, proliferation, protein synthesis, and gene expression. Here we show a functional association between PDK1 and apoptosis signal-regulating kinase 1 (ASK1). The physical association between PDK1 and ASK1 is mediated by the pleckstrin homology (PH) domain of PDK1 and the C-terminal regulatory domain of ASK1. Wild-type PDK1, but not kinase-dead PDK1, negatively regulates ASK1 activity by phosphorylating ASK1 on serine 967. ASK1 phosphorylation by PDK1 significantly inhibits ASK1-mediated JNK and p38 kinase activities. In addition, PDK1-mediated inhibition of ASK1 is induced through the decrease of interaction between ASK1 and its substrate MKK3 and the stabilization of ASK1-14-3-3 complex, as well as the decrease of ASK1 homo-oligomerization. We also demonstrate that PDK1 suppresses ASK1-mediated apoptosis by inhibiting the ASK1-induced capase-3 activity. These results suggest that PDK1 physically interacts with ASK1 in vivo and acts as a negative regulator of ASK1.

Phosphorylation and intracellular distribution of protein kinases ERK1/2 and Src in proteasomeinhibited PC12 cells Introduction: Rat pheochromocytoma (PC12) cells differentiate into a phenotype that resembles sympathetic neurons upon treatment with nerve growth factor (NGF). The signaling events elicited by the activated NGF receptor are extremely complex. The activation of protein kinases ERK1/2 and Src seem to be crucial to change the pattern of gene expression in favour of neuronal differentiation. The phosphorylation of these kinases can also be achieved by alternative treatments like inhibition of the proteasome. Methods: We analysed the activation kinetics and intracellular distribution of active/phosphorylated ERK1/2 and Src isoforms by means of Western blotting and fluorescent immunocytochemistry followed by laser scanning confocal microscopy, after treatment of PC12 cells with NGF or the proteasome inhibitor MG-132.

Results: Compared to the NGF-treated control samples the phosphorylation of ERK1/2 and Src were both biphasic upon MG-132 inhibition, peaking at around one then later at 6 hours. Despite of the robust phosphorylation and prolonged activation kinetics the nuclear translocation of the phosphorylated enzymes and the neuronal differentiation of the cells was significantly weaker compared to that detected after NGF treatment. Conclusion: The weaker nuclear translocation of ERK1/2 and Src could at least in part be responsible for the significantly poorer differentiation seen in proteasome inhibited PC12 cultures compared to NGF-treated controls. Introduction: Clonal cell lines, which possess the phenotype of original cells, are useful for studying the molecular mechanism of cell differentiation and cell death. Temperature-sensitive (ts) mutant of SV40T-antigen is known to be usable to control the cell proliferation and differentiation by temperature shift. We report here the establishment and characterization of noradrenergic chromaffin cell line carrying tsSV40T-antigen. Methods: Transgenic mice carrying tsSV40T-antigen gene (SV40tsA58) under the control of tyrosine hydroxylase gene promoter were produced. From the tumor tissue produced in adrenal medulla, chromaffin cells were isolated and cultured in DMEM containing 10% FBS and G5 supplement. Results: From several clones, we isolated one clone, named tsAM5NE, which produced noradrenaline and secreted it actively into the medium. tsAM5NE cells proliferated at the permissive temperature of 33°C in response to glial cell line-derived neurotrophic factor (GDNF). At a non-permissive temperature of 39°C, GDNF acted to differentiate the cells into neuron-like cells. In the presence of GDNF, the morphological change induced by the temperature shift was associated with up-regulated expression of neuronal marker genes. The temperature shift in the absence of GDNF led to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. These data suggested that GDNF inhibited the TRAIL-mediated apoptosis of tsAM5NE cells, and led to the stable neuronal differentiation. Conclusions: Noradrenergic chromaffin cell line, tsAM5NE cells, are quite useful for studying key molecules involved in GDNF-induced neuronal differentiation.

Glutamate induced ex-cytotoxicity elicits both calpain dependent and independent cell death in naive and NGF-treated PC12 cells Introduction: Mechanisms of increased synthesis of oxygen free radicals injure and damage neurons and are implicated in both acute and chronic neurological diseases. Calcium influx through glutamate receptors and voltage-dependent channels mediate an array of functional and structural responses in neurons. Uncontrollable calcium transfer can trigger potentially lethal biochemical pathways causing excitotoxicity. In the present study we investigated the influence of glutamate induced ex-citotoxicity in neurites using PC12 cells. Methods: PC12-cells were cultured in complete DMEM medium supplemented with 15% horse serum and 5% FBS in the presence of antibiotics at 37°C with 5% CO 2 in a humidified incubator. Glutamate excitotoxicity cell death was affected by exposing naive and NGF-treated PC12 cells to glutamate at two different concentrations (0.5 lM and 10 lM) for 3 hour. We investigated the involvement of calpain inhibition in cell death induced by glutamate toxicity, using a calpain inhibitor MDL28170 (Cbz-Val-Phe-H) at two different concentrations (10 lM and 50 lM). Cell viability was estimated by a colorimetric method using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide. Results: We found both concentrations of MDL28170 protect both naı¨ve and NGF treated PC12 cells from cell death induced by low glutamate concentrations. The comparison between the two MDL28170 concentrations did not reveal any statistical difference. However in higher glutamate concentrations both concentrations of MDL28170 did not seem to have any effect. Conclusions: The mechanisms implicated in cell death by glutamate exitotoxicity seem to differentially engage calpain dependent and independent pathways. Acknowledgements: This work was supported by the European Union/European Social Fund EPEAEK PYTHGORAS II.

Adiponectin inhibited proliferation and protected human non-neoplastic salivary gland epithelial cells from spontaneous apoptosis S. Katsougiannis, R. Tenta and F. Skopouli Harokopio University of Athens, Athens, GREECE Introduction: Adiponectin is a hormone predominantly produced by adipose tissue, which displays immunoregulatory properties. Primary Sjogren's syndrome (pSS) is an autoimmune disease characterized by lymphocytic infiltrates associated with destruction and dysfunction of salivary glands. We have previously shown that salivary gland epithelial cells (SGEC) from pSS patients as well as from controls are a source of adiponectin and they express the two adiponectin membrane receptors AdipoR1 and AdipoR2. SGEC derived from pSS patients, constitutively produce and secrete adiponectin in higher quantities. Therefore, we examined the effect of adiponectin on the proliferation and apoptosis of SGEC of pSS and control individuals. Methods: The rate of cell proliferation was assessed using MTT assay, and apoptosis was measured by a specific ssDNA ELISA which detects apoptotic but not necrotic cells. The intracellular apoptosis pathway-activator camptothecin was used as positive control in both proliferation and apoptosis assays. Results: Adiponectin treatment leaded to a dose-depended suppression of proliferation in SGEC from pSS and control donors, with the maximum effect being observed with 20 lg/ml adiponectin. Intriguingly, SGEC exposure to adiponectin protected cells from spontaneous apoptosis. Conclusion: Adiponectin exerted direct antiproliferative effects on SGEC from pSS and control individuals without inducing apoptosis and protected SGEC from spontaneous apoptosis. These observations connote that this hormone may contribute to a stability of SGEC in this specific inflammatory milieu. Introduction: The cGMP-dependent protein kinase family of proteins (PKG) mediates many of the effects of the NO/cGMP pathway, controlling a variety of cellular responses ranging from smooth muscle cell relaxation to neuronal synaptic plasticity. In the present study we set out to determine the effect of PKG-I inhibition in VEGF-induced angiogenesis both in vivo and in vitro. Methods: In vivo models: the chicken chorioallantoic membrane (CAM) and the corneal assay. In vitro assays: proliferation, migration, tube-like formation assay. Results: Two peptides inhibitors for PKG-I were used: DT2 and DT3. Treatment of CAMs with DT-2 or DT3 reduced angiogenesis in a dose-dependent manner (maximal inhibition of 13.7 ± 0.7% of control at 10 nmol/egg). To investigate the role of PKG-I in growth factor-induced angiogenesis we tested the effect of DT-3 on VEGFstimulated neovessel growth in the corneal assay. DT3, when implanted at the same time with VEGF abolished the angiogenic response, while when implanted 5 days after VEGF reduced angiogenesis by 78%. In addition, DT-3 blocked VEGF-stimulated permeability, in vivo. We also tested the role of PKG-I in angiogenesis-related properties of human endothelial cells. In agreement with our in vivo observations, PKG-I inhibition reduced VEGF-induced signaling (ERK1/2 and p38), and attenuated VEGF-triggered migration and proliferation in these cells. In contrast, DT-3 exerted no effect in the tube-like formation. Conclusion: We conclude that PKG-I activation contributes to VEGF-induced angiogenesis.

Signaling proteins involved in leptin-induced amplification of human monocyte atherosclerosisrelated properties Introduction: Leptin, a 16 kDa cytokine released mainly by the adipose tissue is known to exert a pleiotropic effect, affecting a wide variety of processes ranging from energy homeostasis to angiogenesis and the immune response. In the present study, the effect of leptin on atherosclerosis-related human monocyte properties was investigated. Methods: Monocytes were isolated from whole blood obtained from healthy donors with a normal body mass index (BMI) value. Pharmacological inhibition of specific signaling proteins was implemented. Fluorescence spectrometry and immunofluorescence techniques, as well as ELISA methods, were utilized. Dose-response curves were carried out in order to justify the leptin concentration that was used. Results: Leptin (160 ng/ml) was found to augment monocyte adhesion to and migration through laminin-1 (one of the main components of the extracellular matrix). Additionally, leptin increased CD36 receptor surface expression and moderately oxidized low density lipoprotein (oxLDL 3 ) uptake levels. Conclusions: Leptin amplifies the pro-atheromatic properties of human monocytes through a complex signaling net which involves the Na + /H + exchanger isoform-1 (NHE1), the actin cytoskeleton, phosphoinositide 3-kinase (PI3K), certain conventional isoforms of protein kinase C (PKC) and NADPH oxidase.

PP7A-47 Na + /H + -exchanger-1 is activated by obesity related hormones in obese patient monocytes Introduction: Na + /H + -exchanger-1 (NHE-1) is a membrane protein expressed in mammalian cells and its main role is the maintenance of intracellular pH and the cellular volume. The aim of the present study was to investigate whether NHE-1 activity is influenced by hormones related to obesity. Methods: Monocytes were isolated from 12 obese subjects and 10 healthy age-and sex-matched controls. NHE-1 activity was estimated. Monocytes were incubated in the presence of glucose, insulin, leptin and adrenaline. When necessary, the inhibitors of known signaling molecules were added. Results: Our results indicate that treatment of monocytes with glucose, insulin, leptin or adrenaline cause an elevation in pHi, compared to control value, in both groups studied. In obese subjects d and e isomorphs of PKC and NADPH oxidase were involved, while in healthy volunteers a and b isomorphs of PKC and PI3K seem to play role in the elevation of pHi induced by glucose. Insulin caused an increase of pHi through the involvement PI3K and NO synthase in both groups studied, while in obese NADPH oxidase was also involved. In obese subjects, leptin activates NHE-1 through the involvement of a and b isomorphs of PKC, actin polymerization and NO synthase. Adrenaline also seems to activate NHE-1 through NO sythase, PI3K and actin polymerization, only in healthy subjects. Thyroid hormone (T3) plays a crucial role in processes such as cell proliferation and differentiation, while its implication on cellular apoptosis has not been well documented. Here we examined the effect of T3 on the apoptosis of GH4C1 cells and the mechanisms underlying this effect. We show that T3 produced a significant increase in apoptosis in serum-depleted conditions. This effect was accompanied by a decrease in NF-jB-dependent transcription, translocation of p65/NF-jB to the nucleus, phosphorylation, and transactivation. To further analyze the mechanisms by which T3 induced apoptosis, we examined its effects on the activity of the MAPK family. Our data show that ERK but not JNK or MAPK p38 was activated upon exposure to T3, and inhibition of ERK alone abrogated T3-mediated apoptosis. In addition T3 increased the expression of the MAPK phosphatase, DUSP1, in an ERK-dependent manner. Interestingly, the suppression of DUSP1 expression abrogated T3-induced inhibition of NF-jB-dependent transcription and p65/NF-jB translocation to the nucleus, as well as T3-mediated apoptosis. Overall, our results indicate that T3 induces apoptosis in pituitary cells by down-regulating NF-jB activity through a mechanism dependent on the ERK/DUSP1 pathway.

Angiotensin II regulates transcription of a1adrenoceptors in aorta of C57B/6J and knockout a1D mice The most important systems participating in the blood pressure control are the a 1 -adrenoceptors (a 1A. a 1B, a 1D ) and the Renin-Angiotensin System (RAS). It is suggested that a 1D -adrenoceptors are strongly related with the genesis/maintenance of hypertension and angiotensin II up regulates (cross talk) a 1D -adrenoceptors. We aimed to study the interaction between angiotensin II and the a 1 -adrenoceptors in aorta. We used thoracic aorta from male mice C57B/J6 (WT) and KO a 1D (12 weeks old), the dissected aortas were treated for 1 hour with angiotensin II in DMEM medium/37°C, and the RNA was obtained using Trizol reagent. RT PCR was conducted from treated vessels and controls (pool of 2-3 vessels per group). Quantification of gene expression levels of each a 1 -adrenoceptor was carried out using real-time PCR assay. Results show that angiotensin II up-regulated 4-fold the expression of a 1A and a 1B -adrenoceptors versus control in WT mice, while a 1D -adrenoceptor was up-regulated 1.5-fold. In KO the a 1A was upregulated 2-fold and the a 1B just 0.4-fold versus control (without angiotensin II). Data were normalized using ribosomal housekeeping 18S. Our results support the idea that there is cross talk between angiotensin II and the a 1 -adrenoceptors system since angiotensin II promotes an increased expression not only for a 1D subtype but also for a 1A and a 1B -adrenoceptors. Fever is due to the action of prostaglandin (PG) E2 on the brain and appears to require EP3 prostaglandin receptors (EP3Rs), but the site of action required for fever production has not been definitively identified. We have produced mice with conditional expression of EP3Rs to investigate their role in lipopolysaccharide-induced fever by insertion of flanking loxP sites into the 5'-untranslated region and the first intron of the EP3R gene. To switch the EP3R gene off focally within the brain, we have developed an adeno-associated viral (AAV) vector that causes neurons to express Cre recombinase. In the transduced neurons, Cre recombinase removes the DNA segment flanked by loxP sites, to turn off the EP3R gene expression in pattern dictated by the site of AAV injection. Thereby, we identified the key site in the brain at which PGE2 acts on EP3 receptors on neurons to cause fever [1] . We also found that stereotaxic microinjections of AAV-Cre produce minimal tissue injury and that AAV10 is the superior serotype for expression of Cre recombinase in the MnPO due to enhanced and widespread transduction of neurons as compared to serotypes 2, 8 and 9. AAV transfects neurons very effectively, with small injections caused by AAV serotype 2 and larger ones by AAV serotype 10 (which diffuse more widely). The focal knockout approach used in this study has wide applicability to neuroanatomical problems involving dissection of the effects of different receptors with complex brain distributions. Introduction: Growth hormone secretagogue receptor type Ia (GHS-R1a) transduces signals carried by ghrelin, an acetylated 28-residue peptide hormone, to stimulate the release of growth hormone, and to actively regulate food intake and energy homeostasis. In addition, several studies indicate that ghrelin has other effects in the periphery, including effects on cell proliferation through activation of MAPK and PI3K/Akt pathways, as well as anti-apoptotic effects on various cell types. In this work, we explore the role of b-arrestins in the activation of Akt in line with the role of b-arrestins in MAPK activation by ghrelin. Methods: For this purpose we used the HEK 293 cell line stably transfected with the ghrelin receptor, GHS-R1a, and we made inmunoprecipiation experiments as well as siRNA and also tested different inhibitors. Results: We observed that Akt phosphorylation is mediated by interplay of two signaling pathways. One pathway is mediated by b-arrestins 1 and 2, and requires entry of the receptor into a multiprotein complex associated to the b-arrestins. In this pathway Akt both physically interacts with and is activated by b-arrestins. A second pathway is G i/o -dependent and involves PDK1. Conclusions: Our findings support the view that ghrelin activates Akt pools that differ in their temporal and spatial distributions, and thus probably have different physiological targets as was described for MAPK.

Platelet-derived growth factor receptor transactivation by extracellular sphingosine 1-phosphate requires c-Src kinase activity: involvement in NIH 3T3 fibroblast proliferation Introduction: Spingosine 1-phosphate (S1P) belongs to groups of platelet-derived lipid mediators regulating various biological processes. Extracellular S1P acts through specific G-protein coupled receptors (GPCRs) in various cells including NIH3T3 fibroblasts. In these cells, intracellular S1P is also related to PDGF mitogenic signalling as second messenger and to PDGF-induced cell motility by autocrin mechanisms of S1P receptors stimulation. S1P and some GPCRs ligands transactivate receptor tyrosine kinase (RTK) with important effects down-stream the signalling pathways. Methods: Cell culture, transfection experiments, western blot analysis were performed as reported in the reference (1) . Results: Previously we demonstrated that PDGF receptor (PDGFr) is redox regulated in NIH3T3 fibroblasts and that S1P stimulation induces H 2 O 2 production (1). Concentration-and time-dependent PDGFr tyrosine phosphorylation after S1P stimulation have been demonstrated. Phosphorylation of tyrosine 857 essential for the full activation and regulation of PDGFr kinase activity was observed and similar results were obtained after cell stimulation by dihydro-S1P, S1P analogue that binds GPCRs. In fact, the results demonstrate that PDGFr-S1P transactivation occurred through GPCRs and was specific. S1P effect was mediated by c-Src kinase activation with the involvement of H 2 O 2 production. These events were up-stream of PDGFr transactivation and this study demonstrates that PDGFr transactivation contributes to ERK1/2 activation and NIH3T3 fibroblasts proliferation. Conclusions: These data can be useful to clarify S1P-stimulated transactivation of RTK that can contribute to the modulation of important pathophysiological conditions little known particularly in fibroblasts. We analysed class B scavenger receptors (SR-BI, SR-BII, LIMPII) and hormone sensitive lipase (HSL) protein expression and inmuno-localization in control rat testis, after chemical castration induced by EDS and LGF-treated rats, since LGF improves testicular regeneration after EDS.

Experimental design: Thirty six male rats grouped in non-treated (controls), EDS-treated, LGF-treated, and EDS+LGF. Testes were obtained at days 10 (T1), 21 (T2) and 35 (T3), embedded in paraffin and studied by immunohistochemistry and western Blot.

Results: SR-BI was localized to the surfaces of Leydig cells, acrosome of the spermatides and some spermatogonias in control and LGF-treated testis.SR-BII was localized in spermatocytes.LIMP II was mainly localized in Sertoli cells and less in Leydig cells. EDS treatement distroyed Leydig cells and reduced spermatogenesis. SR-BI Leydig cells immunostaining disappeared after EDS treatement and it was restablished progressivelly with the time.

LGF treatment protects EDS damage and induced a rapid Leydig cell and tubular regeneration, increasing cellular proliferation in the seminiferous tubules. EDS+LGF-treated SR-BI immunopositive Leydig cells were observed earlier than with EDS and SR-BI and SR-BII positive staining was restablished after spermatogenesis regeneration. HSL was localized in some spermatogonias, unlarged spermatides, Sertoli and Leydig cells.

LGF increased positive staining of SR-BII and HSL immunoreactivity of spermatogonias and elongating spermatids.

Acknowledgement: Supported by Fondo de Investigaciones Sanitarias (PI050403). Methods: Testes obtained from control and HSL knockout (HSL-/-) mice were frozen or embedded in paraffin to study protein expression by western Blot and immunolocalization by immunohistochemistry.

Results: HSL (-/-) mice testis completely lacked HSL and presented altered spermatogenesis associated with decreased sperm counts, sperm motility and are infertile. The defects included multinucleation of spermatides, abnormal shapes and reduced elongating spermatids. Many epithelial cells in the seminiferous tubules were vacuolated and the amount of Leydig cells was increased. HSL is expressed in elongating spermatides, from control mice testis. In control testis, SR-BI was localized to the surfaces of Leydig cells and spermatides; SR-BII was in spermatocytes and spermatides, Leydig cells were immunonegatives for SR-BII; and LIMP II was mainly localized in Sertoli cells and less in Leydig cells. HSL (-/-) testis showed increased SR-BI, SR-BII and LIMPII expression and immunostaining in Leydig, germinal and Sertoli cells, respectively.

increase expression in mice testis which allows increased cholesteryl sters uptake. 2 HSL is critical in testis steroidogenesis and spermatogenesis. PRIP (phospholipase C related, but catalytically inactive protein) was identified as a novel inositol 1,4,5-trisphosphate binding protein, whose domain organization is similar to that of phospholipase C-d1 but is catalytically inactive, comprizing two isoforms, PRIP-1 and PRIP-2. To get insight into the biological functions, we generated the double knockout (DKO) mice of both PRIP-1 and PRIP¡V2, followed by the phenotypic analyses. DKO mice grew normally and became fertile. However DKO female exhibited the decreased litter occasion and litter size, indicating the dysfunction of female reproductive system. Then we examined the estrus cycle by cytological analysis of daily vaginal smears, resulting in the irregular cycle in DKO mice. We further noticed that the mutant mice had apparently smaller sized uterus by gross anatomical observation. Basal levels of serum leuteinizing hormone and follicle stimulating hormone were significantly higher in the mutant, the event of which was also confirmed by examining the secretion from the tissue culture of anterior pituitary glands. These results suggest that PRIP is involved in maternal reproduction, through the gonadotropine secretion. The scavenger receptor class B type I (SR-BI) is the natural receptor of high density lipoprotein (HDL) and plays an important role in the reverse transport of cholesterol from peripheral tissues back to the liver for secretion and to steroidogenic tissues for the production of steroid hormones. In the present study we investigated the role of hormone nuclear receptors in human SR-BI gene regulation in hepatic, adrenal and ovarian cells. In hepatic cells we showed that the activity of the hSR-BI promoter was inhibited by the orphan nuclear receptor Hepatocyte Nuclear Factor 4 (HNF-4) as well as by the Small Heterodimer Partner (SHP), which is an established inhibitor of nuclear receptor activity. Chromatin immunoprecipitation assay confirmed the recruitment of HNF-4 to the distal region (-2787/-2301) of the hSR-BI promoter. In agreement with these findings, over-expression of HNF-4 repressed the endogenous SR-BI mRNA levels in HEK293T cells. In the adrenal and ovarian cells the expression of endogenous SR-BI and the activity of the hSR-BI promoter were decreased by dexamethasone administration and were further inhibited by over-expression of the glucocorticoid receptor (GR). The region between -290 and -62 was shown to be responsible for the inhibition by glucocorticoids. Finally, we examined the effect of Corticotropin Releasing Factor (CRF), in the expression of the SR-BI gene in steroidogenic tissues. We found that administration of CRF in ovarian SKOV-3 cells decreased the mRNA levels and the activity of the hSR-BI promoter, suggesting the CRF-induced signal transduction pathways negatively modulate SR-BI gene expression. In agreement with these findings we found that SR-BI mRNA levels were increased in adrenals from CRF KO mice.

Mechanisms of hepatocyte growth factor-induced cholangiocarcinoma cell invasion: an involvement of MAPK pathway A. Menakongka and T. Suthipongchai Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok THAILAND Cholangiocarcinoma (CCA), a malignant tumor of the bile duct epithelium, is one of the major cancers in Northeast Thailand. This disease is difficult to diagnose and has a high mortality rate, thus posing an important public health problem in this region. c-Met, hepatocyte growth factor (HGF) receptor, and its ligand, HGF, regulate diverse biological responses including proliferation, migration and invasion, key features of metastatic cancer. Over-expression of c-Met has frequently been found in cholangiocarcinoma suggesting the role of c-Met in cholangiocarcinogenesis. Here, we studied the biological effects of HGF stimulation on CCA cell lines. We found that two CCA cell lines over-expressed c-Met protein compared to H69 immortalize cholangiocyte. Functional studies demonstrated that HGF strongly induced invasive ability of HuCCA-1 and KKU-M213 via induction of cell motility. As MAPK pathway has been implicated in invasion in many cancer cell types, we have addressed the significance of the role of MAPK in HGF-induced invasiveness by using specific MEK-1 inhibitors, U0126. We showed that inhibition of MAPK pathway slightly affected HGF-mediated HuCCA-1 cell invasion but drastically suppressed KKU-M213 cell invasion induced by HGF. This indicated that HGF induces invasion of two CCA cell lines by distinct signaling pathways.

Novel mechanism that mediates the angiogenic properties of pleiotrophin through a m b 3 integrin C. Mikelis and E. Papadimitriou Laboratory of Molecular Pharmacology, Department of Pharmacy, University of Patras, Patras, GREECE Introduction: Pleiotrophin (PTN) is an 18 kDa growth factor displaying a significant role in tumor growth and angiogenesis. We have recently shown that pleiotrophin (PTN) induces human endothelial cell migration through its receptor protein tyrosine phosphatase RPTPb/e. Integrins are heterodimeric transmembrane receptors that mediate cell attachment to the ECM and interact with cell surface and soluble ligands. Among integrins, a m b 3 is the most abundant and influential receptor regulating angiogenesis. In the present work we studied the possible involvement of a m b 3 integrin in PTN-induced endothelial cell migration. Methods: A combination of methods was used, including immunoprecipitation and western blot analysis, migration assays, siRNA, immunofluorescence and confocal microscopy. Results: A monoclonal antibody against a m b 3 but not a 5 b 1 integrin completely abolished the effect of PTN. PTN interacts directly with a m b 3 but not a 5 b 1 , while RPTPb/e receptor interacts with both integrins. The interaction is RGD-independent but seems to be through a cysteine loop at aminoacids 177-184 that regulates ligand specificity to a m b 3 . RPTPb/e is not required for interaction of PTN with a m b 3 but is necessary for PTN-induced phosphorylation of b 3 . Conclusions: PTN-induced migration of endothelial cells depends on the interaction between a m b 3 and RPTPb/e.

Ganglioside depletion in mammary epithelial HC11 cells alters EGFR phosphorylation and reduces prolactin-induced b-casein mRNA expression S. Milani, S. Zava, B. Berra and I. Colombo Institute of General Physiology and Biological Chemistry 'G. Esposito', Faculty of Pharmacy, University of Milan, Milano, ITALY Introduction: EGFR and ErbB2 are tyrosine-kinase receptors that play a role in the mammary gland differentiation and HC11 cells represent a valid model to investigate their involvement in this process because they can become competent at responding to lactogenic hormones and expressing milk proteins. It is known that lactogenic hormones exhibit their effect antagonizing EGF signaling by the increase of EGFR threonine-phosphorylation and decrease of EGFR tyrosinephosphorylation. It has been also reported that gangliosides are important modulators of EGFR and ErbB2 expression levels and EGFR/ ErbB2 heterodimerization. Here, we provide the first evidence of the involvement of gangliosides in EGFR/ErbB2-mediated mammary cell differentiation. Methods: EGFR/ErbB2 heterodimers and receptor phosphorylation were analyzed by immunoprecipitation and immunoblotting with specific anti-receptor and anti-phosphoaminoacid antibodies, and by alkaline phosphatase digestion. Endogenous gangliosides were removed by treatment with 10 lM [D]-PDMP for 4 days, and HC11 cell differentiation by treatment with 5 lg/ml prolactin and 1 lM desametazone for 6, 8, 16 and 24 hour. b-casein mRNA expression levels was determined by quantitative RT-PCR. Results: Lactogenic hormone treatment of ganglioside-depleted HC11 cells results in a significant decrease of b-casein mRNA expression and analyses of EGFR-phosphorylation status and EGFR/ErbB2 heterodimers show a significant increase of EGFR tyrosine-phosphorylation levels and, interestingly, in the appearance of an alkaline phosphatasesensitive phosphorylated EGFR form that is specifically implied in the heterodimer formation with ErbB2. Conclusions: These novel findings suggest an important crosstalk mechanism between gangliosides and EGFR in mammary gland differentiation that may be relevant for the investigation in the breast cancer behaviour.

The scaffold protein RACK1 regulates Ki-Rasmediated signaling and morphological transformation of NIH 3T3 cells Various Ras mutations are present with high frequency in different human cancers. The mechanism by which mutated Ras contributes to cancer development and morphological transformation of cells is still not completely understood. Ras generates down-stream effects through Raf, Rac, and Rho, affecting cell growth, lamellipodia and stress fiber formation. The subsequent activation of the MAPK (Raf > Mek > Erk) pathway is essential for cell growth and involved in a large number of cell-signaling events. To identify and characterize factors inhibiting the transforming signal of mutated Ki-Ras, we used a retroviral cDNA expression screen in NIH 3T3 cells. We identified a novel potent inhibitor of Ras-mediated morphological transformation encoded by a truncated version of the receptor for activated C-kinase (RACK1). The truncated protein, designated RACK1DWD1, lacked the N-terminal 49 amino acids encoding the first of the seven WD40 repeats in RACK1. RACK1DWD1 blocked the Ki-Ras transforming signal, and the RACK1DWD1 expression restored contact inhibition, stress fiber formation and reduced ERK phosphorylation in Ki-Ras transformed NIH 3T3 cells. We further showed that RACK1DWD1 interacts with wtRACK1 and the protein kinase C (PKC) isoforms a, bI, and d and that the RACK1DWD1 expression inhibits the cellular translocation of these PKC isoforms in response to 12-0-tetradecanoyl-13-phorbol acetate (TPA). The identification of a truncated RACK1 as an inhibitor of Ki-Ras-mediated morphological transformation is intriguing, since RACK1 functions as a scaffold protein and is implicated at several levels of signal transduction ranging from events at the plasma membrane receptors to interactions with transcription factors and regulated translation of mRNA. Our present findings place RACK1 as an important factor in Ki-Ras-initiated signal regulating cell morphology and transformation.

Cortactin regulates insulin-stimulated GLUT4 trafficking and glucose uptake through Rho kinase activity by actin filament re-modeling H. Nazari 1,2,5 , A. Takahashi Insulin promotes glucose uptake in adipose tissue and skeletal muscle by increasing the translocation of the GLUT4, glucose transporter 4, from an intracellular pool to the plasma membrane, but the complete signaling pathway responsible for GLUT4 translocation remains to be defined. We investigated role of Rho kinase in insulin-induced actin/cortactin interaction, GLUT4 trafficking and translocation in L6-GLUT4myc myotube cells. Rho kinase inhibitor decreased insulin-stimulated GLUT4myc translocation, glucose uptake and stress fiber formation. Conversely, lysophosphatidic acid (LPA), a Rho kinase activator, increased insulin-stimulated formation of stress fibers and GLUT4 translocation as well as glucose uptake. Both Rho kinase inhibitor and LPA had no effect on Akt phosphorylation in those cells over-expressed wild type cortactin. On the other hand, Cytochalasin D, an actin depolymerizing agent and Wortmannin, a PI3-K inhibitor, completely blocked insulin-induced glucose uptake, indicating the involvement PI3-K/Akt/Rho kinase/actin/cortactin interaction. The effect of inhibitor was even more pronounced in the presence of over-expressed cortactin suggesting that the same pathway is involved. Knockdown of cortactin by siRNA did inhibit insulin-induced GLUT4 translocation, actin stress fiber formation and glucose uptake. These results suggest that the Rho kinase and actin binding protein cortactin interaction is required for actin stress fiber formation in muscle cells and that this process is absolutely required for translocation of GLUT4-containing vesicles to the plasma membrane. Failure to translocation GLUT4 correctly in states of insulin resistance or diabetes may be linked to changes in actin dynamics and suggest novel potential targets for drug development.

ADIPOQ gene SNP associations with circulating adiponectin, markers of obesity, insulin resistance, and other components of the metabolic syndrome in school-aged children from the GENDAI study I. Ntalla 1 , M. Smart 2 , E. Louizou 1 , M. Yiannakoulia 1 , K. Papoutsakis 1 , P. Talmud 2 and G. Dedoussis 1 1 Harokopeio University of Athens, Athens, GREECE. 2 University College, London, UK Introduction: ADIPOQ gene encodes for adiponectin, an adipocytokline, which is involved in the regulation of glucose and lipid metabolism; and is inversely associated with obesity and insulin resistance. ADIPOQ gene variation has also been associated with obesity, insulin resistance and circulating adiponectin in various populations. The aim of the present study was to search for associations between ADIPOQ gene SNPs and adiponectin concentration, obesity markers, insulin resistance and other components of the metabolic syndrome in children. Methods: The study population was comprised of healthy children aged 11.2y from the GENDAI Study. Two well studied SNPs (+276G/T and -11391G/A) were selected for investigation. High quality genotyping data as well as anthropometrical and biochemical data, and adiponectin levels were available for approximately 700 subjects. Results: Both SNPs were in Hardy-Weinberg equilibrium. The frequency of the minor allele was 0.287 for +276G/T and 0.100 for -11391G/A. Due to the small number of homozygotes for the rare allele for SNP -11391G/A, carriers of the rare allele were tested against homozygotes for the common allele. In univariate analysis of variance, SNP +276G/T was significantly associated with serum triglycerides(mean ± SD: GG = 66.3 ± 24.0, GT = 62.8 ± 23.5, TT = 61.2 ± 21.2 mg/dL; p = 0.026) and adiponectin (mean ± SD: GG = 4.7 ± 2.6; GA = 4.3 ± 2.3, AA = 5.3 ± 3.0 lg/ml; p = 0.045), even after adjustment for known covariates. We failed to find any significant association between SNP -11391G/A and the tested variables. Conclusions: In our population, SNP +276G/T was significantly associated with serum adiponectin and triglycerides. Homozygotes for the rare allele had higher levels of adiponectin and lower levels of triglycerides. Introduction: The V2-vasopressin receptor (V2R) activates the MAPK ERK1/2 through a mechanism involving the scaffolding protein b-arrestin. We recently reported that this activating pathway is independent of Gas/i/q or Gbc and involves c-Src and a metalloproteinase-dependent receptor tyrosine kinase (RTK) transactivation event.

To further characterize the transactivation mechanism leading to V2Rmediated ERK1/2 activation, we sought to identify the RTK involved.

Methods and results: Based on the use of selective pharmacological inhibitors and dominant negative mutants, we report that the V2Rinduced ERK1/2 activation relies on the metalloproteinase-dependent transactivation of the Insulin-like growth factor receptor (IGFR) that becomes phosphorylated upon vasopressin stimulation. In the HEK293 cells studied, we further found that c-Src was required for the metalloproteinase-promoted processing of the ligand precursor. Notably, b-arrestin was not necessary for the metalloproteinase-dependent release of the trans-activating ligand. Given the recent observations that b-arrestin can be recruited to RTK in response to their cognate ligands, we next investigated whether b-arrestin could also be recruited to IGFR following its transactivation by V2R. In co-immunoprecipitation studies, we indeed observed that b-arrestin-1 associates with IGFR in response to V2R activation and that this event is required for the V2R promoted ERK1/2 activation. Conclusion: These findings substantiate the pleiotropic nature of barrestin and suggest that recruitment of barrestin to the transactivated IGFR may be involved in the scaffolding of the down-stream MAPK signaling cascade promoted by V2R. The present study thus brings to light a previously unappreciated level of complexity in the regulation of signaling by these scaffolding proteins.

Role of C-terminal tail of type II BMP receptor in their biosynthesis and processing Also, we generated phosphospecific antibodies for two of confirmed sites and proved the phosphorylation of CoASy in vivo. We are currently examining the role of identified CoASy post-translational modifications and the progress on this project will be presented.

In vivo interactions of preprohepcidin and mature hepcidin with ferroportin Mitochondria undergo continual cycles of fusion and fission, and the balance of these opposing processes regulates mitochondrial morphology. In many cell types mitochondria form tubular structures or networks. But, during apoptosis the mitochondrial network fragments. CTMP (Carboxyl-Terminal Modulator Protein) that is known to negatively regulate the PKB catalytic activity in insulin signaling pathway. CTMP binds specifically to the carboxyl-terminal regulatory domain of PKB at the plasma membrane. Binding of CTMP reduces the activity of PKB by inhibiting phosphorylation on serine 473 and threonine 308.

Here, we showed the subcellular localization of CTMP in U2OS cell line. When GFP was tagged N terminal of the CTMP that localized cytoplasm and plasma membrane, whereas C terminal tagged construct of the CTMP were localized mitochondrion and cytoplasm. The LETM1 encodes for the human homologue of yeast Mdm38p, a mitochondria-shaping protein of unclear function. LETM1 gene was first identified as one of several genes within a chromosomal interval that is deleted in patients with Wolf-Hirschhorn syndrome. Patients with this syndrome can have growth delays, mental retardation, epileptic seizures and they can have facial abnormalities, including facial closure defects. Differences in the combinations of symptoms and their severity are thought to result from the extent of the chromosomal deletion in individual patients. Although the gene or genes that are responsible for the observed pathologies have not yet been positively identified, LETM1 is considered a strong candidate for the epileptic component, because it is a mitochondrial protein and mitochondrial defects often have wide-ranging and varied consequences including epilepsy. Moreover, deletion of the LETM1 gene correlates well with the occurrence of epilepsy in Wolf-Hirschhorn syndrome patients. Here, we investigated CTMP can strong interacts with LETM1 within mitochondria. Over-expression of LETM1 caused mitochondrial fragment and cristae swelling. We also fined that over-expression of LETM1 leading to OPA1 cleavage and sensitive to apoptosis. Taken together, our results suggest that CTMP interacts with LETM1 within mitochondria. LETM1 is a regulator of mitochondrial shape classical OPA1-dependent pathway, and its action possibly is through LETM1. Introduction: Adenosine is an endogenous nucleoside whose immunomodulatory function has been documented by many experimental and clinical observations. Its biological action is mediated by the cell-surface receptors (A1, A2A, A2B, and A3) linked to a variety of signaling systems. Development of diabetes results in altered adenosine metabolism and action in a variety cells including immunological cells. This study was undertaken to examine the insulin and glucose effect on expression of adenosine receptors (ARs) in B lymphocytes. Methods: Lymphocytes were isolated from rat spleen and B cells were separated by the panning method. Purified cells were culturedin RPMI-1640 medium containing different concentrations of insulin and glucose, and ARs mRNA levels were determined by real-time PCR.The AR protein level was analyzed by western blot.

Results: Elevated glucose level decreased the expression of A1-AR in a PKC-dependent manner whereas, insulin increased A1-AR transcript level in a MAPK-dependent way. The expression of A2A-AR was not affected by changes in glucose concentration.A2A-AR mRNA expression was stimulated by insulin in a MAPK-dependent manner.Increaseof glucose or insulin concentration resulted in lowered A2B-AR mRNA level but, high glucose concentration (25 mM) override the insulin effect. SB 203580 a p38 MAPK inhibitor blocked the glucose and insulin effects on A2B-AR mRNA level. The expression of A3-AR was not affected by insulin and was inhibited by increased concentration of glucose in a PKC-dependent manner. Introduction: Mast cells are the major source of TNF-a, which is a multifunctional cytokine and critical component of innate immune response to bacterial infection. However, over activation of TNF signaling result in development of inflammatory disorders. Several lines of evidence indicate that adenosine is able to modulate the mast cell activation and cytokines release however, the involvement of particular adenosine receptor (AR) in this process is not clear. Methods: RBL-2H3 cells were cultured in RPMI-1640 growth medium containing 10% fetal calf serum, and stimulated with 10 lg/ml lipopolysaccharide (LPS). Ten hours later TNF-a was determined in cell culture media using an ELISA kit. AR agonists and antagonist (all at 1 lM concentration) were added 30 minutes before LPS.

Results: Pre-treatment of RBL-2H3 cells with AR non-selective agonist (NECA) increased the LPS-induced TNF-a production by 30%.

Exposures of cells to selective A1-AR agonist (CPA) or an antagonist (DPCPX) have little effect on TNF-a release. Inclusion of a selective A2A-AR agonist (CGS21680) into incubation medium resulted in 41% increase of TNF-a production. Exposition of cells to the selective A2A-AR antagonist (ZM241385) inhibited TNF-a accumulation by 40%. Slight (15%) increase of TNF-a was observed in the culture medium of cells exposed to selective A2B-AR antagonist (MRS1754). Decrease (20%) of TNF-a level was also observed in media of cells pretreated with A3-AR selective agonist (IB-MECA).

Conclusions: These results indicate that adenosine is able to modulate LPS-induced TNF-a production in RBL-2H3 mast cells, and the net effect is an outcome of action of all ARs.

Growth factor-stimulation of intervertebral disc cell-proliferation: involvement of pivotal signalling pathways Increased cell proliferation is frequently encountered in sites of intervertebral disc (IVD) degeneration, probably as a tissue regenerative response. In accordance, many growth factors and their receptors have been shown to be over-expressed during degeneration. Studies concerning growth factor-effects on IVD cell proliferation are limited, however, in part due to the paucity of healthy human tissue samples. We have used primary cultures from bovine coccygeal IVDs as well as from human biopsies, either from disc prolapse or from scoliotic patients. Their proliferative response to three potent mitogens immunolocalized in the disc, i.e. platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF) and insulin-like growth factor-I (IGF-I) was studied using tritiated thymidine and 5-bromodeoxyuridine incorporation, and cell cycle analysis by FACS. The activation of pivotal signalling pathways was investigated using Western analysis as well as specific pharmacologic inihibitors. All three mitogens were shown to induce DNA synthesis in quiescent IVD cells through the activation of extracellular-signal regulated kinases (ERKs), as well as of the phosphatidylinositol 3-kinase/Akt (PI 3-K/Akt) pathway. No differences were observed between human and bovine IVD cell cultures regarding their stimulation by the growth factors or the signal transduction pathways involved, indicating that the bovine coccygeal disc cell model is a suitable surrogate for human lumbar IVD cells, at least for in vitro studies. Our data will contribute to the understanding of IVD tissue repair processes, and, furthermore, to the design of interventional approaches. Acknowledgements: Supported by the AO Fund, Switzerland (05-K68) and the EU (MYJOINT and GENODISC projects).

Serotonin influence on the origination of gonadotropin-releasing hormone producing neurones in rats Introduction: Gonadotropin-releasing hormone (GnRH)-producing neurones originate in embryogenesis in the nasal cranium, and newly formed neurones migrate to the septo-preoptic area of the brain. According to our previous study, the migration and differentiation of GnRH neurones is under the control by serotonin (5-hydroxytryptamine, 5-HT) and catecholamines though it remains uncertain whether their origination is regulated by intercellular signals. This study aimed to test whether 5-HT influences the GnRH neurone origination. Methods: Pregnant rats received either p-chlorophenylalanine (pCPA), the inhibitor of 5-HT synthesis, or saline (control) from the 11th day of gestation to the 17th or 20th day. GnRH-neurones were detected with immunocytochemistry in their fetuses on the 18th and 21st day, respectively. In addition to daily injections from 11th day of gestation to the 20th day of pCPA or saline, some rats received single injection of BrdU on the 11th, 12th, 13th, 14th or 15th day. Doubleimmunofluorescence neurones (GnRH & BrDU) were detected in their pups on the 15th postnatal day.

Results: The number of GnRH neurones in fetuses (18th, 21st day) and pups (15th day) increased under 5-HT deficiency. As follows from the double-immunofluorescence study, the period of the GnRH neurone origin was the same under 5-HT deficiency and in control. However, the maximal number of GnRH neurones appears on the 13th day in control rats and on the 14th day in 5-HT-depletead rats. Conclusion: 5-HT appears to inhibit the proliferation of progenitors of GnRH neurones and stimulate the GnRH neurone origin.

Caveolin-1 reduced expression of the tumor survival gene cyclo-oxygenase-2 and production of prostaglandin E2 via a b-catenin-Tcf/Lef-dependent mechanism Introduction: Augmented activity and/or expression of cyclo-oxygenase-2 (COX2) observed in a variety of human cancers is associated with enhanced production/secretion of prostaglandin E2 (PGE2), as well as increased tumor cell survival and malignancy. Caveolin-1 is a structural protein required for the formation of 50-100 nm plasma membrane invaginations called caveolae that has also ascibed a role as a tumor suppressor in human cancer cells. However, mechanisms by which caveolin-1 functions in this manner remain unclear. In particular, no links to regulation of COX2 have been described.

Objective: To determine whether presence of caveolin-1 in cells reduces COX2 expression/activity and identify the mechanism involved. Results: The human colon (HT29, DLD-1) and breast (ZR75) cancer cell lines express low endogenous levels of caveolin-1. Re-expression of caveolin-1 using an IPTG-inducible plasmid (pLacIOP) reduced in all cases endogenous COX2 mRNA and protein levels, indicating a transcriptional mechanism of control. Likewise, over-expression of caveolin-1 in human embryonic kidney (HEK293T) cells also reduced COX2 mRNA and protein levels. Addionally, in HEK293T and the cancer cell lines augmented caveolin-1 presence was associated with reduced bcatenin/Tcf-Lef (TOP/FOP Flash assay) and COX2 gene reporter activity, as well as reduced liberation of prostaglandin E2 and cell proliferation. Finally, supplementation of the cell culture medium with PGE2 was sufficient to overcome caveolin-1-induced down-regulation of COX2 and reduction of cell proliferation.

Conclusions: Caveolin-1 reduced COX2 expression and liberation of PGE2 associated with enhanced cell proliferation via an 85-catenin/ Tcf-Lef-dependent transcriptional mechanism. Acknowledgements: This work was supported by FONDAP #15010006 (to AFGQ), FONDECYT #11070116 (to JCT), FONDE-CYT #1040390 (to LL), a MECESUP fellowship (to DAR) and a CONICYT PhD fellowship (to GF).

Sex and thyroid hormones relate to brown adipose tissue thermogenic decline during aging A. Valle, F. Jose Garcia-Palmer, J. Oliver and P. Roca Energy Metabolism and Nutrition Group, CIBER Fisiopatologıá Obesidad y Nutricio´n, Instituto Salud Carlos III, University of Balearic Island, Palma de Mallorca, SPAIN Sex and thyroid hormones are among the factors modulating energy metabolism through regulation of mitochondrial oxidative capacity. Brown adipose tissue (BAT) in old female rats has been shown to maintain, better than males, the ability to produce heat when exposed to cold. Considering the decline that takes place in gonadal and thyroid function during aging, the aim of this work was to test whether the age-related hormonal status may be a potential mediator of the gender differences in BAT decline. COX activity, UCP1, mitochondrial respiration rate, mitochondrial DNA (mtDNA), protein and levels of mitochondrial transcription factor A (TFAM) and peroxisome proliferator-activated receptor c co-activator-1a (PGC-1 a) , as well as the serum levels of sex steroids (17 b-estradiol, progesterone and testosterone) and thyroid hormones (T3 and T4) were measured in 6-, 18-, and 24-month-old male and female rats kept at 22°C. Six-month-old female rats showed higher thermogenic features than males. Male rats at 18month showed a decrease in uncoupling activity compared to females. Both genders showed marked signs of atrophy in BAT of 24-monthold rats, characterized by a decrease in total DNA, mitochondrial protein, COX and UCP1, whereas mtDNA was found to increase. Sex steroids were well correlated with BAT parameters when both genders were considered, however, T3 was the hormone with the strongest positive correlations in female rats. In conclusion, our findings provide evidence suggesting that T3 may be a potential mediator of the sexual dimorphism in the effect of aging on the functional decline of BAT. Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that belong to the nuclear receptor superfamily. PPARs play an important role in diabetes and atherosclerosis. There is evidence that activation of PPAR c by specific ligands is able to suppress the growth of different types of human cancer by mechanisms including the growth arrest, apoptosis and induction of differentiation, although the detailed signaling pathways have not been completely elucidated. The aim of our study was to determine whether a novel PPAR a/d/c pan agonists, TIPP-703 (phenylpropanoic acid derivative) could affect the cell growth and expression of some cell cycle related proteins in pancreatic cancer cell lines panc-1 and PT45. MTT assay, flow cytometry and immunoblotting were used for detection of changes in cell growth. Treatment with TIPP-703 exhibited significant inhibition of cell proliferation of human pancreatic cancer cells as suggested by MTT assay. Addition of HX600, which is a RXR specific agonist, could enhance the growth inhibitory action. These results indicate the growth inhibitory effect by TIPP-703 on pancreatic cancer cells is occurred through PPAR receptors. During the process, TIPP-703 significantly elevated the cyclin dependent kinase inhibitor protein p21Waf1/Cip1 level, and reduced cyclin D1. While cyclin dependent kinase inhibitor protein p27Kip1 level were not changed. Cell cycle analysis revealed a decreased proportion of cells in S phase, with arrest at G 0 /G 1 and the percentage of cells in the G 0 /G 1 phase was 90% by treatment of TIPP-703. TIPP-703 may be useful for arresting the growth of some pancreatic cancer cells. The glucocorticoid is a major subclass of steroid hormones that have potential effects of anti-inflammatory and immunosuppressive, autoimmune/allergic and lymphoproliferative functions. This hormone is mediated via an intracellular receptor, glucocorticoid receptor (GR). A human androgen response element (hARE), identified within intron 8 of the human sterol regulatory element-binding protein cleavage-activating protein, interacts with the both glucocorticoid receptor (GR) and androgen receptors (AR). To get better relevant assay system, CMV-driven human GR (pCMV/hGR) and ARE-driven Luciferase reporter (ARE/Luc) were constructed and were transiently transfected into HepG2 cells. In parallel, these genes were also injected into the tail of mice. Subsequently, cells and mice were then treated with increasing concentrations of dexamethasone and its derivatives. Herein, we concluded that cells co-transfected with pCMV/hGR and ARE-TK/Luc showed a decrease in luciferase activity compared to those transfected with ARE/Luc alone. Moreover, co-injection of pCMV/hGR and ARE-TK/Luc gene into mice also resulted in a decrease in luciferase activity relative to that of ARE-TK/Luc gene alone. Thus, in vitro-cell system and in vivo mice model is useful in evaluating antagonists of GR using luciferase expression. These findings further suggest that this assay system allows screening of drug candidates affecting to a signal transduction pathway of the GR and AR and may help in the future discovery and analysis of novel and selection of GR and AR agonists.

A. Shukla, R. Bleiler, K. Becker and D. Mayer Hormonwirkung Und Signaltransduktion, Deutsches Krebsforschungszentrum, Heidelberg, GERMANY Introduction: Insulin analogues are structurally modified insulin, developed to better control blood glucose level of diabetic patients. However, insulin molecule modification may increase the affinity for the IGF-1R, a receptor related to proliferation. Since IGF-1R is expressed in normal mammary glands and over-expressed in many mammary carcinoma cases we studied the mitogenic effects of five currently available analogues on breast epithelial cells.

Methods: Signalling pathways were analysed by investigating the phosphorylation status of signalling molecules by western blotting. Cell proliferation was measured by two independent assays -a colorimetric assay and a BrdU-FACS assay. siRNA was used to knock down the IGFIR or IR and cell proliferation was measured thereafter by BrdU incorporation method. Results: We screened nine different mammary epithelial cell lines and selected non-tumorigenic MCF10A and tumorigenic MCF7 cells. Interestingly, only insulin glargine (Lantus Ò ) activated the Akt/PKB and the MAPK pathway in MCF7 stronger than regular insulin. Cell proliferation experiments also revealed higher proliferative ability of Lantus. Furthermore, we demonstrate by using inhibitors the importance of MAPK pathway in the mitogenic effect of Lantus. By siRNA knockdown, we show that the proliferative ability of Lantus is due to the activation of both IGF1R and IR.

Conclusions: Four of five analogues tested show a similar effect as normal human insulin, in either MCF10A or in MCF7. Lantus elicits a significantly higher proliferative effect but only in MCF7. This high proliferative ability is due the strong activation of the MAPK pathway and involves most probably the activation of both IGF1R and IR.

The role of nicotinic acetylcholine receptors in Ca 2+ uptake by mitochondria G. Gergalova, L. Koval, O. Lykhmus, V. Chernyshov and M. Skok Palladin Institute of Biochemistry, Kyiv, UKRAINE Introduction: Nicotinic acetylcholine receptors are ligand-gated ion channels permeable to mono-and divalent cations including Ca 2+ . Nicotine was shown to protect against neurodegeneration upon Alzheimer and Parkinson diseases, which are accompanied by the changes of nicotinic receptor expression in the brain. One of the protective mechanisms was related to mitochondria functions, although by now the role of nicotinic receptors in this process has been denied. Methods: We studied the presence and functions of nicotinic receptors in mouse liver mitochondria obtained by standard procedure. The purity of preparation was evaluated by flow cytometry with nonyl acrydin orange and by electron microscopy, while mitochondria membrane potential was measured with tetramethylrodamine ether. The presence of nicotinic receptors was studied by the dot-blot, flow cytometry and Sandwich ELISA using specific antibodies. Ca 2+ uptake by mitochondria was studied by flow cytometry with Ca-sensitive fluorescent dye Fluo-3AM.

Results: The preparation was 95% pure and contained non-damaged mitochondria possessing significant membrane potential. The preparation bound antibodies against a7 nicotinic receptor subunit; the binding was strongly decreased in the mitochondria obtained from a7-knockout mice. Nicotine decreased the mitochondria membrane potential. Nicotine, choline and a7-specific antagonist methyllicaconitine attenuated the mitochondria response to Ca 2+ measured by the drop of the membrane potential and decreased the influx of exogenous Ca 2+ according to Fluo-3AM fluorescence.

The results obtained allow suggesting that a7 nicotinic receptors are present on the outer mitochondria membrane and regulate Ca 2+ uptake by mitochondria, possibly, affecting the activity of Ca-uniporter.

Biochemical and structural comparison of two differentially targeted cytokinin dehydrogenase enzymes There are several isoforms of CKX in each plant encoded by a small highly homologous gene family. These isoforms are differentially targeted in plant cells to achieve specific regulatory functions. Differences in local environments affect CKX activity and dictate differences in modes of biochemical action on cytokinin substrates and electron acceptors. Most of the CKX isoforms are localized in apoplast or vacuole; there is generally only one CKX isoform per plant per species that lacks a translocation signal and presumably functions in cytosol. The best characterized CKX from maize is ZmCKX1, known to be secreted into the apoplast, while a gene encoding the non-secreted CKX has not been cloned before. The aims of this work were to clone the gene for non-secreted CKX from maize (nsZmCKX), compare its localization with the apoplastic ZmCKX1 and characterize its biochemical and structural properties. The gene for nsZmCKX was cloned successfully using the sequence information obtained from a survey of maize genome database. For determination of subcellular localization, the genes were fused with fluorescent markers (ZmCKX1:DsRed and nsZmCKX:GFP) and over-expressed in tomato hairy roots. Using confocal microscopy, ZmCKX1 signal was found in apoplast whereas nsZmCKX was detected in cytosol as predicted. The expression of the genes in transgenic tissues was confirmed by RT-PCR and by measuring the enzymatic activity. For further biochemical characterization, the recombinant nsZmCKX protein was prepared using Pichia pastoris expression system.

Compartmentalization-dependent ubiquitination of oncogenic Ras regulates transforming activity S. Terrillon and D. Bar-Sagi Department of Biochemistry, New York University School of Medicine, New York, NY, USA Ras GTPases are key regulators of cell growth, differentiation and transformation. Ubiquitination of Ras modulates its intracellular trafficking and signaling output. Yet, the molecular mechanisms involved in the regulation of this modification remain poorly understood. Here, we took advantage of the Bioluminescence Resonance Energy Transfer (BRET) technology to monitor HRas ubiquitination in living cells and to investigate its relationship to membrane microdomain compartmentalization and oncogenic transformation. We demonstrate that a HRas chimera unable to interact with Caveolin1 is refractory to ubiquitination, indicating that HRas ubiquitination depends on the association with Caveolin1. Consistent with this observation, disruption of lipid microdomains by membrane cholesterol depletion inhibits HRas ubiquitination. The requirement of the integrity of caveolae and Caveolin1 for HRas ubiqutination is futher supported by the observation that the extent of HRas ubiquitination is dependent on Caveolin1 expression levels. Thus, knockdown of endogenous Caveolin1 suppresses ubiquitination of HRas. Conversely, ectopic expression of Caveolin1 leads to an increase in overall ubiquitinated HRas. The ectopic expression of Caveolin1 also inhibits the transforming activity of HRas but does not affect the transforming activity of an ubiquitination-deficient HRas mutant, indicating that Caveolin1 suppresses cell transformation by regulating HRas ubiquitination. Taken together, our data identify HRas ubiquitination as a mechanism by which Caveolin1 can suppress oncogenic transformation. Results: We compared the effects of exposure of bTC-6 cells to intermediate-5 mM, and high-25 mM glucose concentrations with exposure to 1 mM glucose as the appropriate control. We demonstrated that prolonged exposure of bTC-6 cells to increased glucose concentrations resulted in a significant inhibition of insulin signalling, including the phosphorylation of the insulin receptor and IRS-2 as well as the activation of IRS-2-associated PI3-kinase. These changes were also reflected in severe impairment of the sequential Akt-mediated inactivation of FoxO. Glucose-induced down-regulation of IRS-2/Akt/FoxO-mediated signalling was associated with increased IL-1b and SOCS-1 expression. Up-regulation of endogenous cytokine signalling coincided with enhanced caspase-3 activation and increased susceptibility of bTC-6 cells to apoptosis.

The obtained results demonstrated that glucoseenhanced IL-1b expression favours bTC-6 cells apoptosis by inhibiting IRS-2/Akt/FoxO signaling possibly via SOCS-1 up-regulation and suggest that impairment of the insulin survival pathway should play an important role in the loss of pancreatic b-cell mass observed in type II diabetes.

Reactive oxygen species mediate the stimulatory effect of pleiotrophin on human cell migration

Laboratory of Molecular Pharmacology, Department of Pharmacy, University of Patras, Patras, GREECE Introduction: Pleiotrophin (PTN), also called heparin affin regulatory peptide or heparin-binding growth associated molecule, is an 18 kDa secreted growth factor that displays high affinity for heparin. A growing body of evidence indicates that PTN is involved in cell proliferation, migration and differentiation. Reactive oxygen species (ROS) are associated with many aspects of cellular functions. Small quantities of ROS are produced by all types of cells, and several signal transduction pathways in mammalian cells have been reported to be activated by ROS.

Methods: Intracellular ROS production was assayed using the ROSsensitive fluorescent dye, 5(6)-carboxy-2,7-dichlorodihydrofluorescein diacetate. Migration assays were performed in 24-well microchemotaxis chambers. Receptor protein-tyrosine phosphatase b/zeta (RPTPb/e) was knocked down by RNA interference.

Results: Human recombinant PTN significantly increased the production of HP in a concentration and time dependent manner. RPTPb/e appears to be involved in this process, since suppression of the receptor using genetic and pharmacological inhibitors or inhibition of its downstream src kinase activity abolished PTN induced HP levels. The PTN induced production of HP was blocked by NAD(P)H oxidase, but not xanthine oxidase inhibitors, suggesting that superoxide is probably the initial ROS produced by PTN . In addition, apocynin completely abolished the stimulatory effect of PTN in migration of both HUVEC and LNCaP. L-Dopa decarboxylase (Ddc) is a pyridoxal (PLP) requiring enzyme, which catalyzes the decarboxylation of L-Dopa to dopamine and 5hydroxytryptophan (5-HTP) to serotonin. Ddc is found in all aminergic cells in the central and peripheral neurotransmitter systems, as well as in peripheral organs. Ddc is generally considered to be a cytosolic enzyme. However, new evidence indicates the association of the molecule with the membranes. The mRNA transcript encoding for the enzyme has been shown to undergo alternative splicing within its 5'-UTR, as well as in its coding region. Ddc has been implicated in the pathology of neoplastic, neurological and psychiatric disorders. Membrane fraction of K293 cells was prepared as described by Poulikakos, et al., 2001. Membrane-bound Ddc was released into the soluble fraction, following incubation at 37°C. The observed release was pH and time dependent (pH 5.5/20 minutes). Ddc solubilization was inhibited by the cysteine protease inhibitors iodoacetamide and N-ethylmaleimide (NEM), as well as by the serine protease inhibitor leupeptin, whereas aprotinin had no effect in this phenomenon. Release of the membrane-bound molecule was induced by the divalent cations Zn 2+ and Ca 2+ , while Mg 2+ had no effect. Our data indicate for the first time, the regulated solubilization of membrane-bound Ddc and suggest the involvement of an enzymatic mechanism. Knowledge leading to the better understanding of the mechanism of the observed release could also provide us with novel information about the biochemistry of the human enzyme. Introduction: Protein tyrosine kinases (PTKs) transduce extracellular signals mediate events such as proliferation, cytoskeletal rearrangement, and coordination of physiological responses, and also regulate ion channels. The present study was designed to investigate whether PTKs regulate human ether-a`-go-go-related gene (hERG) channels.

Methods: Whole-cell patch clamp technique was applied to record hERG channel current (I hERG ) in HEK 293 cell line stably expressing cloned and mutated hERG channels. Immunoprecipitation and Western blot analysis were used to determine tyrosine phosphorylation level of hERG channels.

Results: The broad-spectrum PTK inhibitor genistein (30 lM), the highly selective EGFR (epidermal growth factor receptor) kinase inhibitor AG556 (10 lM) and the Src-family kinase inhibitor PP2 (10 lM) remarkably inhibited I hERG , and the effects were significantly reversed by the protein tyrosine phosphatase (PTP) inhibitor orthovanadate (1 mM). Immunoprecipitation and western blot analysis demonstrated that tyrosine phosphorylation level of hERG channels was reduced by genistein, AG556, and PP2, and the reduction of the phosphorylation level of hERG channels by genistein, PP2 was significantly antagonized by orthovanadate. Single point mutation(s) of Y475A and/or Y611A dramatically reduced the inhibitory effect of I hERG by PP2 and AG556.

Our results demonstrate the novel information that I hERG is modulated not only by Src-family kinases, but also by ERFR kinases. Y475 and/or Y611 are likely preferred phosphorylation sites.

Signal transduction of glucose in atherosclerosisrelated functions of monocytes Peripheral blood monocytes can bind to the vascular epithelium and to extracellular matrix molecules, like laminin and migrate to the subendothelium space where monocyte CD36 scavenger receptor recognise and bind oxidized low density lipoproteins (oxLDL). OxLDL phagocytosis leads to foam cell formation. The monocyte exposure to oxLDL and oxLDL binding to CD36 activates Peroxisome Proliferator-Activated Receptors c (PPAR-c) transcription. Hormones related to diabetes, like insulin or glucose; activate atherosclerosis-related monocyte functions. In the present study, the effect of high glucose concentrations and PPAR-c activators (rosiglitazone) in atherosclerosis-related functions of human monocytes was investigated. The atherosclerosisrelated functions studied were monocyte adhesion and migration through laminin, collagen type IV or endothelial cells, CD36 expression and oxLDL phagocytosis. We used the inhibitors: cariporide (the inhibitor of Na + /H + exchanger), Go (the inhibitor of PKCa and PKCb isoforms), Gf (the inhibitor of all PKC isoforms) and wortmanin (inhibitor of PI3K) and puromycin (protein synthesis inhibitor). The data of the present study suggests that PPARc activation in monocytes induces atherosclerosis, and that Na + /H + exchanger plays an important role in the beginning of atherosclerosis. The addition of Go, Gf and wortmanin also reversed the effect of glucose or rosiglitazone, indicating the involvement of PKC isoforms and PI3K in the signal transduction leading to atherosclerosis-related functions of monocytes. Besides, puromycin treatment indicated that the increase in the number of CD36 receptors can be attributed to CD36 mRNA transcription. Introduction: Intracellular protein degradation by the ubiquitin-proteasome system releases large amounts of oligopeptides within cells. To evaluate a possible intracellular function of oligopeptides we introduced them into cells fused to a cationic TAT peptide sequence through reversible disulfide bonds. Methods and Results: A mixture containing four of these peptides (20-80 lM) significantly inhibited the increase in extracellular acidification response triggered by angiotensin II (ang II) in CHO-S cells transfected with the ang II type 1 receptor (AT1R-CHO-S). Either in a mixture or alone, these peptides increased luciferase gene transcription in AT1R-CHO-S cells stimulated with ang II and in HEK293 cells treated with isoproterenol. None of the peptides affected GPCR cellular responses without been fused to the TAT sequence, suggesting an effect from the intracellular milieu on GPCR signal transduction. All four functional peptides were shown to interact with thimet-oligopeptidase and three were PKC substrates in vitro. Using individual peptides as baits several proteins involved in GPCR signaling were identified, including alpha-adaptin A, alpha 1 and beta 2c tubulin, and dynamin 1.

Conclusion: These results demonstrate that before complete degradation intracellular peptides similar to those generated by proteasomes can actively affect GPCR cell signaling, likely representing a novel paradigm in cell biology. Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric bHLH-PAS transcription factor responsible for the regulation of a large group of genes mediating the adaptive response to hypoxia. Its inducible subunit, HIF-1a, is controlled by oxygen levels and major signaling pathways, such as PI3-K and MAPK. We have previously shown that p44/ 42 MAPK phosphorylates two Ser residues in the C-terminal domain of HIF-1a and stimulates its transcriptional activity by inhibiting its CRM1-dependent nuclear export (1). Additionally, a second, distinct from MAPK, HeLa nuclear kinase modifies the N-terminal part (amino acids 1-251) of HIF-1a. In order to characterize the phosphorylation and elucidate its function, smaller parts of the bHLH and PAS domains of HIF-1a were expressed as GST-fusions in E. coli, purified and subjected to phosphorylation assays in vitro using HeLa nuclear extracts as a kinase source. Site directed mutagenesis was then used to identify a candidate phosphoacceptor site in the HIF-1a PAS-B domain, which is involved in heterodimerization and/or interaction with Hsp90. The significance of this modification for the activity of HIF-1a is currently under investigation using both yeast and mammalian expression systems. Introduction: Erythropoietin (EPO) is a hormone primarily involved in erythropoiesis but dotted equally with an array of autocrine/paracrine effects. EPO can regulate major cell functions of normal and cancer cell types including breast cancer, a steroid hormone dependent neoplasm. Steroid effects can be nuclear-and/or membrane-initiated, with the latter depending, among others, to a cross-linking with various growth factor receptors. In breast cancer specimens we have reported a correlation of erythropoietin, its receptor (EPOR) and membrane androgen sites. Here we further explore this interaction and the possible mechanism involved. Methods: We assayed the effect of serum deprivation-and testosterone-BSA-induced apoptosis and cell migration in the presence of erythropoietin and explored the signaling pathways involved.

Results: Testosterone-BSA-induced apoptosis and decreased cell migration was reversed by erythropoietin in a dose-and time-related manner. Moreover, the anti-apoptotic effect of EPO was potentiated by the addition of testosterone-BSA indicating an interaction between the two systems. This interaction is not at the membrane-receptor level but is the result of the modulation of specific signaling pathways (switching of p38 and Jnk from pro-to anti-apoptosis and from STAT to Akt and b-catenin signaling), and the enhanced transcription of EPOR by testosterone-BSA.

Conclusions: Erythropoietin could be integrated to the ensemble of growth factors that cross-link with membrane steroid receptors, amending tumor cell survival. Their importance to patients' prognosis and selection of the appropriate therapeutic regimen should be considered. Introduction: Coated platelets are a subpopulation of cells observed after platelet activation, which retain on their surface high levels of alpha-granule proteins, express surface phosphatidylserine and support prothrombinase activity. They have high procoagulant activity, what suggests their importance for thrombosis and/or hemostasis, although their exact function is not presently clear. The aim of the study was to determine the role of platelet secretion in the signalling pathways leading to coated platelets formation. Methods: Gel-filtered platelets activated by either thrombin or thrombin with convulxin (collagen receptor glycoprotein VI agonist) under varying conditions were analyzed by flow cytometry.

Results: The fraction of coated platelets depended on the concentration of platelets in the initial suspension. This suggested a possible role of their secretion. Addition of aspirin and thromboxane A2 analog U46619 had no effect indicating that cyclooxigenase has no role in coated platelet formation. Addition of apyrase dose-dependently decreased the size of coated platelets subpopulation, while addition of ADP increases it. It allowed supposing that appearance of coated platelets is regulated through purinergic receptors. To identify the responsible purinergic receptor, specific antagonists for the receptors P2Y1, P2Y12 and P2X1 were used. We found that antagonists of P2Y1 and P2X1 had no effects, while antagonist of P2Y12 inhibited coated platelet formation. This suggested a central role of purinergic receptor P2Y12 in the development of ADP effect. Conclusions: ADP plays an important role in the regulation of coated platelets formation, and its action is mediated by the P2Y12 receptor. Acknowledgement: This work was supported by RFBR grant 07-04-00146. Introduction: Cholesterol is essential for cell growth and division. In both MOLT-4 and HL-60 cells, we found that cholesterol starvation results in cell cycle arrest at G2/M, followed by polyploid cell formation. In the present work we studied the involvement of stress-activated signal transduction pathways in these effects.

Methods: HL-60 cells were cultured in cholesterol-free medium and treated with SKF104976, an inhibitor of 14?-lanosterol demethylase. Then, the cells were treated or not with SB203580 or SP100625, to block p38MAPK or JNK, respectively, and the progression of the cell cycle was monitored. In other instances, nocodazole was used to synchronize cells at prometaphase and, then, they were released in the presence or the absence of those inhibitors. Cell cycle progression was assessed by measuring BrdU incorporation and DNA content by flow cytometry. Phosphorylated p38MAPK and JNK were analyzed by both western blot and multiparametric flow cytometry.

Results: In prometaphase-synchronized cells, addition of SB203580 at the moment of nocodazole wash-out did not affect mitosis completion and the cells rapidly appeared in G1; in contrast, addition of SP100625 impeded cytokinesis and resulted in the formation of aneuploid cells hours later. Treatment with SKF104976 transiently induced p38MAPK phosphorylation, which was both prevented and reversed by cholesterol. Exposure of cholesterol-deficient cells to SB203580 or SP100625, accelerated the progression into polyploidy. Introduction: Dehydroepiandrosterone protects neural crest-derived cells from apoptosis, via binding on specific plasma membrane receptors, rapid activation of prosurvival kinases Src/PKC a/b /MEK1/2/ ERK1/2, and PI3K/Akt, the induction of transcription factors CREB and NFkB and the subsequent production of antiapoptotic Bcl-2 proteins. In fact, DHEA prosurvival signaling has major common characteristics with this of NGF. We have thus tested the hypothesis that DHEA exerts its prosurvival actions interacting with NGF receptors. [1, 2] . BRAF mutations in CRC occur independent of KRAS and since these two activating mutations do not co-exist, further analysis on their transforming capabilities in the same cell system of colon cancer may explain the reason for this. Methods: Caco-2 cells were stably transfected with KRAS-G12V or BRAF-V600E oncogene. Clones barring either mutation were analyzed for their growth characteristics, cellular morphology and transforming properties both in vivo (tumorigenicity in SCID mice) and in vitro (colony formation in soft agar). Cell cycle was analyzed at protein and mRNA level and implemented by FACS.

Results: BRAF-V600E conferred a spindle like altered cell morphology and higher proliferation rate, as compared to the parental Caco-2 cells and KRAS-G12V clones. Activated BRAF induced far more efficient colony growth in soft agar and tumor formation in SCID mice.

In terms of cell cycle related effects KRAS-G12V expression led to the acquisition of premature senescence related markers as determined by SA-b-Gal and PML staining, that were absent in BRAF-V600E cells.

Conclusions: Taken together, these results suggest that the two oncogenes have different transforming capability. In general BRAF-V600E presents greater potential in mediating tumourigenic effect as compared to KRAS-G12V both in vivo and in vitro. Tamoxifen is an antagonist of estrogen receptor, which is used widely as an breast cancer drug that blocks estradiol binding to estrogen receptor and shut down growth signals. Thus, tamoxifen-treated breast cancer cells finally provoke apoptosis. However, recent studies revealed rather confusing results that tamoxifen induces apoptosis even in estrogen receptor-negative cells. In the present study, we synthesized several tamoxifen derivatives to augment the apoptosis-inducing effect of tamoxifen and evaluated the estrogen receptor-negative apoptosis-inducing pathway. The estrogen receptor-positive human leukemia cell line HL-60 and estrogen receptor-negative human leukemia cell line Jurkat were treated with tamoxifen and synthesized tamoxifen derivatives, and thereafter subjected to cell viability-detection assays. Tamoxifen derivatives, as well as the lead compound tamoxifen, decreased the cell viability despite the expression of estrogen receptor. Among all of the synthesized tamoxifen derivatives, ridaifen-B had more potent Jurkat cell-damaging activity. Ridaifen-B had about 8times potent than tamoxifen. Ridaifen-B fragmented Jurkat cell DNA and activated caspase-3, -8, and -9, suggesting that the ridaifen-B-induced apoptosis pathway is estrogen receptor independent. Moreover, mitochondrial involvement during ridaifen-B-induced apoptosis was estimated. Ridaifen-B significantly reduced mitochondrial membrane potential, and overexpression of Bcl-2 inhibited ridaifen-B-induced apoptosis phenomenon (DNA fragmentation, caspases activation, and loss of mitochondrial integrity). These results suggest that the induction of apoptosis by ridaifen-B is dependent on mitochondrial perturbation without estrogen receptor involvement.

Identification of target regulatory regions in promoter required for hypoxia inducible factor-1ainduced haptoglobin gene expression Although well described in haemopoietic malignancies, its role and regulation in solid tumors remain to be elucidated. Indeed, breast cancer promotion integrates a complex interplay between hormones and cytokines, mediated, among others, through cross-link of membrane initiated steroid signaling with growth factors. Methods: We assayed 52 human breast cancer biopsies by immunohistochemistry for the expression of APRIL as well as its cognitive receptors (BCMA and TACI) and correlated our findings with clinicopathological data and the evolution of the disease. Moreover, utilizing three breast cancer cell lines (MDA-MB-231, T47D and MCF-7) with different phenotypes, we approached by RT-PCR the gene expression profile of this TNF member in breast cancer and the possible transcriptional regulation by membrane androgen and estrogen agonists. Results: APRIL immunoreactive expression was higher in non-malignant than neoplastic breast structures, in contrast to findings in other solid tumors. APRIL expression was associated with more aggressive and undifferentiated phenotypes, correlating with lymph node metastases. Moreover, membrane steroid agonists modify APRIL transcription levels.

Conclusions: Our data show, for the first time, an autocrine secretion of APRIL in breast cancer cells, strongly associated with loss of differentiation and metastatic potency, indicating the emerging role of this TNF-related cytokine in breast cancer biology. The regulatory effect of membrane agonists on APRIL gene regulation reflects another aspect of membrane steroid signaling, providing new insights in the hot link between inflammation and breast cancer promotion.

Abscence of GAPDH regulation in human brain tumors under hypoxic conditions Introduction: Cylindromatosis tumor supressor was initially identified as a suppressor of skin appendage tumor development but it has received wide attention for its involvement in pleiototropic pathophysiological conditions. CYLD is an enzyme with deubiquitinating activity that negatively regulates activation of the transcription factor NF-jB by specific tumour necrosis factors receptors. Methods: In order to unravel the in vivo function of CYLD in T cell physiology we generated conditional knockout mice by using the Cre-loxP system in which the CYLD gene was selectively disrupted in T cells using an lck promoter-driven cre cassette.

ResultsThe number of single positive T cells was dramatically reduced in mice with T cell intrinsic cyld deficiency. Moreover, we observed that cyld-deficient mice exhibit reduced peripheral T cells, that exhibit reduced proliferation upon activation with cd3/cd28. Furthermore, in the periphery we detected a major reduction in the percentage of naı¨ve T cells, whereas effector T cells were increased. At the molecular level, proximal TCR signaling is not affected in cyld-deficient thymocytes. However, PLCc1 phosphorylation is impaired. Further experiments showed that CYLD can preferentially interact with activated PLCc1 and deubiquitinate it. This modification presumably leads to conformational changes that permit the phosphorylation and further activation of PLCc1, which is a key component of TCR signaling. Conclusions: Our data propose that CYLD serves to modulate signals that activate thymocytes and their selection in the thymus, thus acting as a key molecule in T cell development. Moreover, CYLD is implicated in the regulation of activation and homeostasis of peripheral T cells.

Signaling pathways that control the transcription of the human small GTPase RhoB gene in response to the transforming growth factor b Rho GTPases are characterized as molecular switches that control many fundamental processes including signal transduction, cell division, apoptosis and cytoskeleton reorganization. Whereas the classical Rho GTPases are regulated by GDP/GTP cycling, in some cases transcriptional regulation plays also a crucial role in their function. In the present study we show that TGFb increases the steady state mRNA levels of the RhoB gene in HaCaT keratinocytes. Treatment of HaCaT cells with actinomycin D or cycloheximide, showed that activation of the RhoB gene by TGFb occurs at the transcriptional level and that RhoB is a direct target of TGFb-induced signaling pathways. The early transcriptional activation of RhoB gene by TGFb was abolished using a specific MEK1 kinase inhibitor suggesting the involvement of the MEK/ERK pathway in this process. The late activation of RhoB was abolished by an inhibitor of the Type I TGFb receptor (ALK5) indicating the participation of the Smad pathway in RhoB gene regulation by TGFb. By constructing sequential deletion mutants and using transactivation assays, we established that the proximal region of the human RhoB promoter is required for transcriptional activation by the Smads. DNA affinity precipitation and chromatin immunoprecipitation experiments confirmed the recruitment of Smad3 to the proximal RhoB promoter. Nuclear factor Y (NF-Y) shown previously to mediate activation of the RhoB gene by genotoxic stress was not found to serve as a Smad cofactor in RhoB gene regulation. Our findings establish RhoB as a direct transcriptional target of TGFb in human keratinocytes and reveal the TGFb-induced signaling pathways that participate in this regulation.

Synthetic spiro-neurosteroid analogs exerting plasma membrane receptor mediated neuroprotective effects in vitro The human ether-a-go-go related gene (HERG) potassium channel is expressed in a variety of tissues including the heart and the neurons. This channel has elicited intense scientific interest because it is physiologically critical to cardiac repolarization and due to its association with arrhythmia and sudden death. Increasing evidence indicates the involvement of HERG channels in the pathophysiology of cancer. HERG channels are modulated by several intracellular signaling pathways and these provide important mechanisms for regulating cellular excitability. The purpose of the present study is to investigate the expression of HERG protein in thyroid cancer cells; analyze the relationship between the expression of HERG protein and the characteristics of cells proliferation and migration; explore the effects of HERG protein on biological behaviors of thyroid cancer cells.

Methods: The expression of HERG protein in thyroid cancer cells was measured by western blot, RT-PCR and immunocytochemistry respectively. The proliferation, cell cycle, apoptosis and invasive ability of thyroid cancer cells were evaluated by 3H-thymidin incorporation and celltiter assays, cell counting, flow cytometry and the cell migration assay. Electrophysiological experiments were carried out by using the patch-clamp technique. Results: Our results indicated that HERG protein was expressed in thyroid cancer cells. In anaplastic and follicular thyroid cancer cells blockade of HERG channels inhibit cell proliferation and cell migration. Reducing HERG protein could also inhibit thyroid cancer cells entering S phase from G1 phase of the cell cycle.

Conclusions: HERG channel blockade leads to antiproliferative and antimigratory properties and HERG channel blockers can be new pharmacological targets in the treatment of hard-to-treat forms of thyroid cancer.

Essential role of the Rho GTPase Cdc42 in platelet adhesion, spreading, secretion and aggregation revealed by a novel Cdc42-activity specific inhibitor In this study we investigated the role of Cdc42 in regulation of platelet function by investigating the effects of a newly identified chemical inhibitor of Cdc42, CASIN (Cdc42 activity-specific inhibitor), on platelet activation. Incubation of platelets with CASIN, but not with a pharmacologically inactive CASIN analog, blocked collagen induced activation of Cdc42, and phosphorylation of the Cdc42 effector, p21-activated kinase, dose-dependently, without detectably affecting Rac1 activity. Incubation of platelets with CASIN (1-10 lM) inhibited: (i) adhesion of platelets to immobilized fibrinogen, (ii) filopodia and lamellipodia formation, (iii) U46619-induced release of P-selectin and secretion of ATP from the a-and dense granules respectively, and (iv) aggregation induced by diverse agonists. Furthermore, CASIN treatment inhibited: (i) collagen or phorbol 12, 13-dibutyrate induced aggregation in aspirinated platelets in the presence of apyrase, and (ii) collagen induced phosphorylation of Akt. Removal of CASIN from the platelet samples by repeated washings completely reversed inhibition of aggregation by collagen. These data suggest that (i) CASIN is capable of specifically and reversibly inhibiting Cdc42 activity in platelet, (ii) Cdc42 is uniquely involved in the regulation of platelet adhesion, spreading, secretion, and aggregation, and (iii) the Cdc42 targeting small molecule inhibitor, CASIN, may serve as a novel anti-platelet agent.

Hepatitis C virus core protein induces persistent activation of peroxisome proliferator-activated receptor a in transgenic mice T. Aoyama and N. Tanaka Department of Metabolic Regulation, Institute on Aging and Adaptation, Shinshu University Graduate School of Medicine, Matsumoto, JAPAN Persistent infection of hepatitis C virus (HCV) can lead to a high risk for hepatocellular carcinoma (HCC). HCV core protein plays important roles in HCV-related hepatocarcinogenesis, because mice carrying the core protein exhibit multicentric HCCs without hepatic inflammation and fibrosis. However, the precise mechanism of hepatocarcinogenesis in these transgenic mice remains unclear. To evaluate whether the core protein modulates hepatocyte proliferation and/or apoptosis, we examined these parameters in 9-and 22month-old transgenic mice. Although the numbers of apoptotic hepatocytes were similar between transgenic and non-transgenic mice, the numbers of proliferating hepatocytes and the levels of numerous proteins such as cyclin D1, cyclin-dependent kinase 4, and c-Myc, were markedly increased in an age-dependent manner in the transgenic mice. This increase was correlated with the activation of peroxisome proliferator-activated receptor alpha (PPARa), which was closely associated with hepatocarcinogenesis in rodents. In these transgenic mice, spontaneous and persistent PPARa activation occurred heterogeneously, which was different from that observed in mice treated with clofibrate, a potent PPARa activator. We further demonstrated that stabilization of PPARa through a possible interaction with HCV core protein and an increase in nonesterified fatty acids, which may serve as endogenous PPARa ligands, in hepatocyte nuclei contributed to the core protein-specific PPARa activation. In conclusion, these results offer the first suggestion that HCV core protein induces spontaneous, persistent, agedependent, and heterogeneous activation of PPARa in transgenic mice, which may contribute to the age-dependent and multicentric hepatocarcinogenesis mediated by the core protein.

Receptor for advanced glycation end products (RAGE) positively regulates CD36 expression and ROS production in human monocytes in diabetes PP7C-5 New in vitro models for understanding molecular mechanism of oncogenesis C. Bleotu, M. Chivu, I. Alexiu, G. Anton, C. C. Diakonu, A. Neagu and C. Cernescu ''Stefan S. Nicolau'' Insitute of Virology, Bucharest, ROMANIA Abstract: Cell lines provide useful model system for studying biological significance of regulatory processes and molecular mechanisms of oncogenic program induction. Each new cell line should be evaluated in light of its own particular characteristics and aiming for which should be used. Methods: TV cell line was obtained from human laryngeal tissue and maintained like monolayer. Optical and transmission electron-microscopy was used for morphological characterization. The interleukin concentration in cell line supernatant was assesed by ELISA using Endogene Human kits (Pierce). Levels of proliferation marker (CD71 and HLA-DR), transformation (k-ras) and metastasis (EGFR and VEGF) was determined in flow cytometry or RT-PCR.

Results: PCR technique evidenced HPV genomes in TV cells but viral particles can not be observed in electron-microscopy that is normal for cell cultures derived from HPV cancer explants. Our cell line has a high proliferate rate, sustained by markers (CD71 -99.95%; HLA-DR -75.68%). By contrast with HeLa cells, TV culture forms large colonies (>200 lm), with unregulated shape. According to their tumor origin and characteristics (initiated from a laryngeal tumor from a patient which developed later regional and distal metastasis) TV cells produces cytokines that sustain the metastatic phenotype. IL6 and IL8 levels were clearly elevated in TV comparing with HeLa. EGFR and k-ras levels were elevated, but the VEGF value was similarly in TV and HeLa (81.31% vs. 92.2%).

Conclusion: These new models generated in vitro for carcinogenesis studies are very useful and allow us to investigate the events involved in tumorigenesis/metastasis. Elevated blood glucose and free fatty acids induce oxidative stress associated with incidence of cardiovascular disease. In contrast, laminar shear stress (LSS) plays a critical role in vascular health by inhibiting many inflammatory events triggered by reactive oxygen species (ROS). The present study examined how high glucose (HG) and arachidonic acid (AA) induce lipid peroxidation, and whether LSS attenuates lipid peroxidation in human umbilical vein endothelial cells. HG and AA synergistically induced lipid peroxidation, which was attenuated by inhibitors blocking ROS sources as well as by glutathione (GSH) and its precursors. Of special interest, lipid peroxidation was inhibited by L-NAME, a specific inhibitor of NO synthase, implicating 'uncoupled' eNOS might mediate lipid peroxidation by producing ROS instead of NO. In consistent with this notion, tetrahydrobiopterin (BH4), an essential cofactor of NO synthase markedly inhibited lipid peroxidation. LSS also appeared to be a strong inhibitor of lipid peroxidation supporting its vascular protective role. It also increased cellular level of BH4 and GSH content. Inhibition of GSH synthesis by BSO and si-RNA-mediated knockdown of Glutamylcysteine ligase modulatory subunit reduced inhibitory effect of laminar shear stress on lipid peroxidation. Therefore it is suggested that LSS enhances antioxidant capacity of endothelial cells, thereby attenuates oxidative stress caused by cardiovascular risk factors. Introduction: Recent study has demonstrated that Sasa quelpaertensis (Korean Name, Jeju-Joritdae) extracts inhibit cellular melanogenesis implicating potential use in the control of skin pigmentation. The purpose of the present study was to elucidate the active constituents of this plant inhibiting melanogenesis and the associated mechanism.

Methods and Results: When tested in melanoma B16/F10 cells treated with the alpha-melanocyte stimulating hormone (a-MSH), the aqueous ethanol extract of Sasa quelpaertensis culm inhibited the cellular melanogenesis more effectively than its leaf extract. A major active compound was isolated from the culm extract by solvent fractionation and column chromatography, and identified to be para-coumaric acid by spectroscopic and chromatographic analyses. The compound (paracoumaric acid) inhibited a-MSH-stimulated cellular melanogenesis more effectively than arbutin or other structurally similar compounds including 3-(4-Hydroxyphenyl)propionic acid, cinnamic acid and caffeic acid. It also attenuated a-MSH-dependent increase of tyrosinase protein. The anti-melanogenic effect of para-coumaric acid was also verified in neonatal human melanocytes.

Conclusions: The present study identified para-coumaric acid as a main constituent of Sasa quelpaertensis inhibiting cellular melanogenesis. Due to structural similarity, para-coumaric acid may interfere with L-tyrosine action in the control of tyrosinase expression in response to a-MSH.

Overexpression of P-glycoprotein in L1210 cells is associated with massive cell clustering Introduction: Massive expression of P-glycoprotein (P-gp) in collected to multidrug resistance (MDR) by adaptation to vincristine is associated with several changes in cell metabolism that induced alternation in cell architecture. Therefore we studied changes that induce overexpresion of P-gp on morphology of L1210 cells. Methods: P-gp negative L1210 (S), and P-gp positive L1210 cells obtained by adaptation to vincristine (V) or bay transfection with human mdr1 gene encoding P-gp (T) were used. Morphology of cells was assessed by light and electron microscopy. Results: R cells exerted massive cell clustering visible using light and electron microscopy; similar cell clustering could be observed in S cells after treatment with sialidase; Cell clustering in T cells is less pronounced as in R cells. R cells differ from S cells in amount of cell surface acidic saccharide stainable by ruthenium red that is believed to be sialic acid.

Conclusions: Cell clustering of R cells is induced by alteration of contend of acidic sacharides on cell surface -probably sialic acid. Acknowledgement: This work was supported from grants APVT-51/ 027404, APVV 0084-07, VEGA 2/4155, and VEGA 2/6080.

PP7C-9 Investigation of hepcidin secretion in human hepatocytes Iron homeostasis depends on iron absorption and trafficking between plasma and the body reservoirs. Hepcidin, an antimicrobial hepatic hormone, plays a pivotal role in the regulation of iron metabolism by directly controlling both intestinal iron absorption and iron release from macrophages. The hepcidin gene is coding for a pre-prohormone, further processed to a mature form of 20 or 25 amino acids which is secreted to the circulation. However, although hepcidin expression is controlled by diverse stimuli, including hypoxia mimetic agents, it is not known if the prohepcidin maturation process and secretion is posttranslationally regulated. In order to investigate the hepcidin secretion pathway, Huh7 cells were transiently transfected with plasmid pEGFP-N3-hepcidin leading to over-expression of pre-prohepcidin tagged at the C-terminus with GFP. By western blot analysis we detected at least two intracellular forms of hepcidin-GFP, while one distinct form appeared to the culture supernatant. Moreover, as shown by fluorescence microscopy, hepcidin-GFP was confined to cytoplasmic structures reminiscent of organelles of the secretory pathway such as endoplasmic reticulum and Golgi apparatus. Treatment of cells with brefeldin A (which disrupts the Golgi complex) restrained hepcidin-GFP inside the cells. These data suggest that hepcidin-GFP is stable and can be actively processed and secreted by hepatocytes, providing a model for the analysis of hepcidin maturation. We, therefore, established stably transfected Huh7 cell lines expressing hepcidin-GFP. These cell lines are currently used, in combination with specific markers and inhibitors of the secretion machinery, to study trafficking of prohepcidin and its regulation by factors affecting iron homeostasis. Introduction: M3/6 is a dual-specificity protein phosphatase whose main substrate is the c-Jun N-terminal kinase (JNK). JNK is phosphorylated and activated in response to various extracellular stimuli, including stress factors such as arsenite. Dephosphorylation and inactivation of JNK by the M3/6 phosphatase is important for the cellular response to stress. It has been shown that M3/6 itself is phosphorylated by JNK upon stimulation with arsenite. The aim of the project is to investigate the effect of this phosphorylation on the activity of M3/6 and its importance for JNK signaling. Methods: Mass spectrometry was used to map phosphorylated sites on M3/6. These sites were mutated using site-directed mutagenesis. The mutant and wildtype proteins were compared upon arsenite stimulation in transiently transfected cells for their phosphorylation and for their ability to inactivate the JNK pathway.

Results: Phosphorylated serine residues 515 and 520 on the M3/6 protein were identified and mutated to alanines. Upon arsenite stimulation, the mutated M3/6 proteins exhibited decreased phosphorylation compared to the wildtype protein. In addition, time-course stimulations revealed a prolonged inactivation of JNK by the mutant proteins, as well as reduced phosphorylation of the JNK downstream target c-Jun. Therefore, mutation of the M3/6 phosphorylation sites resulted in attenuation of JNK signaling.

We have shown that the phosphorylation state of the M3/6 phosphatase affects the activity of M3/6 in response to stress, and is therefore, important for the regulation of the JNK signaling pathway.

The PKR involvement in amyloid peptide-induced death signalling pathway 1-42) . After PKR inhibition, death signalling markers were analysed in nuclear extracts and cellular lysates by western blotting and immucytofluorescence. Three and six month-old APP SL PS1 KI mice were also examined and compared to control littermates. Results: Immunoblots revealed an increase of TNFa (+406%) and FasL (+255%) levels in hippocampus of APP SL PS1 KI mice at 6 M. PKR and FADD co-immunoprecipitated in cortical mouse homogenates. In SH-SY5Y cells, Phospho-PKR and Phospho-FADD levels increased significantly after 4 hours Ab 1-42 (+126% and +299%, respectively). Physical interaction was observed by confocal staining and confirmed by immunoprecipitation. Treatment with oxindol C16 prevents nuclear Phospho-PKR (-62%) and Phospho-FADD (-45%) expression, and inhibits totally caspase-8 and -3 activities.

Conclusions: These results show the PKR involvement in FADD/caspase-8 signalling pathway and underline PKR as a critical target in neuroprotective strategies in AD.

The extracellular domain of Her2/neu expressed in P. pastoris induces protective antitumor immunity A. Dimitriadis, E. Ntougkos, C. Gontinou and A. Mamalaki Hellenic Pasteur Institute, Athens, GREECE Introduction: Her2/neu is overexpressed in various human cancers of epithelial origin and is associated with increased metastatic potential and poor prognosis. Our goal is to study the effect of the extracellular domain of Her2/neu (ECD/Her2) produced in yeast P. pastoris alone or in combination with an anti-Her2/neu monoclonal antibody (mAb) in tumor prevention. Methods: BALB/c and HHD (C57.HLA.A2.1) transgenic mice were immunized with 25 lg ECD/Her2 with adjuvant. Two boosts with the same amount of ECD/Her2 per mouse were carried out. 10 days after the last immunization, mice were challenged with cancer cells overexressing Her2/neu and tumor development was monitored. When tumors became palpable, 1mg/kg of anti-Her2/neu mAb was administered, twice per week. Blood serum was collected and used in ELISA and immunoprecipitation assays to detect antibodies against Her2/neu. The effect of mice sera in proliferation assays was investigated in the breast cancer cell line SK-BR-3.

Results: ECD/Her2 did not cause any toxicity in mice. Tumor development in mice vaccinated with ECD/Her2 was either delayed or not observed compared to control mice. Specific antibodies against Her2/ neu were detected in sera collected from mice vaccinated with ECD/ Her2 and these antibodies reduced the proliferation rate of SK-BR-3 cells.

Conclusion: These results indicate that ECD/Her2 expressed in P. pastoris is a good candidate for a tumor antigen vaccine. This vaccination in combination with anti-Her2/neu mAb is currently under investigation.

Cox-2 is a target gene of Rho GDP dissociation inhibitor b in breast cancer cells Breast cancer is the leading cause of cancer death in women worldwide. Despite progress in detection and chemotherapy, many women with breast cancer continue to die of this malignancy. Elevated expression of c-Myc is frequently observed and androgens are known to inhibit its growth but the molecular mechanism remains unknown. Androgen receptor (AR) is expressed in large part of breast tumors or normal mammary gland and only limited number of breast cancer cell lines express both estrogen (ER) and AR receptors. We were investigating the role of hormones and c-myc in proliferation (crystal violet assay) and AR, ER, bcl-2, bax, Cyclin D1, c-myc, p27, pRb, cdk2 expression in breast cancer cell lines (MCF-7, T47D) at protein level. The cells were transfected with the small interfering RNA (siRNA) targeting cmyc to reduce its expression (Ambion) and treated by 5alfa-dihydrotestosteron (DHT) or estradiol (E2). T47D cell line express ER and AR and is sensitive to E2 and DHT but MCF-7 express only ER and does not respond to DHT treatment. DHT caused also an accumulation of inhibitor p27. Silencing of c-myc expression reduced its expression in both cell lines up to 85% and decreased the proliferation of cells while promoting apoptosis (Caspase activity kit, bax, bcl-2) The inhibition of breast cancer cell growth by androgen seems to be mediated by p27 and decreasing the c-Myc protein level plays a principal in progression of human breast cancer. N9 microglial cells were activated by LPS and APC was added 1 hour before LPS stimulation. We performed lactate dehydrogenase assay to evaluate LPS-induced glial cell death. TNF-a levels were measured by enzyme-linked immunosorbent assay (ELISA). Results: We found that LPS induced activation of glial cell death and pretreatment with APC attenuated microglial cell death in a dose dependent manner. ELISA revealed that APC also decreased LPSinduced TNF-a production in a dose-dependent manner. Introduction: Inflammation is considered as one of the factors responsible for b-cell death observed in type 1 diabetes mellitus (T1DM). ROL may mediate some of its anti-inflammatory effects by blocking nitric oxide (NO) production by macrophages, through elevation of intracellular cAMP level. The aim of the study was to evaluate whether ROL can improve nitrosative stress and inflammation of pancreatic islet in streptozotocin (STZ)-induced diabetes in rats. Methods: Adult Wistar male rats were divided into three groups: control group, STZ induced-diabetic rats and STZ-induced diabetic rats treated with ROL (10.5 mg/kg) over a period of one week. The degree of insulitis was evaluated histochemically. The islets beta-cell function was estimated immunohistochemically by measuring insulin content of cells using anti-insulin antibodies. Nitrosative stress was evaluated immunohistochemically by measuring nitrated proteins by using antibodies against 3-nitrotyrosine (3-NT).

Results: ROL-STZ group showed a significantly decrease in the degree of insulitis compared with STZ rat-group (1.71 ± 0.13 vs.

2.64 ± 0.10, p < 0.001). The proportion of insulin positive b-cells per islet and per pancreatic sections indicated significant reduction in the STZ rat group (about 92%, 87%, respectively). An increase (p < 0.001) in the number of positive beta-cells and a decrease in the nitrosative stress in the islets of ROL-STZ group compared with STZ group were consistent with the significant improvement in glucose and insulin plasma levels.

Conclusion: In vivo treatment with phosphodiesterase inhibitor type IV restore pancreatic islet morphology and function possibly via improvement in the hyperglycaemia induced nitrosative stress. NO is associated with the mechanism of action of some cardio-vascular drugs. Proceeding from the fact that there exists a close correlation between the cardio-vascular system and the renal functional activity, medicinal preparation, influencing the cardiac performance and vascular condition, may also change the function of the kidneys, it may be assumed that the renal effect of the cardioprotective medicine -Thiotreazolin (Ukraine) may also be mediated via NO. In order to determine a possible participation of arginine -NO -dependent mechanisms of regulating the renal activity, runs of experiments were carried out with a multiple -dose introduction (7 days) to rats of arginine (100 mg/kg) which is a biological substrate and activator for the formation of NO, as well as a simultaneous use of L-arginine and Thiotreazolin . The experiments were run under conditions of functional loading on the kidneys which was carried out via introducing running water into the rat stomach in an amount, constituting 5% of the body mass. L-arginine exerts an influence primarily on the vasculoglomerular apparatus of the nephron, an activation of the mechanism of glomerulo-tubular balance taking place under its action at that an elevation of the filtration load of nephrons with sodium ions by an adequate intensification of their transport in the proximal tubules in case of the activation of sodium -dependent mechanisms of urine acidification. A combined use of Thiotreazolin and L-arginine in case of water load are indicative of the fact that the renal effects of Thiotreazolin do not have a direct dependence on the NO formation in the kidneys. On the contrary, a well-known antioxidant action of Thiotreazolin, probably, blocks NO-dependent intrarenal generation of angiotensin II, favouring the diuretic and natriuretic effects of the drug under study. Methods: Cell viability was tested by MTT test. Type of cell death was determined by AnnexinV-FITC/propidium iodide kit. Levels of proteins of Bcl-2 family were detected by Western blot with appropriate antibodies. Results: We found that R are more sensitive as S to cisPt induced cell death. However, cisPt induced more pronounced apoptosis in S as R cells. Oppositely more R as S cells were entering necrosis in the presence of cisPt. We observed several alternations in the contents as well as time dependency of Bax and Bcl-2 expression by Western blot.

Conclusions: R cells are also resistant to apoptosis induced by cisPt while cisPt is not substrate for P-gp. Integrin-linked kinase (ILK) plays an important role in integrin signaling and its kinase activity is modulated by cell-extracellular matrix. Migration of melanoma cells is dependent on alpha3 beta1 integrin, which binds laminin 5 and regulates the MMP-2 and MMP-9 activity. It was suggested that activation of AKT kinase through PI3-K plays a key role in response to LN-5 signals carried through specific integrin involved in melanoma metastasis. Focal adhesion kinase (FAK) is known to phosphorylate AKT upon integrins activation. Our study identifies the mechanism through which AKT can regulate melanoma cell invasion properties via signaling that is independent of FAK and involves ILK. Studies were carried out on human melanoma cell lines. Secretion of metalloproteinases was analyzed by zymography, and the invasive potential by using Boyden chambers. Expression of cell signaling proteins (AKT, pAKT, ILK, pFAK), was analyzed using western blot, siRNA transfection for ILK and AKT (Ambion). We showed, that silencing of ILK, inhibited the effects of Ln-5 on AKT activation, suggesting a regulatory role of ILK in signaling pathway involved in melanoma invasion. We were also able to show that silencing of ILK gene expression by siRNA process significantly inhibits in vitro invasion of melanoma cells. These results indicate on ILK as critical mediator in signaling pathway necessary for melanoma cell migration and invasion. Methods: The cell lines used were BL41 (Burkitt lymphoma, EBVnegative) and BL41 B958 (Burkitt lymphoma, EBV-positive). Two dimensional (2-D) electrophoresis analysis was used and the proteins from total cell lysates or subcellular fractions were separated in both wide and narrow range IEF (isoelectric focusing) strips. In-solution, narrow range IEF fractionation was also performed. Differentially expressed proteins were trypsinized and identified by LC-MS/MS. Western Blotting and RT-PCR were also used to validate the results.

Results: In the EBV-positive cell line twenty-four proteins were found to be upregulated and twenty-one were found to be downregulated. These were classified into distinct categories according to their molecular function. Included were proteins involved in the ubiquitination pathway (COMMD2 and Ubiquitin carboxyl terminal hydrolase 51) and the caspase pathway (Serpin B9).

Conclusions: A proteomic approach has identified several proteins that are up or down regulated in latency III EBV-infected cells. Some of them may be implicated in the development of lymphomas and therefore may represent novel targets for intervention. Methods: The two cell lines were compared using two-dimensional electrophoresis. The starting material was total cell lysate or subcellular fractions (e.g. nuclear extracts). Narrow range fractionation at the IEF stage was performed as well, in order to reduce sample complexity and improve separation and detection. Differentially We have studied accumulation of novel mitochondria-targeted antioxidants (MTA) in normal and transformed cells. Fluorescent MTA was designed as the conjugate of antioxidant part (plastoquinol) and cationic decylrhodamine (SkQR1). SkQR1 specifically accumulates in mitochondria of the cell due to it positive charge and effectively protects cells against reactive oxygen species (ROS) production and cell death induced by prooxidants (hydrogen peroxide, paraquat and menadione). Uncoupler FCCP which reduces mitochondrial membrane potential prevents accumulation of SkQR1 and abolishes its protective effect. The role of multidrug resistance (MDR) pump in transport of SkQR1 was studied in breast carcinoma MCF-7 cells and erythroleukemia K562 cells with high level of P-glycoprotein selected by doxorubicin cultivation. The inhibitors of P-glycoprotein verapamil and pluronic stimulate accumulation and inhibit the release of SkQR1 in these clones but not in original MCF-7 and K562 cells. These inhibitors stimulate accumulation of SkQR1 in HeLa cells and were less effective in normal human fibroblasts. SkQR1 protects MDR-negative cells against prooxidants at low concentrations (2-20 nM) and was less effective in the cells with high level of MDR. The inhibitors of P-glycoprotein improve the protective action of SkQR1. The same effect was observed with an analog of SkQR1 where decylrhodamine was replaced by decyltriphenylphosphonium (SkQ1). It is suggested that these MTA could serve as the protective agents in chemo-and radiotherapy of MDR-positive tumors.

The role of the dual specificity phosphatase Puckered in Drosophila adult CNS K. Karkali, E. Skoulakis and G. Panayotou Biomedical Sciences Research Center 'Al. Fleming', Athens, GREECE Introduction: Dual specificity phosphatases (DSPs) are cysteine-based protein phosphatases able to remove phosphate groups from both phosphotyrosine and phosphothreonine residues. MAPK phosphatases (MKPs), the key regulators of MAPK activity, are classified as a subgroup of DSPs. In Drosophila six DSPs have been identified, of which Puckered, DMKP1 and DMKP3 have been characterized as MKPs. Basket, the Drosophila homologue of JNK, has been shown to be the substrate of Puckered. The physiological role of Puckered in development is well established, contrary to its role in adult flies, which remains obscure. In this study we address the role of Puckered in the adult Drosophila central nervous system (CNS). Methods: The UAS/GAL4 system was used for expression of reporter genes, disruption of neurotransmission and induction of gene silencing by RNA interference, in a tissue specific manner. Temporal control on the UAS/GAL4 system was achieved upon introduction of the inducible GAL4 suppressor, GAL80. Whole mount tissue preparations were immunostained and pictures were obtained by confocal microscopy. RNA extraction and subsequent RT-PCR was performed. A polyclonal antibody against Puckered was raised, affinity purified and used for Puckered characterization. Lung cancer is one of the most insidious and aggressive neoplasm, and consequently early diagnosis is very critical for clinical outcome and prognosis. It is necessary to discover biomarkers for early detection and prognostic evaluation of lung cancer. The proteins, levels of which are differentially high in the serum of cancer patients can be applicable to the cancer biomarker. For screening candidate potential lung cancer serum biomarkers, we investigated the protein differentially secreted from the transformed lung cell (1198 and 1170I) compared to immortalized normal bronchial cells (Beas-2B) and non-transformed cells derived from Beas-2B treated with beeswax (1799). The proteins were recovered from the conditioned media by TCA precipitation and separated on two-dimensional gel. There was not much difference between 2D patterns of the proteins from Beas-2B and 1799 cells, whereas there was significant difference between the patterns of transformed cells and Beas-2B. Using mass spectrometry and database search, we identified PGP9.5 (UCH-L1), tissue inhibitors of metalloproteinases-2 (TIMP-2), PSMA 3, proteasome activator subunit Reg, TPI, and TCTP among the proteins increased simultaneously in conditioned media of 1198 and 1170I cells.s By means of western blot analysis, we validated that levels of PGP 9.5, TPI, TCTP and TIMP-2 were significantly high not only in the conditioned media of 1198 and 1170I cells compared to those of Beas-2B and 1799 cells, but also in the serum of lung cancer patients compared to normal serum. These results suggest that the four proteins are potential lung cancer serum biomarkers.

Effect of overexpressed cyclophilin B in ROS-mediated cell death The Rab11-family interaction protein 3(Rab11-FIP3), identified from human and mouse and also known as Eferin, is a Rab11 binding protein with RBD(Rab11-binding domain) domain at the C terminus. Rab11 has been shown to play a key role in a variety of cellular processes, including plasma membrane recycling, phagocytosis, and transport of secretory proteins from the trans-Golgi network. Recently mouse Rab11-FIP3 was found to bind to Transient Receptor Vanilloid Potential 1(TRPV1) and it was suggested that Rab11-FIP3 might play a role in trafficking TRPV1 to the membrane. So far, the membrane trafficking of TRPV1 was little known. In the present study, in order to study the membrane trafficking of rat TRPV1, the rat Rab11-FIP3 was cloned from rat brain and characterized. Rat Rab11-FIP3 was presumed to be 50KD with the significant sequence homology with mouse Rab11-FIP3, with up to 90% identity. However, rat Rab11-FIP3 does not seem to have EF Hand motif, but have RBD domain and Glutamic acid-rich region. The role of rat Rab11-FIP3 in membrane trafficking of rat TRPV1 is under investigation by using cell and biochemical techniques.

PP7C-32 5¢-nitro-indirubinoxime induces cell cycle arrest and apoptosis in human KB oral cancer cells Introduction: Indirubin has potent anti-tumor activity on a variety of human cancer cell types, thereby suggesting its potential for application against human cancers. Our previous study showed that novel indirubin derivative 5¢-nitro-indirubinoxime (5¢-NIO) effectively arrested the tumor growth by inhibiting cell proliferation and inducing apoptosis. However, precise molecular mechanisms of anti-tumor activity induced by 5'-NIO were not clear. In this study, we investigated molecular action mechanism of 5'-NIO in human KB oral cancer cells. Methods: Effect of 5'-NIO on KB cell proliferation and cytotoxicity was examined. The kinase activity of CDK and PLK1 was measured and the apoptosis was determined by Annexin-V/propidium iodide staining. MAPK and caspase activity were studied by immunoblotting. Results: 5'-NIO inhibited the proliferation of KB oral cancer cells by inhibiting the G1/S and G2/M phase regulatory proteins. 5¢-NIOmediated cell cycle arrest was mediated by three different mechanisms: (i) decrease the CDK4 and cyclin D1/D3 level by p16, (ii) induce the level of p21, (iii) decrease the activity of CDC2/cyclin B complex by inhibit the PLK1 expression. 5¢-NIO-induced cell cycle arrest is followed by apoptosis. We further identified that 5¢-NIO induced apoptosis is accomplish by mitochondria-dependent activation of caspase cascade.

Conclusions: 5¢-NIO induced cell cycle arrest on G1/S and G2/M phase. 5¢-NIO induced cell cycle arrest is followed by apoptosis which is promoted by mitochondria-mediated caspase activity. These results provide insight into the molecular mechanisms of 5¢-NIO, as well as the potential value as a novel candidate for oral cancer treatment. The new mitochondria-targeted antioxidant SkQ1 (plastoquinone conjugated with decyltriphenylphosphonium) was designed in our laboratory. In isolated mitochondria SkQ1 demonstrated higher antioxidant efficiency then prototypic MitoQ (ubiquinone conjugated with decyltriphenylphosphonium). Fluorescent analog of SkQ1 (SkQR1) accumulated in mitochondria of normal human fibroblasts and various transformed cell lines. SkQ1 and SkQR1 protected the cells at concentrations 2-20 nM when cells were preincubated for 6-7 days. MitoQ was less effective. By the other hand accumulation of SkQR1 in the cells was completed in 1-2 hours. We haven't observed any protection against oxidative stress induced by H 2 O 2 or the other pro-oxidants (menadione, paraquat) in this conditions. MitoQ and SkQ1 also were ineffective. Sub-toxic H 2 O 2 stopped cell cycle of HeLa cells in G2/Mphase and this effect was insensitive to mitochondria-targeted antioxidants as well. The increase of the antioxidants concentration in the range 0.1-1 lM revealed their pro-oxidant properties. In combination with sub-toxic H 2 O 2 these antioxidants caused significant ROS production and cell death. Cationic part was at least partially responsible for this effect since decyltriphenylphosphonium also promoted oxidative stress under these conditions. Probably this effect is related to damage of the mitochondrial membrane due to detergent action. We have suggested that SkQ1 did not accumulate in all mitochondria during short incubation probably due to heterogeneity of the membrane potential. Some mitochondria remained unprotected and initiated ROS production and apoptosis under oxidative stress. Prolonged incubation provided complete protection of mitochondrial population. Introduction: Estrogens play important roles in the development and growth of breast cancer. Although, the action of estradiol (E2) to estrogen receptors (ERa/ERb) and the regulation of target gene transcription is well defined, its involvement in synthesis of matrix is not clearly defined. Studies in our lab indicate that differential gene expression of effective matrix macromolecules, including proteoglycans (PGs) from epithelial breast cancer cells, is associated with malignant phenotype. The aim of this study was, therefore, to examine whether E2 affects the expression of PGs implicated in breast cancer, and whether this is associated with ERs. Introduction: Many physiological and pathophysiological processes are related to changes in cellular level of diadenosine polyphosphates and the most significant is the concentration rate of Ap 3 A to Ap 4 A [1] . Ap 3 A is cleaved by tumor suppressor Fhit protein, one of the enzymes of hydrolases family. It is assumed that the formation of the FhitAp 3 A complex, recognized as a signaling molecule [2] , induces cell apoptosis. Therefore, analogs of Ap 3 A may be of interest as potential therapeutic molecules. The aim of our studies was to determine the cellular uptake of the fluorescent nonhydrolyzable analog of Ap n A (Fl-Ap 3 A analog), which is an analog of inhibitor of Fhit protein [3] . Methods: We used various conditions (with and without Lipofectamine 2000 carrier; concentration from 2.5 to 10 lM) to study the transport of this compound across membrane in A549 and HUVEC cells. Intracellular distribution of fluorescent analogs of diadenosine triphosphate was visualized by fluorescence microscopy.

Results: These studies demonstrate that FI-Ap 3 A analog accumulate in cytoplasmic compartments of the transfected cells. The same localization was observed for other Ap n A analogs with fluorescent residue as ApppBODIPY and GpppBODIPY. Conclusion: Both components of the complex (FHIT protein and Fl-Ap 3 A analog) co-localize in cytoplasmatic compartments of the cell.

Production of soluble glycosylated CD69, a common leukocyte signaling receptor, in normal and "humanized" strains of Pichia pastoris, and its use in animal tumor therapies Introduction: CD69 is the earliest activation antigen of lymphocytes, and an universal activation molecule of the cells of the immune system. From this point of view, there has been an interest in the production of soluble stable forms of this membrane protein for their potential use in immune therapies. Methods: DNA fragment coding for the extracellular part of CD69 has been amplified by PCR, and transferred to Pichia expression vector pPICZalphaA. Normal and 'humanized' Pichia pastoris (X33) were transfected with linearized plasmid. The production of CD69 was induced by Methanol (0.5%). Culture medium was concentrated and protein was purified on Q-Sepharose column. Biochemical stability was monitored for 7 days by SDS-PAGE. Chair of Pharmaceutical Botany, Medical College, Jagiellonian University, Krakow, POLAND, 2 Chair of Medical Biochemistry, Medical College, Jagiellonian University, Krakow, POLAND, 3 Laboratory of Molecular Genetics and Virology, Jagiellonian University, Krakow, POLAND Introduction: High glutathione (GSH) level and elevated gamma-glutamyltranspeptidase (GGT) activity are hallmarks of tumor cells. Toxicity of drugs and radiation to the cells is largely dependent on the level of thiols. Despite well-documented antioxidant action of GSH, under certain conditions GSH/GGT system can produce a moderate oxidative stress, which is a component of signal transduction in the processes of proliferation and apoptosis. In the present studies, we attempted to inhibit GGT activity in human hepatoblastoma (HepG2) cells in order to examine whether the administration of 1,2,3,4-tetrahydroisoquinoline (TIQ) influences cell proliferation and enhances cytostatic action of doxorubicin (DOX) on HepG2 cells. Methods: To find mechanism of the gamma-glutamyl cycle enzymes inhibition we've measure enzymatic activity of GGT, gamma-glutamylcysteine synthase, glutathione synthase and glutathione S-transferase. We also perform Western Blot and RT-PCR analysis. The effects of this TIQ were determined by MTT, BrdU and LDH tests and by estimation of GSH and ROS levels. Additionally, we investigated the changes in caspase-3 activity, which is a marker of apoptosis. Results: The obtained results showed that the GGT inhibitor introduced to the medium alone elicited cytotoxic effect, which was accompanied by an increase in glutathione level in the cells. TIQ concomitantly increased caspase-3 activity. Doxorubicin proved to be cytotoxic, and TIQ augmented this effect. As well doxorubicin radically decreased GSH levels. We also show that HepG2 cells characterized lack of glutathione S-transferase. Conclusions: Therefore, the obtained results confirm that GGT inhibitor can enhance pharmacological action of doxorubicin, which may permit clinicians to decrease their doses thereby alleviating side effects.

The effect of modulation of gamma-glutamyl transpeptidase activity on H 2 O 2 production and signal transduction in HepG2 cells I. Kwiecien 1,2 , M. Iciek 2 and G. Chwatko 3 1 Chair of Pharmaceutical Botany, Medical College, Jagiellonian University, Krakow, POLAND, 2 Chair of Medical Biochemistry, Medical College, Jagiellonian University, Krakow, POLAND, 3 Department of Environmental Chemistry, University of Lodz, Lodz, POLAND Introduction: The membrane ectoenzyme c-glutamyl transpeptidase (GGT) is connected with catabolism of glutathione (GSH), the main low molecular thiol antioxidant. Emerging evidences suggest, that activity of GGT is connected with H 2 O 2 production. The presented studies aimed to elucidate how modulation of GGT activity influences H 2 O 2 production in connection with protein S-thiolation and caspase 3 activity. Methods: H 2 O 2 production was measured using Amplex red hydrogen peroxide assay kit, and caspase 3 activity by fluorimetric assay with Ac-DEVD-AMC as a substrate. The protein-bound fractions of thiols (S-thiolation) were measured by HPLC after precolumn derivatization with 2-chloro-1-methylquinolinium tetrafluoroborate and with UV detection.

The experiments showed that 1-hour stimulation of GGT elevated hydrogen peroxide production, leading to prooxidant conditions. After 24-hour stimulation, hydrogen peroxide concentration was at the control level, while Cys-Gly, Cys and GSH dependent S-thiolation was markedly increased, which was accompanied by a drop in caspase-3 activity. The inhibition of GGT by acivicin led to hydrogen peroxide decrease after 1 hour incubation which still persisted after 24 hours. The inhibition of GGT activity in HepG2 cells in this case was connected with the lowering of S-thiolation with Cys and Cys-Gly. Conclusions: The results of these studies indicate that the modulation of GGT activity in HepG2 cells can be connected with oxidative stress and S-thiolation, what can influence activity of caspase 3. It confirms the hypotesis that the prooxidant reactions induced by GSH catabolism appear to represent a novel mechanism for modulation of cellular signal transduction. Here, we show that treatment with subtoxic doses of Wit A in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in human renal cancer cells, but not in human normal mesangial cells, suggesting that this combined treatment may offer an attractive strategy for safely treating renal cancer. Wit A treatment up-regulated DR5, a death receptor of TRAIL, in a transcription factor CHOP-dependent manner. Furthermore, treatment with Wit A down-regulated the antiapoptotic proteins such as c-FLIP L and c-FLIPs at transcriptional levels through ROS signaling pathways. The lessened c-FLIP expression levels induced by Wit A were recovered by NAC pretreatment and ectopic expression of p65 plus p50. In addition, Withafein A-induced down-regulation of c-FLIP was more enhanced by TRAIL treatment via cleavage of p65 through caspase activation. Taken together, our results show that the activity of Wit A to modulate multiple components in the death receptor-mediated apoptotic pathway is responsible for its ability to sensitize to TRAIL in renal cancer cells. 

Alterations in enzyme activities that catalyse phosphatidylcholine biosynthesis in stretchexposed alveolar type II cells , where it is organised and secreted afterwards into the alveolar space. Major enzyme activities that participate in PC biosynthesis are CDP-choline: 1,2-diacylglycerol cholinephosphotransferase (CPT), an acidic, Ca 2+ -independent phospholipase A2 (aiPLA 2 ) and a lyso-phosphatidylcholine acyltransferase (LPCAT). The aim of the present study was to investigate whether mechanical stretching promotes PC biosynthesis in ATII cells.

Methods: A549 cells, used as model of ATII cells were grown on pet-riPERM dishes with a flexible hydrophlilic growth surface. Afterwards, cells were exposed to an 1 hour continuous stretching using a cell stretch device that applies a force of 35 g/cm 2 . LDH activity was measured as a marker of cell disruption. CPT and LPCAT activity were measured radiometrically and aiPLA 2 fluorimetrically. The PC-fatty acids profile was determined by gas chromatography after transesterification with BF 3 -methanol.

Results: After 1 hour of continuous mechanical stretching, the activities of CPT, PLA 2 and LPCAT were increased by 47, 30 and 20%, respectively. These alterations were consistent to the increase of total PC levels as well as to the remodelling of the fatty acyl-groups profile of the PC fraction. Regulators of G protein Signalling (RGS) constitute a family of proteins that accelerate GTP hydrolysis of Ga protein subunit and act as effector antagonists or scaffolds to recruit cytoplasmic proteins to G protein-coupled receptors (GPCRs). Recent observations from our laboratory have shown that RGS4 directly interacts with the third intracellular loop (i3L) and the C-terminal tail (CT) of l-and d-opioid receptors (l-OR and d-OR), and promotes the formation of ternary complexes between RGS4, Ga subunits and the l-opioid receptor Cterminal (l-CT) [1] . Based on these observations, we sought to determine i) a consensus motif within l-CT and d-CT which is responsible for RGS4 binding and ii) define the role of the N-terminal domain of RGS4 for opioid receptor interaction and signalling regulation. In vitro pulldown experiments using recombinant proteins encompassing truncated versions of l-CT and d-CT have shown for the first time that the juxtamembrane domain, forming helix VIII, of l-CT and d-CT is responsible for RGS4 and RGS4-Ga complex interaction. Consequent co-immunoprecipitation studies have shown that RGS4 confers selectivity to ORs for binding to activated Gai subunits. Introduction: Excitotoxicity and programmed cell death caused by hyperactivation of NMDA receptor are associated with stroke, brain trauma and play essential role in neuronal loss. It becomes evident that cell death realizes through distinctive and mutually exclusive signal transduction pathways in the certain areas of stroke. In present study we assessed a signaling role of NMDA receptor and main sources of energy in cell (oxygen and glucose) in programmed cell death mechanism, and an ability of glycine to prevent such neuronal degeneration. We were especially interested in possible differential effects of glycine under certain conditions. Results: Brain cortex slices were exposed to anoxia in the presents of 5 mM glucose. DNA internucleosomal fragmentation, an apoptosis hallmark, was detected after 24 hours of incubation. However caspase-3 activation and cytochrome release was not observed. DNA internucleosomal fragmentation was prevented by 5 mM glycine, an inhibitory neurotransmitter, and 10 lM of MK-801 (non-competitive antagonist of NMDA receptors). These effects revealed that cell death was mediated via activation of NMDA receptor. Contrariwise, 30 minutes of anoxia followed by 24 hours of reoxygenation caused DNA internucleosomal fragmentation accompanied by cytochrome release and caspase-3 activation. At the same time cell death was prevented neither by MK-801 nor by glycine. Necrosis has been observed in the absence of oxygen and glucose. Conclusion: We conclude that concentrations of oxygen and glucose are the crucial factors in determination of cell death pathway. Glycine is a specific inhibitor of apoptosis induced by NMDA mediated excitotoxicity.

The use of chimeric G proteins for signal dissection and high-throughput screening of G protein-coupled receptors Although only 16 genes have been identified in mammals, several Galpha-subunits can be activated simultaneously by G protein-coupled receptors (GPCRs) to modulate their complicated functions. Because the current GPCR assays, limited to the evaluation of selective Galpha activation, are not allowed the comprehensive pathway prediction and high-throughput screening for any given GPCR, two novel approaches were proposed below. It is known that adenylyl cyclases are directly activated by G(s)alpha and the carboxyl termini of the various Galpha proteins determine their receptor coupling specificities. To reflect the previous uncharacterized pathways of GPCRs, a set of chimeric G(s)alpha where the C-terminal five amino acids were replaced by those of other Galpha proteins was constructed. Two neuromedin U receptors, NMUR1 and NMUR2, were tested using chimeric G(s)alpha for the differentiation of their uncharacterized pathways. In contrast to the G(q) signals mainly conducted by NMUR1, NMUR2 routed preferentially to the G(i) pathways. Further, of, a chimeric reporter plasmid, incorporating cAMP response elements, serum response elements and a chimeric G(qi), was designed to cover G(s)alpha, G(q)alpha, G(12)alpha and G(i)alpha multiple pathways for the screening of ligand-receptor interaction. The constructed plasmid was able to multiply the intensity of reporter signals derived from the activation of NMUR1 or NMUR2. Further cell-line selection with our reporter construct will advance the high-throughput screening of orphan GPCRs. . NMDA receptor hypofunction can be modulated by NMDA receptor agonists and glycine transporter-1 (GlyT1) inhibitors. The aim of this study was to reveal the effects of the atypical antipsychotic risperidone and the GlyT1 inhibitor NFPS to extracellular levels of dopamine, its metabolites and amino acids (glycine, serine, glutamate) in rat striatum after intraperitoneal administration of the drugs. Methods: Extracellular biogenic amine and amino acid concentrations were determined by using microdialysis technique in rat striatum. Dopamine, its metabolites and amino acids were estimated by using HPLC/electrochemical detection. Results: Risperidone increased the synaptic concentration of dopamine in microdialysis but failed to influence the extracellular concentrations of the amino acids measured. Glycine concentration in the vicinity NMDA receptors is regulated by GlyT1. Its inhibition by NFPS led to increased glycine level in the extracellular space but the concentrations of other amino acids were not altered.

Conclusions: Effect of co-administration of risperidone and NFPS on brain extracellular dopamine and glycine concentrations was determined and we conclude that the GlyT1 inhibitor NFPS could be clinically beneficial for patients receiving risperidone treatment.

Gender-dependent response of liver mitochondria to hyperthermic treatment Hyperthermia is applied for cancer treatment although the underlying molecular mechanisms are not properly understood. Mitochondria are important transducers of signal leading to hyperthermic cell injury. We show that the response of liver mitochondria (LM) to hyperthermia is gender-dependent. Fever temperature activated state 3 respiration (V3) in LM from the female rat but strongly (by 26 percents at 41°C) inhibited V3 in male LM. Female LM V3 at 44°C was not different from that at 37°C; inhibition (by 14 percents) was induced only by 44°C (then V3 in male LM was inhibited by 39 percents). However, hyperthermia induced uncoupling was much stronger in female LM. The respiratory chain in male LM is more sensitive to hyperthermia than in female LM, but temperature induced increase in membrane permeability to ions is much larger in female LM. The difference is very obviously pronounced in the supra-physiological range of temperature (42-46oC) commonly used for hyperthermic cancer treatment. Our data imply that gender specific differences and the impact of temperature induced changes in the membrane structure are yet underestimated in the understanding of signaling networks that determine cellular elimination or survival under hyperthermic stress. Microglial cells are the prime effectors in immune and inflammatory responses of the CNS. During pathological conditions, the activation of these cells helps restore CNS homeostasis. However, chronic microglial activation endangers neuronal survival through the release of various proinflammatory molecules and neurotoxins. Thus, negative regulators of microglial activation have been considered as potential therapeutic candidates to target neurodegeneration, such as that in Alzheimer's and Parkinson's diseases. Shikonin, a naphthoquinone pigment from the root of Lithospermum erythrorhizon has been reported to have antibacterial, antitumor, and anti-inflammatory effects. The aim of this study was to examine whether shikonin represses microglial activation. In a study of shikonin and five of its derivatives, isobutyrylshikonin (IBS) and isovalerylshikonin (IVS) were the most effective at inhibiting LPS-induced NO release from microglial cells. RT/real-time PCR analysis revealed that pretreatment of rat brain microglia with IBS and IVS attenuated the LPS-induced expression of mRNAs encoding iNOS, TNF-a, IL-1b, and COX-2. In rat brain microglia, IBS and IVS reduced the LPS-stimulated production of TNF-a and PGE 2 . In addition, IBS and IVS significantly decreased LPS-induced IjB-a phosphorylation and NF-jB DNA binding activity, as well as the phosphorylation of the ERK1/2 and Akt signaling proteins. In organotypic hippocampal slice cultures, PI staining revealed prominent cell death in the hippocampal layer after 72 hours of LPS treatment. Both IBS and IVS clearly blocked the effect of LPS on hippocampal cell death and NO production. These results suggest that IBS and IVS provide neuroprotection by suppressing the proinflammatory pathways from activated microglia.

Farnesylation and nitrosylation change the intrinsic GTP-ase activity of p21Ras Ras proteins perform functional roles in a large number of biological processes leading to changes in cell morphology, gene expression, survival and apoptosis. Ras is regulated by a series of post-translational modifications, including farnesylation, and nitrosylation, but the role of these modifications on the activity of Ras is not fully understood.

Using several chromatographic steps, soluble and membrane-bound Ras protein from bovine brain were purified. To investigate the effects of nitrosylation and farnesylation of Ras-protein the purified preparations were incubated with either S-nitroso-cysteine or farnesyltransferase and farnesyl diphosphate. We have found that in the presence of S-nitroso-cysteine GTPase the activity of soluble Ras was increased, while the farnesylation cause a significant inhibition of GTPase activity. In contrast, the inhibition of GTPase activity was observed after incubation of S-nitroso-cysteine with membrane-derived Ras. These results suggest that different cysteine residues were S-nitrosylated in the membrane-derived and soluble Ras-proteins and these modifications can regulate GTPase activities. The study aims to identify novel immune disturbances associated to heroin consumption and further methadone substitution therapy.

Methods: The immune status of 21 heroin addicts was evaluated as leukocyte counts, lymphocyte subsets, the soluble form of the IL-2 receptor (sCD25), the pro-and anti-inflammatory cytokine pattern and the respiratory burst of granulocytes. Patients were investigated before methadone therapy and 1-6 months thereafter.

Results: Heroin addicts presented high monocyte counts, low proportion of NK cells, partially balanced by B lymphocytes, high values of sCD25, low levels of cytokines, excepting for the Th2 shift towards IL-10, and low response of granulocytes to protein kinase C (PKC) activation. The switch from heroin to methadone consumption (analysed after the first month of methadone therapy) induced an abnormal raise of peripheral lymphocytes, a normalization trend of NK cells, of IL-10 and of the granulocytes response to PKC activation. Meanwhile, sCD25 values remained elevated and the granulocytes respiratory burst induced by E. coli was increased. After 6 months of methadone therapy the immune parameters generally returned to the status found before therapy, though some positive evolution was shown regarding the normalization of mononuclear cells counts and the intense oxygen-dependent microbicidal activity against E. coli.

The interference of miu-opiates (heroin methadone) with the innate immune response, namely NK cells and the respiratory burst of granulocytes, was emphasized. We highlight a novel immunosuppressive mechanism of miu-opiates, related to elevated levels of sCD25, that may hinder the signals delivered by IL-2 to immune cells. The topoisomerase II alpha (TOP2A) protein is the target of the anthracycline class of chemotherapeutic agents, TOP2A can be frequently coamplified with c-erb-B2. The aim of this study was to correlate the test results of IHC and FISH for Her-2 gene amplification in breast cancer patients with TOP2A gene status detected by FISH. Materials and Methods: Assessment of HER-2 gene amplification and TOP2A gene amplification/deletion was made by FISH analysis using the LSI Top2A spectrum orange/HER-2/CEP 17 spectrum green (VYSIS) in formalin-fixed and paraffin-embedded tissue sections of 54 breast cancer patients who were grouped into stages 1+, 2+ or 3+ based on IHC (HercepTest-Dako) observations. Results: None of IHC 1+ breast tumors was HER-2 FISH positive, but three of 18 (17%) IHC 3+ tumors were HER-2 FISH negative. Overall, 53% of the IHC 2+ and 83% of the IHC 3+ cases were HER-2 FISH positive. Only one case with IHC 3+ tumor that was HER-2 FISH positive was found to have TOP2A amplification (>2.0) and no IHC 2+ cases were found to have TOP2A amplification. There were no cases with TOP2A deletion (<0.8) in our series. There were also no cases of HER-2 FISH negative tumors, but IHC scored as 2+ or 3+ (0 of 10), to be found with TOP2A amplification.

Conclusion: This study suggests the low amplification of TOP2A and this can't be considered a prognostic and/or predictive factor for breast cancer patients, when anthracycline-based chemotherapy is a consideration. Introduction: Heart failure (HF) is a composite syndrome, in which inflammation participates. PAF, a potent inflammatory mediator is synthesized via the remodeling and de novo route, key enzymes of which are lyso-PAF acetyltransferase (lyso-PAF-AT) and DTT-insensitive cholinephosphotransferase (PAF-CPT), respectively. PAF-acetylhydrolase (PAF-AH) and lipoprotein-associated phospholipase-A2 (Lp-PLA 2 ) catabolize PAF. Methods: Twelve male newly diagnosed HF patients (63 ± 10.2 years, 83% ischemic) were hospitalized and followed up for 6 weeks. PAF was determined with bioassay. Leukocyte lyso-PAF-AT and PAF-CPT were assayed by the produced PAF. Leukocyte PAF-AH and serum Lp-PLA 2 were determined by the trichloroacetic acid method. Immunonephelometry and ELISA were used for CRP and IL-6 measurements, respectively. For statistical analysis Spearman and Wilcoxon tests were used. Results: Baseline median activities of lyso-PAF-AT, PAF-CPT, PAF-AH and Lp-PLA 2 were 4.1, 68.42, 644.44 pmol/min/mg protein and 51.42 pmol/min/ll serum, correspondingly. Baseline lyso-PAF-AT and PAF-CPT were positively correlated with CRP, IL-6 and with each other. In the follow-up, PAF levels and the activity of biosynthetic enzymes increased, leukocyte PAF-AH remained stable while Lp-PLA 2 decreased. Conclusions: A correlation of PAF-CPT to inflammatory indices was firstly documented. The increase in PAF levels and its biosynthetic enzymes in the follow-up possibly reflect the effects of HF drugs.

Effects of HGS on metastasis ability of mouse melanoma B16 cells K. Ogura, I. Kawashima, M. Sato and M. Shimizu Rinshoken, Tokyo, JAPAN Introduction: GalCer expression factor-1 (GEF-1) is a rat orthologue of the mouse Hgs which is vesicular transport related protein. We took notice of the function as a signal transfer molecule of GEF-1 and reported that GEF-1 induces epithelial-mesenchymal transition (EMT). EMT is a fundamental process in the early stages of embryo morphogenesis induced with signals, such as Wnt, and is also involved in cancer metastasis. In this study, we examined whether Hgs affects on metastasis of the B16 melanoma cells. Methods: B16 cells were transfected with an Hgs overexpression vector (B16/Hgs) or Hgs RNA interference vectors (B16/shRNA). The B16 mutant cells were selected and cloned. The migratory ability and metastasis ability of the B16 mutant cells were examined. Results: B16/Hgs cells expressed Hgs higher than the parental B16 cells, and B16/shRNA cells expressed Hgs lower. Difference of cell-proliferation ability was hardly observed between B16/Hgs cells and B16/ shRNA cells. In the migratory ability and metastasis ability in vitro and in the experimental metastasis examination in vivo, B16/Hgs cells showed higher activities than B16 cells and B16/shRNA cells showed lower activities. Conclusions: The metastasis ability of B16 cell was proportional to the amount of Hgs. Since change of the amount of Hgs expressed affected the metastasis ability of B16 cells, it was suggested that Hgs has the important role for a cancer cell metastasis process, and a possibility of becoming an important target molecule to cancer metastasis.

Effect of oleic acid on lipopolyscharide-induced cyclooxygenase-2 and prostaglandin E2 production in microglial cells

Department of Biochemistry and Molecular Biology, School of Medicine, Kyung Hee University, Seoul, KOREA Introduction: Microglial cells are the major cell type involved in neuroinflammation, releasing several inflammatory mediators, resulting in neurodegenerative disorders. Oleic acid (C18:1), a monounsaturated fatty acid, is known to have both pro-and anti-inflammatory effects in different cell types including macrophages and endothelial cells. Methods: To examine the effects of oleic acid on LPS-induced proinflammatory mediators, primary cultures of rat microglia and murine microglia cell line BV-2 were used. LPS-induced COX-2 expression and prostaglandin E 2 (PGE 2 ) production were analyzed by RT-PCR, Western blotting, and ELISA. MAPKs (ERK, p38, JNK), Akt, and NF-kappaB activation were analyzed by Western blotting and luciferase reporter assay. Results: Oleic acid (OA) inhibited LPS-induced COX-2 mRNA and protein expressions in a dose dependent manner. We found that OA inhibited LPS-induced phosphorylation of Akt and p70S6 kinase, whereas it had no effect on the phosphorylation of ERK1/2 and p38. Finally, we showed that OA inhibited phosphorylation of inhibitor kapaaB kinase (IKK) and activation of NF-kappaB in LPS-stimulated microglial cells. Conclusions: Our results show that OA inhibits COX-2 expression and PGE2 production in microglial cells by inhibiting phosphorylation of Akt and p70S6 kinase and activation of NF-kappaB and suggest that it deserves further study on the therapeutic benefits for suppression of inflammatory-related neuronal injury in neurodegenerative disorders.

PP7C-57 Sensitivity to TRAIL induced apoptosis in vivo via DR5 receptor up-regulation and the role of oncogenes Introduction: The incidence of colorectal cancer (CRC) is increasing while poor diagnoses followed by limited treatment alternatives lead to low survival rate. The need for new adjuvant therapies has been implemented by the recent discovery of TRAIL, an anti-apoptotic member of the TNF family. TRAIL is destined to become an anti-tumoural agent due to its selectivity towards cancer cells. Towards this end, oncogenic mutations accumulating in a step wise manner during CRC carcinogenesis have been described to play a key role in inducing sensitivity to apoptosis. Methods: Cell lines and freshly isolated tumour cells at different stages of CRC were subjected to TRAIL treatment. Induced cell death was analyzed for markers of apoptosis. Observed apoptosis was correlated to genetic background and further investigated in transformed cells over-expressing either KRAS-G12V or BRAFV-600E. Apoptotic cell death was investigated in vivo by intravenous administration of TRAIL in SCID mice.

Results: TRAIL caused apoptosis both in vitro and in vivo [1] , and sensitivity was correlated to DR up-regulation. Cells moderately sensitive to TRAIL seemed to be sensitized in the presence of KRAS G12V via DR up-regulation [2] . RT-PCR analysis performed in 40 CRC biopsies and their respective normal mucosa presented DR5 as the most frequently unregulated receptor while, in the presence of oncogene mutations, DR expression tendency already existed in the tumour samples was amplified.

Conclusions: Sensitization to TRAIL induced apoptosis may result via DR up-regulation suggesting that TRAIL based therapeutic strategies could be used in cases of human CRC bearing oncogenic mutations. Results: Western-blot analysis revealed that the susceptibility of human COLO 205 cells to apoptogenic stimuli resulted from timedependent reduction in cFLIP L and TRADD protein levels. At the same time, the level of FADD protein was up-regulated. Additionally, the combined TNF-a and Bis-IX treatment caused cleavages of Bid and procaspase-9, as well as cytochrome c release.

Conclusions: The evidence of this study indicates that Bis-IX facilitates the death receptor signal mediated by TNF-R1. Moreover, Bis-IX alone initiated intrinsic apoptosis, which could be abolished by Bcl-2 delivery. It heralds the involvement of mitochondria in caspase-8-independent intrinsic apoptosis. In turn, the treatment with bisindolylmaleimide III (Bis III) did not affect TNF-a-dependent apoptosis.

Introduction: Soluble guanylyl cyclase (sGC) is a receptor for the signaling molecule nitric oxide (NO). Binding of NO to the sGC heme moiety leads to increased cGMP synthesis and activation of cGMPdependent protein kinase (PKG). As sGC subunits contain putative phosphorylation sites for PKG, we tested the hypothesis that sGC activity is regulated by PKG.

Results: In vitro kinase assays revealed that the alpha1 subunit of sGC is a PKG substrate and that the phosphorylation site is located within the first 360 residues. In vivo, a constitutively active form of PKG stimulated incorporation of 32 P into the alpha1 subunit. Serine to alanine mutation of putative PKG sites revealed that Ser64 is the main phosphorylation site for PKG. Using a phospho-specific antibody we could demonstrate that phosphorylation of endogenous sGC on Ser 64 increases in cells and tissues exposed to NO. Wild-type (wt) sGC coexpressed with a constitutively active form of PKG exhibited lower basal and NO-stimulated cGMP accumulation, while the S64A alpha1/ beta1 sGC was resistant to the PKG-induced reduction in activity.

Using purified sGC and mutants, we observed that the S64D alpha1 phosphomimetic/beta1 dimer exhibited lower Vmax; moreover, the decrease in Km after NO stimulation was less pronounced in S64D alpha1/beta1 compared to wt sGC. Expression of a phosphorylation deficient sGC showed reduced desensitization to acute NO exposure and allowed for greater VASP phosphorylation.

Conclusions: We conclude that PKG phosphorylates sGC on Ser64 of the alpha1 subunit and phosphorylation inhibits sGC activity, establishing a negative feedback loop.

Development of a functional genomic strategy for the identification of novel cardioprotective genes S. Papathanasiou, P. Panagopoulou, S. Psarras and Y. Capetanaki Biomedical Research Foundation, Academy of Athens, Athens, GREECE

Cardiomyocyte cell death is a major cause of myocardial degeneration and heart failure. A functional genomic strategy has been developed aiming to reveal new genes with protective action against cardiomyocyte death. An adenoviral library expressing a wide range of cDNAs is used for the infection of neonatal rat cardiomyocyte cultures. After cell death induction, microarray analysis of the survived cardiomyocyte subpopulations will identify the responsible for the survival genes. An already constructed normalized plasmid library containing cDNAs synthesized from mRNAs of mouse adult and neonatal hearts, as well as of whole mouse embryo is converted to an adenoviral library following an in vitro recombination system. A goal of the present work is to achieve the optimal functional genomic system by constructing a library with more than 100 times higher transduction activity than the available ones. This will allow the identification of novel cardioprotective genes expressed at very low levels. Additionally, standardization of a suitable cell death induction methodology has established the conditions required for an approximate 80% cardiomyocyte death caused by different stimuli. Finally, a recombinant adenovirus carrying the Insulin-like growth factor 1 gene, known for its beneficial activity in the myocardium has been constructed and used as a positive control and also for further optimization of the cell death conditions. Acknowledgement: Supported by PEP-ATT-49 grant from the Greek Secretariat of Research and Development. tagged PKC I proteins were highly localized in plasma membrane within 5 minutes. According to western blot analysis, the same stimuli also induced ERK phosphrylation. Although, when GFP-PKC I was cotransfected with red fluorescent protein (RFP) tagged-cNELL2, GFP-PKC I did not translocated into the plasma membrane under the same stimuli and ERK phosphorylation also did not detected. Therefore, these results suggested that cNELL2 possibly acts as a negative modulator of PKC I.

Auranofin promotes all-trans retinoic acid-or 1alpha, 25-dihydroxy vitamin D3-mediated cell differentiation of acute promyelocytic leukemia by increasing histone acetylation 

Angiogenesis is an important mediator of tumor progression. As tumors expand, diffusion distances from the existing vascular supply increases resulting in hypoxia. Sustained expansion of a tumor mass requires new blood vessel formation to provide rapidly proliferating tumor cells with an adequate supply of oxygen and nutrients. The key regulator of hypoxia-induced angiogenesis is the transcription factor hypoxia inducible factor (HIF)-1. HIF-1a is stabilized by hypoxiainduced reactive oxygen species (ROS) and enhances several hypoxic gene expressions including angiogenic activator, vascular endothelial cell growth factor (VEGF). In this study, we found that melatonin, a small lipophile molecule that is essentially secreted by the pineal gland, Introduction: Although neuroanatomical left-right (LR) asymmetries are known to mediate important behavioural and cognitive lateralisation, little is known concerning the molecular mechanisms that establish brain asymmetry. We are using the zebrafish as a genetic model system to understand the molecular mechanisms underlying the development of brain asymmetry. The zebrafish epithalamus consist of a left-side parapineal organ and paired habenular nuclei which display asymmetries in neuronal projections and gene expression. 

The effect of chronic NOS inhibition on regulatory proteins in rat hearts Introduction: To better understand the precise molecular mechanism(s) by which genistein exerts its effects on PC3 prostate cancer cells, we utilized a cDNA microarray to interrogate the mRNA levels of 4486 genes and to determine the gene expression profiles of PC3 (androgen independent AR-) prostate cancer cells treated with genistein.

Methods: cDNA microarrays was developed to investigate the relative expression of the corresponding mRNA in PC3 treated with therapeutically doses of genistein compared with untreated PC-3 prostate cancer cells. For generating fluorescently labeled cDNA probes to be used in microarray screening we used the SuperScriptä Indirect cDNA Labeling System (Invitrogenä). The image analysis was performed using a GenePix Pro 41121 and ImaGene. Data analysis was performed by GeneSight and SAM. We involved in silico prediction of wildtype function and variant promoter sequences through assessment of hormone receptor elements and transcription factor binding sites and prediction of likely splice variants through genomic, splicing and EST databases, and multiple sequence alignment packages.

Results: Through differentially gene expression analysis we identified more than one hundred genes that were up-regulated in the PC-3. Investigation of these genes could help us to disclose the molecular mechanism(s) underlying the death of prostate cancer cells treated with genistein. In this study we proposed a novel bioinformatic approach to predict the regulatory gene networks based artificial neural network.

Conclusions: In our studies Genistein suppress prostate cancer cells proliferation related with the induction of cell cycle arrest in phase G 1 and G 2 and/or apoptosis, Akt signaling pathways.

Overexpression of P-glycoprotein in L1210 cells is associated with alternation in cell surface glycosides Methods: The effect of TNF-a on cell proliferation rates was determined using MTT assay. The invasiveness of RMCCA-1 was assessed by Transwell assay, while the MMP-9 activities secreted by these cells were investigated using Gelatin Zymography. The involvement of MAPK signaling was demonstrated by using U0126, a specific inhibitor of MEK1/2, coupled with the Transwell assay and Western Blot.

Results: Treatment with 20-160 ng/ml of TNF-a ?resulted in a significant increase of in vitro invasiveness and MMP-9 activities of RMCCA-1 cells, while cell proliferation rates were minimally affected. Treatment with U0126 severely suppressed the TNF-a-stimulated effects, concomitantly with a reduction of the phospho-ERK1/2 levels.

The in vitro invasiveness and MMP-9 activities secreted by RMCCA-1 cells were stimulated by TNF-a. These stimulations were not due to increased cell proliferation. TNF-a-mediated effects were suppressed by MEK1/2 inhibitor, suggesting that these stimulations were, in part, due to activation of the phospho-ERK1/2 activity. Methods: By focusing on endogenously expressed molecules and using a wide combination of molecular, biochemical and microscopical methods we studied the localization and binding profile of intracellular sCLU.

Results: We report that sCLU depletion in human cancer cells signals stress that induces p53-dependent growth retardation and high rates of endogenous apoptosis. We discovered that increased apoptosis in sCLU-depleted cells, correlates to altered ratios of pro-to anti-apoptotic Bcl-2 protein family members, it is amplified by p53 and it is executed by mitochondrial dysfunction. Stress signals originate from several sites, since sCLU is an integral component, of not only the secretory pathway, but also the nucleo-cytosolic continuum and mitochondria. In the cytoplasm, sCLU-depletion disrupts the Ku70-Bax protein complex and triggers Bax activation and relocation to mitochondria. We demonstrate that sCLU binds, and thereby stabilizes, the Ku70-Bax complex serving as a cytosol retention factor for Bax.

Our results indicate that intracellular sCLU functions in the membranous secretory pathway, the nucleo-cytosolic continuum and mitochondria having a vital role in the maintenance of cellular homeostasis and proteome stability of human cells. Thus, elevated sCLU levels may enhance tumorigenesis by interfering with Bax pro-apoptotic activities and contribute to one of the major characteristics of cancer cells, namely resistance to apoptosis. 

Platelet-activating factor bio-system in human arteries Objective: Platelet-activating factor (PAF) bio-signaling is involved in the pro-inflammatory reactions of atherosclerosis. Methods: We measured several parameters of PAF bio-system in human atherosclerotic aorta and healthy mammary arteries.

Results: Immunohistochemical analysis showed PAF-acetylhydrolase and PAF receptor expression in the intima and the media of the aorta and in the media of mammary arteries. Acetylhydrolase and transacetylase activities were (mean ± SE n = 38): acetylhydrolase of aorta: 2.8 ± 0.5 pmol/min/mg of tissue; transacetylase of aorta: 3.3 ± 0.7 pmol/min/mg of tissue; acetylhydrolase of mammary artery: 1.4 ± 0.3* pmol/min/mg of tissue; transacetylase of mammary arteries 0.8 ± 0.2** pmol/min/mg of tissue. (*p < 0.004 as compared with acetylhydrolase of aorta; ** p < 0.03 as compared with acetylhydrolase of mammary artery). Lyso-PAF and PAF-bioactivity accumulation was observed in the aorta of some patients. RP-HPLC and ESI-MS analysis revealed that 1-O-hexadecyl-2 acetyl-sn glycero-3-phosphocholine accounted for 60% of the PAF-bioactivity and 1-O-hexadecyl-2boutanoyl-sn-glycerol-3 phosphocholine for 40% of the PAF-bioactivity. Conclusions: Mammary arteries show that PAF formation, regulated by PAF-AH, may lead to a regulated inflammatory reaction. In diseased aorta, the accumulation of lyso-PAF and PAF-AH transacetylase activity may lead to unregulated, pro-atherogenic, chronic inflammatory reaction. Introduction: PAF, a potent inflammatory mediator, seems to play a role in the pathogenesis of several AIDS manifestations. In addition, PAFantagonists have been studied in this context with promising results. In order to examine the possible interactions between PAF and antiretroviral therapy, we studied the effect of anti-HIV drugs against PAF activity and its metabolic enzymes.

The inhibitory effect of drugs against PAF-induced aggregation on washed rabbit platelets as well as against PAF-CPT, Lyso-PAF-AT and PAF-AH specific activities of human mesangial cells (HMC) was tested. These enzymes were also assessed in AIDS-patients treated with HAART regimens.

Results: Several drugs and their combinations (in backbones and HAART regimens) inhibited PAF activity with TDF being the most potent one (IC 50 = 0.54±0.16 lg). Inhibition up to 80% of PAF-CPT and Lyso-PAF-AT activities was also achieved when HMC were preincubated with TDF and Lopinavir/r. TDF also induced PAF-AH of these cells. Similar effects were observed in AIDS-patients treated with HAART regimens.

Conclusions: The reported results may offer a new insight in the mechanism of HAART beneficial effect on HIV-patients and could optimize its use.

In vitro functional screening on human neutrophils for development and characterization of potential anti-inflammatory compounds The therapy of inflammatory diseases is far from being solved. The process of inflammation is mostly mediated by phagocytic cells like neutrophils, where the relevant intracellular pathways are critically regulated by kinases. A novel approach, screening of a kinase inhibitor library in primary human phagocytic cell-based assay systems, was used for the identification of molecules with anti-inflammatory properties. In the course of screening, we tested a part of molecular library of Vichem Ltd. on freshly prepared human neutrophil granulocytes using an optimized functional assay. In the primary biological assay, we applied the kinase inhibitors in 10 lM concentration for neutrophil experiments. Then, we investigated the pretreated cells in three different stimulation induced systems: (i) cytokine activation on integrinligand surface. Here, we activated neutrophils placed on the fibrinogen integrin-ligand surface by the inflammation cytokine TNF. This type of cell activation was similar to that which could be observed during inflammation. (ii) Cell activation via immune complexes: this was a model system of the inflammation process during autoantibodyinduced, immune complex-mediated diseases. (iii) Activation by phorbol-ester: This non-physiological activation was to exclude nonspecific effects, focusing on the previous two physiological activation types. Our experiments with the above system indicate that screening of a kinase inhibitor library on primary human phagocytic cells provides a number of potential lead molecules that inhibit inflammation-related but not nonspecific activation of neutrophils. Further detailed characterization of these molecules and their structural modification to optimize their pharmacological characteristics will likely lead to potential drug candidates of inflammatory diseases.

The sporulation of the chytridiomycete Blastocladiella emersonii is dependent on a nitric oxide sensitive-guanylyl cyclase A. Vieira, E. Linares, O. Augusto and S. Gomes Departamento de Bioquıḿica, Instituto de Quıḿica, Universidade de Sa˜o Paulo, Sa˜o Paulo, BRAZIL Introduction: Blastocladiella emersonii is an aquatic fungus of the Chytridiomycete class, which is at the base of the fungal phylogenetic tree. During its developmental stages (germination and sporulation) the cell undergoes a large number of morphological changes, which require the synthesis of many new materials. The present work reports nitric oxide ( NO) production as well as its participation in cGMP signal transduction pathways during the sporulation phase of the fungus. Methods: The fungus was cultivated at 27°C in defined medium. In order to obtain confocal microscopy images, fungal cells were treated with 10 lM of DAF-DA, a nitric oxide marker, for 30 minutes at room temperature before induction of sporulation. Results: The sporulation phase was delayed by addition of L-NAME, suggesting NO production by an enzymatic mechanism from L-arginine. In adition, confocal microscopy experiments using 10 mM L-NAME and 10 mM L-arginine showed a remarkable decrease and increase in NO production, respectively. The intracellular concentrations of nitrite, nitrate and other nitric oxide-derived products were measured using a nitric oxide analyzer. The reagent 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of nitric oxide sensitive-guanylyl cyclase, inhibited sporulation completely when added up to 90 minutes after starvation, suggesting that NO acts as a first messenger in cGMP production. In agreement with these results, microarray experiments showed an increase of guanylyl cyclase mRNA levels after the first hour of starvation. Conclusions: Our results show evidences for the existence of the NO-cGMP pathway during sporulation of the fungus B. emersonii. Cells slow down cell cycle progression in order to adapt to unfavorable stress conditions. Yeast respond to osmotic stress by triggering a G1 and G2 checkpoint delay that is dependent on the MAP kinase Hog1. The high osmolarity glycerol (HOG) pathway is also activated by anticancer drug arsenite and the hog1 mutant is highly sensitive to arsenite, partly due to increased arsenite influx into hog1 cells. Yeast cell cycle regulation in response to arsenite and the role of Hog1 in this process have not yet been analyzed. We found that long-term exposure to arsenite led to transient G1 and G2 delays in wild-type cells whereas cells that lack the HOG1 gene or are defective in Hog1 kinase activity displayed persistent G1 cell cycle arrest. In contrast, hog1 cells exhibited wild-type G2 arrest during arsenite stress. These data demonstrate that arsenite sensitivity of hog1 is not only caused by enhanced accumulation of arsenite but also by inappropriate cell cycle arrest in G1.

In the presence of arsenite, many G1-arrested hog1 cells showed mating projections suggesting cross talk between the HOG and pheromone response pathways. Indeed, phosphorylation of the MAPK Fus3 was stimulated in arsenite-exposed hog1 cells. However, suppressing this cross talk did not abrogate persistent G1 arrest of hog1. Together, our data point to an important role of Hog1 kinase in recovery from stress-induced G1 cell cycle arrest and provide evidence that it is possible to achieve permanent cell cycle arrest by combining drug action with manipulations of signaling pathways. The Wnts comprise a large class of secreted proteins that control essential developmental processes such as embryonic patterning, cell growth, migration, and differentiation. Wnt-5a is a representative ligand that activates b-catenin independent noncanonical pathway in Wnt signaling. Although abnormal activation of b-catenin-dependent pathway is often observed in human cancer, the relationship between b-cateninindependent pathway and tumorigenesis is not clear. In addition MMP-7 is involved in canonical Wnt pathway regulated by nuclear b-catenin. In this study, we investigated the expression of Wnt5a Introduction: Beta-arrestins (beta-Arrs) are known to be associated with transforming growth factor-beta1 (TGF-beta1), P53/murine double minute (MDM2) and NF-kappaB tumor signaling pathways in cells. However, less is reported about beta-Arr in tumor progression in vivo.

Methods: Beta-Arr transgenic mice were generated, then subcutaneously inoculated tumor cells, and dynamically observed the tumor initiation time and tumor volume. Matrix metalloproteinase 9 (MMP9) activity, vascular endothelial growth factor (VEGF) concentration in plasma and small blood vessel formation in tumor tissues were measured. MMP9 inhibitors were also injected in the mice. And the human microvascular endothelial cells (HMEC-1) were then applied as the cell model for further studying the mechanism. Results: The xenograft tumor initiated earlier and grew more rapidly in beta-Arr1 transgenic mice than in both the beta-Arr2 transgenic and wild-type mice after inoculating murine liver cancer Hepa1-6 cells or lymphoma EL4 cells. Moreover, plasma MMP9 activity, VEGF concentration and small blood vessel formation in tumor tissues were enhanced in beta-Arr1 transgenic mice compared with those in control mice. In addition, injection of MMP9 inhibitors in beta-Arr1 transgenic mice abrogated all these effects and suppressed rapid tumor progression. Similar results were observed in HMEC-1 cells. Furthermore, phosphatidylinositol 3-kinase (PI3K) inhibitors could suppress beta-Arr1-enhanced MMP9 activity and the C-terminal 181-418 amino acids of beta-Arr1 was largely responsible for this effect. Conclusions: Our data reveal a functional role for beta-arrestin1 in tumor progression in vivo, in which overexpression of beta-Arr1 promotes MMP9 activity and tumor angiogenesis by providing a suitable microenvironment for tumor progression. Academia Sinica, Taipei, TAIWAN 2 National Taiwan Ocean University, Keelung, TAIWAN Introduction: The spermatozoa active fertilizing competence when the sperm cells presents in the female tract and subsequent capacitation. Several studies suggested that capacitation is associated with phosphorylation of membrane protein. However, the quantitation and qualification of membrane protein phosphorylation during capacitation is still less understood. In this study, a more universal and efficient strategy for the study of membrane phosphoproteome are applied for the study of sperm capacitation. Several new phosphoration sites correlated with sperm capacitation were identified and discussed in this study.

The cell was first subjected to the two phase partition to enrich the membrane proteins. The extracted membrane proteins were further subject to the tryptic digestion and the phosphopeptides were selectively purified using Fe-IMAC. The purified phosphopeptide from capacitated and non-capacitated sperm cell were analysis by LC-ESI-MS and followed by the MS/MS database search and peptide quantitation.

Results: After the purification of membrane protein, about 250 proteins were identified and about 80% of the identified proteins were determined to be membrane associated. After the Fe-IMAC purification, about 131 and 96 phosphorylation sites for the capacitated and noncapaciated were identified and quantified. Using HK1 as an internal control, about 18 phosphorylation sites were identified to be increased in phosphorylation level during capacitation.

Conclusion: This approach provides a site specific quantitation result for the protein phosphorylation during sperm cell capacitation. The results will help to improve our understanding of the protein phosphorylation that associates with hyperactivited motility and redistribution of the membrane component during capacitation. We have confirmed that cytokeratin-1, but not vimentin, is present on the plasma membrane of neuroblastoma NMB7 cells as both a RACK1, Receptor for Activated C Kinase, and integrin a V b 3 -associated protein Methods: In our studies, cytokeratin-1 bound specifically to integrin a V b 3 in the presence of RACK1 and Taxol blocks cytokeratin-1 binding. Using a b-tubulin peptide specific for Taxol (PEP Taxol ) and RACK1 binding, we constructued an affinity column HiTrap-PEP Taxol . When HiTrap-PEP Taxol was loaded with warm 2% Triton X-100extracted membrane preparation of NMB7 cells, the cytokeratin-1, integrin a V b 3 and RACK1 were bound in the column and were eluted with 0.5 M NaCl. The identity of cytokeratin-1 was confirmed by mass specrophotometry. By using fluorescence microscopy, RACK1 was evidenced to support cytokeratin-1 binding to integrin? a V b 3 . Of interests, the cytokerain-1/ RACK1/integrin a V b 3 was found to be actively reactive with actin as well. Results: Cytokeratin-1 was isolated as a partner of RACK1 from the plasma membrane of neuroblastoma NMB7 cells. This molecular complex of cytokeratin-1/RACK1 was found as an associate with integrin a V b 3 and actins. We are interested to apply them to examine the interaction with organelles (Endoplasmic Reticulum, mitochondria, lysosome and Golgi apparatus) upon cytokine stimulation, especially during hypoxia. Introduction: Quercetin (QUE) is a natural flavonoid currently known for its antioxidant effects and its ability to attach itself to cellular membranes. Its reported effects suggest also interference in cellular signalling pathways, including effects on ionic channels (CFTR chloride channels, ryanodinic calcium receptors). It shows both neuroprotective and neurotoxic effects, in a concentration dependent manner. In our study, we researched the QUE interference on the nicotinic pathways, since no studies of its effects on nicotinic receptors have been reported so far. Materials and method: We performed whole-cell patch-clamp measurements on the TE 671 cell line (ECACC No. 89071904). We observed the effect of different concentrations of QUE (applied in the bath solution) on the peak current amplitude, the current decay phase and on recovery from desensitization as assessed by a sequential stimulation protocol with 10 lM ACh. Results: Quercetin decreases the peak current amplitude at 20 minutes of exposure, in a concentration dependent manner. The current decay phase of the whole-cell currents showed a bi-exponential kinetic, compared to the single-exponential channel closing kinetic in the control experiments. Recovery from desensitization was slower in the presence of QUE, with no differences observed for different exposure times. Conclusions: Our results suggest that QUE modulates the activity of the muscle-type nicotinic receptors by influencing both the activationinactivation kinetic of the nAChR and the nicotinic receptor desensitization. A possible mechanism suggested by our data might be the increasing, due to QUE, of the opening probability of a non-diligated state of the nicotinic receptor. One of the well-known protein modules regulating molecular interactions is pleckstrin homology (PH) domain whose best-characterized ligand is inositol phospholipid, and thus leading the recruitment their host proteins to the cellular membrane. Upon cellular stimulation, by sensing the changing of local concentration of the lipids or by intra-or intermolecular regulatory mechanism of accessibility to phospholipids, the modules then translocate to different subcellular compartment. We have investigated the subcellular localizations of the PH domains from PRIP (PLC-related, but catalytically inactive protein) with a domain organization similar to phospholipase C-d1 (PLC-d1). We previously showed that PRIP distributed throughout the cell cytoplasm but did not localized to any membranous structures, despite showing comparable binding affinity to PtdIns(4,5)P 2 with PLC-d1 whose PH domain is well known to localize to the plasma membrane by binding to PtdIns(4,5)P 2 . In the present study, we used recombinant PH domain from PRIP with or without the N-terminal extension comprising of about 100 amino acids present in PRIP but not in PLC-d1. The PH domain constructs showed different subcellular distribution when they were expressed as GFP-fused protein in MDCK cells. GFP-PRIP-PH localized to plasma membrane in resting cells similarly to GFP-PLC-d1PH while GFP-PRIP-PH with N-terminal extension did not show the plasma membrane localization. The results raised the possibility that the N-terminal extension preceding the PH domain of PRIP has inhibitory role in membrane [PI(4,5)P 2 ] binding of the PH domain.

Inactivation of the endocannabinoid 2arachidonylglycerol in rabbit platelets. Identification and characterization of monoacylglycerol lipase (MAGL) E. Gkini, D. Anagnostopoulos and A. Siafaka-Kapadai Department of Chemistry, University of Athens, Athens, GREECE Introduction: The endocannabinoid 2-arachidonylglycerol (2-AG) exerts its action through G protein-coupled cannabinoid receptors and is inactivated through transport into cells followed by enzymatic degradation. In this study we investigated the enzymatic hydrolysis of 2-AG in rabbit platelets and characterized the responsible enzyme(s). Methods: Washed rabbit platelets were homogenated and monoacylglycerol lipase (MAGL) activity was determined using [ 3 H]glycerol and the respective fatty acid in a time and protein concentration-dependent manner. In the presence of the specific FAAH inhibitors, though, hydrolysis was inhibited up to 60% in a concentration-dependent manner indicating the involvement of FAAH in 2-monoacylglycerol hydrolysis. MAGL was further characterized using [ 3 H]2-OG in the presence of URB 597 and it was found that its hydrolysis followed Michaelis-Menten kinetics. Subcellular fractionation of platelets homogenate showed that MAGL activity was present in both the cytosolic and membrane fractions. Finally, immunoblot analysis revealed the presence of both MAGL and FAAH. Conclusions: The endocannabinoid 2-AG is a substrate of both FAAH and MAGL; the latter was characterized for the first time in rabbit platelets. An apparent K m of 8.71 lM and V max of 0.76 nmol/ min*mg and an optimum pH 8 were determined. The molecular mass of MAGL and FAAH were found~47kD and~57kD respectively. Acknowledgement: This work was supported in part by the University of Athens Special Account for Research Grants (70/4/3351). Cell chemotaxis in a gradient of external stimuli is important in many biological processes. Class I PI3Kinases have key roles in the mechanism by which chemoattractants stimulate cells to move and to become preferentially activated at the leading-edge. This results in the lipid product of Class I PI3Ks, the second messenger PtdIns(3,4,5)P 3 , accumulating at the leading-edge where it signals by recruiting proteins containing PH domains to the plasma membrane. It is already clear that the dephosphorylation of PtdIns(3,4,5)P3 is an important step in this cascade. However, in neutrophils there is debate about which of the enzymes potentially responsible, PTEN or SHIP I, is implicated and whether this step is dynamically regulated. We have attempted to address this problem by analyzing the distribution of PtdIns(3,4)P2, the characteristic product of SHIP but not PTEN activity. We used a retrovirus to introduce a PtdIns(3,4)P2 reporter (TAPP1 PH domain fused to cherry-FP) in stem cells isolated from transgenic mice expressing a reporter for both PtdIns(3,4,5)P3 and PtdIns(3,4)P2 (PKB PH domain fused to GFP). These cells were used to reconstitute in vivo neutrophils expressing both reporters allowing us to define by confocal microscopy the relative distribution of PtdIns(3,4,5)P3 and PtdIns(3,4)P2 during chemotaxis. Our data show that, unlike PtdIns(3,4,5)P3, PtdIns(3,4)P2 does not localise at the leading edge of the cell. This study strongly suggests that in the context of chemotaxis both PTEN and SHIP I are active and furthermore are probably dynamically regulated to contribute to the patterning of PtdIns(3,4,5)P3 signals.

Oxidative slow-twitch skeletal muscle fibres express the ascorbic acid transporter SVCT2 during chick myogenesis M. Low, D. Sandoval, E. Aviles, F. Nualart and J. P. Henriquez Departamento de Biologıá Celular, Facultad de Ciencias Biologicas, Universidad de Concepcion, Concepcion, CHILE Introduction: The reduced form of vitamin C (ascorbic acid, AA) functions as a potent antioxidant as well as in cell differentiation. AA is uptaken by mammalian cells through specific sodium/ascorbate cotransporters (SVCT1 and 2). Although skeletal muscle contains about 50% of the whole-body vitamin C, the expression of SVCTs has not been addressed in this tissue. In this work, we have studied the expression pattern and function of SVCT2 during embryonic myogenesis using the chick as model system. Methods: The expression of SVCT2 was followed by RT-PCR, Western blot and immunohistochemistry in embryonic (HH27-45) chick limb skeletal muscles. AA transport kinetic analyses were developed in cell cultures overexpressing chick SVCT2. Results: We have cloned the chick ortholog of SVCT2 sharing a 93% identity with the mouse transporter. Functional kinetic analyses in transfected cells in vitro corroborated its identity as SVCT2. Chick SVCT2 mRNA is expressed during chick embryonic muscle development. In addition, Western blots using a specific antibody against rat SVCT2 showed an immunoreactive band of 65kDa during chick myogenesis. Interestingly, immunohistochemical analyses in chick limb cryosections showed that SVCT2 expression is preferentially located to type I slow-twitch oxidative muscle fibres. Double immunostaining using specific antibodies against the chick slow muscle markers myosin (S58) and Ca-ATPase (SERCA2) displayed a high degree of co-localization with anti-SVCT2. Conclusions: SVCT2 is preferentially expressed by slow-twitch skeletal muscle fibres. We conclude that SVCT2-mediated uptake of AA is relevant to the oxidative nature of slow-twitch skeletal muscle fibres. Introduction: Ryanodine receptors (RyR), by amplifying neuronal calcium entry signals via calcium-induced calcium release (CICR), participate in some types of synaptic plasticity and spatial memory formation. We have shown previously that reactive oxygen and nitrogen species (ROS/RNS) induce RyR-mediated CICR. Here we investigated if RyR contribute to the expression of proteins involved in synaptic plasticity and memory, such as brain-derived nerve factor (BDNF) and its receptor TrkB, PKM e, and the RyR isoforms RyR2 and RyR3. Methods: Expression of RNA and proteins was determined in primary cultures of hippocampal cells by RT PCR, Western blot assays and immunocytochemistry. RyR function was selectively inhibited by preincubating cells with 50 lM ryanodine for 60 minute. Results: Neurons incubated for 6-h with BDNF displayed increased expression of PKM e at the mRNA level, and of TrkB, RyR2 and RyR3 at the mRNA and protein level. RyR inhibition abolished these stimulatory effects, and in some cases decreased basal expression levels. Conclusions: We propose that RyR-mediated CICR generates calcium signals that stimulate the expression of genes underlying activitydependent morphological changes required for long-lasting synaptic plasticity, memory and learning. Among the 48 human ABC-transporters the multidrug-resistance (MDR) proteins (P-glycoprotein/MDR1, MRP1, and ABCG2) are peculiar in their unusually broad substrate recognition. They can transport a wide variety of structurally unrelated, mostly hydrophobic compounds. Since these nonpolar molecules are known to partition into the lipid bilayer and accumulate to high concentration in membranes, the 'classical pump' model for the transport mechanism of MDR proteins has been challenged. The proposed alternative models, i.e. the 'hydrophobic vacuum cleaner' and the 'floppase' models suggest substrate recognition within the lipid bilayer. Although this idea was supported by a number of drug-binding experiments, unambiguous evidence for the direct extrusion from the membrane has not provided as yet. In the present work, we employed the fully functional GFPtagged ABCG2 in confocal microscopy studies to determine the kinetics of drug distribution in the plasma membrane and other cellular compartments using the fluorescent anti-cancer agent, mitoxantrone. We generated kinetic models describing the transport processes for both the classical pump model and the alternative models, and compared them with our experimental observations. Our results clearly indicate that ABCG2 extrudes mitoxantrone directly from the plasma membrane. Melanocortin 4 receptor (MC4R) is a G protein-coupled receptor, which is involved in the regulation of central energy homeostasis. MC4R displays a constitutive activity, increasing basal cAMP production in the absence of ligand. Among the clinically reported mutants of MC4R, we investigated the functional characteristics of mutants where the mutations in the third intracellular (i3) loop of MC4R have been reported, such as A219V, I226T, P230L, G231S, G238D, N240S, and A244E. Using a CRE (cAMP responsive element)-mediated luciferase reporter gene assay, we observed that mutant A219V showed significantly decreased basal activity, whereas mutant P230L displayed significantly increased basal activity. When the dominant negative Gs mutant was co-expressed, the majority of the mutants showed reduced basal activity. These results suggest that the i3 loop of MC4R is essential not only for the functional activity but also for the regulation and maintenance of an optimal constitutive activity of MC4R in association with G protein coupling. Identification and characterization of proteins involved in interaction with the i3 loop of MC4R is currently undertaking to understand the molecular environment of MC4R signaling in control of energy homeostasis. Discoidin domain receptor 1 (DDR1), nonintegrin collagen receptor, is a receptor tyrosine kinase (RTK) with a unique extracellular domain homologous to discoidin 1 of Dictyostelium discoideum. DDR1 is known to play a role in cell attachment, migration, survival, and proliferation. However, little is known about the molecular mechanisms underlying the role of DDR1. A recent paper has reported that p53 induction and activation of DDR1 kinase counteract p53-mediated apoptosis and influence p53 regulation through a positive feedback loop. This study was designed to determine the role of DDR1 in nitric oxide (NO)-induced apoptosis. NO plays important roles in physiologic processes including nerve system and inflammation. NO generation initiates apoptotic cell death in different experimental system. In our study, the administration of S-nitroso-N-acetyl-DL -penicillamine (SNAP), a NO donor, decreased the cell viability and induced apoptotic cell death in J774A.1 macrophages. Interestingly, the expression of DDR1 was decreased by treatment of SNAP. On the contrary, the inhibition of expression of DDR1 by siRNA and blocking peptide increased cell viability and reduced NO-induced apoptosis. These results demonstrate that DDR1 has pro-apoptotic effect against NOinduced apoptotic cell death in macrophages by regulating the signaling molecules. We suggest that modulation of DDR1 signaling in macrophages may be useful in controlling apoptosis signal transduction.

Identification of an Atg18p/Svp1p containing complex that may regulate PI(3,5)P2 synthesis T. Kobayashi and S. Dove School of Biosciences, The University of Birmingham, Birmingham, UK Introduction: Phosphatidylinositol (3,5) bisphosphate (PI(3,5)P 2 ) is the most recently identified member of the class of signaling lipids known as the phosphoinositides (PPln). PPln are regulatory lipids that orchestrate a wide variety of cellular events. PPln are made by PPln kinases and degraded by lipid phosphatases. PPln control cellular events via binding to and activating effector proteins. PI(3,5)P 2 is implicated in the regulation of endosome-lysosome trafficking, vacuolar pH control, and stress responses. In S cerevisiae, the synthesis of PI(3,5)P 2 is carried out by the Fab1p lipid kinase, and also requires two accessory proteins, Vac7p and Vac14p. Degradation of this signal is mediated by the Fig4p lipid phosphatase, but this process also requires Vac14p. Atg18p/Svp1p is an effector for PI(3,5)P 2 and binds this lipid with high affinity. Atg18p/Svp1p is required for PI(3,5)P 2 mediated vacuole size control and correct membrane trafficking and also seems to feedback inhibit synthesis of this lipid as atg18D cells display elevated levels of PI(3,5)P 2. This complex set of inter-relationships is known as the Fab1p pathway but the nature of the protein-protein interactions among the various players in this signaling cascade remains undefined. Results and conclusions: In this study, we investigate inter-protein interactions in the Fab1p pathway by co-immunoprecipitation and demonstrate a core complex centered around the PI(3,5)P 2 effector Atg18p/Svp1p. We found that Atg18p/Svp1p bind to Vac14p and Fig4p in vivo and we are currently investigating interactions of this complex with Fab1p.

Cytokinine secondary hormone and 14-3-3 proteins activate Ca-ATP-ase from aleuron layers of wheat seeds Z. S. Kudiyarova and M. K. Gilmanov M.A. Aitkhozhin's Institute of Molecular Biology and Biochemistry, Almaty, KAZAKHSTAN Investigation of signal transduction is a one of the hot point of modern biology. One of the important phytohormone is cytokinine. In cytokinine signal transduction one of the main questions is existence of cytokinine secondary hormone. In this reason the main task of our investigation is the isolation, purification and investigation of cytokinine secondary hormone (CSH). At first time the CSH was purified by chromatography column with the new nanostructured carbon-mineral sorbents. Purified CSH shows it's physiological and biochemical effects at concentrations 100 times less than cytokinin. It was shown that CSH is very close by its properties to fusicoccine. Also, in recent decades it was established that 14-3-3 proteins are a one of the important component of the signal transduction systems. Namely 14-3-3 proteins are molecular targets for hormonal and other signals. One of the interesting effects of CSH is its ability with 14-3-3 proteins to activate Ca 2+ -dependent ATP-ase of plasmatic membranes from aleurone layer of wheat seeds. The activation of this enzyme let to increasing the level of the cytosolic Ca 2+ ions. It's well known that cytosolic Ca 2+ is very important intracellular signal for activation of protein kinases and many biochemical and physiological functions. Thus we have discovered the new fusicoccine like phytohormone cytokinine secondary hormone which activates the Ca dependent ATP-ase from plasmatic membrane of aleuron layer of wheat seeds. Herpes simplex virus type 1 (HSV-1) after host infection stay latent and during the acute infection undergo reactivation. It can lead to the modification of cell redox systems activation mechanisms. The aim of present study was to evaluate the changes of cell redox state during HSV-1 infection. C6 glioma cells, an astroglial cells, where HSV-1 is known to replicate, were used. Redox state of the cells was investigated using menadione-induced lucigenin-enhanced chemiluminescence (CL), and for registration of NO production we applied Griess assay. Before we have shown that the addition of menadione to monolayers of C6 glioma cells leads to the ROS generation. It was revealed that menadione-induced ROS generation in astroglial cells during HSV-1 infection is changed depending the time of HSV-1 inoculation. Integral intensity of CL was higher than in control sample in the period of 100-270 minutes after HSV-1 inoculation, whereas maximal value of this parameter was observed through 180 minute. It was established that at this period of time NO production was increased. The received data permit to conclude that HSV-1 inoculation to C6 glioma cells leads to modification of cell redox state and increased superoxide anions and NO production. Acknowledgement: This work was supported by BRFFR (projects B07MC-064). The C2 domain, composed of about 130 amino acids, has been found in many signal proteins involving in membrane trafficking, generation of lipid-second messengers, activation of GTPase, or control phosphorylation or other signaling processes. Most C2 domains specifically interaction with particular lipid molecules in a Ca 2+ -dependent manner, and some C2 domains play a crucial role in cellular processes via a direct interaction with a specific protein. In human, total 175 C2 domains have been identified in 225 different proteins. We have cloned about 150 human C2 domains using the Gateway cloning system. For in vitro lipid binding analysis, we converted all C2 domain genes to the bacterial GST-fusion expression vector. All C2 domains were successfully expressed in DH5 a E. coli cells and purified to near homogeneity using Glutathione-agarose affinity chromatography. All purified C2 domains were subjected to lipid binding assay using strips spotted with a variety of lipid species (PIP-Stripsä). Many proteins specifically bind to particular lipid species such as phosphoinosites (PI3-P, PI4-P, PI5-P) in a Ca 2+ -dependent manner. From these analyses, we defined C2 domains into several groups and their functional relation among C2 domains. These studies might provide information about how each domain is responsible for protein function and the functional significance of C2 domain in many different cellular processes.

Immunotherapy for bladder carcinoma using interleukin-27 and recombinant BCG vaccines Introduction: Cytosolic PLA 2 (cPLA 2 ) can hydrolyze lung surfactant phospholipids giving rise to potent inflammatory lipid mediators. Previous studies in our laboratory showed that cPLA 2 was activated in alveolar epithelial cells type II (AEC II) in response to mechanical signal. Activation of cPLA 2 has been correlated with the direct phosphorylation of the enzyme by members of the mitogen-activated protein kinase (MAPK) cascade. In this study we investigated the effect of two specific MEK inhibitors on the stretch-induced cPLA 2 phosphorylation in AEC II. Methods: A549 (a model for AEC II) cell cultures were pre-incubated with the inhibitors of MEK1/2, U0126 and PD98059. The cultures were then subjected to continuous mechanical stretching for 15 minutes Unstretched cells were used as control. Detection of cPLA 2 , phospho-cPLA 2 (activated form of cPLA 2 ), ERK 1/2 and phospho-ERK 1/2 was performed in cell extracts by immunoblotting using specific antibodies. Results: Mechanical stretch induced (within 15 minute) an increase in cPLA 2 phosphorylation in AEC II. PD98059 and U0126 inhibited the activation of the ERK MAP kinases in AEC II. Both inhibitors prevented the stretch-induced cPLA 2 phosphorylation. Conclusions: cPLA 2 phosphorylation in mechanically stretched A549 cells is mediated via a MEK/ ERK pathway.

Changes in the diabetic rat brain antioxidant status and acetylcholinesterase activity: modulation by L-cysteine A. Zarros 1,2 , C. Liapi 1 , P. Galanopoulou 1 , K. Marinou 2 , Z. Mellios 2 , N. Skandali 1 , H. Al-Humadi 1 , F. Anifantaki 1 , E. Gkrouzman 1 and S. Tsakiris 2 1 Department of Pharmacology, Medical School, University of Athens, Athens, GREECE, 2 Department of Physiology, Medical School, University of Athens, Athens, GREECE Introduction: Uncontrolled diabetes is known to affect the cholinergic system. The aim of this study was to investigate the effect of the antioxidant L-cysteine (Cys) on the changes caused by adult-onset streptozotocin (STZ)-induced diabetes on the rat brain total-antioxidantstatus (TAS) and the activity of acetylcholinesterase (AChE). Methods: Thirty-eight male Wistar rats were divided into six groups: C1 (8-week-control), C2 (8-week-control + 1-week-saline-treated), C+Cys (8-week-control + 1-week-Cys-treated), D1 (8-week-diabetic), D2 (8-week-diabetic + 1-week-saline-treated) and D+Cys (8-week-diabetic + 1-week-Cys-treated). All diabetic rats were once treated with an i.p. STZ injection (50 mg/kg body weight) at the beginning of the experiment, while all Cys-treated groups received i.p. Cys 7 mg/kg body weight (daily, for 1-week, during the 9th-week). Whole rat brain parameters were measured spectrophotometrically. Results: Diabetic rats exhibited a significant reduction in brain TAS ()28%, D1 vs. C1; )30%, D2 vs. C2) that was reversed after 1-week-Cys-administration into basal levels. Diabetes caused a significant increase in AChE activity (+27%, D1 vs. C1; +15%, D2 vs. C2), that was further enhanced by Cys-administration (+57%, D+Cys vs. C2). The C+Cys group exhibited no significant difference compared to the C2 group in TAS (+2%), but showed a significantly increased AChE activity (+66%, C+Cys vs. C2). Conclusions: The present data revealed a protective role for Cys towards the oxidative effect of diabetes on the adult rat brain. Moreover, an increase in whole brain AChE activity due to diabetes was recorded (not repeatedly established in the literature, since contradictory findings exist), that was further increased by Cys. The latter could be helpful in coping with the diabetes-induced brain dysfunction and is useful in identifying the mechanism(s) involved.

Electrophysiological characterization of sensory neurons from double transgenic mice with type I diabetes compared to normal mice Results: There are significant differences between the electrophysiological parameters of sensory neurons from dTg mice compared to Balb/ c mice. In our study we have recorded, the intensity and amplitude of the action potential, the intensity and amplitude of the posthyperpolarisation, and the effect of ATP, protons and capsaicin. Conclusions: The algesic profile of peripheral sensory neurons from dTg mice is very different from that of the Balb/c mice. Introduction: Epithelial-mesenchymal transition (EMT) describes the differentiation switch between epithelial cells and motile mesenchymal cells, and facilitates cell movements and generation of new tissue types during carcinogenesis and fibrosis. Many secreted polypeptides are implicated in the EMT process and their corresponding intracellular transduction pathways form highly interconnected networks. Transforming growth factor (TGFb1) has a major role in inducing EMT in pulmonary fibrosis and EBV lytic phase activation, which proteins have been found in lung tissue of patients with idiopathic pulmonary fibrosis. Here we present a gene expression array-based method to investigate altered signalling networks in pulmonary epithelial cells. Methods: Extracted RNA from primary human small airway epithelial cells (SAECs), alveolar epithelial cells type II (A549) and EBV infected A549 (VAAK) were utilized to investigate altered gene expression in response to TGFb1 10 ng/ml for 4h by Affymetrix Human Genome U133A microarrays. Resulted data from multicompare analysis were clustered and processed by Genomatix BiblioSphere Pathway (genomatix.de) and David 2.1 Functional Annotation clustering (david.abcc.ncifcrf.gov). Results: Multicompare analysis produces a list of 50 altered transcripts in SAECs, A549 and VAAK. 18 transcripts were associated with signal transduction (p = 1.30E-03) and related to transcription factor activity further, presenting binding sites for c-Jun, FOXA2 CEPD and SOX4. A significant correlation between reduced expression of tumor suppressor gene, FOXA2, and increased expression of c-Jun has been reported by functional annotation clustering and BiblioSphere Pathway. Conclusions: By using a web-based computational biology method, we propose reduced FOXA2 as a novel mediator of TGFb1-induced EMT in pulmonary fibrosis. Methods: We tested in vitro on Jurkat T lymphoblasts the new nucleoside analogs by investigating cellular viability in association with membrane integrity (LDH release), apoptosis (membrane asymmetry disorder), thymidine and uridine metabolism (the radioactive method), and drug uptake (using tritium-labeled variants). Results: We showed that the uracil-34 compound (U-34) was cytotoxic for Jurkat cells above 10 lM and this deleterious action started early with membrane integrity disturbance. The remaining cells presented a moderately elevated population of apoptotic cells. U-34 exerted an early, persistent and dose-dependent inhibitory effect on uridine uptake by tumor cells. The action of U-34 on thymidine uptake was initially stimulatory, but turned into a late inhibition. U-34 inhibited uridine and thymidine uptake by tumor cells even at non-cytotoxic concentrations. Jurkat cells incorporated only small amounts of tritium-labeled U-34. Early uptake was mediated by hENT receptors sensitive to small concentrations of dipyridamole, but other receptors seemed to be involved in later phases. Addition of non-cytotoxic amounts of non-labeled U-34 particularly inhibited the uptake of the radioactive variant. Conclusion: Our in vitro results pointed out that U-34 may exert in vitro anti-tumor activity on Jurkat lymphoblasts by inducing cytotoxicity and disturbance of uridine metabolism. Drug uptake is mediated by particular nucleoside receptors and is sensitive to non-cytotoxic concentrations of U-34. 

Abstract Withdrawn PP7-27 Advances in clarifying prion protein functions in PC12 cells B. Pantera, C. Bini, G. Camici, G. Manao and A. Caselli Department of Biochemical Sciences, University of Florence, Florence, ITALY Introduction: The cellular prion protein (PrPc) is a ubiquitous glycoprotein, localized at the cell surface via a glycosylphosphatidylinositol anchor, which function remains poorly characterized. It may participate in synaptic structure, neurite formation, copper metabolism, and signal transduction. It has also a protective role in models of neurodegeneration due to oxidative stress. Several cellular partner of PrPc have been proposed, but to date there is no functional ligand of PrPc which has been clearly identified and widely accepted. In this study, we analysed the signaling cascade activated after PrPc antibody-mediated cross-linking in PC12 cells.

Methods: PC12 cells were cultured in RPMI with 10% horse serum and 5% foetal calf serum. Differentiation was induced with 50 ng/ml of NGF. Starved cells were treated with 10 (g/ml anti-PrP c . Lysates and immunoprecipitates obtained with specific antibodies, were analysed by SDS-PAGE/western blotting. Lipid rafts were separated on a 5-35% discontinuous sucrose gradient.

Results: Antibody-mediated ligation of PrP c promotes the formation of a signaling platform, which associates PrP c , caveolin-1, Fyn and other related proteins. The interaction is strictly dependent from caveolin-1 phosphorylation. Downstream from this complex, PrP c antibodies trigger to the transient activation of ERK1/2. This process is integrins-dependent and also leads to FAK activation. The phosphocaveolin induction conduces to its association with csk and to the inactivation of Fyn.

Here we demonstrated a fundamental role for caveolin-1 phosphorylation in the PrP c -mediated signalling and detailed the molecular partners involved in this transduction pathway.

Ultrastructural insights into cellular uptake of S4 13 PV peptide Cell penetrating peptides (CPP) are gaining widening popularity as vehicles for cellular delivery of molecules of pharmacological interest but with very limited cellular uptake. The exact mechanism(s) that CPPs use for cell entry and transduction of cargo molecules, however, has remained elusive and controversial so far. The cellular translocation of CPPs and their payloads has been described by fluorescenceand activity-based methods mostly, leaving the more detailed characterisation on the ultrastructural level almost out of attention. We tagged S4 13 PV and its analogues with 1.4 nM Nanogold particles by forming a covalent bond between the peptide and label. We mapped the interaction mode of labelled peptides with the plasma membrane, uptake by HeLa and CHO cells and intracellular targeting by using transmission electron microscopy. S4 13 PV peptide and its analogue with reversed NLS sequence, which are known to be taken up very efficiently, interact with cell surface in cooperative manner by assembling into spherical structures on/in the plasma membrane. The formed structures contain several peptide molecules on the electron-dense background and accumulation of these in the plasma membrane induces the uptake into cells by vesicular mechanism mostly. In vesicles the spherical structures are not dissociated but retain their organisation and remain associated with the membrane of vesicles. High concentrations of peptide impair the integrity of the vesicle's membrane and induce escape into cytosol. Our study demonstrates that CPPs interact with the plasma membrane at specific regions and by forming regular structures introduce disturbances in the plasma inducing vesicular uptake by cells.

A yeast-based screen for the functional study of mammalian Akt In mammalian cells, protein kinase B (PKB/Akt) is the main transducer of signalling dependent on phosphatidylinositol-3,4,5-trisphosphate (PIP 3 ) in multiple pathways related to cell proliferation, survival, insulin signalling, angiogenesis, metabolism and migration. Recognition of PIP 3 by the PH domain of Akt and phosphorylation in its activation loop by upstream kinases is the key for its activation. We have reconstituted this signalling pathway by heterologous expression in the unicellular eukaryote Saccharomyces cerevisiae and have found that yeast cell growth was inhibited by Akt activated in vivo by co-expressed nontoxic wild type p110. Akt toxicity requires its kinase activity and induces the formation of plasma membrane and cell wall invaginations supported by actin polarization. We have screened a collection of 4thinsp;787 haploid S. cerevisiae single gene deletion mutants to identify non-essential genes associated with Akt toxicity on S. cerevisiae. This led to the identification of yeast genes that either counterbalanced growth inhibition induced by Akt on yeast or displayed enhanced sensitivity to in vivo activation of Akt. Most of these genes have human homologues, so by extrapolating our results to mammalian cells, this study can contribute to our knowledge about the role of Akt in cancer and other pathologies. In summary, we show here that the yeast model is a useful tool for the functional analysis of Akt. Infection by influenza virus causes the cascade of biochemical processes, which lead to immunity suppression and in particular to the decrease of neutrophils phagocytosis. The role of oxygen-dependent redox systems of neutrophils in influenza virus inoculation is not known. Mechanisms of ROS generation during the initial stage of the interaction of influenza virus A (FPV H7N1) with neutrophils of human blood were studied in present work. ROS generation and MPO activity of neutrophils were investigated by luminol dependent chemiluminescence (Lum-ChL). Cells were activated by adhesion to glass. To determine the influence of virus of different ROS types generation in neutrophils specific scavengers of ROS were used. Secretory degranulation was studied by lysozyme release into extracellular space by spectrophotometry. It was established that virus significantly reduced ROS production. It decreased the yield of hydrogen peroxide, superoxide and NO in cells and did not influence the quantity of hydroxyl radicals. Virus diminished lysozyme and thereby MPO secretion by neutrophils from cells. In model system MPO/H 2 O 2 /luminol virus inhibited MPO activity by 90% that was mainly associated by OClscavenging. Data supposed that virus revealed immunodepressed effect on neutrophils possibly by modification of cells redox system.

Helicobacter pylori infection down regulate Signaling pathways that depend on TP53 or NFjB transcription factors are essential components of cellular responses to stress. TP53 activated primarily upon genotoxic stress is involved in regulation of either cell cycle or apoptosis. NFjB activated by many different factors, including cytokines like TNFa, frequently exert anti-apoptotic functions. Both regulatory pathways apparently interfere each other, yet molecular details of such interactions remains to be elucidated. Here we have analyzed effects of activation of NFjB on apoptosis, either TP53-dependent or TP53-independent, induced by UV radiation in human colon carcinoma cell lines HCT116 and RKO. TP53 was inactivated in such cells by either biallelic knock-out or expression of inhibitory viral EPV E6 protein, respectively. Cells were treated with TNFa to activate NFjB pathway and irradiated with UVC to induce apoptosis (at several time combinations), and then number of apoptotic cells was assessed by flow cytometry at different time points. We have observed that treatment with TNFa resulted in 2-4-fold reduction in number of apoptotic cells in TP53-proficient lines. Interestingly, an inhibitory effect was highest when cells were treated with TNFa after UV irradiation. In marked contrast, treatment with TNFa after UV irradiation resulted in significantly increased number of apoptotic cells in TP53-deficient lines. We concluded that TNFa-dependent activation of NFjB signaling differentially affects TP53-dependent and TP53independent apoptotic pathways. Methods: In vivo labeling of protein phosphorylation events during signal transduction indicated the rapid phosphorylation of several proteins. Substantial differences and de novo LPS-induced phosphorylation were also observed with two-dimensional analysis. Qualitative and quantitative changes in the phosphoproteins of Nicotiana tabacum suspension cells during elicitation by LPS were analyzed using two-dimensional electrophoresis in combination with a phosphoprotein-specific gel stain. Trypsin digested phosphoproteins were analyzed by MALDI-TOF/MS and nano-ESI-LC/MS/MS. Results: The identified phosphoproteins indicate that LPS-induced signal perception/transduction involves G-protein coupled receptor signaling, Ca 2+ /calmodulin-dependent signaling pathways, H + -ATPase regulation of intracellular pH, thioredoxin-mediated signaling and phosphorylation of 14-3-3 regulatory proteins. Other targets of LPS-responsive phosphorylation included NTP pool maintenance, heat shock proteins, protein biosynthesis and chaperones as well as cytoskeletal tubulin. Conclusion: The results provide a broad picture of the mechanisms of LPS perception and signal transduction. Many of the identified LPSresponsive phosphoproteins are known to be interconnected at many levels through a complex web of activation/deactivation, complex formation, protein-protein interactions, and chaperoning reactions. This study thus provides novel insights for the biochemical action mechanism of LPS as a resistance elicitor, a PAMP and triggering agent of defense responses associated with innate immunity. Introduction: Some labdane-type diterpenes show significant antimicrobial and cytotoxic activities. The resin found in the glands of Cistus creticus ssp. creticus, a plant native to Crete, contains high concentrations of several labdane-type diterpenes. The metabolic pathway leading to the biosynthesis of this class of labdane-type diterpenes remains unclear. Here we report the cDNA isolation, heterologous expression in E. coli and functional characterization of a diterpene synthase from C. creticus trichomes involved in labdane biosynthesis. Methods: The full length cDNA of a putative diterpene synthase was isolated from a C. creticus trichome cDNA library and the coding region was spliced into an E. coli expression vector. Crude protein extracts from bacterial cultures expressing the gene were used in enzymatic assays with GGDP as substrate. The compounds produced, with or without hydrolysis, were analyzed by gas chromatography-mass spectrometry. Results: The deduced amino-acid sequence of this protein is highly similar (59-70%) to type B diterpene synthases that catalyze a protonationinitiated cyclization in other angiosperm species. Nevertheless, motifs related to ionization-initiated cyclizations, similar to those performed by type A diterpene synthases, were identified in the protein primary structure. The E. coli-produced enzyme catalyzed the formation of the tricyclic labdane-type diterpenes manoyl-oxide and epi-manoyl oxide as well as another unidentified compound in two sequential cyclization steps, with diphosphate labda-13-en-8a, 15-diol as a stable bicyclic intermediate. Therefore the encoded protein can be named manoyl-oxide synthase (MAOS). Conclusions: MAOS is the first bifunctional type A/B diterpene synthase identified among the angiosperms and its activity sheds new light on labdane-type diterpene biosynthesis. This knowledge is critical for biotechnological applications in the production of these pharmacologically active compounds.

The roles of 1-aminocyclopropane-1-carboxylate synthase isogenes in the flower and fruit development in tomatoes R. Fan and W. K. Yip School of Biological Sciences, University of Hong Kong, HK SAR Introduction: Plant hormone ethylene regulates many aspects of plant growth and development. The regulatory enzyme in ethylene biosynthesis, ACC synthase (ACS), is encoded by a multi-gene family whose members are differentially regulated by different developmental, hormonal and environmental conditions. Nine ACS isogenes has been reported in tomato and based on the transcript expression profiles of these genes, four ACS isogenes (LE-ACS1A, LE-ACS2, LE-ACS4, LE-ACS6) are found to be involved mainly in flower and fruit development. Methods: To elucidate the roles of these genes in reproductive growth, gene specific ACS silenced lines were first generated by employing antisense RNA and/or RNAi method. Double and triple ACS genes silencing lines could then be generated by cross breeding afterwards. Results: Suppression of LE-ACS1A, LE-ACS2 and LE-ACS4 in transgenic tomato lines showed different degree of morphological and anatomical change in flower and fruit: increase in petal number and locule number, abnormality in the placenta formation, increase in fruit size and weight etc. as compared with the wild type UC82B. When applied ACC to these transgenic lines during very early stage of flower development (1 mm), the locule number of fruits could partially restore to normal. In contrast, LE-ACS6 suppressed lines did not show phenotypic changes during reproductive growth, but showed a larger leaf area compared to wild type plants.

Conclusions: The phenotypic changes in the transgenic lines correlate with the suppression of these genes' expression. Therefore, LE-ACS1A, LE-ACS2 and LE-ACS4 play important roles during tomato reproductive growth. 

Introduction: Pistacia lentiscus Var. Chia plants growing on the island of Chios are present in different morphological varieties, which exhibit moderate differences in respect to the their products's quality. An attempt was made, to investigate whether the morphological differences correspond to taxonomic ones. Methods: Samples were collected from five individuals of each of the following local varieties Vottomos, Marouliotis, Mauroschinos, Stenofyllos and Fardyphyllos growing on the island. They were initially examined by RAPD using decameric primers. To verify RAPD results, individuals from each of the above varieties were further examined by cloning and sequencing of their Intergenic Regions (IR) located between 18S and 26S rDNA genes. Sequences were aligned and a phylogenetic tree was constructed. Results: RAPD results indicated that all varieties differ among themselves based on RAPD pattern generated with a specific primer. Within each variety, Vottomos, Stenofyllos and Fardyphyllos do not comprise of homogeneous populations, as further substantiated by IR results. To the contrary, varieties Marouliotis and Mavroschinos appeared to consist of a homogeneous population according to RAPD results, a fact not supported by the IR sequencing data. Some of the above varieties proved to be 100% identical to Pistacia saportae and all of them 98-99% identical with Pistacia lentiscus. Intoduction: Perennial plant species, including peach trees have to withstand adverse environmental conditions. Thus, at the end of each growing season they move into dormancy producing floral and vegetative buds to protect their meristems from freezing. Trees accumulate chilling hours to count the duration of winter preventing growth under non-favorable conditions. With respect to their Chilling Requirements (CRs) peach varieties are either suitable for warm regions (Low-CRs) or for cold regions (High-CRs). Understanding the molecular mechanisms that modulate CRs could determine the prerequisites for tree species adaptation in a changing climate. Therefore farmers could choose to cultivate productive and profitable elite cultivars. The identification of genes involved in flowering time could serve as a platform for conventional plant biotechnological sorting of peach varieties. Methods: We have collected vegetative and floral buds of the 'Evert' variety from two locations in Greece with different bud break periods. Samples were analyzed by two-dimensional polyacrylamide gel electrophoresis. Results: Comparison of vegetative and floral bud proteomes revealed high degree of colinearity between the two bud types. Furthermore, the protein profile of each bud-type from both geographical regions showed high degree of colinearity. Protein spots representing either differential or co-expression among the two bud-types and/or locations were selected for sequence determination by MALDI-TOF Mass-Spectrometry. Conclusions: Proteins showing differential expression between each bud-type could indicate developmental specificity, while geographical differential expression may discriminate the advancement to bud breakage. Consequently, proteomics allows the identification of genes involved in bud development that may serve as biomarkers for conventional biotechnological breeding of tree species. Intoduction: Measurements of atmospheric CO 2 show a gradual rise enhancing the greenhouse effect that results in global warming. Mankind has to cope with the consequences of climate change and identify environmental benign ways to meet the expanding energy demands while decreasing greenhouse gas emissions. Through a variety of 'renewable' sources available, the form of solar energy that can contribute substantially to fuel needs at competitive level to fossil fuel is the one captured by photosynthesis. Thus, understanding plant cell wall synthesis (biomass) is of crucial importance, as it is the greatest carbon sink in the biosphere. Methods: Performing a genetic screen of an EMS mutagenized Arabidopsis, we isolated thanatos (than), a semidominant-negative mutation with impaired cellular architecture. Intriguingly, than homozygous seedlings are embryo lethal while growth of heterozygous plants is dramatically decreased. FTIR-Microspectroscopy and cell-wall component analysis revealed reduction of cellulose content of than plants compared to wild type. Results: Positional cloning revealed a single-nucleotide transition causing the amino acid substitution of Proline 578 to Serine of Arabidopsis Cellulose synthaseA3 (AtCesA3) gene. AtCesA3 (P578S) mutant gene when introduced into wild-type plants resulted in acquisition of than semidominant-negative mutant phenotype. Multiple sequence alignment revealed that Proline 578 is conserved among plant and non-plant CesA genes being part of the catalytic UDP-glucose substrate-binding domain. Conclusions: These data suggest that the incorporation of a than defective CesA3 subunit into the rosette complex most likely prevents the production of b-(1,4)-glucose chain, which could in a way stall cellulose microfibril synthesis. Aurones, the 6-glucosides of aureusidin, are plant flavonoids that provide the bright yellow colour of some important ornamental flowers. Despite the potential significance of aureusidin biosynthesis in biotechnology, the enzymatic details of its synthesis have not been analysed. A new enzyme, aureusidin synthase was proposed to play a key role in aureusidin biosynthesis. This enzyme was identified as a chalcone-specific homolog of polyphenol oxidase (PPO). In this study, we analyze the enzymatic formation of aureusidin by PPO, an enzyme which catalyses two different reactions involving oxygen: the o-hydroxylation of monophenols to o-diphenols (monophenolase activity) and the subsequent oxidation of o-diphenols to o-quinones (diphenolase activity). 2',4',6',4-Tetrahydroxychalcone (THC), a suggested precursor for aurone aglycons, has been used as substate of PPO. Enzymatic oxidation of THC was followed by recording spectral changes with time. Maximal spectral changes were observed at 415 nm. The oxidation rate which varied with pH was highest at pH 4.5.Time-course of THC oxidation by PPO showed a lag period, characteristic of monophenolase activity. The length of the lag phase increased with THC concentration but decreased with enzyme concentration. The lag was shortened by the addition of increasing amounts of o-diphenol or hydrogen peroxide. The following kinetic parameters were determined: V m = 13 lM/min, K m = 0.12 mM, V m /K m = 0.1/min. THC oxidation was efficiently inhibited (K I = 1.8 lM) by tropolone, a specific PPO inhibitor. The results obtained confirmed that PPO is capable of oxidizing directly THC, the precursor of aurone biosynthesis. Objective: The aim of this study was to determine the anti-coagulant and anti-diabetic activities associated with Leonotis leonurus extracts and marrubiin, a compound isolated from this plant. Methods: Aqueous and organic (acetone) extracts were prepared from Leonotis leonurus. Human platelets were incubated with different concentrations of the aqueous and organic extracts (0.5-2 mg/ml) and commercial marrubiin (0.01-0.1 lg/ml). For the platelet studies; the platelets were activated with thrombin and adhesion to coated plates was evaluated with light microscopy and a phosphatase assay. Expression of GIIb/IIIa in platelets was used to evaluate platelet aggregation with flow cytometry. For the diabetic studies; rat islets were exposed to the extracts for 48 hours to determine the effect of the extracts to insulin release under hyper and normal glucose levels.

Results: For the antiplatelet activity; the aqueous (2 mg/ml), organic extracts (2 mg/ml), and marrubiin (0.1 lg/ml) inhibited platelet adhesion by 55%, 60%, and 72%, while GIIb/IIIa expression was also inhibited by 69%, 86%, and 46%, respectively (n = 5). The antidiabetic studies displayed a stimulatory index of islets cultured under hyperglycemic conditions which significantly increased by 450% and 500% relative to the control (P < 0.01; n = 5) in islets exposed to the organic extract and marrubiin (150 ng/ml), respectively. Conclusion: The in vitro studies clearly reflect that Leonotus leonurus has the potential to serve as a multi-therapeutic anticoagulant and antidiabetic agent. Introduction: While serine proteases perform a wide range of functions essential to life, they can also be dangerous and must be controlled. One of the many control mechanisms is their specific inhibition by protein protease inhibitors. Protease inhibitors in plants, also present in their seeds, participate in defense mechanisms and their production is induced by herbivory or wounding. For the first time we isolated a canonical serine protease inhibitor from Acacia schweinfurthii seeds.

Methods: The purification includes 0.15 M NaCl extraction of the seeds, fractionation of the extract by 80% (v/v) acetone, followed by ion exchange-and affinity chromatograpy and then by reverse phase HPLC.

Results: Both HPLC analysis and SDS-PAGE indicated that inhibitor with a molecular mass of approximately 21 kDa and reducing conditions revealed, however, that the native inhibitor is composed of two polypeptide chains with molecular masses of approximately 14 and 8 kD, respectively. Our inhibitor was shown to inhibit both trypsin and chymotrypsin, with an inhibitory constant (K i ) estimated to be in the range of 1-6 · 10 -10 M.

Conclusions: The specificity of inhibition together with the two-chain composition of the inhibitor protein and results of N-terminal sequencing of the two constituent polypeptide chains showed that this new inhibitor is homologous to a serine protease inhibitor previously isolated from Acacia confusa. Based on this homology the new serine protease inhibitor from Acacia schweinfurthii appears to be a Kunitz-type canonical inhibitor. there are few studies considering the isolation, characterization and purification of GSTs from forest trees, no information were available about the Pinus brutia, Ten (Kizilcam) in the literature. In this study, the GST activity for the conjugation of xenobiotic substance (CDNB) was isolated from needles of Pinus brutia, Ten. trees from a nursery in Yalıncak area/METU (Ankara-Turkey). Trees that exhibited healthy appereance were selected from the same altitude profile. GSTs activities in the cytosolic fractions prepared from Pinus brutia needles were determined spectrophotometrically by using 1-chloro-2,4-dinitrobenzene (CDNB), 2,3-dichloro-4-(2-methylene butyryl)-phenoxy acetic acid (Ethacrynic acid; EA), 1,2-Dichloro-4-nitrobenzene (DCNB), 1,2-Epoxy-3-(p-nitrophenoxy) propane (EPNP), and p-Nitrobenzyl chloride (p-NBC) as substrates at 25°C. There was at about 68% decrease in the GST activities after 25°C. Only CDNB (160 ± 10 nmoles/min/mg) and DCNB (2.30 ± 0.38 nmoles/min/mg) activities were detected and the rest were found as negligible. Therefore, CDNB was used as the substrate during purification of GSTs from needles of P.brutia. The sequential application of cytosol to gel filtration column chromatography on Sephadex G-25, anion exchange DEAE cellulose column chromatography, and S-hexylglutathione agarose affinity chromatography were performed for purification of GSTs. After the final step of purification procedure, CDNB conjugating activity of P.brutia cytosolic GSTs was purified about 15.45 fold with 1.95% yield. SDS-PAGE results showed that distinctly purified GST isozyme had an Mr of 24 kDa. In this study, we report for the first time the GST isozymes in a gymnosperm, P. brutia. Ubiquitously distributed multifunctional superfamily of Glutathione Stransferases (GST, EC.2.5.1.18) generally constitute a dimeric enzymes and catalyze the conjugation of the thiol group of the glutathione (GSH) to diverse electrophilic centres on lipophilic molecules with the formation of rather less active end products. Plant GSTs had an attention because of their roles in herbicide detoxification. Therefore, GSTs have been identified and characterized with a differential and overlapping substrate specifities mainly from the agricultural crop species. However, there is almost no information about molecular characterization of this superfamily in gymnosperms, except for a very recent study from Pinus tabulaeformis and our previous studies by using Pinus brutia, which is the climax vegetation of the Mediterranean Region in Turkey. As conifers have wide distributions and have to cope with several environmental stresses, the definitions of detoxification enzymes like GSTs in conifers is very important for their adaptations. Due to this purpose, it was decided to examine the molecular cloning and expression of GST-Tau isoenzymes in E. coli. In this study, the total RNA was isolated from Pinus brutia needles and subsequently cDNA was prepared with RT-PCR strategy. PbGST- Introduction: Olive mill wastewater (OMW), a byproduct of olive oil extraction, constitutes a significant agroindustrial waste and an important environmental pollutant due to its high organic load, low pH and presence of biotoxic substances (polyphenols) that are difficult to degrade. A specific group of the OMW phenolic profile called secoiridoids includes oleuropein, tyrosol and hydroxytyrosol, which have antimicrobial activity and act as antioxidants, thus having a healthbeneficial role. Methods: A group of bacterial strains that grew with ease on untreated OMW were isolated based on their ability to bioremediate and decrease total organic carbon (Folin-Ciocalteau method) and phenolic concentration (e.g. vanillic, p-coumaric feroulic and syringic acids; GC spectrophotometer) in diluted OMW. Furthermore, they were found to produce significant amounts of hydroxytyrosol and tyrosol (Reverse-phase HPLC). The selected strains were characterised based on their 16S rRNA gene sequence and the microbial flora of OMW was investigated by means of 16s rDNA array probing. Results: OMW is a substrate for a number of bacterial phyla, with emphasis to Cyanobacteria and c-Proteobacteria -mostly Enterobacteria. The microbial load varies depending on OMW treatment/geographical area. An uncultured isolate of Enterobacteria and a Gram+ species were able to decrease the total organic load, in some cases as much as 69% in 24 hours and significantly decreased phenolic concentration. In addition, a 7 hour incubation in diluted OMW yielded an almost 100% increase in its tyrosol/hydroxytyrosol content. Based on remediation treatments and the phylogenetic status of these bacteria we provide alternative tools for OMW management. 

Ribes nigrum anthocyans acting as antitumoral agents through an antioxidative mechanism in C32 melanoma cells PP8A-20 Virp1, a host factor necessary for replication of nuclear viroids The use of metal accumulator plants to clean-up soil and water contaminated with toxic metals is the most rapidly developing, environmental friendly and cost-effective phytoremediation technology. From our previous research (1) we found Brassica nigra as a Cu and Cd accumulator. We developed an efficient regeneration system for Brassica nigra which could be used for our genetic transformation studies. Thirty days old plants were divided into shoot, apex, and hypocotyls and these explants were cultured in MS media containing 20 g l-1 sucrose, 1 ml l- Activity of b-amylase is very important for mobilization of polycarbohydrates in early stage of germination of cereal seeds. We used different types of chromatography for purification of b-amylase. The treatment by triton X-100 liberates b-amylase from membrane bound state. The treatment by papaine changes the electrophoretic spectra of b-amylase by causing appearance of catodic isoenzymes and dissapereance of anodic isoenzymes. This phenomenon can be explained by splitting of charged peptides from molecules of b-amylase. Our data are confirmed by treatment of b-amylase by endoproteinases which was isolated by affinity chromatography on hemoglobin-sepharose. The treatment by endoproteinases causes the same effect as papaine treatment. In this case there is appearance of catodic isoenzymes and dissapereance of anodic isoenzymese. In contrast of treatment by papaine, endoproteinases treatment causes also the essential increasing of bamylase activity. The treatment of wheat grain by protease inhibitors of cystein proteinases: leupeptine and antipaine prevents the changes of electrophoretic spectra of b-amylase. Our results speak about very important role of endoproteinases in regulation of activity and electrophoretic spectra of b-amylase in wheat seeds during early stages of germination. The regulation of b-amylase is very important for biotechnological control of preharvest germination of wheat seeds in the ears which causes damage to quality of seeds.

Search for molecular markers associated with yellow rust resistance in Turkish bread wheat genotypes Methods: Vector was delivered into plant cells using agroinjection method. The agrobacteria suspension, earlier transformed with construct containing G-CSF gene, was inoculated in leaves. G-CSF accumulated in plants was extracted using Ni-NTA column for further testing its physiological activity: bone marrow was treated with plantmade G-CSF and a week later the number of newly formed neutrophilic granulocytes colonies was counted.

Results: This expression system appeared to be effective, it provides up to 1-2 grams of G-CSF per kilogram of fresh leaf biomass which is up to 20% of the total soluble protein. Plant-made G-CSF developed a high level of physiological activity comparative to the commercial G-CSF (Neupogen) drug.

Conclusions: Currently we are planning to improve the technologies of G-CSF extraction and purification and that is expected to increase its specific activity. The plant-made G-CSF may be used for development of a cheap pharmaceutical. Methods: A hydrophobized inorganic support was used as absorbent and a less polar solvent system was taken to induce both enzyme reversible partial unfolding and effective substituted ligand exposition. Absorptive immobilization of the enzyme was achieved when SDH was dissolved in an appropriate ratio concentration of dioxan, and in the same way the matrix was presented at the same conditions. Enzyme kinetic properties were also studied to provide comparisons between soluble and adsorbed enzyme.

Results: As a result hydrophobic interactions between exposed hydrophobic pockets from the protein side and octadesyl chains from the matrix-side are facilitated. The immobilized enzyme was approved to be stable when used at discontinuous catalytic operations. The immobilized preparation represents more than 2-fold remaining activity in comparison with the free enzyme.

Conclusions: In this study we are reporting for the first time an effective method for adsorptive immobilization of proteins (enzymes). In spite of the high level of purification techniques and detailed analysis of different components of zootoxins, the mechanism of the interaction between venom and intact tissues in the living organism is still not clear. A fact that has received some attention is the difference between the toxic activity of purified venom components and that of the same components in the whole venom. BLMs and GUVs represent an adequate model of the lipid moiety of biomembranes, which makes it possible to reconstruct the changes of some features characteristic of natural bilayer membranes under snake venom influation. Model membranes (BLM) were formed from the total lipid fraction from bovine brain on a teflon aperture by the method of Muller. The electrical parameters of the BLMs were determined on an electrometric devise equipped with a Keithley 301 differential feedback amplifier (USA) in a voltage-fixation mode. The electroformation method, developed by Angelova and Dimitrov (Dimitrov and Angelova, 1987) was used to prepare the vesicles. Venom was added to the sample chamber before the vesicles were formed and we investigated the electrical parameters of BLMs after their merging with venom modified GUVs. The presence of viper venom in BLMs media has not shown a noticable differences of plastic properties compare the control, while the merging of BLMs with GUVs modified by venom lead to appearance of channel activity. It was recognized early that the vipers venom components preferred an organized lipid substrate near the lipid's phase transition and were particularly active against micellar lipids. Introduction: A primary intent in drug targeting and pharmacological studies is the understanding of driving forces involved in the transport and/or distribution of drugs within cells. At the same time, transport studies require quantitative elaborations and vitality assessment. Synchrotron radiation X-ray fluorescence microscopy (lSR-XRF) allows the simultaneous determination of different chemical elements with nanometric resolved localization (1), while phase contrast microscopy probes cell morphology. With this technique we investigated transport and intracellular localization of a Gd-based MRI contrast agent (B22956/1) by following gadolinium internalization in hepatic cells.

Methods: Chang liver cells grown on SiN 4 windows were incubated overnight with 500 lM of B22956/1 and fixed. Cells were then analyzed under ID-21 Scanning X-ray Microscope, at ESRF, Grenoble, France, by applying a 7.3 keV monochromatic X-ray beam.

Results: Gd was detected in the perinuclear region and quantification (2.76 ± 0.8 lg Gd/g cell) was evenly distributed among viable cells. Early apoptotic cells revealed 10 times higher accumulation (22.1 lg Gd/g cell) of the agent and Gd presence also inside the nucleus. Contrast agent accumulation was associated with changed gradients of endogenous elements, particularly Ca and K.

Conclusions: Results confirmed previously reported high accumulation of B22956/1 in hepatic cells (2) and, for the first time, the cytoplasmic perinuclear localization of the drug was revealed at nanometric resolution. Single cell analysis under lSR-XRF can be applied for pharmacological studies to inspect simultaneously transport mechanisms and targeting in a homeostatic cellular state. Nano-biotechnology is an extremely powerful emerging technology, which is expected to have a substantial impact on nanotechnology now and in the future. Although, in this area many applications are still in their infancy, it seems that nanoparticles would have a great role in future prescription. Albeit, nanoparticles have been known for decades, most of their properties and characteristics especially encounter with biological systems are still unknown. In this study, Gold Nanoparticles (GNp) which have exciting properties were adapted to biological system. For fabricating bio-nano gold particles the reducing agents such as NaBH4 and citrates were replaced with UV irradiation and stabilizing agent such as Triton X-100 and Sodium Dodecyl Sulfate (SDS) and other surfactants which are not adjust to biological system were omitted. At least GNP in small size and in biological PH with non intrusive agents has been made in photochemical synthesis way. The color of GNP solution and their absorption in UV spectrometry studies and TEM images showed the producing small GNP. Following to understand the interaction of GNP to protein, microtubule protein (MTP), extracted from sheep brain, has been chosen. Microtubules are cytoskeletal proteins that, among other functions are essential for, cell transport and cell division and are present in all eukaryotes. They are highly dynamic polymers and consist of tubulin dimers. Interaction of gold nanoparticles with microtubial protein was characterized by using UV-Visible spectroscopy (UV-Vis) and fluorescent spectroscopy. UV spectrometry has shown decreasing in microtubule polymerization and protein activity. It seems that they can interact with microtubule protein and inhibit their activity. Fluorescence spectrometry showed a significant changes in tubulin structure. According to these results we suggest that GNP interaction with microtobule protein result to dimer conformational changes of protein, therefore, decreasing polymerization. We use statistical mechanics and a grand canonical ensemble to study the conformational properties of a long DNA chain capable to bear the formation of a certain amount of loops. We use a microscopic model where the DNA chain is described by a continuous line. The probability of formation of a loop, after the contact of two sites away along the chain, is based on Gaussian statistics which have been extensively used in the past. Beyond its persistence length it includes also a small length parameter which can describe the local structure of the DNA chain like its helical behavior. Based on this probability an optimum size of the loop is determined in accordance with previous more sophisticated theories and the results of enumerations of ring closures on a DNA chain. The average number of loops which goes as the probability of contact of two sites as well as the average size of the DNA chain are analytically described in terms of the size of the chain the chemical potential of formation of a loop and the largest possible number of loops. Cases with a small number of loops like in DNA various processes where a few sites are brought in close proximity and interact can properly be described. Cases with a larger number of loops leading to an overall expansion or condensation of the chain after the addition of condensing agents, can also be properly described. A comparison with existing experimental data based on single chain observations is included.

Proteoliposome production regulation by detergent ratios and proteoliposome fates after cellular uptake K. E. Lee and S. S. Han School of Life Sciences and Biotechnology, Korea University, KOREA Introduction: Liposomes are the most commonly used biomaterials for drug or nutrient delivery due to the incorporation property of the molecules. Liposomes are applied to artificial regulation systems of gene expression, protein or lipophilic delivery drug, the stabilization of nanoparticles and liposomal vaccines. Especially proteoliposomes products are very useful for target delivery system. Methods: Proteoliposomes were produced by detergent mediated reconstitution from membrane proteins and liposomes. The processes of proteoliposome formation with various detergent ratios were monitored by the optical density and cryo-TEM. The protein amount of reconstitution in proteoliposome was confirmed by cryo-TEM.

Results: The detergent ratios had effects on the structure of the liposome and the amount of membrane proteins that were reconstituted into the liposomes. Vesicular crystals were aggregated from saturated detergentsolubilized lipid micelles containing membrane proteins, and low-density liposomal proteins were formed at low detergent to lipid ratios when liposomes were ruptured into open structure. Different types of proteoliposomes have different fates inside cell after endocytosis or phagocytosis.

Conclusions: High-density proteoliposomes kept their structure stable in the phagosome then they were migrated to the cytoplasm. If any drug will be loaded inside the high-density proteoliposomes, it may operate at the cytoplasm. From these results, differently produced proteoliposomes will be able to have wide applications to delivery system by different distribution inside of the cells.

Characterization of lysine-tagged Bacillus stearothermophilus aminopeptidase II immobilized onto carboxylated gold nanoparticles Introduction: Aminopeptidases are widely distributed exopeptidases that selectively remove the N-terminal amino acid residues from peptides and proteins. In this study, we demonstrate the manufacture of a highly water dispersive gold nanoparticles that can efficiently immobilize the lysine-tagged Bacillus stearothermophilus aminopeptidase II (BsAPII). Methods: The experimental procedures include expression plasmid constructions, AuNP synthesis, MHA modification of AuNP, and enzyme immobilization. Results: Bacillus stearothermophilus aminopeptidase II tagged C-terminally with either tri-or nona-lysine (BsAPII-Lys 3/9 ) was constructed and over-expressed in Escherichia coli M15 (pRep4). The recombinant enzymes were purified to homogeneity by nickel-chelate chromatography and their molecular masses were determined to be approximately 45 kDa by SDS/PAGE. Surface modification of colloidal gold with 16-mercaptohexadecanoic acid was employed to generate the carboxylated nanoparticles. BsAPII-Lys 9 was efficiently immobilized onto the carboxylated gold nanoparticles (AuNP-COOH) and the obtained bioconjugate showed excellent biocatalytic activity in the immobilized form. Additionally, the bioconjugate material exhibited a significant enhancement in temperature stability and could be reused over five successive cycles.

Conclusions: In summary, poly-lysine-tagged BsAPII can be successfully immobilized on the surface of carboxyl-terminated alkanethiol modified AuNPs. The substrates are easily accessible to the immobilized enzyme, thus mimicking the free enzyme in solution for all practical purposes. As compared with free BsAPII, the lysine-tagged enzyme in the bioconjugate system shows enhanced stability toward more harsh temperature condition. Moreover, the enzyme-nanogold bioconjugate material exhibits excellent reuse characteristic. Based on these advantages, the BsAPII/AuNP-COOH bioconjugates can be expected as catalytic nanodevice to construct nanoreactors based on LAP reaction for biological applications. Liposomes are biocompatible and biodegradable systems for local targeting of therapeutic agents to the site of interest while reducing systemic toxicity and minimizing side effects. The aim of our study was to investigate whether the entrapment into negatively charged pH-sensitive liposomes of some compounds such as lactoferrin (Lf), a glycoprotein from bovine milk or of chemotherapeutic drugs, dacarbazine and cisplatin, could improve their antitumoral activities. The experiments were performed in vitro using human melanoma cell line SK-mel 28. MTS proliferation assay revealed that free Lf as well as the synthetic compounds significantly reduced the number of living cells. The IC 50 evaluated after 24 hour of treatment was 350 lg/ml for Lf, 2000 lg/ml for dacarbazine and 400 lg/ml for cisplatin. The effect on cell viability was associated with morphological changes visualized by optical microscopy. FACS analysis of cells treated with the antitumoral compounds revealed a decreased in the percentage of cells in S-phase of cell cycle. Lf or anticancer drugs entrapment into liposomes resulted in two to three fold better efficiency in inhibiting cell growth and affecting cell morphology as compared to free agent treatment. These results demonstrate that liposomes containing antitumoral agents could represent a base for a new strategy in cancer prevention and/or treatment. Acknowledgments: This work was supported by MATNANTECH research program, project CEEX 57/2006 (NANOCONTER).

Bioelectrochemistry of drug metabolising human flavin containing monooxygenase S. Sadeghi, R. Meirinhos and G. Gilardi Department of Human and Animal Biology, University of Turin, Turin, ITALY Introduction: Flavin-containing monooxygenase (FMO) is a widely distributed enzyme that catalyses the NADPH-dependent oxygenation of a large variety of drugs to benign metabolites. To date, five functional genes of human FMO (hFMO) have been identified with FMO3 as the predominant one present in adult human liver. This enzyme is responsible for the detoxification of important drugs including cancer therapy drugs. Methods: The gene encoding the hFMO3 was cloned and the resulting soluble recombinant protein expressed in E.coli and purified. Redox potential and drug metabolism measurements were carried out using electrochemical techniques of cyclic voltammetry and chronoamperometry. Results: Here we report the first direct electrochemical characterisation of hFMO3 using both non-oriented entrapped protein on glassy carbon electrode (GC) and covalently linked protein on gold electrode. The enzyme was initially immobilised on GC electrode using a cationic surfactant and cyclic voltammetry carried out under anaerobic conditions. The voltammograms showed a single redox couple with a midpoint potential of -0.42 V (vs. Ag/AgCl). The peak current was linear with scan rates up to 150 mV, indicating that the protein was adsorbed on the electrode surface. Cyclic voltammetry was also carried out with enzyme immobilised on gold electrode after functionalisation with sulfhydryl-reactive cross-linkers showing similar measured redox potential. Finally, the ability of the immobilised enzyme to catalyse the turn over of two important drugs, benzydamine and Tamoxifen was followed electrochemically and Km values measured. Conclusion: The findings constitute the first step towards the creation of electrochemical sensors for HTS of drug metabolism.

Drug-drug interactions of cytochrome P450 3A4 studied by electrochemistry S. Sadeghi, S. Ferreo, G. DiNardo and G. Gilardi Department of Human and Animal Biology, University of Turin, Turin, ITALY Introduction: Mammalian cytochrome P450s are the key enzymes involved in Phase I metabolism of clinically relevant drugs and contribute to the metabolism of a huge number of xenobiotics. Cytochrome P4503A4 (CYP3A4) is the major cytochrome P450 in human liver responsible for more than 60% of drug turn over. Whenever two drugs are administered together, the possibility of drug interactions exists if both drugs are metabolised by the same P450 enzyme. It is therefore very important to use in vitro screening methods to evaluate potential drug-drug interactions. Here we report the first such electrochemical interaction measurement for CYP3A4. Methods: Redox potential, drug metabolism and IC50 measurements were carried out using electrochemical techniques of cyclic voltammetry (under anaerobic conditions) and chronoamperometry. Results: The recombinant CYP3A4 was immobilised on glassy carbon electrode (GC) using a polyelectrolyte polymer (diallyldimethylammonium chloride) and cyclic voltammetry carried out under anaerobic conditions. The voltammograms showed a single redox couple with a midpoint potential of -0.097 V (vs. NHE). The peak current was linear with scan rates up to 150 mV/s -1 , indicating that the protein was adsorbed on the electrode surface. Chronoamperometric experiments were then carried out in the presence of erythromycin as a substrate. Three different known inhibitors of CYP3A4 namely ketocanozole, cimetidine and diclofenac were then tested and their respective IC50s measured. The latter data are in good agreement with microsomal data. Conclusion: The findings constitute the first step towards the creation of an in vitro electrochemical platform for drug-drug interaction measurements. Introduction: Molecular biomimetics approaches have been used to select short peptides that can bind to inorganic target material specifically and with high affinity. These material-specific peptides, called GEPIs, can be utilized as molecular linkers for biological and material applications. Biomolecules can be linked with any material via peptidebiomolecule constructs in which peptide is the inorganic-specific moiety and the protein is the biofunctional member. Methods: pETBlue-2 expression system was chosen for the construct formation. The construct contains QBP-1 which was generated and fused to the N terminal of the functional proteins, alkaline phosphatase (AP) and GFPuv. Tuner (DE3) placI cells were used for the expression of tagged proteins, and expressed proteins were purified using silica columns. For comparison, 6xHis tag was fused to N-terminal of functional proteins and expressed proteins were purified using Ni-NTA column chromatography. Results: The vector constructs were successfully formed for each functional protein and transformed into Tuner DE3 pLacI host strains for the expression of the proteins. The expressed tagged proteins were passed through silica column to show the applicability of the QBP1 as tag peptide and the results were compared to that of the same functional proteins having 6xHis as tag peptide at their N terminals. Conclusion: Our designed vector systems make available the use of QBP-1 as the tag peptide for the isolation of any desired protein through silica columns. We here show one of the possible applications of GEPI peptides in the medical and nanobiotechnological applications, which is here an alternative tagging system for the purification purposes.

Construction and expression of a bi-functional peptide by using genetic engineering methods for bio-nanotechnologies Introduction: Combinatorial biology-based molecular libraries, phage display or cell surface display libraries, are increasingly becoming one of the major methods to obtain inorganic specific peptides. Selected peptides can be further engineered, modified through genetic engineering methods and synthesized to utilize as molecular tools with controlled functionalities. Peptides selected by the display libraries are generally short ones, between 7-14 amino acids. To increase binding affinity, tandem repeats of the sequence can be generated by genetic engineering. Repeated structures of short peptides may show a better affinity and provide a controlled assembly process. The increased surface recognition sites could result in an enhancement in binding strength and/or specificity of the peptide (Sarikaya et al., Nat. Mater. 2003; 2: 577-585). Methods: In this study, three tandem repeats of both quartz-binding peptide (QBP1-PPPWLPYMPPWS) and gold-binding peptide (GBP1-MHGKTQATSGTIQS) was used to develop the bi-functional construct. Two peptide sequences were linked by a flexible linker, composed of 10 amino acids (GGGGSGGGGT). For the expression of the peptide, an E. coli-based expression system TAGZyme pQE-2 is used. Results: The vector was constructed and the bi-functional peptide was expressed in LB medium. Expressed proteins were purified by using Immobilized Metal Affinity Chromatography. Both functionalities of the peptide are conserved after linking two well-characterized peptides. Conclusions: The genetically engineered in vivo construct is expected to be a versatile molecular tool for bionanotechnology applications. Bifunctional peptide can be used to immobilize gold nanoparticles on silica for sensing applications. Introduction: Controlled drug release from the biodegradable matrix provides planned delivery of the cytotoxic substances to the specific places in the organism. To ensure the correct delivery, the biodegradable matrix should possess certain unique properties, such as high mechanical strength, elasticity, biocompatibility and low immunogenicity. Spidroins I and II (SPI, SPII)-two components of the spider dragline silk -are of the specific interest because they obtain all the listed properties. We studied the nanostructure of the fibers and films obtained from the recombinant analogs of SPI and SPII. Methods: We expressed both proteins in P.pastoris and purified them to 95% purity using FPLC. The concentrated protein solution was rapidly dehydrated to produce 'as spun' fiber that was subsequently manually stretched. The films with the various pore sizes were produced using shift, casting and electrospinning. Morphology of fibers and films was observed using electron and atomic force microscopy. Results: We observed that both proteins spontaneously form nanofibrils in solution. TEM and negative contrasting revealed that the single nanofibril exhibits the amyloid-like helical structure. The electrospun nanofibrils possess a somewhat similar morphology. Stretching of the 'as spun' fiber leads to the rebuilding of its sponge-like matrix and to the appearance of microfibrils. The mechanical properties of the stretched fibers depend on the properties of the microfibrils, comprising the fiber. Conclusions: The obtained results dissect the relationship between the structure and mechanical properties of artificial fibers and films that could be further used as matrixes for controlled drug release or stem cells cultivation. Introduction: Our research focuses on the in vitro antimicrobial activity evaluation of some new original compounds, 2-phenylthiomethylbenzoic acid amides. Methods: The aforementioned amides were obtained in four stages: the 2-phenylthiomethyl-benzoic R1-substituted acids synthesis, their acid chlorides, synthesis of the N-(2-diethylaminoethyl)-anilines, and finally the new amidic compounds. The qualitative screening of the susceptibility spectra of various microbial strains to these compounds was performed by three adaptated diffusion methods: paper filter disk impregnation with tested substances solutions, the disposal of tested solutions in agar wells and the spotting of tested solutions on solid medium seeded with microbial inoculums. The in vitro antimicrobial testing was performed by binary microdilution method, in 96 multi-well plates, in order to establish the minimal inhibitory concentration (MIC), against Gram-positive: Staphylococcus (S.) aureus, Bacillus (B.) subtilis, Gram-negative: Pseudomonas (P.) aeruginosa, Escherichia (E.) coli, Klebsiella (K.) pneumoniae), as well as Candida (C.) albicans and Aspergillus (A.) niger, using both reference and clinical, multidrug resistant strains. Results: New original amides were synthesized, their physico-chemical properties, structures and purity were determined by means of elemental analysis, TLC methods, IR, 1 H-NMR and 13 C-NMR spectra. The compounds were microbiologically investigated against reference strains, Gram-negative and positive clinical strains, also fungus. All the compounds showed valuable antimicrobiolal effects. Conclusion: The new amides of 2-phenylthiomethyl-benzoic R1-substituted acids were tested for their in vitro antimicrobial activity proving to be active against the reference strains and also against Gram-negative clinical strains; all of them exhibit antifungal activity.

The human-kallikrein related peptidase expression pattern in colorectal cancer Kallikrein-related peptidases (KLKs) constitute a large multigene family of serine proteases. In this study we examined the mRNA expression of KLK6 and 10 in malignant and normal colorectal tissues using RT-PCR method. Additionally we quantified the differential expression of 9 KLKs using ELISA. Results: KLK6 was expressed significantly higher in the cancer samples (P = 0.001). Tumors of advanced TNM and Duke's stage showed high KLK10 expression (P = 0.028, P = 0.08 respectively). Nearly all KLKs highly effectively separated cancer from normal tissue (P < 0.001). KLK5, KLK6, KLK10, KLK13, and KLK14 were univariately significantly (P £ 0.05) associated with the OS. For the DFS, none of the KLK markers was significant at 0.05 level. An increment in AUC under the time-dependent ROC curve was only observed at year 1 for the DFS. But the increment was less obvious than the OS. This study showed a clear differentiation in expression of the most of markers measured between tumour and its paired normal tissue. KLK6 and 10 seem to be involved in colorectal cancer progression and may serve as new markers of unfavorable prognosis.

Nanosensing measurement using novel monoclonal antibodies generated by B-cell targeting Introduction: Many practical methods have been shown for sensitive measurement of various functional materials. Among them, the usage of monoclonal antibodies (mAbs) is one of the promising methods. However, it poses difficult to raise aimed mAbs with high affinity and specificity. To address this problem, we developed a new technology to selectively generate specific mAbs with high affinity based on antigenbased B-cell targeting. Methods: Based on B-cell targeting, mAbs were generated against a di-2-ethylhexyl phthalate (DEHP) that is suspected to be one of the endocrine disruptors. Firstly, the sensitized B lymphocytes were selected by DEHP-streptavidin conjugates, and combined with biotinylated myeloma cells by strong and specific interaction of streptavidin to the tethered biotin. The B lymphocyte and myeloma cell complexes were then selectively fused by electrical pulses. Results: The hybridoma cell (2F4) producing high-affinity mAb against DEHP was successfully obtained as evidenced by Kd values between 10 -9 M and 10 -11 M. It is first shown that the anti-DEHP mAb was directly fixed on a 96-well assay plate for quantitative analysis of DEHP. Interestingly, the fixation of the anti-DEHP mAb to magnetic beads markedly enhanced its sensitivity by 10-100 times, realizing the detection of DEHP at 0.01-0.001 mg/L. Although 2F4 showed relatively wide cross-reactivities with other phthalate esters, we have lately succeeded in generating highly specific mAb against DEHP. Conclusions: We could succeed in selective production of novel mAbs against lower antigenic compound by B-cell targeting. This new technology may be applicable for highly sensitive measurement of biomarkers such as cancer and other diseases. Recent advance in biotechnology enables us to find the peptides with affinity for nonbiological materials and with function of mineralizing inorganic materials. The use of the functional peptides is attracting a growing interest for bottom-up fabrication approaches of nanoscale devise. Zinc oxide (ZnO), a semiconductor with a wide direct band gap, possess unique optical, acoustic, and electronic properties, so that it is one of most widely studied metal oxides for solar cells, ultra violet nanolaser, blue light-emitting diode and so on. This wide variety of applications requires various fabrications of morphologically and functionally distinct ZnO nanostructures. Here, the peptides which are selected from the phage-displayed peptide library with a 12-mer on the surface, can bind the ZnO particle but not other metal oxides particle, and further, the ZnO-binding peptides play an important role on the crystal growth of ZnO in its synthesis. We describe the biomimetic ZnO synthesis using the artificial peptide with affinity for ZnO.

Study of osteoblast interaction with iron oxide nanoparticles coated with dextrin in cell culture Introduction: Nanoscale magnetic structure offer great potential for advancements in electronics, optoelectronics, magnetic storage and biomedical applications. The benefit of such materials is that they could be designed with biocompatible surface stabilizers for new biomedical applications (retinal detachment therapy, cell separation methods and radioactive therapies). In this study, cell viability/citotoxicity was determined by the interaction between primary osteoblast cell line and bioceramics. Methods: MTT test was used to assess cell viability of the osteoblasts. Cells were incubated with D15S bioceramic at 1% concentration for 2, 4, 12 and 24 hours. The viability cell number is directly proportional to the production of formazan. Results: There is a general tendency of depletion in osteoblast number which is quite normal after 2 hours of exposure to bioceramic. This effect might be due to cells adaptation following interaction with nanostructure. After 12 and 24 hours of interaction between osteoblasts and bioceramic, we detected an increase in cell growth relating to control. The cell viability of the osteoblasts in contact with biocera-mic support presented in time a linear increase trend between 2 hours (56.14%) and 24 hours (126.32%) comparing to control. Conclusions: According to our results, cells' incubation in the presence of iron oxide coated with dextrin can modify parameters of cell growing (augmentation or depletion of viability relating to control), exposure period (2, 4, 12 and 24 hours) representing an significant factor in osteoblasts' growing. Hydroxyapatite (HA) can be found in different sizes and shapes in mammalian hard tissues as the main component. In the formation and growth of hydroxyapatite crystals, huge number of proteins play important roles. Phage or bacterial display technology is a practical approach for screening a large number of random peptide for any inorganic surface. Peptides that can bind inorganic substances specifically and with high affinity have been identified and characterized previously by our group, through the usage of this technology [1] . Among them, hydroxyapatite-binding heptapeptides that offer a route for controlling biomineral formation was used in this study [2] . Fluorescent proteins which were recently developed for different spectral properties and enhanced brightness provide advantage to label non-living and living subjects. One strong and one weak-HA binding peptide which have seven amino acids Cys-Cys constrained sequence, HABP1 and HABP2 respectively, and also linear (without Cys-Cys) form of HABP1 were fused with Green Fluorescent Protein (GFPuv) which works as a fluorescent prob. These bifunctional proteins have been expressed in E. coli and the binding affinities of the purified proteins on HA powder were compered by fluorescence microscopy. It was observed that HABP1-GFPuv fusion protein has a much higher affinity to HA powder comparing with HABP2 and the linear constructs. Our bifunctional constructs present fluorescent sensory systems for real time monitoring of controlled in vivo and in vitro biomineralization which may be utilized for hard tissue engineering studies. Introduction: Bacillus subtilis forms the source of most of the enzymes that are of industrial importance. Amylase has an important place among these enzymes. There have been a lot of studies related to the isolation and characterisation of Bacillus amylase. Amylases are enzymes which hdrolyse starch molecules to give diverse products including dextrins and progressively smaller polymers composed of glucose units. These enzymes have a great significance with extensive biotechnological applications in bread and baking, food, textile, and paper industries. Material and Method: The thermotolerant bacterium was isolated from the Ç ermik hot spring (Southeastern Turkey) and identified as Bacillus subtilis. B. subtilis was produced in various media, in which the growth of bacteria and the amylase activity was the best was determined. a-Amylase activity was determined according to Bernfeld method. SDS-PAGE (Polyacryilamide gel electrophoresis) of these samples was carried out. Optimum temperature and pH were determined. The effects of glucose, EDTA, Ca 2+ and some heavy metals (Hg, Mn, Cu, Zn) on a-amylase activity were investigated. Results: It was found that the best growth and the best activity occurred in starch added LB (Luria Broth) medium. The optimum temperature was determined to be as 65˚C, and the optimum pH as 7.0 for the enzyme. It was seen that Ca 2+ increased the enzyme activity, and increasing glucose and EDTA concentrations caused enzyme inhibition. Discussion: The results we have obtained so far from our study are compatible with the findings of other researchers.

Effect of aged garlic extract on glycaemia and oxidative stress in Saudi Arabian type 2 diabetics Introduction: Diabetic patients with hyperglycaemia show oxidative stress and increased formation of advanced glycation endproducts (AGEs), which increases their susceptibility to complications. Aged garlic extract has antioxidant properties and prevents the formation of AGEs in vitro. This study investigated the effects of dietary intervention with aged garlic extract on glycaemia and oxidative stress in 48 diabetics. Methods: Blood was collected from patients on recruitment, after 1 month and then monthly following an intake of 1000 mg of aged garlic extract daily over 3 months. Samples were analysed for glucose and glycated haemoglobin using automated analyses. Low molecular weight AGEs were measured by a fluorimetric method. Malondialdehyde concentrations were determined using an accepted colorimetric method. Results: Intervention with aged garlic extract did not effect blood glucose or glycated haemoglobin concentrations. However, aged garlic intake did reduce levels of serum AGEs although this was not significant over the 3 month period. There was a significant reduction of serum malondialdehyde after only 1 month of therapy. Conclusion: Serum malondialdehyde, an indicator of oxidative stress, was reduced following intake of aged garlic extract, in Saudi Arabian type 2 diabetic patients. Further investigations are needed to assess the reliability of these data. Introduction: Natural compounds, usually occurring food and, especially, in food supplements, may act as modulators of the expression of key protein in drug transport and metabolization may be used in a controlled fashion in order to improve therapeutic efficacy and reduce adverse effects of medicines. Two potential areas of application were investigated: the influence on long term medication (addressing diabetes, inflammatory or cardiovascular diseases) and on intensive (aggressive) medication (as in the cases of anticancer or antimicrobial therapies). Methods: In vivo and in vitro models were used to investigate both changes in levels of expression of hepatic metabolization enzymes, as well as effects on drugs metabolization and drug toxicity. Farmacokinetic effects were estimated using LC-MS, for 5 -fluorouracil, indometacin and glibenclamide. Results: Controlled administration of plant poly-phenol complexes was demonstrated to modify the level of expression and activity of members of the cytochrome family. Relevant changes in drug toxicity and pharmacokinetics were found in the case of 5 FU, and changes were also associated with changes in the expression of liver metabolizing enzymes.

Conclusions: The use of polyphenol complexes (usually used as food supplements and as therapeutic adjuvants) represents also a simplified model, for the potential changes induced by variations in nutritional intake of such products. Based on the transient changes in gene expression, a strategy to optimize the 'pharmacogenomic' profile of the patients may be further developed, thus ensuring improved risk-benefit ratio for therapies. Introduction: The aim of this study was to characterize the regulation of cytosolic alanine aminotransferase (cALT) expression in liver of gilthead sea bream (Sparus aurata) under conditions associated with enhanced gluconeogenesis. Methods: RT and RACE PCR assays allowed us to isolate a novel ALT isozyme (cALT2) generated from alternative splicing of cALT gene in S. aurata. Cytosolic localization of cALT2 was confirmed in HEK293 cells transfected with constructs expressing cALT2 as a C-terminal fusion with the enhanced green fluorescent protein. To address the molecular functions of cALT1 and cALT2 in liver of S. aurata, we examined tissue distribution, expression of piscine cALT isozymes in Saccharomyces cerevisiae, and regulation of hepatic cALT1 and cALT2 expression in various metabolic conditions. Results: Expression of S. aurata cALT isozymes in yeast indicate that cALT2 is more efficient in catalysing the conversion of L-alanine and 2-oxoglutarate to pyruvate and L-glutamate than cALT1. In S. aurata, starvation increased cALT2 expression and decreased cALT1 mRNA levels in liver. Opposite effects were found in regularly-fed fish at postprandial time 4-8 hours, and after 6 hours of treatment with glucose or insulin. Conclusions: We conclude that cALT2 expression is associated with increased gluconeogenesis in liver, while cALT1 is predominant during postprandial utilization of dietary nutrients. Since increased ALT activity is associated with insulin resistance and the development of type 2 diabetes, further research should focus on the usefulness of hepatic cALT2 expression as a marker in the prognosis of diabetes. Introduction: Conjugated Linoleic Acid consists of a group of isomers of linoleic acid. The biological effects are associated with anti carcinogenesis, diabetes and obesity. However, the most studied aspects regrd the alterations in lipid metabolism. Changes in lipid metabolism are closely related with associated protein gene expression modification, which is in many cases, mediated by mechanisms dependent on the activation of peroxisome proliferator activated receptors (PPARs). PPARa is highly expressed in the skeletal muscle and plays an important role in the regulation of fatty acid uptake, activation, mitochondrial transport and b-oxidation. Our purpose was to verify the relationship between CLA supplementation and gene expression of some important proteins related to lipid metabolism in the muscle and the possible role of PPARa Introduction: Olive leaves are well known for their effect on metabolism in particular as a traditional anti-diabetic herbal drug. Until now these properties are only attributed to oleuropein that is the major component of olive leaves. The present study was designed to investigate the effects of the olive leaves extract (OLE), olive polyphenolic fraction (PF) and oleuropein (OL) on free radical metabolism and insulin secretion in insulinoma cell line. Methods: Cell viability was investigated withMTT colorimetric assay after a short time (45 minute) H 2 O 2 (0-0.15 mM) exposure. Intracellular reactive oxygen species (ROS) was measured using DCF-DA that is converted to the fluorescent dichlorofluorescein. Insulin release was evaluated with ELISA. Results: After 0.035 mM H 2 O 2 exposure, cell viability was measured as LD 50 50.8 ± 9.6% of control cells. H 2 O 2 injury in INS-1E cells was accompanied by increase of intracellular ROS production (308.1 ± 56.7% of control). All tested samples showed a significant decrease in ROS value with a maximum effect at the highest concentration used, 226.1 ± 24.1, 233.0 ± 14.5, 238.9 ± 16.7% of control for OL, OLE and PF, respectively. The H 2 O 2 -stress led to decrease in insulin release by 44.1 ± 6.1% of control cells. Incubation with OL and PF resulted in the stimulation of insulin release by 92.6 ± 1.0%, 228.9 ± 30.0% of control cells respectively. However OLE has no effect on insulin secretion. Conclusions: The results of this study suggest that olive extracts can protect the function of insulinoma cells against H 2 O 2 -induced oxidative damage.

Intestinal epithelial cell production of beta defensin-2 by catalytic abzymes from breast milk L. Thomas, G. Barrera, R. Portillo, A. Mijares, M. Rocafull and J. del Castillo Instituto Venezolano de Investigaciones Cientificas, Artos de Pipe, VENEZUELA Background: Defensins are small cysteine-rich cationic proteins with antibacterial, antifungal and antiviral propierties. They are produced by different cells incluiding enterocytes. Breast milk contains catalytic antibodies (abzymes) with proteolytic activity. It has been demonstrated that such abzymes may regulate signal transduction in enterocytes by cleaving and activating the protease activated receptor-2 (PAR-2). However, the physiological rol of these proteolytic abzymes have been not determined. In this work we investigated if the abzymes secreted in breast milk may induce the intestinal expresio´n of beta defensin-2 (BD2) through PAR-2 activation. Methods: Cultures of human intestinal epithelial cell line HT-29 were treated with LPS (1 lg/ml), trypsin (10 nM), and F(ab)2-fragments (5 lg/ml) obtained from proteolytic sIgA alone or mixed with the protease inhibitor PMSF (1 lg/ml), additionally the catalytic F(ab)2-fragments were assayed on HT-29 cells preincubated with B. pertussis toxin. As negative control, the cells were incubated with F(ab)2-fragments (5 lg/ml) obtained from non-proteolytic seric IgG. The treatments were performed by 24, 48 and 72 hours. BD2 secreted in supernatans was evaluated by ELISA. The mRNA expression was evaluated by RT-PCR. Four independient experiments were performed. Results: We found a significant F(ab)2-sIgA effect on the expression of BD2 (P < 0.05), B. Pertussis toxins and PMSF having an inhibitory effect compared to F(ab)2-IgG and media alone. LPS and trypsin increased the BD2, as had been reported. Conclusions: F(ab)2-fragments from sIgA seem to modulate the expression of BD2 in intestinal epithelial cells. It could represent a new protective mechanism associated with lactation.

The anti-apoptotic effect of deoxynivalenolinduced oxidative stress in Hek293 cell line Introduction: Deoxynivalenol (DON), a trichothecene produced by Fusarium species is one of the most common mycotoxins, food and feed contaminants. It is a potent inhibitor of eukaryotic protein synthesis and exerts cytotoxic effects in a time and concentration depending manner on some types of monolayer kidney cells. We investigated oxidative status parameters, p53 and caspase 3 expression in the Hek293 cell line after DON mediated induction of oxidative stress. Methods: Biochemical parameters were assessed after 6, 12 and 24 hours of cell treatment with 2.5 lM DON. The superoxide ion, GSH, lipid peroxidation level and superoxide dismutase, catalase and glutathione peroxidase specific activities were assayed. The p53 and caspase 3 protein expressions were evaluated by immunohistochemical methods. Results: The variation of the biochemical parameters and gene expression modulation after 6, 12 and 24 hour highlighted: i) the correlation of the profile of superoxide ion with the SOD activity, ii) the lipid peroxidation pattern identical with that of GPX activity, iii) slow initial decrease of GSH and later increase with 36%, and iv) the abolishment of p53 and caspase 3 expression starting with 6 hours of intoxication. Betacyanins are red-violet plant pigments widely used as food colorants under the E-162 code. They are highly soluble in water and non toxic even at high concentrations, being used for coloring meat products, ice cream, jam and fruit conserves. Furthermore interest in these natural pigments has grown since their radical scavenging capacity was characterized. In the present work, the transformation of the structural unit of betacyanins, betanidin, by tyrosinase (EC 1.14.18.1) is studied, as well as other parameters affecting the pigment stability. The enzymatic reaction was characterized and followed by HPLC and spectrophotometry. Betalains were obtained from natural sources and purified by anionic exchange chromatography. Pigments were identified by HPLC with PDA and ESI-MS detection. Tyrosinase showed activity against betanidin, and yielded an unique product identified as a stable quinone, revealing a K m = 0.66 mM. pH dependence on betanidin degradation presented a first-order degradation kinetics, and the corresponding degradation constants, k d , were calculated. Stability was enhanced in the pH range 3.0-6.0, observing a quick color loss at higher values. The presence of oxidizing agents and high ionic strength negatively affected the stability of betanidin solutions. Ascorbic acid preserves the pigment integrity in natural sources. Since color plays a key role in consumer acceptance of food it is necessary to understand the physical parameters and biochemical steps involved in pigments stability. Betanidin degradation kinetics demonstrated that pH values over 6.0 and high ionic strength reduce the pigment stability, and tyrosinase plays a major role in the metabolism of betacyanins.

T. Nomikos, E. Fragopoulou, H. C. Karantonis and S. Antonopoulou Department of Science of Nutrition-Dietetics, Harokopio University, Athens, GREECE Introduction: The mesangium plays an important regulatory role in inflammation-mediated glomerular diseases. Studies have revealed that the consumption of olive oil could be beneficial for human health. Aim of this study is the elucidation of the inflammatory processes induced by IL-1b on HMCs and the modulation of its actions by OPL. Methods: An established HMC line was used. The cells were incubated with IL-1b (1-5 ng/ml) in the absence or presence of OPL (0.002-0.2 microg/ml) and a) the levels of PAF, IL-6, MCP-1 b) the activity of PAF's biosynthetic enzymes and c) the levels of cytoplasmic GSH were measured. Results: IL-1b induces a transient decrease of GSH and elevation of intracellular PAF levels within 1 hour, while IL-6 and MCP-1 secretion was observed at longer incubation time points. The activation of PAF biosynthetic enzymes precedes the increase of PAF levels. Pre-incubation of HMC with OPL resulted in inhibition of IL-1b-induced PAF increase while co-incubation of IL-1b with OPL slightly attenuate the activation of PAF synthesis. Co-incubation of OPL with IL-1b resulted in the enhancement of IL-6 secretion while pre-incubation of the cells with OPL resulted in the partial inhibition of IL-6 secretion and does not affect MCP-1 secretion. Conclusions: Intracellular PAF, IL-6 and MCP-1 mediate IL-1b actions on HMC. OPL could modulate IL-1b actions on HMC and may confer protection to renal inflammation.

Fat should be removed before microbiological folate assay in foods containing high levels of fat Microbiological assay using Lactobacillus casei has been widely used for the determination of total folate content in foods. As fats and fatty acids were known as growth factors of Lactobacillus bacteria, it is necessary to confirm whether fat should be removed before microbiological assay in foods containing high levels of fat. We compared folate contents in 19 foods containing various amounts of fat (0-33% by weight) before and after removing fat. Fat was removed by adding hexane to the weighed food samples, mixing well, centrifuging, decanting off hexane/fat supernatant, and drying in oven to evaporate hexane. Trienzyme (a-amylase, protease, folate conjugase) was treated to food samples for folate extraction before and after removing fat, and folate content was measured by microbiological assay using L. casei. Folate contents in 6 food samples containing less than 10% fat were not much different before and after removing fat. The decrease rates ranged from 0% to 4.4%. But folate contents in 13 food samples containing more than 10% fat such as fried chicken, French fries, and pork (belly) decreased from 8.1 to 31.0% after removing fat from the food samples. The decrease rates of folate content after removing fat were significantly correlated with the percentages of fat in foods. Our results suggest that fat should be removed before microbiological folate assay in foods containing more than 10% of fat.

Co-super-induction system of lipase and n-alkane emulsifying protein from Pseudomonas-like bacteria M. Ishizuka 1 and K. Ushio 2 1 Chuo University, Tokyo, JAPAN, 2 Niihama National College of Technology, Niihama, JAPAN Stable and high stereo-selective lipase and esterase have attracted much attention from viewpoints of industrial usage. In the course of our studies searching for efficient inducers for lipase, among various additives, we have found that fatty alcohols act as the most effective super-inducers for induction of lipase production by several Pseudomonas-like bacteria. The addition of fatty alcohols brought about more than several hundred-fold enhancement of the lipase activity compared to the case with no additive. This means several dozen-fold enhancement of lipase activity compared with olive oil grown case. Gram per litter lipase production has been capable. We also found that when several Pseudomonas-like bacteria were grown on fatty alcohols, not only lipases but a large amount of an extracellular 15 kD protein (P15) was strongly induced. This induction of P15 seems to be simultaneous with that of lipases. P15 is a n-alkane emulsifying protein, activator-protein of n-alkane oxidation and utilization system. Productions of those two proteins are possibly under the same regulation system. Under oxygenlimited condition, an extracellular 17 kD protein was induced instead of P15. Expressed lipases, deduced from the cloned nucleotide sequences, have high homology among Pseudomonas-like lipases including lactonizing lipase. Lactones can be synthesized from the corresponding omega-hydroxy fatty acids, an inducer of cutinase in a fungal system, and above strains possess lactone-specific esterase. Although omega-hydroxy palmitic acid did not induce lipases from these bacteria, these lipases induced by fatty alcohols seem to be directed to cutine degradations. The genus Allium, has potent antitumor activity against many types of tumors, however, its molecular target and mechanism of anti proliferative activity is not clear. The present study aims at defining the antimicrotubule activities of A. hirtifolium which belongs to genus Allium. The effects of A. hirtifolium extracts and it's Allicin on the proliferation of HeLa (cervical cancer), MCF7 (human,caucasion,breast,adenocarcinoma) and L929 (mouse,C3H/An,connective) cell lines and also on nerve cell microtubules has been examined in this study. A. hirtifolium showed growth inhibitory activity against HeLa and MCF7 cells with IC (50) value of 20 and 24 lg/ml for 72 hour. Cell growth inhibition was measured by MTT after treatment with A. hirtifolium. Microtubule protein (MTP) was prepared from sheep brain through two cycles of polymerizatinon-depolymerization in a high molarities buffer. Inhibition of MTP polymerization induced by A .hirtifolium was determined by a turbidity measurement and a sedimentation assay. A. hirtifolium was tested for its ability to bind to tubulin as a ligand through turbidimetry assay and changed the time and form of MTP polymerization then investigated by Transion Electron microscopy. A. hirtifolium clearly showed a cell growth inhibition on several human cancer cell lines at non-toxic concentration (lower than 1 mg/ml).With respect to the cell lines studied,IC50 values varied from 20 in Hela to 24 lg in MCF-7 . The necessary concentration of A. hirtifolium to inhibit the assembly of microtubule proteins by 50% was 1.2 mg/ml, while an inhibition higher than 80% was observed in the presence of 4 mg/ml A. hirtifolium.

Diversity in genes responsible for lifestyle-related diseases in the Asia-Pacific region Nonenzymatic glycation results in the formation of Advanced Glycation End products (AGEs). AGEs have been implicated in diabetes related complications such as retinopathies, nephropathies and neuropathies. Glycation induces an alpha helix to beta-sheet transition in secondary structure of proteins. These cross beta-structures are recognized by receptor of AGE (RAGE) on the surface of some cells, such as microglial cells and induce inflammation. In an attempt to find a natural pharmaceutical, which might inhibit glycation of proteins alpha helix to beta-sheet transition, a water-soluble fraction was obtained from Melissa officinalis which has shown to have clinical benefit. We incubated Bovine Serum Albumin (BSA) with glucose in the presence and absence of Melissa officinalis extract and the effects of this fraction on the formation of advanced glycation end-products and structure of protein were studied. Introduction: Iodine is essential for normal growth, mental development, and survival of infants. The main source of iodine for breastfeeding infants is the iodine found in human milk.

Objective: This study was carried out to evaluate iodine sufficiency in lactating mothers in Iran and effect of breast milk iodine on infant growth. Methods and Materials: A total 130 healthy and non-smoker lactating mothers and their singletons, full-term, with appropriate weight for gestational age infants (71F, 59M) participated in the study.The women completed a 3 day dietary record, and iodine content of consumed food was determined. Infants' anthropometric data were checked monthly from one month to 6 month old. At the first admission, 10 ml of each mother's milk, for the measurement of iodine concentrations, were collected, and frozen immediately at -20˚C until analysis was carried out. Data were analyzed by SPSS 11.5 software.

Results: The mean (±SD) of iodine content of mothers' nutrition were 317 lg/day (±42.81), and the mean (±SD) of the breast milk iodine concentration were 183.5 lg/l (±178.3). Breast milk iodine concentration was less than 50 lg/l in 30% of subjectes. No statistically significant difference was observed between infant growth and iodine breast milk, except for head circumference at the age of 1-3 months (P < 0.05).We did not find any relationship between of the iodine breast milk level and mothers' nutrition iodine level. Introduction: Alcohol abuse is a main cause of liver fibrosis and cirrhosis. Therefore, some health care food may be use as therapeutic (or preventive) reagents for alcoholic liver disease. The hard clam (Corbicular fluminea) was thought have hepatoprotective effect for several years ago in Taiwan, but its function has not validate yet. The intragastric ethanol infusion Tsukamoto-French rat model has proved that alcohol induced progressive alcoholic liver injury including fatty liver, alcoholic hepatitis and liver fibrosis and cirrhosis that can be used for the study of alcoholic liver disease. We will investigate how the extracts of clam to improve hepatic damage in alcoholic liver disease by Tsukamoto-French rat model. Methods: The clam extracts were derived from solution of heating hard clam. By using Tsukamoto-French rat model, first fed ethanol (8 g/kg/day) to rat for 12 weeks, then fed extracts of clam (2 ml/day) for 2 weeks. Pathological diagnosis of liver and hematology teat was investigated by sacrificed rat. Results: In histochemical analysis, the extracts of clam could improve the level of pathology in alcoholic liver. Furthermore, the hematologic data showed that the extracts of clam could recover the white blood cells on alcohol-induced liver injury in rat.

The results of functionality assessment suggest that the extracts of clam could have hepatoprotective effect and promote immunity in alcoholic liver disease. Results: The enzyme is not effected by concentrations of glucose (1-128 mM), while it is greatly inhibited by its reaction product galactose (tested between 1-128 mM). Linewever-Burk plot analysis showed that galactose has non-competative inhibition effect on b-galactosidase. The purified enzyme is activated by Mg 2+ (13% at 20 mM), while inhibited by Ca +2 (33% at 10 mM), Zn +2 (86% at 8 mM) and Cu +2 (87% at 4 mM). Moreover, b-mercaptoethanol and DDT at 8 mM were found to increase the enzyme activity by 22 and 20%, respectively. b-galactosidase is slightly inhibited by EDTA (15% at 4 mM) and iodoacetamide (15% at 10 mM), while the enzyme was completely inhibited by p-chloromercuribenzoic acid (91% at 1 mM; 99.7% at 2 mM) and PMSF (19.8% at 1 mM; 71.9% at 10 mM). The enzyme was not affected by N-ethylmaleimide and 1-10-phenanthroline.

Conclusions: The present study shows that various inhibitors and activators have effects on enzyme activity, which may clarify the nature of the catalytic machinery of family GH42. Introduction: Previously our research group suggested a role of grapeseed derived procyanidins (GSPE) on glucidic metabolism. GSPE had an antihyperglycemic effect in streptozotocin-induced diabetic rats [1] and after 15 hours of treatment with GSPE, there was no resistance to the stimulation of glucose uptake in adipocytes in culture by an acute dose of insulin [2] . Now, we analyze the GSPE effects on an insulinresistant profile induced by a cafeteria diet. Methods: We fed female Wistar rats with a cafeteria diet during 13 weeks. Then animals were divided in two groups: cafeteria diet rats and cafeteria rats treated with a daily intake of 20 mg of GSPE/day during 10 days. A week before killing animals we did an intraperitoneally glucose tolerance test (IPGTT). At the end of the treatments we sacrificed the animals to obtain plasma and tissue samples. All the procedures were approved by the Experimental Animals Committee of our University.

Results: After IPGTT we showed that this GSPE treatment did not change significantly the impaired glucose tolerance due to an insulin resistance on muscle an adipose tissue of cafeteria diet. But GSPE modified some mRNA levels that suggested an improvement of this situation. Muscle HKII mRNA was normalized by GSPE. The liver was, in fact, very much insulin responsible since glucose-6-phosphatase was down-regulated and GK up-regulated. And mesenteric white adipose tissue GLUT-4 mRNA levels remained lower than control but higher than cafeteria group. Conclusion: This GSPE dose improves muscle and mesenteric white adipose resistance and also favours liver glucose uptake. The mRNA changes observed were not enough to correct glucose tolerance on these animals but they would be enough to justify the lower HOMA index of these GSPE-treated animals on fasting situation. 

Role of glutaminergic-NO pathway in antinociceptive effect of ascorbic acid F. Nasirinezhad and S. Safarpour University of Medical Sciences, Tehran, IRAN Ascorbic acid (AA) is present in high concentration in nervous system and is believed to act as a neuromodulator and inhibits nitric oxide synthase which is important for pain induction in neuropathic pain models. This study investigates the involvement of nitric oxide (NO) pathway in the analgesic effect of ascorbic acid in CCI model of neuropathic rats. Neuropathic pain induced by chronic constriction injury of sciatic nerve. Paw withdrawal thresholds were assessed by radiant heat, Randall Selitto test and Von Frey filaments. To investigate the analgesic effect of ascorbic acid, 2 weeks after CCI ascorbate (1, 5 and 10 mg/kg) or saline was injected intraperituneally. Behavioral tests were done 15 and 30 minute after injection. In attempt to provide evidence concerning the involvement of glutaminergic-NO pathway in antinociceptive effect of ascorbic acid, on the second week after CCI, 30 minute after injection of saline or AA, animals received intraperituneally injection of glutamate (10 3 nmol), L-arginine (500 mg/kg) and L-NAME (20 mg/kg). Animals were tested 20 minute after injection. The results indicate that acute injection of 5 and 10 mg/kg but not 1 mg/kg of ascorbic acid increase mechanical and thermal threshold in the second week after CCI which confirm the dose dependent antinociceptive effect of AA that seems to be medicated through its interaction with glutaminergic-NO pathway.

Antithrombotic and antiatherogenic properties of fish polar lipids Caloric restriction (CR) is the only intervention known to be able to increase lifespan and delay the rate of aging in many species, including rodents. Previous studies in rodent liver, heart and skeletal muscle have revealed that CR reduces the generation of free radicals by mitochondria, prevents the age-associated decline in mitochondrial function and promotes mitochondrial biogenesis. However, little is known about the effects of CR on a tissue in which the mitochondria have no ATP-producing purpose but show a high degree of uncoupling, namely brown adipose tissue (BAT). Our objective was to determine which effects long-term CR has on the decline in BAT mitochondrial function in old rats. To tackle this aim, we analysed parameters of BAT thermogenic capacity in young (6 months) and old (24 months) male rats and compare these with that of age-matched old rats subjected to 12 months 40% CR. BAT composition, mitochondrial protein and mtDNA, COX activity, mitochondrial oxygen consumption and UCP1 content were analysed. CR induced resistance to lose total and mitochondrial protein, COX activity, and uncoupling capacity with advancing age. In summary, our study demonstrates that BAT thermogenic machinery is preserved by CR in old male rats.

The omega-3 index in the Turkish population .44 ± 0.78%, 81.53 ± 2.08%, and 6.10 ± 0.64% of total FAs, respectively. Omega-3 index and mono-polyunsaturated FAs were very low, whereas saturated FAs was very high in comparison with populations exposed to a diet rich in fish. Conclusion: We conclude that this FA composition and low omega-3 index arise from low fish consumption and the dietary habits in this region thus contributing to the high incidence of CHD in the Turkish population.

8C. Nutritional Biochemistry Introduction: Ascorbic acid (AA) is best known for its role as an essential nutrient in humans and other species. Because AA cannot be synthesized within the brain, high levels in this organ are achieved by specific uptake mechanisms. Two different isoforms of sodium-vitamin C cotransporters (SVCT1 and SVCT2) have been cloned. Both SVCT proteins mediate high affinity Na + -dependent L-ascorbic acid transport and are necessary for the uptake of vitamin C in many tissues. In the adult brain the expression of SVCT2 has been observed; however, there is no data regarding SVCT2 expression and function in post-natal brain.

Methods: We analyze SVCT2 expression in mouse post-natal developing brain using RT-PCR, Western blot, in situ hybridization and immunohistochemistry (confocal analysis). SVCT2wt-YFP and SVCT2sh-YFP (negative dominant) were used to overexpress the transporter in N2a cells to obtain functional data. Results: SVCT2 is highly expressed in the ventricular and subventricular area of the rat brain. During post-natal life (pn1-5 days) SVCT2 expression is concentrated in deeper brain cortex neurons. Using N2a cells and FRET analysis we demonstrated intracellular interaction between SVCT2wt and SVCT2sh. The kinetic parameters led us to observe that SVCT2sh inhibits AA uptake in N2a cells.

Conclusions: Our data demonstrates that brain cortex neurons, ventricular stem cells, neuroblastoma cells lines express the Na + -dependent L-ascorbic acid transporter SVCT2. The protein is first induced in deeper neurons of the brain cortex during post-natal life and the transporter activity may be relulated by SVCT2 hetero-oligomerization. Acknowledgements: Supported by CONICYT-World Bank ACT-02, CONICYT PhD Thesis Grant (KS). Introduction: In contrast to aqueous enzymology, biotransformations in water-organic solvent mixtures offer unique industrially attractive advantages. The removal of enzymes from their natural environs results in reduced reaction rates and low stabilities. The aim of our work was to modify the primary amino groups of a-chymotrypsin with different organic acid anhydrides (acetic, propionic, succinic, citraconic and phthalic) possessing different chemical structure to enhance the conformational stability of the enzyme and to compare the stabilities of the acylated enzyme forms. Methods: For the measurement of a-chymotrypsin activity, N-acetyl-L-tyrosine ethyl ester was used and the decrease in absorbance at 237 nm was followed. Near-UV CD measurements were performed with the enzyme samples previously incubated for 30 minute. The band intensities were expressed as mean residue ellipticity [Q] MR . Results: Acylation of a-chymotrypsin with different anhydrides increased its stablity in aqueous organic solvents. In 60% ethanol, acetonitrile and 1,4-dioxane the modified enzyme forms preserved about 50% of their activities during a 2 hours incubation while the controll enzyme was inactivated within 20 minute. The modification of a-chymotrypsin by phthalic anhydride was accompanied by an enormous activation. The acylated enzyme forms did not show significant structural changes with the exception of phthalic anhydride modified one. The improvement of stabilities might be related to side chain reorientations of aromatics upon modifications especially caused by phthalic anhydride.

Conclusions: Our results demonstrate that the stability of a-chymotrypsin may be increased by acylation of amino groups thereby improving the efficiency of the biocatalyst in water organic solvent mixtures. Introduction: Glycation and the subsequent formation of advanced glycation endproducts (AGEs) and free radicals produced during hyperglycaemia are major factors in the complications of diabetes and ageing. The development of clinically effective antiglycation compounds would be of obvious benefit in the management of such conditions. Soy and its products, soy sauces, have been used in East Asian countries since the 15th century. More recently, their anticarcinogenic, antioxidant, antimicrobial, antiplatelet, hypoallergenicity and antiallergic activities have been demonstrated. Methods: The antioxidant properties of light and dark soy sauces were investigated using the ABTS method, by following the decolourization of the radical monocation of 2,2¢-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid). Their capacities to prevent the formation of AGEs were examined using polyacrylamide gel electrophoresis to determine the extent of protein cross-linking and polymerization.

Results: All the brands of soy sauces investigated exhibited antioxidant properties, with the dark having more potent actions than that of light. All soy sauces produced a dose dependent inhibition of protein cross-linking and AGE formation, although in this case, the light sauces were the more effective.

Conclusions: The antioxidant properties and antiglycation effects of soy sauces means some of their components may be of clinical value in the prevention of diabetic complications and age-related diseases, and therefore warrant further investigations.

Anti-atherogenic activity of procyanidins is driven by inhibiting CD36 and inducing ABCA1 expression, thus reducing lipid accumulation in foam cells 

Relationship between plasma acute phase proteins and lipid peroxides in diabetic foot patients Introduction: The principal culprits in type II diabetes mellitus pathogenesis are oxidative stress and inflammation. The aim of this study was to assess the relationship between acute phase proteins and oxidative stress plasma markers in diabetic foot patients. Methods: Two groups, Romanian (n = 35) and Belgian (n = 20) diabetic foot patients with stages 3 or 4 foot ulcerations (according to Wagner classification) were enrolled. Twenty controls were also involved. The patients were asked to complete a 3 weeks day food consumption questionnaire.

Results: Plasma concentrations for dROM (determinable reactive oxygen metabolites), acute phase proteins, uric acid and blood glutathion were higher in diabetic patients versus controls. Comparing the two groups of diabetic patients, plasma concentrations of dROM (P < 0.001), C reactive protein (P = 0.02), ceruloplasmin (P = 0.001), haptoglobin (P = 0.03), blood glutathion (P = 0.01), total plasma thiols (P < 0.001), glycated hemoglobin (P < 0.001), total cholesterol (P = 0.02), atherogenic index (P < 0.001) were significantly increased in Romanian versus Belgian patients. Body mass index, plasma uric acid and total antioxidant capacity levels were similar in both groups. Influence of contamination of the environment with heavy metals is widely studied. Analysis of the impact of heavy metals, especially Cd and Pb individually and in combination in the form of inorganic salts corresponding to daily intake in experimental diets on oxidative stress in experimental animals was studied. Metallothioneins (MT) are considered to be important in homeostatic control and their role is examined in connection with oxidative stress. Relationship between the level of hepatic MT and the exposure of the rat to heavy metals has been shown. Quantity of the rat MT was performed by differential pulse polarography. Determination of the specific activity of oxidative stress enzymes was performed in heparinized plasma. Peroxidase, superoxiddismutase, glutathione reductase, glutathione-S-transferase and catalase were selected for our studies. In the case of glutathionreductase, the increase of enzymatic activity is considerable after intraperitoneal application of cadmium solution in comparison with cadmium application in diet or tap water. The influence of the genetic background of the animals has been studied on the transgenic recombinant strains of rat. Peaches contain phenolic compounds that have potent antioxidantactivity, free-radical scavenging abilities, and protective cardiovascular effects and so they have been linked with the health-promoting properties of this fruit. Phenolic compounds can however be oxidized by the endogenous enzyme polyphenol-oxidase (PPO) that catalyses two different reactions involving oxygen: the o-hydroxylation of monophenols to o-diphenols (monophenolase-activity) and the subsequent oxidation of o-diphenols to o-quinones (diphenolase-activity). These quinones are highly reactive to other quinones, amino acids or proteins producing a considerable nutritional loss. The lability of monophenolase-activity during the purification of plant and fruit PPOs is well-known being necessary the use of gentle methods based on non-ionic detergents to preserve it. In this study, we analyzed the monophenolase-activity showing that the use of Triton X-114 preserves this activity. Enzymatic oxidation of 3-(p-Hydroxyphenyl)-propionic acid in presence of 3-Methyl-2-Benzothiazolinone-hydrazone was followed spectrophotometrically at 500 nm. PPO was in a latent state and was activated by acid shock and by SDS although when used jointly inhibited the enzyme. A substrate inhibition was observed for the higher substrate concentrations with an apparent ksi of 6.1 mM. Results: Expression plasmids for AcsA and His-tagged AcsA were constructed and both were expressed in Escherichia coli. Each of the enzymes was purified to homogeneously. Both types of enzymes showed same specific activity and kinetic properties for acyl-CoA synthesis. Using the highly purified His-tagged AcsA, we detected a new reaction product during the examination of the ability to catalyze the ligation of various acids and CoA analogs. The reaction product was identified by NMR. We measured molar amount of reaction products formed and accompanying decreases in the substrates at several time points, and determined the reaction stoichiometry. Based on these findings, we found a novel substrate specificity of acyl-CoA synthetase involved in nitrile degradation.

The inferred network in progression of hepatocellular carcinoma by graphical chain model Many HCV carriers develop chronic hepatitis C (CH-C), finally complicated with hepatocellular carcinoma (HCC) in a liver with advanced stage CH-C. In this study, we analyzed the expression profiles of CHC and HCC based on GCM. The genes characteristically expressed in CHC and HCC were selected, and the profiles of genes thus selected were subjected to the GCM. The inferred association which was presented by a network between clusters was interpreted by the knowledge on gene interactions and by the gene ontology with GO terms. The interpretation of the inferred network by biological knowledge provides a snapshot of the progression of gene systems in HCC. From the inferred network, the progression of HCC from CHC are divided into two major ways. One way is the reduction of apoptosis related genes and the other is the induction of the development related genes at HCC stage. Actually, the reduction of apoptosis related genes and the induction of development genes are well known characteristic gene expressions at HCC stage. Thus, these characters at HCC tumor are considered to be regulated by other genes at CHC stage.

Abstract Withdrawn

The effect of in vitro alpha tocopherol and ascorbic acid supplementation on inflammatory characteristic of HDL Introduction: The purpose of this study was to investigate the dosedependent anti-inflammatory effects of a-tocopherol and ascorbic acid supplementation on HDLs which are isolated from 30 healthy young subjects' plasma in vitro. Methods: Plasma HDLs were isolated by centrifugation and increasing dosages of a-tocopherol at 7.5, 15 and 30 mg/dl and 1.4, 2.8 mg/dl of ascorbic acid were added to tubes for interaction with HDLs (10 lg/ ml) and incubated for an hour. The ability of the individuals' HDL to prevent oxidation of normal LDL was measured. Values > 1.0 after the addition of HDL indicated pro inflammatory HDL; values < 1.0 indicated anti inflammatory HDL.

Results: In vitro a-tocopherol supplementation was increased proinflammatory HDL which was not depend on concentration. Low dose ascorbic acid (1.4 mg/dl) had no effect on inflammatory characteristics of HDL. However high dose (2.8 mg/dl) ascorbic acid supplementation was increased pro-inflammatory HDL (P = <0.001).

Conclusions: In conclusion, in vitro a-tocopherol and ascorbic acid supplementation were not decreased the inflammatory characteristics of HDL which may be due to lack of interaction between HDL, a-tocopherol and ascorbic acid. The Atcay gene encodes a protein, Caytaxin, which is defective in human Cayman-type ataxia and hereditary ataxia/dystonia of mice and rats. We show that the N-terminal region of Caytaxin binds to kinesin light chain-1 (KLC-1) by yeast two-hybrid assay and immunoprecipitation. Here, we report evidence indicating that Caytaxin is an adaptor that tethers cargoes to kinesin1. Analysis with deletion and replacement mutants demonstrates that KLC-1 binds to the ELEWED (115-120 aa) sequence of Caytaxin. This sequence is conserved among Caytaxin/ BNIP family proteins and shows high similarity to KLC-1 binding motifs found in a neuron-specific vesicular membrane protein, Calsyntenin, which is bound to and transported by kinesin. When expressed in rat primary hippocampal neurons, GFP-fused Caytaxin shows a granular distribution pattern and each granule moves in neurites at 1 micrometer/second, a speed coincided with the movement of kinesin motors. Endogenous Caytaxin is distributed both in axons and dendrites in a similar granular pattern. Some part of Caytaxin is colocalized with vesicles and mitochondria in neurites and some is condensed in the presynaptic region. Caytaxin is predominantly detected in the molecular layer of the cerebellum and in the inner plexiform layer of the retina. Because these two regions consist mainly of axons, dendrites and synapses, these staining patterns support the idea that Caytaxin is transported from the neural cell body to neurites and concentrated in synapses. All together, it is highly plausible that Caytaxin is a kinesin adaptor which bridges cargoes and kinesin motors.

Protective effects of some Iranian medicinal plants against free radical-mediated protein oxidation A. Ardestani, S. Bahramikia and R. Yazdanparast Institute of Biochemistry and Biophysics, University of Tehran, Tehran, IRAN Introduction: The role of oxidative protein damages in the pathophysiology of human diseases is currently a topic of considerable interest as oxidized proteins has been implicated in a wide spectrum of clinical disorders.

Methods: In the present study, the antioxidant activities of four Iranian medicinal plants namely, Teucrium polium, Cyperus rotundus, Anethum graveolens and Nasturtium officanale against metal-catalyzed protein oxidation were evaluated by proxidant model (Fe +2 /ascorabate) in the rat liver homogenates. In addition, total phenolic and flavonoid contents of each plant extracts were estimated.

Results: The addition of Fe 2+ /ascorbate to the liver homogenate significantly increased the extent of protein oxidation such as protein carbonyl (PCO) formation and loss of protein-bound sulfhydryl (P-SH) groups. Furthermore, the rate of reactive oxygen species (ROS) formation and lipid peroxidation was also increased. All the plant extracts showed inhibitory effect against PCO formation, P-SH oxidation, ROS formation and lipid peroxidation in varying degrees. Among of the four plants, T. polium has been found to possess the highest inhibitory capacity against protein oxidation. Based on the data, the order of antioxidant activity of different plants against protein oxidation was found to: T. polium > C. rotundus > A. graveolens > N. officinale.

The protective effects of plant extracts could be due to their polyphenolic content which able to chelate redox active metals as well as to the trapping of some ROS. In that respect, the T. polium extract with higher polyphenol contents was also has powerful antioxidant activity against protein oxidation.

Leishmania major: effects of proteophosphoglycan on reactive oxygen species, IL-12, IFN-c and IL-10 production in healthy individuals , and molecular oxygen. In nature, bioluminescence emission by beetle luciferases is observed in different colors ranging from green (530 nm) to red (630 nm), yet all known luciferases use the same luciferin substrate. Due to its high sensitivity and extreme specificity for ATP, luciferase has been used to determine the amount of ATP in various biological samples. Furthermore, luciferase is now being used in various applications, including food testing, BLI, BERT, bioluminescence immunoassay and for genetic reporter assays in molecular biology.

Methods: In this study we made a set of red-shifted multicolored mutant by site directed mutagenesis in an Iranian luciferase (Lampyris turkestanicus). The mutated cDNAs were cloned in pET28a plasmid. After confirmation by sequencing, and protein expression we purified each mutant protein and calculated their kinetic properties. Results: Bioluminescence emission spectra indicated that saturation mutagenesis at position 354 produced luciferases that emit light with various colors. The characterizations of mutants and the comparison of results with native luciferases showed that mutations of firefly luciferase resulted in the shift of the bioluminescence to the red region. Characterization of mutant luciferases compared to native luciferase using intrinsic and extrinsic fluorescence, CD spectropolarimetry revealed mutation brought about changes in folding and structural properties of L. turkestanicus luciferase. However, evaluation of kinetic properties also revealed changes in stability and activity of luciferase upon mutation at higher temperatures.

The results of present investigation indicate the effect of specific residues on structure and color of light emission in firefly luciferase.

A study for determining the direct effect of myeloperoxidase enzyme on tamoxifen C. Arioz and H. Kuzu Department of Bioengineering, Yildiz Technical University, Istanbul, TURKEY Introduction: 'Myeloperoxidase-H 2 0 2 -Cl -' is a potent antibacterial and cytotoxic system of leucocytes. As a peroxidase, myeloperoxidase generates free radicals by disintegrating drug molecules into fragments [1] . Tamoxifen is the class representative of a group of nonsteroidal triphenylethylene antiestrogens and has been widely used for the treatment of advanced breast cancer. Tamoxifen activation by peroxidases could lead to the formation of hazardous radical species. In this study, myeloperoxidase was purified at laboratory scale for the first time. This abled to examine the intensive effect of myeloperoxidase on Tamoxifen.

Methods: Myeloperoxidase was purified from human blood samples and its purity was evaluated by HPLC and SDS-PAGE electrophoresis. The effect of the purified myeloperoxidase obtained from leucocytes on Tamoxifen at 37°C and in physiological pH (pH 7.2-7.3) was evaluated by using UV-Vis. spectrophotometer, FT-IR and LC-MS systems.

Results: A prominent alteration in recorded UV-Vis. spectra implied that a reaction between Tamoxifen and myeloperoxidase was occurred. The occurrence of a very drastic reaction between Tamoxifen and myeloperoxidase was clarified by evaluating FT-IR and LC-MS spectra.

The obtained data provided us that Tamoxifen was disintegrated effectively by myeloperoxidase to give free radicals. Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is responsible for the production of PGD 2 , a potent endogenous sleep substance, and is secreted into the cerebrospinal fluid (CSF) as beta-trace, a major protein of human CSF. L-PGDS is a member of the lipocalin gene family, which consists of small secretory transporter proteins, and binds various lipophilic ligands, such as retinoic acid, retinal, biliverdin, bilirubin, gangliosides, and amyloid-beta peptides with high affinities (Kd = 30 to 200 nM). As PGD 2 is chemically unstable in aqueous solution and is non-enzymatically converted to the J-type PGs, such as PGJ 2 , D 12 -PGJ 2 , and 15-deoxy-D 12,14 -PGJ 2 in vitro, we hypothesized that PGD 2 may be bound to L-PGDS/beta-trace in the CSF and transported to its receptors, DP 1 and DP 2 . Surface plasmon resonance analysis revealed that PGD 2 bound to L-PGDS with high affinities (Kd = 20 nM). When PGD 2 was incubated in the presence of 5 lM L-PGDS, the J-type PGs were not detected for six hours, indicating that L-PGDS stabilized PGD 2 by binding. In contrast, albumin accelerated the conversion of PGD 2 to the J-type PGs. On the other hand, when J-type PGs were incubated with L-PGDS, they were covalently bound by L-PGDS within 30 minutes. Moreover, we identified the key residues of PGD 2 -binding by NMR analysis of PGD 2 -LPGDS complex and by site-directed mutagenesis. We, therefore, propose that PGD 2 is stabilized via binding to L-PGDS providing a stable transport towards its receptors. Moreover, L-PGDS consecutively acts as scavenger for J-type PGs through covalent binding providing a balance in the biological PGD 2 system. A calorimetric study on the interaction of different PEG containing diblock copolymers including SA400, SA600, SA1500 and OA1500 (stearyl and oleyl esters of polyethylene glycol with 400, 600, 1500 molecular weights respectively) with bovine serum albumin (BSA) was carried out using isothermal titration calorimetry (ITC) and circular dichroism (CD). ITC data show that SA400, SA600, SA1500 and OA1500 bind to BSA, with association constants of (14.5, 3.16, 50.7 and 17.6) ·103M-1 respectively. Results also show that the binding is enthalpically driven, disfavored by conformational entropy.Thermal denaturation studies show that these polymers cause anincerment in melting temperature about 1 to 3 centigrade. It can be concluded that diblock copolymers bind to BSA better than their constituent PEGs causing an increment in its Tm; SA1500 is showing the strongest effect.

Oxy-radical induced protein damage and loss of fluorescence of GFP Small amounts of highly reactive oxygen species (oxy-radicals) are normal by-products of cellular metabolism. However, under certain conditions large amounts of oxy-radicals are generated inside cells. These oxy-radicals are known to cause extensive cellular damage by causing DNA-breakage, lipid (membrane) oxidation, and extensive protein damage. Not surprisingly, a large number of disease states have been linked to oxidative stress, including cancer, diabetes, Parkinson's disease, Alzheimer's disease, and heart disease. Previously, we had shown that fluorescence spectroscopy could be used to study the pH-dependence of GFP denaturation with various agents. In this report, we show that GFP readily undergoes oxidative damage (increased protein carbonyl formation) upon exposure to oxy-radicals leading to concomitant loss of fluorescence. Therefore, using GFP fluorescence spectroscopy, it is possible to study oxy-radical induced protein damage. We have evaluated the various parameters which could affect the sensitivity of this GFP-based oxy-radical induced protein damage assay, such as concentration H 2 O 2 used to produce oxy-radicals, pH of the buffer, as well as UV intensity. Surprisingly, we found that pH had a very dramatic effect on oxy-radical induced GFP damage. GFP was found to be most susceptible to oxy-radical induced damage at pH 6.5, and least susceptible at pH 8.5. This is the first demonstration that GFP looses its fluorescence upon exposed to oxy-radicals. Furthermore, the data presented here suggest that GFP could be used to develop assays to assess protein oxidative damage as well as to screen antioxidants. Short shelf life is of the upmost importance in fruit conservation. As living organisms, fruits maintain metabolism after harvesting, and its shelf-life can be extended by reducing respiration rates. This can be done by controlling factors such as temperature, light, humidity and gas composition (ethylene, O 2 and CO 2 ). To control these, an important strategy is the use of modified atmospheres, with low caloric galactomannan coatings. Collagen coatings have, also, already been used on meat and sausage to reduce gas and/or water vapor permeability (WVP). In this work the preparation novel edible coatings, based on the mixture of galactomannan, collagen and glycerol, was investigated, and their influence in gas transfer rates in mangoes and apples evaluated. The gas permeability properties of the coatings, presenting the best values of wettability (previously determined), were tested. Mangoes coated with an Adenanthera pavonina seed endospermic galactomannan-collagen-glycerol solution were studied and the gas transfer rates compared with no coated mangoes. Gas transfer rate was calculated and )28% O 2 consumption and )11% CO 2 production were observed in coated mangoes. The same procedure was done (using Caesalpinea pulcherrima galactomannan) in apples. The CO 2 production and O 2 consumption were approximately 50% lower in coated apples. These results suggest that these coatings, reducing gas transfer rates, may be important tools to extend fruit shelf-life. Acknowledgement: Supported by CAPES/CNPq, VALNATURA, FCT, RENORBIO.

Galactomannan edible films influence of glycerol Fruit and vegetable overall quality and shelf life are usually reduced by several factors including water loss, enzymatic browning, texture deterioration, senescence processes and microbial growth. Synthetic polymer coatings have been used to reduce these deleterious effects in minimal processing. The use of edible galactomannan coatings can provide alternative semi-permeable barriers, to extend shelf life by reducing moisture and solute migration, gas exchange, oxidative reaction rates and suppressing physiological disorders on fresh-cut fruits with no deleterious effects caused by the plastic materials. In this study the influence of glycerol plasticizer on the galactomannan coating characteristics for packaging design was investigated. Caesalpinia pulcherrima seed endosperm galactomannan films were prepared and the influence of the plasticizer presence on the absorption of humidity evaluated. The water uptake was higher as glycerol concentration increases, with apparently no significant influence of the galactomannan concentration.

Maximum absorption was obtained with 2.5 to 3.5% glycerol and 0.4 to 0.5% galactomannan. A high correlation of the water vapor permeability with the glycerol concentration was observed. The overall tendency, when galactomannan concentration varies, was similar. Combining the two variables highest levels of WVP were found at glycerol and galactomannan concentrations of 3.0% and 0.75%, respectively. The data showed that, at low galactomannan concentrations, the glycerol effect on the WVP was higher. Acknowledgement: Supported by: ALFA-VALNATURA, CNPq/ CAPES, RENORBIO.

Mytilus galloprovincialis Lmk. and Rapana venosa lipid extracts with nutraceutical application Materials and Methods: A protease named as miliin was purified from the latex of Euphorbia milii by a combination of acetone precipitation followed by ion-exchange and size-exclusion chromatography.

Results: The pure enzyme has an estimated Mr and pI of 79 kDa and 4.3, respectively. Using casein as substrate optimal temperature was 60ordm;C and showed two peaks of higher activity at 7.5 and 11.0. The enzyme was effectively inhibited by PMSF, 2,2 dithiopyridine and HgCl 2 , suggesting that milliin is a thiol-dependent serine protease. The effect of optimal pH on the hydrolysis of Abz-KLLFSKQ-EDDnp by miliin was observed above pH 9.0 and metals decreased the activity in the order MgCl 2 > HgCl 2 > AlCl 3 > LiCl > ZnCl 2 . Miliin prefers substrates containing hydrophobic residues. The N-terminus 10 residues is similar to those reported for other Euphorbia proteases. Conclusions: We found a new protease that is being analyzed functionally and structurally. We report the biochemical characterization of proteases isolated from Aelia rostrata that produce gluten degradation in wheat flour. Firstly, we determined the dough extensibility by measuring W and P/L parameters with the Chopin Alveograph. We also analysed the peptides released after gluten hydrolysis in wheat flour samples by SDS-PAGE and capillary electrophoresis. Then, we determined in A. rostrata (salivary glands and midguts from adults and whole-body nymphs) samples, the biochemical activities of aspartyl proteases (optimum pH 3) and aminopeptidases (optimum pH 7), using specific substrates (hemoglobin and LpNa, respectively) and inhibitors (pepstatin-A and CdCl 2 , respectively). Other protein activities were evaluated such as cystein, trypsin, chymotrypsin and elastase proteases, without finding any activity. Likewise, only aspartyl protease (optimum pH 3) and aminopeptidase (optimum pH 6.5), activities were identified in wheat flour samples. Finally, the proteolytic activity have been analysed by 2Dzymography using hemoglobin, gelatin and gliadin as substrates. We observed a band of activity in the native zymogram using hemoglobin and incubated at 35°C and pH 3 in A. rostrata (larvae) and wheat flour samples, which correspond to a M r between 66 KDa and 132 KDa. These results are in concordance with previous studies that establish molecular weight of insect aspartyl proteases is in between 60 KDa and 90 KDa. We are isolating the protein from PAGE gels for MALDITOF characterization.

Comparison of serum phospholipid fatty acids as a biomarker of prostate cancer and benign prostatic hyperplasia In an attempt to explore the molecular mechanisms these associations we have performed a yeast two-hybrid screening a cardiac cDNA library. We showed that the desmin head domain (N58 aa -103 aa) binds to a 413 kDa protein, myospryn, which is expressed in cardiac and skeletal muscle. Myospryn is a TRIM-like protein which has been found to interact with dysbindin, which is a component the soluble fraction Biogenesis Lysosomes Related Organelles Complex 1 (BLOC-1) which is involved in protein trafficking and organelle biogenesis. We determined that Myospryn co-localizes with desmin at the periphery the nucleus mouse neonatal cardiomyocytes and in intercalated discs and costameres in adult cardiac muscle. Desmin seems to be essential for the appropriate perinuclear localization myospryn since its pattern is altered in cardiomyocytes from des-min null neonatal mice. The association myospryn with intermediate filaments, suggests a potential role desmin in vesicle trafficking. The in vivo role myospryn is under investigation using the zebrafish model. Using a splice morpholino for knocking down myospryn transcript, we observed abnormalities in heart development. The morphant embryos examined at 52hpf demonstrated mild structural abnormalities, pericardial edema and 'ventricular hypoplasia'. They, also, had a slower heart rate than wild type embryos. These data demonstrate that desmin binding partner play a pivotal role in heart development.

Evaluation of levels of some tumor markers, acute phase proteins, sialic acid and lipid bound sialic acid before and after treatment in stomach cancer patients CA 19-9, CEA, sialic acid, lipid bound sialic acid, CRP and fibrinogen differences between control and before chemotherapy cancer group were P < 0.001, P < 0.01, P < 0.05, P < 0.001, P < 0.001, P < 0.001, P < 0.001, P < 0.001 were respectively between control and after chemotherapy were P < 0.05, P = 0.494, P = 0.054, P < 0.01, P < 0.001, P < 0.01, P < 0.001, P <0.01, respectively. The comparison of some parameters at before and after chemotherapy had been shown P < 0.01, P < 0.01, P < 0.05, P < 0.01, P < 0.001, P < 0.001, P = 0.054, P < 0.01 statistical importance respectively. As conclusions, all analyzed parameters had increases in cancer group before chemotherapy. They all were declined significantly after chemotherapy. Increases of CEA, CA 19-9, CA 125 were determined but surprisingly 40-fold increase in CEA level was estimated. Also increases of sialic acid, lipid bound sialic acid and 36-fold increases of CRP, changes of fibrinogen levels in gastric cancer patients were important for the diagnosis and prognosis. This diet enhances mitochondrial respiratory activity and reduces the production of reactive oxygen species. In order to determine if increased respiration is the cause of beneficial effects of CR, we used low doses of 2,4-dinitrophenol (DNP) to promote mild mitochondrial uncoupling. female Swiss mice were treated with DNP added in the drinking water. Tissue respiratory rates, body temperatures and the levels of 8-oxo-7,8dihydro-2´-deoxyguanosine (8-oxodGuo), carbonyl proteins and oxidized and reduced glutathione in heart, liver and brain were measured after 4 or 25 weeks of treatment. DNP increased life span in 10% and reduced body weight significantly. Four weeks of treatment was enough to increase respiratory rates and reduce 8-oxodGuo in DNA at all tissues. After 25 weeks, carbonyls proteins and oxidized glutatione were significantly decreased. The reduction of the 8-oxodGuo levels was striking, reaching 300% in brain. In conclusion, mild mitochondrial uncoupling was able to promote many beneficial effects of CR, including increased life span and improvement of redox state.

Kinome profiling annotation of pre-osteoblast cells differentiation process by using arrays of kinase consensus substrates We compared the transcript level of Bax inhibitor-1/testis enhanced gene transcript (BI-1/TEGT) among immature and in vitro-matured oocytes and 2-4 cell stage embryos and found that it is highly expressed at immature stage, but significantly decreased after in vitro or in vivo maturation. The forced expression of BI-1/TEGT under elongagion factor (EF) promoter resulted in reduced stress-induced apoptosis in HEK 293 and TEGT MEF cells. We could also observe that BI-1/ TEGT suppressed etoposide-induced ROS production concomitant with the activation of extracellular signal-regulated kinase (ERK) signaling pathway. Through the effect of specific MEK inhibitor or BI-1/ TEGT siRNA, we could confirm that ERK activation is important for the anti-apoptotic function of BI-1/TEGT. To study the effect of the forced expression of BI-1/TEGT in vivo during oocyte and embryo development, we generated transgenic mice overexpressing BI-1/TEGT or BI-1/TEGT-DC. MEF cells from BI-1/TEGT transgenic mice showed higher ERK phosphorylation than those from the wild-type or BI-1/TEGT-DC transgenic mice. During breeding of the respective transgenic lines, we recognized that litter sizes were dramatically reduced in BI-1/TEGT-overexpressing transgenic mice compared to that of the wild-type or BI-1/TEGT-DC transgenic mice. Taken together, our results strongly suggest that downregulation of BI-1/ TEGT during early development is important for normal development process, probably by regulating apoptotic signaling pathway during development.

Sustained ERK activation is involved in the kaempferol-induced apoptosis of breast cancer cells and is more evident under 3-D culture condition Kaempferol was demonstrated to induce sustained ERK activation concomitantly with MEK1 and ELK1 activation, and this kaempferol-induced apoptosis was suppressed by treatment with PD98059 or the overexpression of a kinase-inactive ERK mutant. Kaempferol treatment was shown to profoundly induce the generation of fluorescent DCF in the MCF-7 cells, and treatment with N-acetyl cysteine suppressed kaempferol-induced PARP cleavage. Moreover, because breast cancer is associated with increased collagen synthesis and accumulation, we utilized a collagen-based 3D culture method. Under the 3-dimensional culture condition employed herein, kaempferol treatment was shown to result in a significant reduction in cell viability, an effect which occurred in a dose-dependent manner. Compared with what was observed under conventional 2D culture condition, we observed more evident apoptotic cell death and ERK activation as the result of kaempferol treatment in a collagen-based 3D culture environment. Similar to the case of conventional 2D cultured cells, the addition of PD98059 significantly suppressed intracellular ROS production. Collectively, these results show that the sustained activation of the ERK signaling pathway is markedly involved in kaempferol-induced apoptosis of breast cancer MCF-7 cells, and that this effect is more evident under 3D culture condition. Epithelial cells from proximal tubule and small intestine actively transport Na + by two different mechanisms: the Na + /K + -pump, associated with the ouabain-sensitive Na + /K + -ATPase, and the second sodium pump whose enzymatic expression has been attributed to the ouabaininsensitive Na + -ATPase. Recently, this last enzyme has been isolated, purified, cloned and silenced, differentiating it from the Na + /K + -ATPase. In this paper, we describe the preparation of chicken IgY polyclonal antibodies against the Na + -and Na + /K + -ATPase and evaluate the differential distribution of these enzymes in intestinal and renal tissues. Additionally, the effects of these antibodies on the Na +and Na + /K + -ATPase activities and active Na + -transport were evaluated. The anti-Na + -ATPase IgY specifically inhibited both ouabaininsensitive Na + -transport and Na + -ATPase activity without any effect on ouabain-sensitive Na + -extrusion nor Na + /K + -ATPase. In contrast, the anti-Na + /K + -ATPase IgY specifically inhibited ouabain-sensitive Na + -transport and Na + /K + -ATPase. The anti-Na + -ATPase IgY recognized the a and b subunits of the Na + -ATPase but did not interacted with the purified Na + /K + -ATPase. On the other hand, the anti-Na + /K + -ATPase IgY only recognized the Na + /K + -ATPase. The Na + -ATPase was identified, by immunoblot and RT-PCR, in renal cortex, jejune, ileum, proximal and distal colon, being absent in renal medulla. Immunohistochemistry and immunofluorescence analysis shown that the Na + -ATPase is preferentially expressed in villous, surface colonic and proximal renal tubule epithelial cells, but absent in renal medulla, contrasting with the uniform distribution of the Na + / K + -ATPase. The preferential location of the Na + -ATPase at small intestine villi and renal proximal tubules could reflect the physiological relevance of this enzyme in intestinal and renal sodium and water absorption.

Grade inflation results in a biostatistics course when using a virtual internet method as compared to a traditional one used as control Methods: A biostatistics course was given to pharmacology students at the Autonomous Metropolitan University at Mexico City using two different methods. One corresponded to a traditional course and the other corresponded to a virtual course via internet. The subjects covered were Confidence interval, t-test, One Way ANOVA, Simple Linear Regression, Factorial and Nest and Logit Analysis. The course was distributed by the professor himself to two different groups. The time allotted to the course was 4 hours weekly during three months. Half of the time of each class was dedicated to either method, For the virtual course, PCs were available for the students. Statistical analysis consisted of a 2 · 2 factorial design. Evaluations were made one in the traditional way and the other via internet.

Results: Significant differences were observed between the two evaluation methods and between groups (P < 0.05). No significant differences were found in the interaction between the evaluation of the two groups and both methods (P = 0.93). The grades obtained with the virtual evaluation via Internet were higher than those attained with the traditional evaluation.

Conclusions: We think that the evaluation via internet should be taken as a light weighted criterion for the final results because information is easily exchanged among the students.

PP8-44 CRF and urocortins induce catecholamine production from adrenal chromaffin cells via activation of transcription factor NFAT through specific signalling pathways Introduction: Corticotropin Releasing Factor (CRF) and the Urocortins induce catecholamine production in human and rat adrenals chromaffin cells and in PC12 pheochromocytoma. Here, we demonstrate that the stimulatory effect of CRF and Urocortins is mediated via transcription factor NFAT (Nuclear Factor of Activated T cells). Methods: Norepinephrine (NE) measurements-PC12 cells were exposed to CRF receptor agonists and NE concentration was measured in the supernatant by HPLC. Measurement of NFAT activity-PC12 cells were transiently transfected with a construct containing NFAT binding site linked to the luciferase gene (NFAT-Luc) and co-transfected with dominant negative mutants of p38 MAPK, AKT-K, Tpl-2 or Ha-ras N17.

Results: Treatment of PC12 cells with CRF, UCN1, UCN2 or the synthetic CRF 1 receptor agonist Cortagine, induced NE production which was inhibited by Cyclosporin A, a Calcineurin inhibitor that blocks NFAT activation. All CRF family peptides induced NFAT transcriptional activity which was abolished in the presence of pharmacological inhibitors of p38MAPK (SB203580), ERKs (PD98059) or PI3K (Wortmannin) as well as by dominant negative mutants of p38 MAPK, AKT, Tpl-2 or Ha-ras. Conclusions: CRF 1 and CRF 2 agonists induce NE production in adrenal chromaffin cells via activation of transcription factor NFAT. CRF receptor signals activate NFAT on a calcineurin-dependent manner via convergence of signaling pathways p38MAPK, ERK1/2, AKT, Tpl-2 and Ha-Ras.

Preparation and chromatographic characterization of molecularly imprinted polymer for the determination of glutathione Introduction: Molecular imprinted polymers (MIPs) are tailor-made materials with high selectivity for a target molecule. Creating MIPs involves the assembly of functional monomers around a template molecule in a solution containing a high of cross-linker, polymerization of the resultant mixture and removal of the template. Glutathione, an ubiquitous thiol, plays a major role in maintaining intracellular reductionoxidation (redox) balance and regulating signaling pathways augmented by oxidative stress. In this study preparation and selective determination of glutathione by imprinted polymers were aimed. Methods: MIPs for the recognition of glutathione (GSH-MIP) was prepared by a bulk polymerization method using 1-vinyl imidazole as the functional monomer, ethylene glycol dimethacrylate as the crosslinker, dimethyl sulfoxide as the porogen. Following the termal polymerization, the polymers were crushed, ground and sieved. Glutathione was removed from the polymer by methanol-acetic acid extraction. Selectivity of GSH-MIP was tested by glutathione, oxidized glutathione and Gly-Leu-Tyr in two solvent systems. Reusability of MIP particles was also investigated. Furthermore blood specimens were applied to the GSH-MIP followed by HPLC analysis.

Results: GSH-MIP showed higher selectivity for GSH. GSH-MIP enabled the selective extraction of glutathione from a complex matrix such as blood, demostrating the potential application of molecularly imprinted solid phase extraction. MIP particles found to be stable and maintain glutathione adsorption capacity after seven usage.

Conclusions: In this study, GSH imprinted polymer was prepared and characterized for selective determination of glutathione.

Stearoyl-CoA desaturase 1 deficiency alters substrate utilization in the heart Stearoyl-CoA desaturase (SCD) is a regulatory enzyme in lipogenesis, catalyzing synthesis of monounsaturated fatty acids. SCD1 deficiency activates metabolic pathways that promote fatty acid b-oxidation and decrease lipogenesis in liver. In the present study, we show that fatty acid (FA) transport and oxidation are decreased, whereas glucose uptake and oxidation are increased in the heart of SCD1-/-mice. The level of fatty acid translocase/CD36 and fatty acid transport proteins were significantly lower (by 50 and 70%, respectively) in the heart of SCD1-/-mice compared to SCD1+/+ mice. Both carnitine palmitoyltransferase 1 protein level and activity were significantly decreased (by 40 and 31%, respectively) in the heart of SCD1-/-mice in comparison to SCD1+/+ controls. Consequently, the rate of palmitoyl-CoA oxidation was decreased by 37.1% in the heart of SCD1-/-mice. mRNA levels of PPARa, a key transcription factor controlling genes of fatty acid oxidation, were significantly reduced in SCD1-/-mice, whereas phosphorylation and protein levels of AMPK-a were not affected by SCD1 deficiency. Phosphorylation of both insulin receptor and insulin receptor substrate-1 (IRS-1) after insulin injection were about 2-fold higher in the heart of SCD1-/-mice. Also the association of ?p85 subunit of PI3kinase with IRS-1 was significantly higher under both basal and insulin stimulated conditions in SCD1-/-hearts. This increased insulin sensitivity translated to a 1.8-fold greater 2-deoxyglucose uptake and 2-fold higher rate of glucose oxidation in the myocardium, compared to SCD1+/+ counterparts. The results suggest that SCD1 deficiency causes a shift in cardiac substrate utilization from FA to glucose by upregulating insulin signaling, decreasing FA availability, and reducing expression of FA oxidation genes in the heart. . Despite of its distinctive advantages, the limitation in the thermostability of the enzyme required to be enhanced. The wild type FDH from Candida methylica (cmFDH) does not contain disulphide bridges, but does possess two cysteine residues buried in separate hydrophobic pockets. Cysteine residues are particularly susceptible to atmospheric oxidation at elevated temperatures. One approach to increase thermostability is to replace both cysteine resides by valine, C23V and C262V. The second approach is stabilizing via engineering disulphide bridges into the protein: I239C (to partner C262) and A153C (to partner C23). If a disulphide bridge can be engineered into the native fold without introducing an enthalpic cost (strain), such a bridge would stabilize the native state by decreasing the entropy of the protein's unfolded state 3 .

C23V, C262V, C23V+C262V, A153C, I239C, A153C+I239C mutations were designed by using Insight II program on homology model of cmFDH based on Pseudomonas sp.101. The mutations have been introduced into cmFDH gene in 6xHis-tag pQE-2 expression vector by using Invitrogen Gene Tailor TM site-directed mutagenesis system. Constructed mutants were overexpressed in JM105 E. coli strain and purified. Kinetic and thermostability studies have been performed for both wild type and mutant cmFDHs. Results: Mutants A153C and A153C+I239C lost FDH activity and mutants C23V, C262V, C23V+C262V and I239C had almost similar activity with wild type cmFDH and less thermostable than the original enzyme. Smoking is one of the most important cause of cardiovascular disease and known as a preventable risk factor. Oxidized LDL increases the risk for cardiovascular disease. Normal high density lipoproteins (HDLs) protect LDL from oxidation; proinflammatory HDLs do not. The aim of this study was to determine whether the young smokers have more proinflammatory HDL which predispose them to cardiovascular disease than nonsmokers. Methods: Twenty-five young smokers (SM), who have smoking 8-10 years and twenty-five healthy nonsmokers (CNT) were included in this study. Blood samples were collected after overnight fast and absence of smoking. The ability of the cases' HDL to prevent oxidation of normal LDL was measured. Values >1.0 (the value assigned for LDL oxidation in the absence of HDL) after the addition of HDL indicated proinflammatory HDL. Total-C, TG, HDL-C, LDL-C, Apo A, Apo B, hsCRP and Lp (a) levels were also determined in our clinical laboratory using routine standard methods. Results: The smokers had more proinflammatory HDL than nonsmokers (median score 1.01, vs. 0.92, P < 0.001). A higher proportion of smokers had proinflammatory HDL: 68% of smokers versus 20% of nonsmokers had scores >1.0. Conclusion: We concluded that smoking impairs HDLs anti inflammatory functions. This may indicate that proinflammatory HDL have a role in atherosclerosis process of smokers.

Refolding of denatured alkaline phosphatase assisted by a new synthetic stripping agent The 'artificial chaperone method' developed by Rozema and Gellman [1] is an elegant method for protein refolding with higher yields compared to other methods. From industrial point of view, this method suffers from the tedious steps of purifications which will finally affect the production costs. Replacement of the soluble stripping agent with immobilized CDs or CD polymer beads might elevate some of these problems. Regarding this fact, we synthesized and evaluated the application of various cyclodextrin-bonded silica gels to improve the refolding efficiency of denatured alkaline phosphatase in artificial chaperone approach. Our results indicated that, under this condition, refolding of denatured alkaline phosphatase raised from 30%, in the absence of chaperone, to about 65% in the presence of 70 mg/ml of the b-cyclodextrin-bonded silica gel and to 74% in the concomitant presence of the this stripping agent and MgSO 4 , a yield near to stripping by soluble b-CD. This new stripping approach, compared to the common method, will reduce the protein refolding costs at the industrial level. However, further improvements in this technique are demanded either through synthesizing better stripping agents or by optimizing better refolding environments. The endocannabinoid system is characterized in a number of mammalian cells. In previous studies we reported that Fatty Acid Amide Hydrolase (FAAH), the enzyme which is responsible for the inactivation of anandamide (arachidonoylethanolamide, AEA), is present in Tetrahymena pyriformis. The ciliate Tetrahymena is a model organism for molecular and cellular biology and its genome sequence is available. In this study, using Tetrahymena thermophila cell homogenate, we found that 2-arachidonylglycerol ([ 3 H]2-AG), as well as 2-oleylglycerol ([ 3 H]2-OG), were hydrolyzed to [ 3 H]ethanolamine and respective fatty acid, by the action of two enzymes, FAAH and a monoacylglycerol lipase (MAGL)-like enzyme. The latter, was partially characterized in the presence of the specific FAAH inhibitor URB597. Our results show that 2-AG was metabolized by T. thermophila cell homogenate in a time and concentration dependent manner, by the action of two enzymes (FAAH and MAGL), since in the presence of the inhibitor, its degradation was significantly decreased, indicating the involvement of FAAH. MAGL activity was maximal at pH 8-9 and kinetic experiments demonstrated that the enzyme had an apparent K m of 14.1 lM and V max of 5.8 nmol/min.mg. Subcellular fractionation of T. thermophila homogenate showed that MAGL activity was present in the cytosolic and membrane fraction. Similar results were obtained using a second specific FAAH inhibitor, AM374. In conclusion, MAGL and FAAH as well, could inactivate endogenous signaling lipids which might have important roles in Tetrahymena as suggested in mammals and invertebrates; their presence in protists suggests the importance of the endocannabinoid system throughout the evolution. Acknowledgements: This research project (PENED) is co-financed by E.U. -European Social Fund (75%) and the Greek Ministry of Development-GSRT (25%).

Identification of the essential catalytic residues and selectivity-related residues of maltooligosyltrehalose trehalohydrolase from the thermophilic archaeon Sulfolobus solfataricus ATCC 35092 Background: Maltooligosyltrehalose trehalohydrolase (MTHase) catalyzes the release of trehalose by cleaving the a-1,4-glucosidic linkage next to the a-1, 1-linked terminal disaccharide of maltooligosyltrehalose. Methods: Mutations were constructed by site-directed mutagenesis at residues D255, E286, and D380 to identify the essential catalytic residues of MTHase, while mutations at residues W218, A259, Y328, F355, and R356 were constructed to identify selectivity-related residues of the enzyme. Results: The specific activity of the purified D255A, E286A, and D380A MTHases were only 0.15, 0.09 and 0.01%, respectively, of that of wild-type MTHase, suggesting these three residues are essential catalytic residues. Compared with wild-type MTHase, A259S, Y328F, F355Y, and R356K MTHases had increased selectivity ratios, which were defined as the ratios of the catalytic efficiencies for glucose formation to those for trehalose formation in the hydrolysis of maltooligosaccharides and maltooligosyltrehaloses, respectively, while W218A and W218F MTHases had decreased selectivity ratios. When starch digestion was carried out at 75°C and wild-type and mutant MTHases were, respectively, used with isoamylase and maltooligosyltrehalose synthase (MTSase), the ratios of initial rates of glucose formation to those of trehalose formation were inversely correlated to the peak trehalose yields. Conclusions: We have confirmed the catalytic roles of D255, E286, and D380 and suggested the existence of two hydrogen bonds between substrate and the enzyme at position W218. The lower ratios of the initial rate of glucose formation to that of trehalose formation from the digestion of soluble starch predict high trehalose yield. The venom is a rich source of biochemically active enzymes, proteins, peptides and low molecular weight compounds. The scorpion venom peptides are particularly interesting for pharmaceutical, agrochemical and protein engineering purposes because of their remarkable diversity, molecular targets, and their flexible loops despite rigid scaffold. In this report, after separation of mucopeptides with ultracentrifugation peptidic components of Mesobuthus eupeus eupeus crude venom were fractionated into 5 parts by gel filtration chromatography and Tris-Tricine SDS-PAGE. The peptides of fourth part were isolated to more than 95% purity by means of repeated chromatography including cation exchange chromatography and repeated RP-HPLC. Then two peptides were characterized by amino acid analysis, molecular weight and N-terminal sequence determination. Using these information, one of these peptides showed high similarity of molecular weight and N-terminal sequence with the toxic peptide BeI4 (Insectotoxin4) from this scorpion venom. The structure of BeI4 is not known but it has high similarity to BeI5A; so we decided to model them with MODELLER8v1 software from BeI5A template which has 97% sequence similarity and identity based on sequence alignment data from CLUSTALW. After energy minimization, analysis of the H-bond pattern and secondary structures with STRIDE and Swiss PDB viewer softwares showed high accordance of BeI5A template for BeI4 with RMSD < 0.57 for a-carbon. This structure will be relaxed and refined by doing molecular dynamic simulation with explicit water solvation. To unravel the binding modes and details of interaction with potassium channels, the next step will be docking simulation of the modeled peptide. Amburana cearensis A.C. Smith is a tree species of economic importance found in northeastern semiarid region of Brazil, where it has been under intensive pressure due to its use as rare, fine and high quality wood by the furniture industry. On the other hand, A. cearensis is a medicinal plant commonly known as 'cumaru', important for the native people on the preparation syrup out of crushed seeds which is used for the treatment of respiratory tract diseases. Seed development is genetically programmed and correlated with changes on metabolite levels. Differentiation occurs successively starting from the maternal tissues followed by the filial organs (embryo/endosperm), which later become later specialized storage tissues at seed maturity. The mobilization of storage materials such as carbohydrates, lipids and proteins by activation or de novo synthesis of key enzymes may underlie the mechanism of seed imbibitions and germination, and seedling establishment in places such as the semiarid where the availability of water is limited. The objective of this research was to evaluate the effects of water restriction on the germination of A. cearensis, under various levels of osmotic stress. Levels of carbohydrates, lipids and proteins were analyzed in parallel with western blot analysis of the accumulation of tubulin during seed imbibition and germination, under various level of osmotic stress. Changes in metabolite levels as signals in seed metabolism and developmental processes under stress were evidenced and analyzed in the perspective of multiplication protocols aimed at sustainable use of A. cearensis. Jatropha curcas (Euphorbiaceae) is a native multipurpose oilseed plant species which is well adapted to heat and water restriction in the northeastern semi-arid region of Brazil. Its seeds is nowadays the most valuable end product as a source of oil and, therefore, as an alternative crop for renewable energy. The species has also many traditional medicinal uses among the local population against diseases such as dysentery, haemorrhoid, gonorrhoea, small pox. Storage proteins, carbohydrates and oils are synthesized and accumulate as source of reserves during seed development and maturation, which are then subject to hydrolytic breakdown upon seed imbibition as source of nitrogen and carbon skeletons for the germinating seed and seedling growth. The objective of the present study was to study the mechanisms underlying the germination of J. curcas under water restriction. Seeds of J. curcas obtained from three different semi-arid provenances were submitted to biochemical and molecular analysis during germination under various levels of osmotic stress. Additionally, the expression of stress-associated genes was monitored during germination and seedling growth under stress. Oil was extracted using n-hexane and mixture of other solvents. The extracted oils were immediately analyzed for physicochemical properties and mineral composition. The overall analysis demonstrated differences among seeds from different provenances as well during seed imbibition under stress conditions, allowing a better understanding of the metabolic and molecular mechanisms underlying the germination process, aimed at multiplication protocols for sustainable use of J. curcas under the stress conditions of the Brazilian northeastern semi-arid. Peripheral nerve surgery has always been a challenging problem in the context of scar formation. Suppressing the formation of this disturbing tissue is therefore a holly grail in treating peripheral nerve injuries. When studying the scar-suppressing potential of a given substance or treatment, investigators usually deliberately induce the formation of scar tissue. Depending on the intervention (physical or chemical), the resulted scar varies in quantity and quality which is usually less intensive and durable than that of humans. Physical intrusions seem to be more relevant to clinical situations than chemical ones. The aim of this study is to evaluate scar formation by four physical interventions: slashing, crushing, mincing and burning of the muscular bed of sciatic nerve. Thirty-two female Wistar rats were divided into four groups, each sustaining one of the mentioned intrusions. Animals were followed for 4 weeks, and with one week intervals, by macroscopic, microscopic and functional assessments. While the results of macroscopic and microscopic evaluations reveal the higher capacity of mincing procedure in producing a more intense and lasting scar tissue, functional assessment showed no differences among the groups in hampering sciatic nerve function; however all the groups demonstrated significant difference with the normal-functioning nerve. Our data suggest that except one intervention (mincing), others were almost equipotent in scar induction. It can be concluded that of these four interventions, and in the context of severity and persistency, the mincing procedure induces a scar tissue more comparable to what occurs in humans. Effects of such scar models on histology of the nerve along with injury-bearing nerves subjected to scar formation are issues to be investigated by further studies.

Investigating the molecular mechanisms of moisture induced solid-state instability of protein pharmaceuticals G. Flores, K. Griebenow and R. Soll University of Puerto Rico, Puerto Rico, USA Introduction: The employment of proteins as biotherapeutic agents has gained increased interest due to their chemical selectivity and catalytic efficiency when compared to small molecules. Solid protein pharmaceuticals often suffer from instability problems due to moisture adsorption thus hampering their successful therapeutic application. One frequently formulated hypothesis is that partial protein hydration from moisture adsorption increases protein structural motions leading to protein unfolding and aggregation. Our goal is that, once understanding the molecular mechanisms by which residual moisture influences the solid state stability of protein pharmaceuticals, it will become possible to develop rational stabilization strategies to prevent these degradation processes. Methods: To test the hypothesis, lyophilized insulin and a-chymotrypsin powder was incubated at various relative humidities for different time intervals. Formation of buffer insoluble and soluble aggregates was monitored for these samples. To detect unfolding/refolding events occurring to the solid protein, these were analyzed by FTIR and Circular Dichroism after incubation. Results: Results showed that the protein forms aggregates upon storage in humidity chambers. Furthermore long-term storage of the proteins at increasing levels of residual moisture increases the formation of aggregates with this effect also increasing with storage time. Moreover results showed that the protein looses its native-like structure after storage for 1 week. This suggests that high moisture levels cause detrimental events in the protein such as, structural perturbations. Conclusions: This study has showed that residual moisture plays an important role in the denaturation and aggregation of protein. Stabilization strategies as PEGylation and glycosylation are going to be studied to reduce or prevent these phenomena.

Redox properties of the ATP-sensitive K + channel in brain mitochondria Brain mitochondrial ATP-sensitive K + channel (mitoK ATP ) opening by diazoxide protects against ischemic damage and excitotoxic cell death. We studied here the redox properties of brain mitoK ATP . MitoK ATP activation during excitotoxicity in cultured cerebellar granule neurons prevented the accumulation of reactive oxygen species (ROS) and cell death. Furthermore, mitoK ATP activation in isolated brain mitochondria significantly prevented H 2 O 2 release by these organelles, but did not change Ca 2+ accumulation capacity. Interestingly, the activity of mitoK ATP was highly dependent on redox state. Thiol reductant mercaptopropionylglycine prevented mitoK ATP activity, while exogenous ROS activated the channel. In addition, the use of mitochondrial substrates that lead to higher levels of endogenous mitochondrial ROS release closely correlated with enhanced K + transport activity through mitoK ATP . Altogether, our results indicate that brain mitoK ATP is a redox-sensitive channel that controls mitochondrial ROS release.

PP8-59 Drug delivery system for hydrophobic drugs using lipocalin-type prostaglandin D synthase A. Fukuhara 1 , H. Nakajima 1 , K. Inoue 2 , Y. Miyamoto 1 , S. Nishimura 1 , T. Takeuchi 1 and T. Inui 1   1 Osaka Prefecture University, Osaka, Japan, 2 Spring-8/JASRI, Hyogo, JAPAN Introduction: Lipocalin-type prostaglandin D synthase (L-PGDS) is a unique dual functional protein acting as both a PGD 2 -synthesizing enzyme and an extracellular transporter protein for hydrophobic ligands. L-PGDS possesses the broad ligand selectivity and binds to a large variety of hydrophobic ligands, such as retinoids, thyroids, and bile pigments with high affinities in vitro. So far many drugs have been dropped out from the development because of their poor water solubility, although they have high pharmaceutical activities. In this study, we performed the concept validation of drug delivery system in vivo using L-PGDS for diazepam (DZP), a benzodiazepine anxiolytic and poorly water-soluble drug. Results and Discussion: L-PGDS improved the solubility of DZP in a concentration-dependent manner, and in the presence of 500 ?M L-PGDS the solubility was 7-fold higher than that in PBS. DZP is well-known to enhance the anesthetic effect via activating GABA A receptor. DZP of 10 mM was mixed with L-PGDS (25, 100, and 250 ?M in 5 mM Tris/HCl, pH 8.0) and passed through a filter to remove DZP left insoluble. L-PGDS/DZP complex was administered intracerebroventricularly in mice 10 minutes before giving an intraperitoneal injection of pentobarbital sodium (35 mg/kg), and duration of pentobarbital-induced loss of righting reflex (LORR) was measured. Mice treated with complex showed significantly 1.5-fold longer duration of LORR than those treated with DZP in buffer. These results show that L-PGDS delivers DZP to GABA A receptor as a solubilizer and the concept of DDS for hydrophobic drugs using L-PGDS is succeeded to validate.

The effects of phytoestrogens genistein and genistein-8-C-glucoside in vitro A. Rucinska 1 , S. Kirko 2 and T. Gabryelak 1 1 Department of General Biophysics, University of Lodz, Lodz, POLAND, 2 Institute of Pharmacology and Biochemistry, National Academy of Sciences, Grodno, BELARUS Introduction: In recent years, scientific and public interests on phytoestrogens has focused on their potentially beneficial health effects as anticarcinogens, cardioprotectans and as alternatives to hormone replacement therapy in menopause. Numerous in vitro cell culture studies have also reported genistein to be cytotoxic, DNA damaging and proapoptotic. The aim of this study was to investigate the effects of the two isoflavones: genistein and genistein-8-C-glucoside (G8CG) in combination on cultured human ovarian cancer SK-OV-3 cells and immortalized Chinese hamster ovary CHO cells. In the current investigation we used genistein and glycosylated genistein G8CG, which we isolated from flowers of lupine (Lupinus luteus L.). The possible cytotoxic and apoptotic properties of genistein and G8CG in combination was examined in vitro. Methods: The calcein AM/propidium iodide assay was used to assess cytotoxicity and Hoechst 33258/propidium iodide staining technique was employed for the induction of apoptotic cell death. The morphological changes of cells were also examined in inverted confocal microscopy. Mitochondrial membrane potential changes, in genistein-G8CG treated cells, were monitored using fluorescence probe JC-1 by fluorescent microscopy and flow cytometry. The cells were exposed to three different concentrations (10, 50 and 100 lM) of combination genistein-G8CG and incubated for 2, 24 and 48 h. Results: Our results indicate that the mixture of isoflavones inhibits cell viability, induces apoptotic cell death and causes mitochondrial membrane potential alteration in cells in a dose-and time-dependent manner. Conclusions: Our preliminary in vitro studies, provide the first evidence that the combination of genistein and G8CG could demonstrate anti-cancer properties.

PP8-61 Interstitial telomeres in continuous in vitro growth In mammalian tumors telomere length is almost universally maintained through telomerase (TERT) or by a much less common alternative lengthening of telomeres (ALT) which involves telomeric recombination. However, cancers develop in the absence of telomerase and their proliferation potential can be substituted by recombinatorial mechanisms. We applied telomeric PNA FISH, in a panel of 12 telomerase positive, and six ALT continuous human cell lines to investigate types and frequencies of chromosome rearrangements involving telomeric repeats, to quantify the length of the telomeric tracts involved, and to identify mechanisms responsible for their generation. Interstitial telomeric repeats were present in both types of telomere restoration but were pronounced in the ALT cells. The orientation of interstitial telomeric repeats by CO-FISH and M-FISH/SKY showed three categories of interstitial telomeres: (a) clonal, inverted intra-chromosomal duplications, (b) clonal, originating by variable length telomere-to-telomere inter-chromosomal fusions and (c) random dicentric or polycentric chromosomes with interstitial telomeres at their junction points. We evaluated the generation of interstitial telomeres in the ALT VA-13 cells and a TRAP positive derivative of the same cell line (VA-13+hTERC+hTERT) and after induction of DNA breaks by with c-radiation, or depletion of telomerase and the telomere protecting factor POT1 by RNA interference. Our data indicate that telomerase and POT1 activity suppressed random inter-telomere recombination whereas telomerase inhibition leads to chromosome fusions without measurable interstitial telomeres. Introduction: a-Amylase (a-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1), which catalyzes the hydrolysis of amylose, amylopectin, and related carbohydrates with endo-acting property on 1,4-glycosidic linkages, is among of the most important industrial enzymes with widespread applications. A Bacillus sp. WHO, which produces an extracellular a-amylase, was isolated from the high natural radiation area in Ramsar. Material and Methods: The a-amylase gene from this Bacillus strain was cloned in vector pTZ57R/T and its nucleotide sequence was determined. It was subcloned in the expression vector (pET21a) and transferred to BL21 strain of E. coli. The recombinant enzyme was purified under denaturing conditions by Ni-NTA Agarose. Results: The gene has an open reading frame composed of 1563 bases, which encode 520 amino acid residues and shows high sequence homologies with other microbial amylase, such as Bacillus megaterium and Bacillus sp. WS06 (97 and 96% identity). The deduced amino acid sequence contains four highly conserved sequences (regions I, II, III, IV) of the a-amylase family. The molecular mass of the recombinant enzyme was 64 kDa as determined by SDS-PAGE. The enzyme was Ca 2+ -dependent and the optimum pH and temperature was 7.0, and 50°C, respectively. Conclusion: This enzyme with low homology with BLA and BAA is an interesting candidate for studying of its thermostability. Future investigations will focus on enhancing the thermostability and determination of amino acids has effects on it by site-directed mutagenesis method.

Biochemical characterization of L 14 -amylopullulanase from an Iranian Thermophilic bacillus species M. Ghollasi, K. Khajeh, N. Mollania and S. Zareian Department of Biochemistry, Faculty of Basic Science, Tarbiat Modares University, Tehran, IRAN Introduction: A unique pullulanase type II (L 14 -APU) was isolated and purified. The biochemical characterization such as the effects of metal ions, inhibitory effect of acarbose and determination of the active site numbers has been studied. Methods: L 14 -APU was purified by ion exchange and gel filtration chromatography. Amylolytic and pullulytic activities were measured at 65°C and pH 5.5 by DNS method and in another way using EPS as a substrate. In addition, the effect of acarbose, various metal ions and some reagents on the enzyme was determined using the standard assay.

Results: Acarbose is a strong competitive inhibitor of L 14 -APU enzyme with respect to pullulan and starch. This results showed the enzyme hydrolyzes both a-1,4 and a-1,6 glycosidic bonds at the same active site. Divalent cations such as Ni 2+ , Cu 2+ inhibited the amylolytic and pullulytic activities while Ba 2+ had no effect. Against Ca 2+ , Mg 2+ , Mn 2+ and Co 2+ enhanced both activities of the enzyme. The L 14 -APU activity on 1% (w/v) pullulan and starch was not influenced after preincubation of the enzyme with a-cyclodextrin (0.1%) which is generally known as possible competitive inhibitors of pullulanase type II. In the presence of 1 mM SDS pullulanase activity increased by 46%. As observed for most pullulanase, both L 14 -APU activities was completely eliminated by low concentration of N-boromosuccinimide (0.01%) which oxidizes tryptophan and histidine residues, suggesting the crucial involvement of these residues at the active site. Conclusion: In general, abolishment of both activities of the enzyme by NBS, similar effects of metal ions and reagents on the enzyme activity, identity of the pattern of acarbose inhibition and reduction in the rate of EPS hydrolysis in the presence of pullulan prove that one single active site is responsible for the cleavage of a-1,4 and a-1,6 glycosidic bonds in this report. We have developed a new bioregulator based on PI designated Rhizoginin-S which stimulates plant cell division very effectively. The invention of Rhizoginin-S was only possible after development of IMBIB participants a simple and effective method of preparation PI, U.S. patent (4,977,091). Thus, developing new culture systems without protein supplements from animal sources will generate safer vaccines. The purpose of our work was studying opportunities of received 'Rhizoginin-S' for acceleration of growth transplantable cultures of cells used in virology. Activity of a preparation studied on its action on culture BHK-21. It were established in the result of carried out investigations that cell growth at 'Rhizoginin-S' addition was increased for 3.2 times. Whereas in medium with serum and buffer, and in medium with usual nutrient medium and serum this index was 2.5 and 2.7 accordingly. Proliferation index at the process of cells growing in medium without serum with addition of 'Rhizoginin-S' was 1.2. Proliferation index in control was 0.8 and 1.0 accordingly. It was marked stimulating effect at cytomorphological investigation BHK-21 cell cultures properties with addition of 'Rhizoginin-S'. This effect was expressed by presence of well-evident nucleolus. While it was shown that in cells untreated by 'Rhizoginin-S' there were no nucleolus. Thus, the data on moderate stimulation proliferation properties of cells and presence of well-defined nucleolus confirm about positive effect of 'Rhizoginin-S' on DNA synthesis in cells. NK cells play a critical role in the immune response against cancer and infected cells. UL16 binding proteins are ligands of NKG2D, which is an activating receptor expressed by NK and T cells. ULBPs show a restricted expression in normal tissues, but they are frequently overexpressed in cancer and infected cells, marking them to be eliminated by the immune system. Here, we characterized that the epigenetic mechanisms play a key role in the regulation of ULBP1-3 expression. The promoters of ULBP1-3 are not methylated, but the structure of chromatin is an essential regulator of their expression. HDAC3 plays a key role in their regulation since it is bound to ULBP1-3 promoters and represses their transcription. Over-expression of HDAC3 decreases ULBP1-3 expression, whereas knockdown of HDAC3 expression using siRNA significantly induced the expression of ULBPs. We also characterized that HDAC3 is over-expressed in colon cancer and our results suggest that this is responsible of the repression of ULBPs expression in these tumors. The promoter analysis coupled with the chromatin immunoprecipitation assays showed that the repression of transcription of ULBP1 is mediated, at least in part, by the interaction with Sp1/ Sp3. Our results also indicated that HDAC2 plays an opposite role in the regulation of ULBPs since its inhibition significantly up-regulated the expression of ULBP1-3. Thus, the opposite effect of several HDACs in ULBPs expression clearly suggest that the development of a specific inhibitors of each HDAC is needed in order to improve the therapeutic efficacy of the HDAC inhibitors.

Dissociation, deflavination, and reconstitution studies of a novel multimeric protein containing different alcohol oxidases produced by Aspergillus terreus A. Kiran Kumar and P. Goswami Department of Biotechnology, Indian Institute of Technology Guwahati, Assam, INDIA Introduction: The importance of alcohol oxidases in biosensor and bioprocess applications is realized and further research to understand these complex proteins is warranted for advancing their applications. Methods: Protein was purified by DEAE-sepharose ion-exchange column and the M r was studied by SDS-PAGE. The size of the protein particles was measured by transmission electron microscopy. For dissociation and deflavination the protein sample was treated with the test reagent and passed through Gel filtration column using UV and fluorescence detector simultaneously. The average size distribution was measured by light scattering detector. LC-MS was used to analyze fatty acids. Alcohol oxidases were assayed by HRP-ABTS method.

Results: The short chain alcohol-, long chain alcohol-, aryl alcohol-, and secondary alcohol-oxidases present in the microsome of A. terreus were found in a multimeric protein entity with M r of the associated proteins were 85-, 63-, 43-, and 27-kDa. The diameter of the smallest protein entity was 10.2 nm, while the average particle size distribution in the highest soluble state of the protein was 180 nm. This multimeric protein was deflavinated and dissociated by treating with (b-mercaptoethanol) b-ME. These dissociated proteins were reconstituted by removing b-ME and subsequently activated by incubating with alcohol substrates. Lipoprotein nature of the protein entity containing oleic acid and palmitic acid was established and correlated with the highly aggregating property of the protein.

Conclusions: A novel multimeric lipoidal protein entity containing different alcohol oxidases was dissociated and deflavinated by using b-ME and subsequently reconstituted to a catalytically active multimeric form.

Cloning, expression and characterization of cyclooxygenase gene from amphipods Introduction: In animals, the first two steps in the biosynthesis of important signaling molecules, prostaglandins and tromboxanes, are catalyzed by cyclooxygenases (COX). In the current study, we describe molecular cloning, characterization and functional expression of COX cDNA from caprellid and gammarid amphipods, the first cyclooxygenases so far identified in arthropod species. Methods: Using homology based degenerate primers and RT-PCR strategy, the protein sequences of the amphipods COX were determined. The recombinant COX proteins from expression in baculovirusexpression system were incubated with arachidonic acid and the products were identified by HPLC. The overlapping fragments of COX gene were amplified from genomic DNA using PCR and the sequences coding COX gene were identified.

The amphipod cyclooxygenases share about 40-43% amino acid sequence identity with the human cyclooxygenases. The proteins contain key residues known to be important for cyclooxygenase and peroxidase activities. However, significant structural differences can be found in the N-and C-termini of the proteins. Expression of the amphipod COX cDNA afforded functional cyclooxygenases. The COX gene from Gammarus is 9.6 kb long and COX gene from Caprella is 6.4 kb long.

Conclusions: First COX enzymes from the organisms belonging to the phylum Arthropoda were cloned and characterized. The recombinant COX proteins were able to convert the exogenous arachidonic acid into prostaglandins. The COX gene structures in amphipods were characterized.

Novel amide (peptide) bond synthetic activity observed in the superfamily of adenylate-forming enzymes Zearalenone (ZEN) is one of the most widely distributed fusarial mycotoxins. ZEN was associated with different reproductive disorders in animals.The aim of the current study was (i) to find out whether oxidative stress and DNA damage could be relevant for ZEN induced toxicity using Balb/c mice and (ii) to evaluate the safety and efficacy of cactus cladodes Opuntia ficus to prevent the deleterious effects of ZEN. To this end, the effect of a single dose of ZEN (40 mg/kg b.w.) alone and with extract of cactus cladodes (25, 50 and 100 mg/kg b.w.) on the induction of oxidative stress was monitored in kidney and liver by measuring the MDA level, the protein carbonyls generation, the Catalase activity and the expression of the Heat Shock Proteins (Hsp). For the genotoxicity assays, we have performed the DNA fragmentation, chromosome aberrations and micronuclei induction. Oxidative damage seems to be a key determinant of ZEN induced toxicity in both liver and kidney of Balb/c mice. The combined treatment of ZEN with the lowest tested dose of cactus extracts (25 mg/kg b.w.) showed a total reduction of ZEN induced oxidative damage for all tested markersSimilar results were found for the DNA damage, Cactus extracts revealed a significaant decrease in DNAG fragmentation chromosome aberrations mainly breaks and the induction of micronuclei. It could be concluded that cactus cladodes extract was effective in the protection against ZEN hazards. This could be relevant, particularly with the emergent demand for natural products which may counteract the detrimental effects of oxidative stress and therefore prevent multiple human diseases.

Innovation of antibodies with high affinity for inorganic materials for bio-interface application Proteins which recognize the surface of bulk inorganic materials are candidates as a building block tool for manipulating nanomaterials. In this study, we constructed the antibody fragments with binding affinity and specificity for bulk surface of nonbiological ZnO material. To create material-binding antibodies, we first functionalized the camelid heavy chain antibody fragment (VHH) by grafting a ZnO-binding peptide into the complementarity determining region (CDR) of VHH. The grafted VHH fragment showed similar CD spectrum to native VHH and has the same quantitative affinity for ZnO as the inserted peptide itself. Further, phage display selection technique was applied to promote the affinity of the peptide-grafted VHH fragment. The selected clone 4F2 VHH from the library had the dissociation equilibrium constant (K D ) of 9 nM and showed no binding to other materials, indicating that we maturated the grafted VHH to highly affinitive and selective VHH. In addition, we attempted to create the artificial antibody fragment with bispecificity for ZnO and Au materials by fusing anti-Au VHH and 4F2 VHH via hinge-linker to demonstrate the application of material-binding antibody as bio-interface molecule.

A circular dichroism study of the stability of guanine quadruplexes of thrombin DNA aptamers at presence of potassium and sodium allowing them to bind specifically to various molecules. Increased interest to the aptamers is connected with their high potential for medical therapy and for biosensor development. The DNA aptamer against fibrinogen binding site of thrombin was among the first developed. It has following sequence 5¢-GGT TGG TGT GGT TGG-3¢ (FIBRI). Later the aptamer for heparin binding site of thrombin was synthesized: 5¢-GGT AGG GCA GGT TGG-3¢ (HEPA). It was suggested, that that the later aptamer should contain also large supporting part in order to provide their stability. Both aptamers form typical G-quadruplexes, which stability substantially depends on the ion composition and concentration. In this work we used circular dichroism (CD) to study the stability of both aptamers at presence of potassium and sodium ions. Methods: We measured CD spectra using Jasco JASCO J-810 spectropolarimeter (Japan) at presence of sodium and various concentrations of potassium and as a function of temperature. The concentration of both aptamers was 10 lM.

The CD studies showed that the complexes of FIBRI-K + were slightly more stable than HEPA-K + . However, lower stability was observed for HEPA-K + at presence of Na + in comparison with FIBRI-K + . The analysis of CD melting curves suggests differences in thermal stability of both aptamers at presence of K + . The melting temperatures, T m , and changes in van't Hoff enthalpy for HEPA-K + complexes were lower in comparison with those for FIBRI-K + . With increasing HEPA concentration the T m value increased, but did not change with increasing FIBRI concentration. This suggests formation of HEPA aggregates that could stabilize quadruplex structure, while FIBRI aptamers were in monomeric form.

Conclusion: Using the CD spectroscopy we studied the stability of guanine quadruplexes at presence of potassium and sodium ions for two 15-mer DNA aptamers that selectively bind the thrombin to fibrinogen (FIBRI) or heparin (HEPA) binding sites. Despite the substitution of certain nucleotides in HEPA by purines, both aptamers form high ordered guanine quadruplexes at presence of potassium ions. However, sodium has unfavorable effect on binding of potassium for HEPA. In contrast with FIBRI, HEPA forms aggregates at higher concentrations.

Different modes of interaction of safranal in comparison to picrocrocin with GC and AT rich oligonucleotides R. Houshyar and S. Z. Bathaie Clinical Biochemistry Department, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, IRAN Introduction: Saffron (Crocus sativus L.) shows different biological properties. Since specific functions have been reported for safranal (a monoterpene aldehyde as saffron aroma and deglycosylated derivative of picrocrocin), the present study aimed to investigate its interaction with calf thymus DNA (ctDNA), oligo(dG-dC) 15 and oligo(dA-dT) 15 in comparison to picrocrocin. Methods: The circular dichroism (CD) and fluorometric results show the non-intercalative or minor groove binding of the mentioned ligands with ctDNA. Results: The CD plots indicate that picrocrocin interacts with DNA and oligonucleotides at lower concentrations than safranal, which is accompanied by B-to C-DNA transition; at higher concentrations, it induces more conformational changes, i.e. unstacking of DNA and oligonucleotides bases as we also previously reported for saffron carotenoids. The similar results are obtained for safranal interaction with ctDNA (56% A.T) and oligo(dA-dT) 15 ; but the CD results indicate a two step mechanism of binding for safranal with oligo(dG-dC) 15 . At lower concentrations of ligand, CD pattern is similar to others; by adding more safranal, the positive peak in the range of 260-280 nm is deconvoluted to two separate peaks. One small peak appears at 260 nm and a higher peak at 285 nm, which are the characteristic peaks of triplehelix formation. A new binding mechanism is suggested for these observations. Conclusions: Results indicated picrocrocin cause more distortion on oligonucleotide structure and induces B to C transition in poly(dG.dC) 15 and poly(dA.dT) 15 . Whereas safranal induces the same effects on poly(dA.dT) 15 , the B-to H-DNA transition is observed due to its interaction with GC reach sequences. Introduction: Glucocorticoid receptor-interacting protein 1 (GRIP1), one of the p160 family co-activator proteins, primarily mediates transcriptional activation by nuclear receptors via three activation domains that recruit at least three secondary co-activators, CBP/p300, co-activator-associated arginine methyltransferase 1, and coiled-coil co-activator (CoCoA), which exert histone acetyltransferase and/or arginine methyltransferase enzyme activity. The regulatory mechanisms underlying the GRIP1 co-activation functions involve its post-translational sumoylation by promyelocytic leukaemia protein (PML), but are not well understood.

Results: Here, our findings demonstrate that PML, similar to UBC9, preferentially acted on subregions within the GRIP1 C-terminal region. The mechanisms of enhancement by PML and UBC9 both suppressed the repressive effect within the GRIP1 C-terminal region and depended on the sumoylation status of PML, but not on the sumo-conjugating enzyme activity of UBC9. Daxx is not the primary target of PML in the regulation of GRIP1 co-activator functions. Therefore, the activity of PML might be mediated through the regulation of GRIP1 transactivation activity via protein-protein interaction to enhance the nuclear receptor co-activator functions of GRIP1.

Conclusions: In this study, we provide new insights into the sumoylation status of PML involved in the regulation of the GRIP1 co-activator functions, both in the conformation change of GRIP1 and in forming nuclear foci through PML-related nuclear bodies in nuclei.

The multiple activities of polyphosphate kinase of Mycobacterium tuberculosis H37Rv Glutathione is a tripeptide (Glu-Cys-Gly) that exists ubiquitously in food. In general, most glutathione is present as a reduced form GSH in food. GSH shows no basic taste (sweetness, saltiness, acidity, bitterness, and umami), but is believed to affect some flavor characteristics, such as thickness, mouthfulness, and continuity. This suggests that GSH is one of taste modifiers in food. The purpose of this study is then to reveal physiological roles of GSH on mammalian taste reception and the influence of GSH on calcium response in monosodium glutamate (MSG). The lingual epithelium of c57BL mouse was peeled off enzymatically. Circumvallete and foliate papillae were collected from the epithelium under microscopic observation and applied to in situ calcium imaging using Calcium Green 1. Fluorescein-5-maleimide-labelled GSH was alternatively loaded on the mouse lingual epithelium to monitor GSH distribution. Licking response of GSH was measured using conditioned MSG aversion mouse. GSH slightly elicited intracellular calcium responses in MSG responsive taste receptor cells and enhanced the calcium response induced by MSG. Taste buds were well stained with the fluorescein-labelled GSH. The conditioned mouse decreased licking to GSH with increasing MSG concentration. These results suggest that mammalian taste cell might express GSH receptor proteins on cell membrane and receive it as a taste modifier.

Identification of putative antigenic candidates to an antielapidic serum based on analysis of Micrurus corallinus transcriptome (coral snake) L. Iwanaga Lea˜o, P. L. Ho and I. Junqueira de Azevedo Centro de Biotecnologia, Instituto Butantan, Sa˜o Paulo, BRAZIL

The analysis of the transcriptome from Micrurus corallinus venom gland makes possible the identification of some antigenic candidates for an antielapidic serum based on DNA immunization. After an analysis of the obtained ESTs, five cDNAs coding for toxins were selected from the databank as candidates, due to their overall representation and known role in the envenoming. They were named: Atg1, Atg2, Atg3, Atg4, which are four distinct possible neurotoxins holding the three-finger toxins scaffold, and Atg5, which are an abundant presynaptic type A2 Phospholipase. These five selected candidates were cloned in pSecTag2A, a vector for protein expression in mammal cells. For testing the construction, the candidates were transfected in COS-07 cells (transient transfection). Then, we started the DNA immunization in mice, injecting 100 mg of each candidate cDNA. For the analysis of the immunological response, we produce and purify the recombinant proteins in E. coli. The recombinant proteins were used in the ELISA coating to react with the primary serum originated from the DNA immunization, in order to evaluate the immunological response. The results showed that the serum of immunized animals were able to recognize the recombinant antigens. This indicates that the DNA immunization in mice could be a good alternative to crude venom immunization, which is costly and relies on venom availability. It is a promissory result since the antibody production could be achieved via intramuscular immunization, which is practical for producing purposes. Therefore, we are optimistic and ready to proceed with the next experiments, which will be to evaluate two other possible immunization methods: the Gene-Gun immunization and the liposome. Aflatoxins are a group toxic metabolites produced by fungi Aspergillus flavus and A. parasiticus. These toxins frequently contaminate food and feed crops and have been classified as group I carcinogens by the International Agency for Research on Cancer. Antibody-based assays are among the most effective techniques available for the measurement and detection aflatoxins. The performance these methods is influenced by the specificity and the sensitivity the antibodies used. The present work is the first step the development recombinant antibodies against aflatoxins with increased specificity and sensitivity and their expression in foreign hosts. By using indirect ELISA 10 monoclonal antibodies (mAb) against aflatoxins B1, B2 and G1 were selected as potential candidates for the protein engineering studies. Their affinity and crossreactivity to the different types aflatoxins were characterized by indirect and competitive ELISA. The hybridoma cells producing these mAb's were used for the total RNA isolation, subsequent cDNA synthesis and amplification gene fragments coding for immunoglobulin heavy and light chain variable regions. The fragments obtained were cloned and sequenced. Sequence analysis showed that the mAb's have different heavy chains, but only two types light chains. It can be inferred from these findings that the difference in mAb's affinity and cross-reactivity is provided by their heavy chain structure. These data could serve as a basis for the development recombinant antibodies characterized by improved affinity and specificity parameters. Introduction: Preparation polyelectrolyte complexes natural and synthetic macromolecules appears to be a great importance for medicine and biotechnology in particular with respect to design immunogenic compounds for vaccine innovation (1) . In this study copolymers vinyl pyrrolidone/acrylic acid (VP/AA) were used as a carrier polyelectrolyte. As a novel part this work, microwave energy was used for conjugation reaction between polyelectrolye and synthetic peptides. Methods: Synthesized viral peptides VP1 135-161 (WSKYSTT-GERTRGDLGALAARVATQLPA) and VP1 200-213 (WDRHKQRI-IAPAKQLQ) defined from foot and mouth disease viruse's (FMDV) capsid protein sequences. Two different types conjugation method between peptides and VP/AA was used in this study. Water media conjugation was proceeded in Phosphate-buffered saline (PBS). EDC was used for carboxyl group's activation at pH 5. Reaction takes 36 hours in 4°C. Organic conjugation was proceeded in DMF. HBTU, HOBt was used for carboxyl group's activation. Reaction takes only 1.5 hours. Microwave used 25 W for maximum 75°C. LC-MS system (Shimadzu LC-MS 2010 EV) with ESI (Electrospray Ionization) method and GPC Viscotek TDA 302 with four detectors were used for characterization synthetic peptides and their conjugates. Results and Conclusions: Figure 1 indicates that microwave assisted organic conjugation peptide and copolymer gives more efficient labeling according to water media conjugation. Also polydispersity index the organic conjugation smaller than the water media conjugation. This indicates that organic conjugation more homogenious than water media conjugation. Introduction: The HCV, GBV-B and GBV-C RNA viruses, all members the Flaviviridae family, exhibit a great similarity on their genome organization encoding for a precursor polyprotein that is processed to give the structural and the non-structural viral proteins. Initiation translation, in these viruses, is cap-independent and is driven by an internal ribosome entry site (IRES), located mainly within the 5¢ non-coding region. Recent data suggested that different viral proteins can regulate the activity the HCV IRES. Among them, HCV NS5A has been shown to down-regulate the HCV IRES in a specific and dose dependent manner. The aim this study is to investigate whether the above property HCV NS5A is conserved across the members the Flaviviridae family, and to examine whether the effect HCV NS5A can be exchanged with that the homologue proteins. Methods: To address these issues, transient transfection was carried out using two different plasmid vectors. The first expresses a bicistronic transcriptional unit carrying the chloramphenicol acetyltransferase (CAT) and the firefly luciferase (LUC) genes separated by the HCV or GBV-B IRES sequences. The other, expresses separately the NS5A HCV, GBV-B, and GBV-C.

Results: Here we present data concerning the comparison translation initiation efficiency, HCV and GBV-B IRESs, in different cell lines -hepatic and non-hepatic -as well as the effect NS5A homologues on the HCV and GBV-B IRES. Our data suggest that NS5A GBV-C, similar to HCV NS5A, represses the HCV IRES activity whereas the GBV-B NS5A has a milder effect. Recombinant viral vectors and virus-like particles are considered one the most promising vehicles to deliver antigens in prophylactic and therapeutic vaccines against infectious diseases and cancer. As part evaluation vaccine candidates, we analyse the transcriptome dendritic cells (DCs -the most potent antigen presenting cells) from vaccinated mice. We aim to define molecular signatures relevant to both safety and efficacy evaluation. We performed microarray analysis on spleen DCs, isolated from mice vaccinated with two types viral vectors delivering the same gp33-41 epitope LCMV as a model antigen. The vaccine vectors tested were a defective adenoviral vector and a VLP formed by the coat protein the RNA phage AP205, covalently linked to the epitope. RNA was isolated from CD11c+ cells vaccinated and control mice and transcriptional priles were assessed with the CodeLink mouse whole genome microarrays. Acquired images were normalized and inspected for spot irregularities. Data was filtered according to quality spot identifiers and genes affected more than two-fold, compared to the respective controls, were selected. A third filter was applied to identify common upregulated or downregulated genes, following vaccinations with the adenoviruses and VLPs. The differentially expressed genes were further classified according to gene ontology identifiers, in order to select genes involved in immune regulation, apoptosis and other characterised pathways. The outcome this microarray analysis, together with the evaluation the specific immune responses to the model antigens will be presented. Such analyses should help in the standardisation the vaccine assessment.

Anti-metastatic activity of a new anti-tumour enzyme L-lysine-alpha-oxidase derived from Trichoderma sp Biochemistry have been involved, in particular, in investigation cancer metabolism for a long time. As an example biochemical approaches to the cancer therapy might be using bacterial origin enzymes (for instance, L-asparaginase) in cancer chemotherapy schemes. Together with enzymotherapy undeniable practical interest for oncology has enzymodiagnostic which galvanizing further research at the area tumour markers. Thus, medical enzymology (enzymotherapy + enzymodiagnostic) recently started to play an important role in the biochemistry carcinogenesis and tumour growth at whole. It is important to pointed out that enzymes in comparison with cytostatics carter for main requirements chemotherapy -selectivity anti-tumour effect due to high level specifity to substrate. Clinical experience L-asparaginase usage shows that these particular enzymes has some side-effects which prompting a nessasity searching a mew enzymes Oncological usage. One a new promising anti-cancer enzyme is L-lysine-alpha-oxidase (LO) -EC 1.4.3.14. This part studies have been conducted jointly with the Institute Oncology Problems (Kiev, Ukraine). It is important to stress up that the results on anti-invasive effect LO in vitro logically fulfilling data obtained during investigation anti-metastatic activity LO in vivo. Taking into a consideration with established fact anti-invasive effect LO in vitro our main goal was investigation influence LO on metastatic process on mice with a spreading Lung Lewis Carcinoma (3LL) and also activity AD and 5¢-N in alveolar macrophages and a content polyamines in erythrocytes. Decreasing volume and quantity metastases (MTS) 3LL have been detected even under administration LO in dose 10 IU/kg and maximal anti-metastatic effect -after LO-injection in dose 50 IU/kg. Surgical removal primary tumour (on 15-day after transplantation) causes drastical increasing both volume and quantity MTS as well in lungs in comparison with unoperated animals. Reason for that may lay down in a stress-reactions galvanized a body after tumour removal. In addition to that, LO anti-metastatic effect was confirmed by using two independent biochemical approaches: Polyamine test (level putrscene, spermene and spermedene in erythrocytes) as a tumour markers using for the complex assessment the effectiveness chemotherapy Determination functional status alveolar macrophages -measuring activity two enzymes purine-metabolism -AD and 5¢-N.

In addition to all above, therapy with LO (50 IU/kg) causes a considerable increasing in life-span mices with 3LL (primary tumour removed). Even 200 days later after tumour transplantation -administration LO caused preservation 50% animals in experimental group. For example, it seems that the numbers hydrophobic residues in coxsackievirus B 3 2A protease is much more in a way that reduces its thermal stability. In addition docking studies reveal that this hydrophobic region can make a strong interaction with the active site and decrease its stability remarkably. The protein structure comprises a two-domain structure; the N-terminal domain comprises several b-strands (barrel) while C-terminal domain shows a a-helix and a b-sheet. The domains are connected by a loop. The active site consists a catalytic triad formed by His21, Asp39 and Cys110 which can serve as an important prerequisite for drug design. Docking studies reveal a substrate-induced fit that explains the specificity the active site residues and support a direct cleavage eIF4G that continues to be the subject much debate. The experimental investigation through western blot analyses was in agreement with our computational study. In the absence a crystal structure, our model facilitates design inhibitors against 2A protease.

Production of 5-aminolevulinic acid by propionibacteria Faculty of Science, Mahidol University, Bangkok, THAILAND 5-Aminolevulinic acid (5-ALA) is a first intermediate in biosynthesis pathway heme, chlorophyll and viaminB 12 in living organisms. 5-ALA can be used as plant growth regulators and in photodynamic cancer therapy. Recently, 5-ALA has been produced from photosynthetic bacteria but no report its production in Propionibacterium species which is a vitamin B 12 producer. In this study, yields 5-ALA synthesized from classical Propionibacterium species including the effects various precursors and ALA dehydratase inhibitor on 5-ALA accumulation were investigated. We found that P. acidipropionici produced 5-ALA at 34.1 mg/l which was 3-5 folds higher than that produced by the other species. The production 5-ALA in P. acidipropionici was enhanced to 38.7, 39.6, and 47.2 mg/l in the presence 1 g/l levulinic acid, 1 g/l glutamate, and 4 g/l glucose, respectively, while glycine exhibited the strongest ability to enhance 5-ALA production. In addition, P. acidipropionici produced 5-ALA at 80-104 mg/l which was 10 fold higher than that produced by the other species when 12 g/l glycine were added. Furthermore, in the presence 1 g/l levulinic acid and 12 g/l glycine, the production 5-ALA in P. acidipropionici was enhanced to 173.6 mg/l which was 17 fold higher than that produced by the other species. Protein flexibility and plasticity makes the drug design task for the particular target more difficult. Although in particular cases where high specificity is required this characteristic could be beneficial. Cyclindependent kinases (CDKs) are important anti-cancer targets shown those characteristics. Wide variety CDK2 inhibitors complex structures having been characterized in inactive form. In addition, crystallographic data exists for only one ATP-competitive inhibitor in both the active cyclin-bound and inactive CDK2 forms. We have solved the structures six inhibitors in both the monomeric CDK2 and binary CDK2/cyclin A complexes and demonstrate for the first time that significant differences in binding CDK2 ligands occur depending on the activation state. The binding mode two ligands varies substantially as a result binding site differences induced upon CDK2 activation. Furthermore, an energetic analysis CDK2/cyclin complexes demonstrates that those differences could be used beneficial in the design new inhibitors. These results confirm that structures solved in complex with the monomeric CDK2 do not fully reflect the active conformation bound inhibitors. This analysis reveals significant implications for inhibitor design towards active structures since these are distinct from the inactive CDK2 and also suggests that the monomeric CDK2 conformation could be selectively inhibited.

Cell-free expression for production of eukaryotic proteins in a soluble form The cell-free systems are successfully used for protein production together with expression in E.coli. A unique feature the cell-free systems is their openness that allows to modify the reaction conditions in order to protect or stabilize the synthesized recombinant proteins. For example, detergents can be added to the translation mix for maintenance membrane proteins in a soluble form or red-ox potential can be regulated for the formation a disulfide bonds in proteins. We expressed human nicotinic acetylcholine receptor (nAChR) a7 and extracellular domain nAChR in E.coli continuous-exchange cell-free system. The yields proteins were 1-2 mg/ml translation mix. In the case extracellular domain the addition reduced and oxidized glutathione in certain ratio resulted in formation disulfide bonds and considerable fraction protein remained soluble. Also nAChR was synthesized in soluble form in the presence nonionic detergents 0.5% Brij 35 and Brij 58. Interestingly, many the nonionic detergents decrease the yield target protein in a bacterial cell-free system, but it emerged that they do not affect wheat germ cell-free system. 1% Brij 35, Digitonin, Nonidet P40, Triton X100 only slightly influence the productivity wheat germ cell-free system. That let us synthesize human peptidoglycan recognition protein Tag7 in soluble state in the presence Brij 35. It is also notable that green fluorescence protein synthesized in the presence listed detergents is less active than in their absence whereas for dihydrolate reductase this is not the case. Vinca Institute of Nuclear Sciences, Belgrade, SERBIA, 2 Istituto Nazionale di Fisica Nucleare, Laboratori Nazionali del Sud, Catania, ITALY Introduction: Considering that deregulation cell cycle and apoptosis commonly occur during tumorigenesis, effects combined treatments with fotemustine (FM) and proton irradiation on these processes as well as on relevant gene expression were investigated. Methods: Subconfluent HTB140 human melanoma cells were treated with FM (100, 250 lM) 24 hour prior to proton irradiation (12 Gy, 16 Gy). Assays were performed 48 hour after irradiation. Cell proliferation was monitored by bromodeoxyuridine (BrdU) incorporation, while cell cycle distribution and apoptosis were measured by flow cytometry. Variation p53, cyclin A, cyclin B and cyclin E mRNA levels were evaluated by RT-PCR. Results: All analyzed single or combined treatments induced significant inhibition cell proliferation, ranging from 24 to 53%. Level apoptosis increased after treatments with FM, protons or their combination, with apoptotic indexes varying from 1.54 to 6.43. The highest values were obtained for irradiated samples pretreated with 250 lM FM. Flow cytometric analysis showed that 250 lM FM induced G2 phase arrest. Other applied treatments did not affect cell cycle distribution. In samples exposed to proton irradiation pretreated with 250 lM FM, p53 gene expression was up-regulated. Cyclin A was slightly down-regulated in all analyzed samples, while expression cyclin B and cyclin E genes were unaffected. Conclusions: All analyzed treatments reduced cell proliferation. The best effects on apoptosis and up-regulation p53 expression were obtained after combined treatments with higher FM concentration.

PP8-91 p16 methylation among high risk, cigarette smokers and heavy metal-exposed individuals Introduction: Methylation in the p16 promoter, a cell-cycle regulator, is frequent in lung and other carcinomas and has been proposed as a marker for the disease. p16 promoter methylation is also observed in myelodysplastic syndrome. Methods and Results: We presently analyzed 40 sputum samples from smokers visiting a smoking cessation clinic (14 male, 26 female), 104 cervical scrapes (54 from regular smokers), 12 blood samples from Pb-exposed individuals, (7 cases >10/100 lg/ml) and 12 samples blood from healthy non-smokers for p16 promoter methylation using a nested, sensitive methylation specific PCR assay (MSP). The frequency p16 methylation was frequent in the sputum smokers (22.5%) and higher in cervical smears smokers (38.33%) compared to non-smokers (20%) and correlated with the tobacco exposure in both cases (P = 0.05). Similarly, p16 methylation was frequent among individuals with present or past Pb-overexposure (8/8) and correlated with the severity exposure (2/2 negative with lower Pb blood levels). However p16 methylation was not detected among healthy non-smokers. Temperature melting analysis 50 p16 MSP products either from sputum or smears, revealed that higher thermal stability (Tm at 89°C vs Tm 86°C) is observed in MSP products from heavy smokers (5/12 cases, with >12.5 pack/years tobacco consumption) indicating a greater extent p16 promoter methylation with increasing tobacco exposure.

Conclusion: This data shows that p16 methylation is not rare among non-patients over-exposed to tobacco and heavy metals, and that the degree methylation is related to genotoxic factor exposure.

Identification of two 1-hydroxy-2-naphthoate dioxygenase genes in Arthrobacter sp. strain Here we report the purification and characterization 1-H-2-N dioxygenase from strain Sphe3. Purification 1-H-2-N dioxygenase to homogeneity showed a homotetramer a 45-kDa subunit. MS analysis the 45 kDa protein band species enabled the identification oligopeptide fragments covering up to 29% the whole sequence with 100% homology to the relative fragments 1-H-2-N dioxygenase from Nocardioides sp. Whole genome sequencing the Sphe3 strain led to the identification two ORFs, named diox1 and diox2, coding for 387 aa proteins, respectively. Both ORFs exhibited 1-H-2-N dioxygenase activity following PCR amplification, subcloning and overexpression using pET29c. The predicted protein products the two 1-H-2-N dioxygenase ORFs shared 93% homology, while they shared 84% homology with the Nocardia 1-H-2-N dioxygenase. In addition, ORFs diox1 and diox2 shared 90% homology to each other at the DNA level, implying a gene-duplication. Introduction: Polycyclic aromatic hydrocarbons (PAHs) are major constituents crude oil, creosote and coal tar. Their presence in soil and sediments poses a significant risk to the environment since they have toxic, mutagenic and/or carcinogenic properties. Microbial biotransformation is a major environmental process affecting the fate xenobiotics as PAHs in soil and aquatic ecosystems. Arthrobacter sp. Sphe3 isolated from a creosote-polluted Greek soil, utilizes phenanthrene as a sole source carbon and energy. Methods: Identification proteins involved in the degradation phenanthrene was performed by two-dimensional gel electrophoresis (2-DE) in combination with mass spectrometry (MALDI-TOF MS and MS/ MS) * .

Results: Several PAH-degrading proteins were identified by proteomic analysis, including 1-hydroxy-2-naphthoate dioxygenase and protocatechuate dioxygenase. These proteins were solely expressed at growth on phenanthrene and were not detectable on 2D gels cells grown on glucose. In addition, growth on phenantrene induced the enzymes responsible for the conversion 1-hydroxy-2-naphthoate to 2-carboxybenzaldehyde, NAD-dependent 2-carboxybenzaldehyde dehydrogenase and protocatechuate oxygenase, but not NAD(P)H-dependent Mantle cell lymphoma (MCL) is a distinct subtype of B cell lymphoma composed of small-to medium-sized lymphoid cells, probably originated from follicle mantle B cells. This lymphoma is characterized by the (t11; 14) (q13; q32) translocation, which results in deregulated aberrant expression of cyclin D1. The aim of our study was to determine whether the PI3K/Akt signaling pathway contributes to the pathogenesis of MCL. To accomplish our goal, we examined the change in gene copy number and the mutation frequency of PI3KCA gene, the phosphorylation status of Akt and the loss of expression of PTEN, in a set of primary MCL samples and MCL cell lines. We found that 14 of 23 (58%) primary MCL cases with amplifiable GAPDH and two of two cell lines harboured a gain (> 3) of PI3KCA gene copy number. PI3KCA gene mutations were not identified. In the immuno-histochemical analyis, 12 of 33 (36%) MCL samples showed high levels of pAkt expression, whereas increased levels of pAkt were shown in the cell lines and in 5 of 14 (36%) frozen MCL samples by immunoblotting. In addition, amplification of PI3KCA correlated with the status of Akt phosphorylation in 7 of 12 (58%) primary MCL cases and the two MCL cell lines. Inhibition of PI3KCA induced increased apoptosis in the MCL cell lines. PTEN protein expression was present in all 14 frozen MCL cases and cell lines, whereas five of 33 (15%) cases tested by immunohistochemistry had loss of PTEN protein expression. In conclusion, we demonstrated that PI3K/Akt pathway is activated in MCL and that PI3KCA gene amplification may contribute to PI3K/Akt oncogenic activation. Therefore, PI3KCA is an attractive molecular marker and a promising therapeutic target in MCL.

Mutation screening of exon 20 of BRCA1 gene by high-resolution melting curve analysis P. Vorkas 1 , C. Kroupis 1,2 , K. Christopoulos 1 and E. Lianidou 1 1 Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, Athens, GREECE, 2 Laboratory of Clinical Biochemistry, Attikon University Hospital, Medical School, Athens, GREECE Introduction: 5382 insC frameshift mutation in exon 20 of the BRCA1 gene is the second most frequently reported mutation in this gene according to the Breast Information Core (BIC) database. This mutation along with the 5331 G>A (G1738R) missense mutation (also found in exon 20) are relatively frequent amongst the Greek breast and ovarian cancer population. Our goal was to develop a novel, reliable and rapid screening method for mutation screening of the whole BRCA1 exon 20 by using high-resolution curve analysis. Methods: After extraction of genomic DNA, exon 20 of the BRCA1 gene was amplified by flanking specific primers. We developed methodology based on real-time PCR (LightCycler, Roche), and high-resolution melting curve analysis in the presence of LCGreen I dye (HR-1 Melter, Idaho Technology). Our methodology was first optimized and evaluated by using 5382 insC and 5331 G>A positive and wild type genomic DNA samples as verified by DNA sequencing (ABI 310, Applied Biosystems). Sensitivity studies showed that mutated DNA could be reliably detected even at a 10% ratio with wt DNA. Results: Genomic DNA was isolated from 102 FFPE breast tumors, 15 normal breast tissue samples, as well as from 40 peripheral blood samples. The developed method managed to successfully identify one BRCA1 5382 insC and two 5331 A>G mutated samples, that were further verified by DNA sequencing.

Conclusions: The combination of real-time PCR and high-resolution melting curve analysis provides an inexpensive, simple and rapid approach to successfully screen exon 20 of BRCA1 gene for these clinically important and frequent mutations. The influence of octanoate concentration and fermentation parameters on polyester biosynthesis and accumulation and the correlation between biomass and PHA concentration were determined by monitoring the following fermentation media characteristics: optical density, octanoate content (GC method), dry cell weight (gravimetric method), MCL-PHA content as polyhydroxyoctanoate -PHO (GC-MS and FT-IR method). The recovery of intracellular PHA was performed using enzymatic or NaClO digestion and/ or solvent extraction.

Results: Maximal production of MCL-PHA (52% from DCW) and bacterial biomass (50 gDCW/L) were achieved in fermentation medium with 0.4% Na-octanoate, after 48 hours of cultivation, at 35°C. Maximal percentage of MCL-PHA (95% purity) recovery from biomass (85%) was obtained using NaClO digestion, extraction with hot chloroform and precipitation with cold methanol.

Conclusions: (i) The mutant strain tested as PHA producer has the capacity to convert octanoate into MCL-PHA at a maximal level of 52% from DCW, with a productivity of 0.54 g/l/hour; (ii) Time profiles of PHA biosynthesis and biomass growth are similar and (iii) NaClO digestion of biomass leads to a better recovery of the polymer than the enzymatic digestion.

Effective bacterial expression of three-finger neurotoxins from snake venom a-Neurotoxins from snake venom are small b-structural proteins with classical 'three-finger' fold. Different neurotoxins block distinct subtypes of nicotinic acetylcholine receptor (nAChR). Short-chain a-neurotoxins (60-62 a.a., four disulfide bonds) preferentially inhibit muscletype nAChRs, whereas long-chain a-neurotoxins (66-75 a.a., five disulfide bonds) block both muscle-type and a7 homooligomeric neuronal nAChRs. For structural investigations of neurotoxin/receptor interactions the effective production system of neurotoxins is required. In search for such system we concentrated our attention on bacterial expression, as giving highest yield of recombinant proteins and allowing straightforward isotope-labeling. Bacterial expression of disulfide rich proteins like a-neurotoxins is a difficult task. The cytoplasm expression leads to formation of the inclusion bodies. To solve this problem, we developed the system for the secretion of snake neurotoxins as a fusion with the signal peptide of E. coli heat-stable enterotoxin II. After induction with IPTG, toxins (processed during the secretion process) begin to accumulate in periplasm and growth medium. As a result, recombinant neurotoxins with correctly formed disulfide bonds and native N-terminus are obtained. We tried developed system for the production of short-chain neurotoxin II from Naja oxiana venom and its several mutants (including isotope-labelled variants and five disulfide analogue of long-chain neurotoxin). The final yield of purified neurotoxins was up to 20 mg per liter of bacterial culture on minimal medium. To the best of our knowledge, this is the highest yield obtained for the heterologously expressed three-finger toxins.

Generality of marine allene oxide synthesis by the allene oxide synthase-lipoxygenase fusion protein pathway H. Lohelaid, R. Jarving and N. Samel Department of Chemistry, Tallinn University of Technology, Tallinn, ESTONIA Introduction: Lipoxygenases (LOX) are widely distributed in animals and plants and the LOX products (fatty acid hydroperoxides) have a wide range of biological functions as diverse signal molecules, oxidants and modifiers of membrane structures. The conversion of fatty acid hydroperoxides to allene epoxides is catalysed by a cytochrome P450 in plants. In contrast, in the coral Plexaura homomalla, a catalase-related hemoprotein fused to the lipoxygenase (LOX) was found to function as an allene oxide synthase (AOS). The aim was to identify the presence of this pathway in the Arctic soft coral Gersemia fruticosa. Methods: G. fruticosa allene oxide synthase-lipoxygenase (AOS-LOX) cDNA was cloned by using homology-based RT-PCR, sequenced and the full-length fusion protein and LOX domain were expressed separately in E. coli. The incubation products were identified by reverse phase-HPLC and mass-spectrometry, the stereochemistry of the LOX product was identified by chiral-phase HPLC.

Results: The G. fruticosa mRNA encodes for a protein with 84% sequence identity to the P. homomalla AOS-LOX. The expression of G. fruticosa AOS-LOX in E.coli at 16°C gave a fully active fusion protein that converted arachidonic acid into an allene oxide. In aquous media the latter is non-enzymatically converted into a-ketol (8-hydroxy, 9keto-5Z, 11Z, 14Z-eicosatrienoic acid), as a major product and a minor cyclopentenone, and 8-hydroeicosatetraenoic acid (the known reduced product of the 8-lipoxygenase). The lipoxygenase product identified was 8R.

Conclusions: Our data clearly indicate that the AOS-LOX fusion protein pathway of allene oxide synthesis is widespread in coral and P. homomalla represents no exception. 2A protease of the pathogenic coxsackievirus B 3 is a major enzyme involved in the etiology of heart disease. Despite this obvious importance, this enzyme remains poorly characterized by a lack of large quantities of purified enzyme. In this study structural features were determined through designing a high yield purification process. CD analyses showed that the protein contains mostly B-strand elements and requires Zn 2+ ions as structural component. Using intrinsic fluorescence analyses to evaluate the effect of different kinds of cations on structural integrity showed that among all the metal ions, Zn 2+ is inhibitory if added exogenously whereas the effects of other salts (NaCl, CaCl 2 and MgCl 2 ) were less pronounced. This effect is probably due to shielding and interaction of some critical areas and structural residues with the excess zinc ions. Under such an environment, the protein finds almost rigid conformation and loses its structure somehow. As 2A pro is a cysteine protease, it requires the presence of a free thiol group as the nucleophile and the inhibitory effect of zinc ions could result from binding of the metal to the cysteine residue in the enzyme active site. The maximum enzyme stability was achieved at 4°C and pH 7. Turbidity measurements showed that increase of temperature caused a large amount of aggregation which can be the result of a high content of B-structure Measurement of free SH groups using Ellman test showed that both basic and acidic pH oxidizes the protein and induced disulfide bands which decreases the protein stability remarkably.

The alternative oxidase of Aspergillus fumigatus plays an important role in oxidative defense A. fumigatus is a human pathogenic fungus capable of inducing a range of disease states in immuno-compromised patients. Fungal respiration exhibits peculiar features and involves an alternative oxidase pathway not yet fully elucidated. In this sense, we have silenced an aox gene in A. fumigatus (Afaox) by RNA interference. Inverted repeats of Afaox coding sequence were cloned in the pALB1 plasmid, which contain an alb1 gene cloned. In xylose medium, the Afaox/alb1 and alb1 transformants had the same phenotype, a green color, as the wild strain, indicating that the interference was not induced. However, in maltose medium the Afaox/alb1 transformant grew in white color indicating an entire silence and a Real-time PCR revealed a decrease of 95% in RNAm levels. It has been suggested that AOX may be important for fungal survival in macrophages, based on accumulating evidence for its antioxidant defense. ROS production was monitored using fluorescent probe CM-H 2 DCFDA. In Afaox/alb1 transformants, ROS production increased 12.5 and 2-fold when compared with wild-strain and alb1 transformants, respectively, suggesting that AOX can prevent ROS formation. The survival rate of silenced strain in macrophages was measured by MTT assay. Afaox, double silenced strain, revealed a highest survival when compared with the other strains. Conidia were incubated with different ROS donors and conidia viability was measured by flow cytometria with FUN-1 and PI uptakes. These results indicate that the alternative oxidase has an important role in oxidative defense in Aspergillus fumigatus Acknowledgements: Supported by: FAPESP and CNPq.

Comparison of the effect of an inorganic arsenical compound, AS 2 O 3 , with an organoarsenic such as dimethylarsinic acid on HeLa cell Arsenic trioxide (As 2 O 3 ) is an effective drug for treatment of acute promyelocytic leukemia (APL) and exhibits differential toxicity to cancer cell. Dimethylarsinic acid (DMA) is a major metabolite of organoarsenic in mammals including human. The acute toxicity of organoarsenic compounds are much lower than that of inorganic arsenicals. We compared the effect of As 2 O 3 and DMA on HeLa cell proliferation. HeLa cells were cultured in RPMI with 10% fetal bovine serum (FBS) and then incubated in As 2 O 3 (10 lM-5 mM) and DMA (700 lM-5 mM) solution at 37°C for 24, 48 and 72 hours. Cell proliferation was measured by 3-(4,5-dimethylthiazolyl)-2,5-diphenyl-tetrazolium bromide (MTT). The results showed that 700 lM As 2 O 3 has maximum effect (96.4%toxicity) on HeLa cell at 72 hours. Toxicity remained constant by increasing As 2 O 3 concentration up to 5 mM. Effect of 700 lM DMA on HeLa cell at the same condition showed 22.4% toxicity which means As 2 O 3 has 4.3-fold efficacy over DMA. According to our data, it seems that arsenic trioxide diffused very slowly into the cell membrane upto critical concentration but while entered the cell induced cytotoxicity very fast. On the other hand DMA was able to enter the cell much faster. We proposed that the lower toxicity rate of DMA compared to As 2 O 3 demonstrate a different mechanism of action on cell death, mainly interaction with microtubule protein, the main constituent of mitotic arrest.

PP8-117 Adsorption and biophysical characterization of alcohol dehydrogenase on diamond nanoparticles for the development of biomedical fuel cells Glioblastoma is a severe type of primary brain tumor and its highly invasive character is considered to be a major therapeutic obstacle. Phospholipase D (PLD) isozyme is overexpressed in various human tumor tissues and involved in tumorigenesis. However, the molecular mechanisms by which PLD enhances glioma invasion are not known.

In this study, we found that elevated expression of PLD isozyme and its enzymatic activity stimulate the secretion and expression of matrix metalloproteinase-2 (MMP-2) and induce the invasiveness of glioma cells. Upregulation of MMP-2 induced by phosphatidic acid (PA), the product of PLD, was mediated via protein kinase C (PKC), protein kinase A (PKA), NFjB, and Sp1, but not ERK, p38, or JNK, and enhanced the invasion of glioma cells. PA activated PKC and PKA, and induced nuclear translocation and transactivation of NFjB. PA also increased binding of NFjB and Sp1 to MMP-2 promoter. Mutation of NFjB-or Sp1-binding sites significantly attenuated MMP-2 promoter activity. This is the first report revealing NFjB and Sp1 as essential transcriptional factors linking PLD to MMP-2 upregulation, providing evidence that PLD isozyme contributes to glioma progression by enhancing MMP-2 expression and thus tumor-cell invasion via PKC/PKA/NFB/Sp1-mediated signaling pathways. Phospholipase D (PLD) is essential for cell survival and its activity is required for protection against apoptosis. However, the regulatory mechanism of PLD during apoptosis remains unknown. Here we report that stress stimuli induce caspase-mediated cleavage of PLD1. Cleavage occurs at the DDVD 545 residues, found within the loop region of the polypeptide, generating an N-terminal fragment (NF-PLD1) and a C-terminal fragment of PLD1. During apoptosis, the ectopic expression of the caspase-resistant mutant-PLD1 significantly prevented the loss of enzymatic activity and apoptosis compared to that of wild type PLD1. NF-PLD1 itself caused apoptosis even in the absence of apoptotic stress. The pro-apoptotic effect of NF-PLD1 may be attributable to a dominant-negative action through interaction with endogenous PLD1 and inhibition of its activity. NF-PLD1 suppressed E3 ubiquitin ligase MDM2 levels and elevated p53-protein stability, and thereby increased the expression of p53 target genes. Furthermore, NF-PLD1-induced apoptosis was dramatically suppressed in p53-deficient cells, suggesting that the pro-apoptotic action of NF-PLD1 is a p53-dependent event. Taken together, our results reveal a novel regulatory mechanism based on the caspase-dependent cleavage of PLD1, which contributes to apoptosis via the p53-mediated pathway.

Synthesis and structure-activity study of GnRH analogues In another way recent studies suggest that about 50% of breast cancer and 80% of ovary cancer cell lines express high-affinity binding site for GnRH and their analogues, therefore we investigate direct anticancer effect of GnRH analogues. Methods: The GnRH analogues synthesized manually with Fmoc/tBut strategy in Wang resin and purified with RP-HPLC method (AKTA Purifier). Amino acid contents analyzed with amino acid analyzer (Amino Nova).the structure of these analogues investigated with NMR 500 MHz and anticancer effect carried out with proliferation assay on some cancer cell lines such as T47D, MCF-7, OVCAR-3 and CAOV4.

Results: The purity and amino acid content of analogues satisfy with RP-HPLC and amino acid analysis. The anticancer activity has proven and was dose and time dependent.

Conclusions: The finding suggests the GnRH receptors that are expressed by human ovarian and breast cancer cell lines mediate direct Antiproliferetive effect of GnRH analogues and might be used for novel antiproliferative therapeutic approach.

Comparative antibiotic resistance profiles in enterobacterial strains isolated from surfaces in the hospital environment and different clinical samples PP8-125 p68/Ddx5 RNA helicase is a key regulator of de novo DNA methyltransferases in mouse embryonic stem cells DNA methylation is of fundamental importance for mammalian development. However, little is known about the molecular mechanisms responsible for the establishment of the specific genomic methylation patterns, a putative molecular fingerprint characterizing individual cell types that is formed during cell differentiation. Since de novo DNA methyltransferases, the enzymes that create these patterns, do not posses specific DNA recognition affinity, it is reasonable to assume that these enzymes are tethered to specific genomic regions via their interactions with proteins acting as de novo DNA methylation regulators. To identify these potential regulators, we have inserted cDNAs encoding tagged versions of de novo DNA methyltransferases into the ROSA locus of mouse ES cells. By immunoprecipitation and Mass spectrometry we have identified p68/Ddx5 RNA Helicase as interactions partner of Dnmt3a2 de novo methyltransferase. Ddx5/p68 RNA helicase coimmunoprecipitates with Dnmt3a2 and Thymidine glycosylase (Tdg) an enzyme that is implicated in base-excision DNA repair, from ESCs extracts. We have explored the physiological and functional significance of these interactions. We have found that Ddx5 interacts via multiple domains with Dnmt3a/3a2 in vivo and in vitro. Moreover, Ddx5 represses Dnmt3a2 activity in an ATP dependent manner, in vitro. We provide evidence that Ddx5/p68 forms a complex with the de novo DNA methyltransferase in vivo and regulates Dnmt3a/a2 activity at specific genomic sites. Bifidobacteria are thought to be important for human health and their participation in the functions of the intestine, taxonomy and ecology had been reviewed. Several reports concerned the composition of the bifidobacterial cell wall and the structure of cell wall polysaccharides. It was shown that the polysaccharide components present an antitumour and immunopotentiating activities. In this study we purified and analysed the extracellular polysaccharides of Bifidobacterium adolescentis 94 BIM, B. bifidum 791 and B.longum 379 M. Morphological studies of B. adolescentis 94 BIM, B. bifidum 791 and B.longum 379 M strains showed that cells are either long (0.5 · 8 nm), sometimes branched and have wide ends, or short and coccoid . We observed, using the electron microscopy, the existence of the intercellular links with the aid of different extracellular structures -cell wall evaginations, capsule-like material or microfibrillae, similar to the other strains of this species. Aiming at understanding of the molecular basis for the intercellular links, we have been investigating surface polysaccharides of these strains. Cells grown in various media produced similar polysaccharides with the same sugars, but some differences occurred in their molar ratios. These polysaccharides differs from previously reported polysaccharides of B. adolescentis YIT 4011 and B. adolescentis M101-4. The first one contained glucose and 6-deoxytalose with small amount of glycopeptide and the second one had glucose and galactose as major constituents and a galactofuranose as minor compound. Cell wall polysaccharides composed of glucose and galactose were also found in B. infantis and B. longum. Variation of dietary environment of bifidobacteria might change the structure of surface polysaccharides, thus modulate their antigenicity and possibly other, including probiotic activities.

Anti-bacterial activity of thionin Introduction: Bovine mastitis is mainly caused by Staphylococcus aureus and the anti-microbial therapy is commonly used for its control. As consequence the frequency of resistant staphylococci has been increased in last decades. Thus, alternative therapies are desirable and the anti-microbial peptides represent attractive control agents. We expressed the anti-microbial peptide thionin Thi2.1 cDNA from Arabidopsis thaliana in the bovine endothelial cell line BVE-E6E7 and evaluated its activity against bovine mastitis S. aureus isolates. Methods: Thi2.1 cDNA from A. thaliana was cloned into the mammalian expression vector pTracer-EF/V5-His-B to obtain the construction pThi2.1, which was used to transfect BVE-E6E7 cells. Expression of thionin Thi2 Results: Clones showed a high anti-bacterial activity (> 95%) against S. aureus ATCC 27543 strain in relation to the activity of the polyclonal population. C8 clone showed the highest antibacterial activity (> 99%) and was used to evaluate its CM against eleven bovine mastitis S. aureus isolates. 2.5 lg of total protein from CM of C8 clone inhibited the growth of S. aureus isolates (> 40%) in relation to the CM from BVE-E6E7 cells used as control. The growth inhibition of S. aureus isolates was dose dependent, showing a total inhibition at concentrations higher than 3.12 lg/ml. Conclusions: These results suggest that thionin Thi2.1 is an anti-microbial peptide that could be use in the treatment of bovine mastitis.

The effect of some biologically active compounds from marine organisms on the status level of the enzymes involved in articulary degradative processes The actual approach is towards obtaining natural biological compounds with efficacy/toxicity profile superior to synthetic drugs. There were extracted lipidic fractions, eicosanoids poliunsaturated fatty acids, glycerophospholipids, etc., from Mytilus galloprovincialis and Rapana venosa, and associated in active structural groups, with applicative potential in pharmaceutics and dermatocosmetics. Articular cartilage degradation/regeneration mechanisms represent elements of high topicality in research and medical practice due to the ever increasing incidence of articular degenerative disorders. There were studied the effects of biological active compounds onto the enzymes involved in cartilage metabolism, as hialuronidase, elastase, colagenase. In physiological conditions these enzymes promote the cartilage matrix degradation. The experimental data sustain the function of some biological active fractions in reducing the level of oxygen free radicals and modulating the enzyme activities which interfere in synthesis and degradation of articulary cartilage. The in vitro tests suggest a significant biological activity of these biocompounds for using them in pharmaceutical preparations.

Investigating the role of protein structural dynamics in regulating the catalytic mechanism of subtilisin-like proteases M. Pagon, R. J Solai and K. Griebenow University of Puerto Rico-Rio Piedras Campus, San Juan, PUERTO RICO Introduction: Subtilisin-like proteases constitute one of the principal structural classes of enzymes belonging to the serine protease family. Members of this enzyme class are directly involved in the development of various diseases (e.g., obesity, diabetes) when their enzymatic activities become deregulated. While at the biochemical level, many of the active-site chemical and structural elements involved in the catalytic mechanism of this class of enzymes are known, the molecular factors responsible for their catalytic regulation are not well understood. Although it has been suggested that the enzyme's intrinsic structural dynamics might be a critical variable involved in regulating the catalytic cycle of these enzymes, the molecular mechanisms by which this phenomena occur remain unknown. Here we investigate whether subtilisin structural dynamics directly influence the kinetics of enzyme catalysis. Methods: Chemical protein glycosylation was employed to sequentially modulate the structural dynamics of subtilisin. We quantified the changes in protein dynamics and thermodynamic stability by measuring amide hydrogen/deuterium exchange kinetics and thermal unfolding temperature (T m ). The catalytic behavior (k cat , K M ) of subtilisin after chemical glycosylation was determined from the hydrolysis of the peptide substrate N-succinyl-Ala-Ala-Pro-Phe p-nitroanalide. Results: The reduction in subtilisin structural dynamics correlates with the increased thermodynamic stability (T m ) and reduced rate of enzyme catalysis (k cat ) without affecting substrate binding (K M ) after chemical glycosylation.

Conclusion: The experimental results presented here provided fundamentals insights about the influence of glycosylation on subtilisin biophysical properties and also support the hypothesis that for subtilisin the global structural dynamics directly influence the kinetics of the enzyme catalysis. Motility in many bacteria is provided by flagellar organelles. Flagellin, the subunit protein of the flagella was found to be strongly associated with the extracellular polyhydroxybutyrate (PHB) depolymerase in T. thermophilus. T. thermophilus HB8 (DSM Acc No 579) was grown in minimal salt medium (MSM) containing sodium gluconate as carbon source [1] . PHA depolymerase activity was assayed using PHB as substrate [2] . Flagellin was concentrated from cell free supernatant by precipitation with trichloroacetic acid. Purification of flagella was performed as described previously [3] . Immunoblots were carried out using flagellin antibody (anti-FliC; Salmonella H antiserum rabbit). During the growth of T. thermophilus in MSM progressive increase of PHB depolymerase and flagellin was obtained. Analysis by SDS-PAGE and western blot of purified flagella revealed a single band of 62 kDa. Two stable flagellin fragments of 51 and 32 kDa were observed after incubation at 100°C for 20 minutes or after acid pH treatment, respectively. Flagellin was isolated in a complex form with PHB-depolymerase either by precipitation with PHB or by co-immunoprecibitation with protein-A sepharose/anti-FliC. Furthermore, the fragment of 32 kDa was characterized as flagellin by N-terminal and intenal sequence analysis. The extracellular PHB depolymerase of T. thermophilus forms complex with flagellin of the type III export system. These results indicate that T. thermophilus HB8 is a motile bacterium, capable of sensing environmental changes or escaping from adverse conditions.

Global translation alterations as a biomarker of pollution by heavy metals vectors are capable of generating very high levels of target protein in infected cells. However, its widespread use has been prevented due to the costs involved in its production. Specifically, in order to make SFV vectors, RNA is first transcribed in vitro using SP6 RNA polymerase and is subsequently electroporated into producer cells. Methods: We have previously constructed temperature sensitive SFV vectors harboring two point mutations in the viral nsP2 gene that resulted in an increased safety profile, associated with prolonged gene expression in target cells. We have further developed this tsSFV mutant vector into a two-layered expression system, whereby the SFV replicon is first expressed from a CMV or TRE promoter in situ that subsequently allows for the expression of EGFP reporter gene from the SFV subgenomic promoter.

Results: Using this system we have observed expression of EGFP to levels two orders of magnitude higher than those witnessed from a simple CMV-EGFP expression cassette. Moreover, we demonstrated that the inclusion of the SFV replicon in a TRE-based expression cassette results in tetracycline regulated gene expression with increased stringency. We have further generated plasmid constructs where the tumour specific MUC1 promoter controls replicon expression and demonstrate increased transgene expression compared to MUC1-GFP. Conclusions: We present data demonstrating increased stringency and efficacy of transgene expression compared to standard plasmid-based systems and effective tumour-restricted gene expression.

Glycosaminoglycans from marine organisms for pharmaceutical industry From marine organisms (small sea fish from Black Sea) we have extracted glycosaminoglycans by a patented technology. We have obtained a bioactive complex which contains: 40-60% sulphated glycosaminoglicans and other substances -essential aminoacids, essential fatty acids (linoleic and arachidonic acids), eicosanoids, vitamins (myoinositol), etc. The complex was analysed by gas chromatography coupled with mass-spectrometry, gel electrophoresis in different systems (SDS-poliacrilamide, cellulose acetate), potentiometric titration, high performance liquid chromatography. The chemical composition provides multiple therapeutically properties: anticoagulant, antithrombotic, antilipidemic, tissue restoration, antioxidant, anti-inflammatory activities. We investigate the in vitro antioxidant activity and regenerative activity of collagen fibriles. The obtained complex is rich in biological active compounds which could be used to make pharmaceutical preparations with high efficiency and minimal side effects. The bioactive complex will be useful to prevent the unsettlement of the macromolecular structure of ground substance and keep the functionality of the extracellular matrix from conjunctive, cartilaginous and bone tissue. 1.8) is a serine hydrolase. Its real physiological function is still unknown but it has been shown that BChE can serve as a co-regulator of cholinergic neurotransmission. Therefore, inhibition of BChE appears to be of an interest in treating diseases having symptoms of reduced neurotransmitter levels, such as the Alzheimer disease. We evaluated the BChE inhibition by seven selected flavonoids (galangin, kaempferol, quercetin, myricetin, fisetin, apigenin, luteolin) belonging to a large family of biologically active polyphenolic compounds found in many plants and plant-derived products that are components of everyday human diet. Methods: Inhibition potency was determined on BChE containing native human plasma samples. The enzyme activity was measured spectrophotometrically according to the Ellman procedure and kinetic parameters were calculated from experimental data obtained in at least three experiments.

Results: All flavonoids reversibly inhibited BChE. The determined enzyme-inhibitor dissociation constants (K i ) ranged from 10 lM to 166 lM. The inhibition potency increased in the following order: luteolin < fisetin £ myricetin £ quercetin < kaempferol £ apigenin < galangin. Conclusions: Differences in flavonoid inhibition potency were connected with the number of OH groups on their ring B. With the lowest observed K i value of 10 lM, galangin was pointed out as a promising lead in the search for new BChE inhibitors. Introduction: Liver injury after ischemia, followed by reperfusion, remains one of the major obstacles in liver surgery and transplantation. Various mechanisms have been implicated in liver ischemia/reperfusion injury, including reactive oxygen species generation, lipid peroxidation and mitochondrial dysfunction. Methods: Wistar albino rats were assigned into three groups: (i) Group A: sham, (ii) Group B: hepatic ischemia was formed by clamping on the superior mesenteric arther (SMA) for 1 hour and reperfusion for zero, 2, 24, 48 hours and then subjects were sacrified, and (iii) Group C: pre-conditionary ischemia was obtained by clamping on hepatic pedicules twice for 5 minute and reperfused for 10 minute. Then, the clamping and reperfusion processes that used in Group B were repeated. Liver, intestine and lung malondialdehyde (MDA) levels and lung myeloperoxidase (MPO) activity were evaluated.

Results: Data revealed significantly elevated levels of MDA in liver, intestine and lung tissues in Group B compared to Group A and Group C. In addition to those results, also the MPO activity of lung tissue was increased in Group B compared to Group A and Group C. In all tissues just like lung MPO activity, MDA levels were also reached to the highest level at reperfusion period of 2 hour and then a tendency to decrease was observed. The decreased MDA levels were measured in Group B and Group C after 24 hour. These results were also supported by histopathologically. Conclusion: It can be suggested that the pre-conditionary hepatic ischemia may have a protective effects against intestinal ischemia/reperfusion injury.

Tritrichomonas foetus glycosidases J. Sakyiama, P. Greenwell and D. Perr School of Biosciences, University of Westminster, London, UK Introduction: Tritrichomonas foetus, which causes bovine trichomoniasis, is a parasitic protozoan with worldwide distribution and is particularly rampant in America, Australia and Africa where natural breeding is practised. It is of economic importance because of the substantial financial loss it causes to countries involved in commercial scale beef and milk production. The pathogen has been implicated in feline diarrhoea and research has shown Tritrichomonas foetus and Tritrichomonas suis in swine to be the same organism in different hosts. Tritrichomonas foetus is able to evade the bovine defense mechanism to cause spontaneous abortion and one potential virulence factor is the secretion of hydrolases into the extracellular environment especially glycosidases, DNAses and cysteine proteases. Methods: Chromatographic methods like gel filtration, ion-exchange chromatography, affinity chromatography and chromatofocusing were used for isolation of glycosidases. Concanavalin A chromatography was used to check for glycosylation. Glycosidase activity was determined using the para-nitrophenyl sugar assays.

Results: The glycosidases appeared to be activated by different metals and possessed activity over a pH range of 4.5-9.0 with an optimum activity observed between 5.0 and 6.0. Most bound to Concanavalin A. The purified enzymes were active over a temperature range of 25 o C-60 o C. Conclusion: The properties of the glycosidases suggest they will be active in the harsh environment they colonise possibly due to glycosylation. Bovine trichomoniasis has no effective diagnosis and treatment and an understanding of these glycosidases could lead to more effective ways of combating the pathogen.

PP8-167 Improvement in the teaching of biochemistry Introduction: Three main factors are involved in the teaching-learning process: the system, the students, and the professors. For a good formation of university students it is indispensable to strive for a solid learning, useful for the future professional practice of the students. In addition to knowing their disciplines, professors must count upon the resources, personal and didactic attributes to transmit knowledge to their students. Professors must search for the best way to attain a significant learning, since occasionally due to their array of activities, attitudes, time dedicated to the class or its preparation, the professors themselves contribute to not reaching the desired learning.

Methods: Forty-two professors of Biochemistry of some universities located in different states of Mexico (Baja California, Campeche, State of Mexico, Michoacan, Puebla, Sinaloa, and Yucatan) and from four different universities in Mexico City participated. The work was organized as an educational research. The professors applied surveys to their students to know their demographic characteristics and previous formation. They evaluated the difficulty of the academic contents and tried different didactic and dynamic techniques suitable for the corresponding populations. Each professor had to show respect for the students, impartiality and honesty in grading their performance. Results: Two annual meetings of the participants have been held in Mexico City, the results indicated that 40% of the students exhibit a better learning with this approach. Conclusions: If the professor assumes his (her) commitment as a teacher, a better learning will be achieved.

Oncogenic B-RAF increases NFjB activity through the ERK/MSK1 pathway I. Sa´nchez-Herna´ndez, P. Baquero Valls and A. Chiloeches Ga´lvez Departamento Bioquıḿica y Biologıá Molecular, University of Alcala, Alcala, SPAIN B-RAF protein is included in the Ras-RAF-MEK-ERK pathway that regulates cellular proliferation, differentiation and apoptosis. This pathway is hyperactivated in cancer with activating mutations in B-RAF occurring in approximately 7% of cancers. It has been identified the V600E B-RAF mutation in about 30-70% of thyroid cancers. In this study we analyze the role of oncogenic B-RAF on the NFjB pathway in thyroid cancer cells, since this transcription factor also plays an important role in carcinogenesis. First of all, we observed that overexpression of B-RAF mutants with different kinase activities increased the NFkB-dependent transcription, with a higher increase in the case of the V600E B-RAF mutant. This increase in NFjB activity was observed in both basal and TNFa-stimulated conditions. However, V600E B-RAF did not increase IjB degradation and p65/NFkB nuclear translocaction. We also found that inhibition of MEK with the specific inhibitor UO126 blocks the NFjB-induced transcription, indicating a role of the MEK/ERK pathway. NFjB can also be activated by increasing the p65/NFjB transactivation potential after phosphorylation of S276 of p65 by the ERK-substrate MSK1. Thus, we observed that overexpression of a kinase-dead mutant of MSK1 blocks the NFjB activity induced by V600E B-RAF. This data indicate that oncogenic B-RAF increases NFjB transcriptional activity without affecting IjB degradation and p65/NFjB nuclear translocation, but increasing the transactivation potential of p65/NFjB through an ERK/MSK1 dependent pathway.

The biochemical properties of non-stimulated tears from camels (Camelus dromedarius)

A. Al-Hommadi, F. A. Shamsi and A. A. Al-Rajhi Research Department, King Khaled Eye Specialist Hospital, Riyadh, SAUDI ARABIA Introduction: Camels resist harsh climatic conditions including extremes of light and temperatures. Knowledge of the normal camel tears components and comparison with humans can have clinical implications for the management of certain ocular surface disorders that effect humans. Objective: To analyze the biochemical contents of the periocular tear film from camels. Methods: The tears were collected from domesticated female camels by sterile capillaries. The spectroscopic characteristics of the tears were analyzed. Osmolarity, protein content and antioxidant in were measured by standard assays. Electrolytes, Ca 2+ , Mg 2+ , PO 4 -3 , Fe 3+ , CO 2 , LDH, glucose and cholesterol were measured on the Dimension Ò chemistry system. Results: The camel tears showed UV absorption peaks between 200-290 nm. Some absorption was noted in the UV-B region; however, nearly 100% UV transmittance was observed in the UV-A and visible range. Distinct fluorescence excitation/emission maxima were observed in camel tears and the osmolarity was 318 ± 14.69 mOsmM. The protein concentration was 4.56 ± 2.47 mg/ml, while glutathione ranged from 1.39 to 82.28 nmoles/ml with a mean of 33.30 ± 44.29 nmoles/ ml. The N + /K + /Cl + concentrations were 168.63 ± 5.92, 11.37 ± 2.32 and 156.21 ± 4.64 lmoles/ml respectively. Magnesium was 1.47 ± 0.41 lmoles/ml, while concentrations of Ca 2+ (0.46 ± 0.25 lmoles/ ml), PO 4 -3 (0.13 lmoles/ml) and Fe 3+ (0.57 ± 0.52 lmoles/ml) were low. The CO 2 concentration was 11.2 ± 0.92 lmoles/ml. LDH was not detected in some samples, while glucose and cholesterol were £0.1 ± 0.3 lmoles/ml. Camel tear components values tested were higher then those reported for humans. Conclusions: These results suggest that the transmitted light not absorbed by tears can cause corneal abrasions. Our preliminary results suggest that understanding of the properties of camel tears may help in the evaluation of pathophysiology and treatment of dry eye syndrome.

Peroxidase-like activity in monoclonal antibody 7D9 Introduction: The first monoclonal catalytic antibodies against a chemically stable analog of the transition state of a reaction were obtained in 1986 and termed abzymes (Abzs). Numerous Abzs discovered afterwards in sera of patients with various autoimmune diseases are capable of hydrolyzing proteins, DNA, RNA, polysaccharides, or nucleotides. On the other hand, some natural catalytic antibodies were prepared by normal immunization using ground-state antigen. We prepared a monoclonal antibody against beta human chorionic gonadotropin (hCG) that show a peroxidase-like activity. Methods: The 7D9 MAb analyzed with respect to its specificity for hCG and b-hCG by immunoblot and ELISA. This antibody was purified by sequential chromatography on protein G/A-Sepharose, gel filtration and ion-exchange. The presence of peroxidase activity of 7D9 MAb was confirmed by zymogram and enzyme assay in presence of substrates including, 3,3'-diaminobenzidine(DAB),tetra methylbenzidine (TMB) and L-DOPA. Results: Immunoblot analysis and ELISA showed that this MAb reacts with beta hCG and did not cross-react with other glycoprotein hormones such as FSH, TSH and LH. Our data reveal that this MAb is very unstable and has auto degradation characteristics. Zymogram and spectrophotometer analyses show that 7D9 mAb has oxidase like activity against different substrates such as DAB, TMB and L-Dopa. Conclusions: 7D9 mAb has oxidase-like activity against different substrates such as DAB, TMB and L-Dopa. Antioxidant peroxidase activity of Abzs can also play an important role in the protection of organisms from oxidative stress as well as in oxidation of toxic compounds.

Lipid oxidized products interact with taste glutamate receptors?

A. Shinoda, S. Yamaguchi, R. Nagasaka, H. Ushio and Y. Ninomiya Tokyo University of Marine Science and Technology, Tokyo, JAPAN Basic taste modalities are classified into sweetness, saltiness, sourness, bitterness, and umami. The five basic taste modalities express individual and unique taste sensations and play important roles in taste reception. Together with the five basic tastes, some kinds of compounds, such as proteins, lipids, and polysaccharaides, bring us complicated impressions. Umami is a taste modality for monosodium glutamate (MSG) that was discovered in Laminaria seaweeds. It is generally accepted that glutamate receptors receive MSG in taste reception cells. The goal of this study is to seek the materials that enhance mouse MSG taste response and reveal their interacting modes for the reception of MSG. The oxidized compounds of polyunsaturated fatty acids are used and measured the sensitivity for MSG through behavioral and electrophysiological technique. Licking response of MSG is measured using MSG aversion mouse. Corda tympani nerve, one of the taste nerve, response is also measured. Oxidized compounds of arachidonic acid raised the sensibility for MSG. These results suggest that oxidized compounds might interact with lingual glutamate receptors. This paper will discuss about the possible interacting modes among glutamate receptors, MSG, and lipid oxidized compounds.

Amino acid models to building protein molecules: a new tool to teaching protein structures A. R. Silva, N. R. S. Bossolan and L. M. Beltramini Instituto de Fıśica e Informatica, USP, Sa˜o Paulo, BRAZIL Introduction: Representation of three-dimensional structures of DNA and RNA with ludic models have supplied to the students and teachers a successfully experience to better visualize and understanding it (BAMBED, 34, 187, 2006) . In this sense it was developed an amino acid kit to building protein primary and secondary structures. The kit allows demonstrate different characteristics of each amino acid residues (represented by colors, shapes, sizes), its interactions through peptidic bonds, visualization of secondary structures by binding through hydrogen bonds. The models were designed for Ca, amino and carboxyl groups, hydrogen, lateral chains and hydrogen bound representation. Methods: CAD software was used for models development. Design and applicability were discussed with researchers and teachers, who also tested the plastic prototypes, which were made by milling process. The next step was the mould construction for production in large scale. The evaluation of the impact of this tool in the learning/teaching process is being conduced in high school, under graduation and graduation classes by means of notes, classes filming, and written questionnaire. Results: The kit containing plastic pieces and instructions manual was completed. It will be presented at this meeting for the community of biochemical as well as some results from the evaluation process made with graduate students from the discipline 'Biomolecules: structure and functions.' Conclusions: The main achievement of this work was the involvement of scientific knowledge in areas as structural biology, computational, mechanical and science education. The steps for kit production were finish, a patent is in requesting in Brazil and we are in evaluation phase as a teaching/learning tool. Acknowledgements: Supported by Brazilian Agencies: FAPESP, CNPq.

PP8-177 Interaction between the local anaesthetic articaine and membranes: a theoretical and experimental study E. A. Skjevik and I. B. Pedersen Department of Biomedicine, University of Bergen, Bergen, NORWAY Introduction: Articaine is a local anaesthetic widely used in dentistry. Its primary effect through inhibition of sodium channels in the nerve cell membrane is well known. In recent years important secondary effects of local anaesthetics have been demonstrated, including anti-inflammatory and antibacterial effects. However, the mechanisms related to these effects are rather unclear. One plausible explanation for the antibacterial potency might be membrane intercalation. In this study, interaction with model membrane systems is investigated. Articaine is marketed as a racemic mixture, making the study even more relevant, as the molecular dynamics part deals with stereospecific interaction.

The main method used in this study is molecular dynamics simulation. A neutral palmitoyl-oleyl-phosphatidylcholine (POPC) membrane was simulated for 25 ns. Subsequently, four different forms of articaine were inserted and four separate simulations carried out. The same procedure was employed for a negatively charged membrane consisting of palmitoyl-oleyl-phosphatidylserine (POPS). Molecular docking was utilized to find probable binding configurations for articaine to the membranes. In order to validate the simulations, the Langmuir monolayer technique was used to find area per lipid for POPC and POPS. In addition, articaine interaction with POPC and POPS was studied by this method.

Results: Langmuir experiments revealed that articaine intercalates in the lipid monolayers and with different kinetics depending on the monolayer. Molecular dynamics simulations provided stereospecific information on the interaction at an atomic level of resolution.

The results of this study suggest that membrane interaction and intercalation is a possible mechanism for the antibacterial effect of articaine. The radioligand binding assay is one of the most convenient methods for DAT assay, but its reliability depends on properties of radioligands used. In this study we used rat striatal membrane fragments as source of DAT and compared properties of two radioligands suggested for such assay. The commercially available and widely recommended ligand [ 3 H]GBR12935 (K D = 2.0 nM) was compared to the [ 3 H]PE2I (K D = 3.5 nM), which has originally been proposed as DAT tracer for PET analysis, but was also suggested for use in in vitro assay [Stepanov, et al., Appl. Radiat. Isot. 2007; 65: 293]. The kinetic properties of GBR12935 and PE2I in binding to DAT are similar, as are their affinities. Our analysis has revealed that incubation time around 30 minute is sufficient for the conventional equilibrium binding and displacement assays for both ligands, with [ 3 H]GBR12935 revealed 15-20% less of DAT sites due to the faster off-rate. However, the major difference between these ligands also lies in the levels of non-specific binding. We have found that [ 3 H]GBR12935 exhibited unacceptably high level of nonspecific binding to membrane preparation, as well as to GF/B filters, even after optimization of the assay conditions. The non-specific binding was considerably less pronounced in the case [ 3 H]PE2I.

This difference may be explained on the basis of hydrophobicity of these ligands, characterized by log P values 4.7 and 5.5, respectively. In conclusion, for in vitro quantification of DAT, the [ 3 H]PE2I is clearly superior to [ 3 H]GBR12935 due to lower hydrophobicity and more favorable kinetic properties.

Calmodulin-dependent protein kinase IV regulates insulin secretion under the glucotoxic condition in INS-1 rat insulinoma cell line Chronic hyperglycemia causes glucotoxicity with impaired insulin secretion in type 2 diabetes. However, the molecular mechanisms of impaired insulin secretion and signal transduction of glucotoxicity are poorly understood. In this study, we examined protein kinases that correlated with insulin secretion in INS-1 cells using Multi-PK antibodies, which could detect a wide variety of protein kinases. When expression patterns of protein kinases in INS-1 cells under varying glucose concentrations were analyzed by Western blotting with Multi-PK antibodies, a kinase of 63 kDa was significantly reduced concomitant with the decrease of insulin secretion under the glucotoxic condition. To identify 63-kDa kinase, we employed a unique two-dimensional gel electrophoretic technique and MicroRotofor electrophoresis. From the molecular sizes of native kinase/CNBr-fragment and pI value, the 63-kDa protein kinase was predicted to be CaMKIV and it was confirmed by Western blotting analysis with anti-CaMKIV antibody. Decrease in CaMKIV expression under glucotoxic condition was recovered to original level after changing the medium to normal glucose concentration. Recombinant CaM-KIV was degraded in a Ca 2+ -dependent manner by incubation with cell lysates from INS-1 under glucotoxic condition, and degradation was protected by calpain inhibitor III. Transfection of constitutively active CaMKIV in INS-1 cells under glucotoxic condition induced four-fold increase in insulin promoter activity. Furthermore, the expression of CaMKIV was found to be decreased in the pancreatic islets of diabetic OLETF rats, while that of non-diabetic LETO rats was not. These results, taken together, suggest that CaMKIV regulates insulin secretion and may be degraded by calpain under the glucotoxic condition.

Genistein promotes apoptosis and cell cycle arrest via mitochondrial pathways and Akt signaling pathways through nuclear receptor family in prostate cancer cells PC3 Introduction: An increased understanding of molecular biological properties of anticancer agents will lead to the development of mechanism-based chemopreventive and/or therapeutic strategies. cDNA microarray analysis permits the simultaneous analysis of the expression of tens of thousands of genes, and, in turn, provides an opportunity for determining the effects of anticancer agents on prostate cancer cells.

Methods: The use of genomics technology as cDNA microarrays does not exclude hypothesis driven studies. To understand molecular models we used microarrays from HGMP-UK. We try to answer the questions related with the transcription factors if it could have an essential and general function in transcription (affecting all the genes) or a specific function (affecting only some genes -selectively). This approach has been used to analyze differentially gene expression in prostate cancer cells PC-3 treated with Genistein compared with untreated cells in order to understand initiating of the transcription process, apoptosis, and cell cycle arrest. Results: One exciting result of microarray technology has been the demonstration that patterns of gene expression can distinguish between Genistein treated and untreated PC3. Modification of existing statistical methodologies is needed for gene expression analysis. We proposed an evolutionary neural network that classifies gene expression profiles of PC3 cells. Such algorithms can play an important role in molecular profiling of underlying pathways. Conclusions: Neural networks can assist the discovery of new Genistein-suppressing, enhancing target genes, identify patterns of expression correlated with biologically significant endpoints, and distinguish preclinical meaningful outcomes (e.g., apoptosis, cell cycle arrest, Akt signaling pathways, nuclear receptor family).

Symbolic computation approach for dynamics of gene regulatory network modules Introduction: A large number of biological networks, such as metabolic pathways, signal transduction pathways, genetic networks, etc., have been reported. Generally such networks are represented by graphs consist of nodes and edges. Although networks reflect parts of an inside structure of a cell or a organism, many of these networksdo not have enough information about dynamics. Since biological phenomena are observed as changes in time and are dynamic essentially, information about dynamics, such as model schemes and its parameter values are necessary to understand life phenomena. Many kinds of mathematical models have been developed and established today. Since mathematical models for biological networks are complex generally, the idea of 'network module' is useful. Small modules are easily solved mathematically, and can be applied iteratively to upper streams in a network from observable variables and make it possible toanalyze dynamic behaviors.

Methods: A simultaneous differential equation model is defined representing linear relationships among genes, under the assumption that time courses of several gene expression levels can be observed. Hidden variables except for 'Kinetic parameters' and observable variables are eliminated, therefore we obtain the way how to calculate kinetic parameters. Results: We chose the 'bi-fan' (shown in the figure) structure for the case study. We obtain formulae in which ratios of kinetic parameters.

Conclusions: To obtain the each value of kinetic parameters, the module should be extended. Numerical reliability must be confirmed for calculated parameter values. It is carried out directly by observation of hidden variables, or blind test. It should be a next future work. Introduction: MEL cells are used as a model system to uncover the cellular and molecular events of hematopoietic cell differentiation. We investigated the question whether induction of erythroid diiferentiation is accompanied by apoptosis. Methods: Cultured cells were treated separately with none, or with each one of the two inducers of erythroid differentiation (DMSO,HMBA) and/or etoposide (ETO), an apoptotic promoter. At different times (0-96 hour), cells were assessed for differentiation and apoptosis by flow cytometry, cell cycle profiling, DNA analysis, detection of hemoglobin, viability, and Western immunoblotting using mabs against caspase-3, Bcl-x. Bcl-2, CdK2, Fas ligand, cyclin D3 and other protein markers. Cytochrome c exit from mitochondria and changes in morphology were also examined.

The data indicate that, in contrast to ETO that caused an extensive apoptotic damage in more than 90% of the cells without inducing any erythroid differentiation, DMS0 and HMBA induced apoptosis in 20-30% of terminally differentiated hemoglobin producing cells as shown by flow cytometric analysis using Annexin V(+) and other markers.

Conclusion: These findings indicate that induction of erythroid differentiation is accompanied by differentiation-dependent apoptosis (DDA) presumably as an integral part of hematopoiesis. The potential role of DDA in the homeostatic regulation of hematopoiesis is discussed.

Structural and functional characterization of Micrurus fulvius venom proteins with focus on a myoglobinuria-inducing phospholipase A2 We have purified three phospholipases A 2 (PLAs), four threefinger toxins (3FTxs) and a natriuretic peptide (NP) from the venom of North America coral snake, Micrurus fulvius (abbreviated as Mfu). Their mass, N-terminal sequences and functions were characterized. Specific primers for the polymerase chain reactions were designed from known homologies, and the cDNAs encoding these proteins were successfully cloned and sequenced from venom glands. Full amino acid sequences were deduced, which match the Mass data of the purified venom proteins and some of them were confirmed by peptide mass fingerprinting. A very abundant basic PLA in this venom (designated as Mfu-PL-I) is non-neurotoxic but highly lipolytic and lethal with an i.v. LD 50 of 0.18 mg/kg, and causes myoglobinuria (dark urine) in mice. The in vivo effects of Mfu-PLI on mice suggested that it may cause hemolysis and general myotoxicity. Amino acid sequences of many group I venom PLAs eliciting dark urine were aligned and compared with those without such effects; we found Lys69 and Trp 19 or Trp31 are important for the PLA to interact with cell membrane and elicit myotoxicity. Additionally, neurotoxicities of two of the 3FTxs were confirmed by assays on chick biventer-cervicis tissue. Venom NP peptides of 34amino acid residues was also identified and its sequence is 71% identical to the NP from Micrurus corallinus venom. Molecular phylogenetic trees of these venom proteins confirmed that Mfu is a rather unique elapid species.

Using trehalose as an enhancer for the transgene expression mediated by DNA-PEI complexes Background: Using enhancers to improve the transfection efficiency of polyethylenimine (PEI) can circumvent the needs of chemical modifications as well as subsequent purification and characterization of the modified PEI. Methods: Flow cytometry was used to measure the cellular fluorescence of ethidium monoazide labeled plasmid and green fluorescent protein, which represented the plasmid entry and transgene expression, respectively. We examined the effects of various trehalose concentrations on the enhancements of transfection efficiency as well as the effects of the timing of trehalose treatment and the duration of trehalose treatment on the plasmid entry and transgene expression. Results: We found that incorporating trehalose into the transfection reagent could improve the transgene expression mediated by DNA-PEI complexes. Such enhancements were not observed when other disaccharides were used. In an effort to explore the mechanisms, the treatments with trehalose for 5-120 minute prior to transfection could cause drops in transfection efficiency by 30-50%; such treatments, however, hardly affected the amounts of intracellular plasmid. The transfection efficiency remained almost unchanged when the transfected cells were treated with trehalose after the removal of transfection reagents. Despite the enhanced transgene expression, the presence of trehalose during transfection showed inhibitory effects on the internalization of DNA-PEI complexes. Additionally, the extent of enhancement in transgene expression strongly depended on the duration of trehalose.

Conclusions: As the above observations suggested, only during the transfection process when complexes and trehalose coexisted, trehalose became an effective enhancer of transgene expression mediated by DNA-PEI complexes possibly by affecting the mechanisms of intracellular trafficking.

In vivo flux control coefficient of glycogen synthase in frog oocytes

Glycogen synthesis is generally considered to be regulated by glycogen synthase (GS). However, in vivo studies have cast doubts on this view.

In vivo experiments in frog oocytes, based on metabolic control analysis, show that the control coefficient of hexokinase is 0.5-0.6 suggesting that this is the main controlling step. Control coefficients of phosphoglucomutase and UDP-glucose pyrophosphorylase are 0.19 and 0.12, respectively. Because of the summation theorem, a maximal value for GS control coefficient should be around 0.1-0.2. The lack of homogeneous GS has prevented microinjection in oocytes to measure its control coefficient. Instead, we have chosen to modify GS endogenous activity by microinjection of glucose-6-P, a known activator of GS. We report that glucose-6-P effectively raises GS endogenous activity. Experiments were performed microinjecting unlabeled glucose-6-P followed, after 8 minutes, by 3 mM UDP-[U-3 H] glucose. After 12 minutes incubation, glycogen radioactivity was measured. Maximal activation of GS was about 2-to 3-fold at 2 mM microinjected glucose-6-P. The ester effect was transient being maximal between 5 and 10 minutes. Therefore, the control coefficient determination was performed by microinjecting different glucose-6-P concentrations. After 8 minutes of incubation, oocytes received [U-14 C]glucose (6 nmoles). After 12 minutes incubation, glycogen radioactivity was measured. Flux coefficients were calculated and found to be 0.01, i.e. almost zero. We conclude that GS does not control the flux of glycogen synthesis in oocytes. Acknowledgement: Support: FONDECYT 1040886.

Studies concerning the obtaining and characterization of new pharmaceutical products from Ginkgo biloba species Increased incidency of afections with the effect of reducing the cerebral capacity among the population, on a global level, has led to the conduct of interdisciplinary research for the obtainement of bioactive substances and products used in the therapy, but especially in the prevention of the estabishement of these afections. The current tendencies are oriented towards the elaboration of a strategy for the prevention of disease with the help of nutritional supplements and bioactive products of vegetable origin, etc.

Purpose: This study had as a purpose the obtainment and characterization of a new natural pharmaceutical products of the Ginkgo biloba L species used for the improvement of cerebral activity. Materials and Methods: The vegetal material consisted in the leaves of the Ginkgo biloba L species. For the obtaiment of the total extracts the clasique methods were used and for the enriched fractions the selective extracting methods and purifications or the hydrolysis and purification were used. For the obtaining of pharmaceutical products, the total extracts have been conditioned as capsules (pelletes). The obtained products have been characterized by performant analysis techniques(HPLC, HPTLC, UPLC-MS).

Results: The purified extracts of Ginkgo biloba leaves contains: 20-26% total ginkgoflavonoides (quercitin, kaempherol, isorhamnetin) and 5-7% biflavonoids (amentoflavon, bilobetin, ginkgetol). Conclusions: A new methods was developed from obtaining and characterisation of new natural pharmaceutical products used in for the improvement of cerebral activity. 7) is a b-fructofuranosidase which cleaves b-(2,1) linkages between fructose residues in inulin, a storage polysaccharide accumulated in the underground organs of plants such as chicory, dahlia and Jerusalem Artichoke. The single step production of fructose by inulinase makes this enzyme an appropriate candidate for production of High Fructose Syrups in the food industry. Inulinase has been produced in various microbial sources, among which Aspergillus species are one of the most common choices for commercial applications. In this work, Aspergillus strains were studied for their inulinase production capacity. Methods: The strains were purchased from the Persian Type Culture Collection and grown on inulin (extracted from chicory root and Jersusalem Artichoke) or sucrose as the sole carbon source. Presence of inulin in the extracts was detected by TLC. Inulinase and sucrase activities were determined by measuring the amount of released reducing sugar. Enzyme production was also verified by SDS-PAGE. Results: A. awamori showed the highest Inulinase activity (3.63 U/mg) and Inulinase/Sucrase activity ratio 3.7. It seems that sucrose is a better carbon source for enzyme production.

Conclusions: Aspergillus awamori (PTCC:5097, ATCC:16877) could be a suitable candidate for production of inulinase for use in the production of High Fructose Syrups.

Chemical proteomic characterization of the novel synthetic quinolone compound repressing the transition from metaphase to anaphase In contrast, MCF-7 cells exhibited a lower susceptibility to colchicine and paclitaxel. Importantly, CHM-1 is less toxic to normal fibroblasts (NIH3T3) cell. The effect of CHM-1 on cell cycle progression was examined by flow cytometry. CHM-1 caused a time-dependent accumulation of MCF-7 cells in the G2-M phase. Moreover, CHM-1 interacted with tubulin, markedly inhibited tubulin polymerization, and disrupted microtubule organization. In contrast, colchicine caused cellular microtubule depolymerization with short microtubule fragments in the cytoplasm, paclitaxel resulted in microtubule polymerization with an increase in the density of cellular microtubules. However, the biologic and physiologic significance for the anticancer effects of CHM-1 is not clear. Identification of new proteins binding with CHM-1 should help in the design of new strategies to prevent cancer. We used the MCF-7 cell line to identify novel proteins that bind with CHM-1. Proteins from MCF-7 cell lysates were subjected to affinity chromatography using CHM-1-Sepharose 4B. Fractions containing proteins binding with CHM-1 were analyzed by two-dimensional electrophoresis and MALDI-TOF to identify proteins that directly bind with CHM-1. Our preliminary results indicated that CHM-1 binds with the eIF4a and GRP78. Finally, we carried out xenografts with MCF-7 cells in athymic SCID mice. CHM-1 induced a dose-dependent inhibition of tumor growth. Tumors were removed, the weights of tumors from CHM-1-treated groups were also smaller than vehicle-treated group. These results suggest that the anti-tumor activity of CHM-1 may against MCF-7 breast cancer cell line in both in vitro and in vivo assays.

The targeted generation of free oxygen radicals promoting apoptotic death of cancer cells A number of human tumours require high amounts of hormones for sustained growth. The most common types of these tumours are breast cancer and prostate cancer. About 60% of breast tumours are rich in estradiol receptors (ER) and these tumours are hormone-dependant.

Administration of antiestrogens such as tamoxifen helps to inhibit the tumour growth. Despite the advantages, tamoxifen primarily acts as a cytostatic and not cytotoxic agent. As a result, most patients ultimately experience disease recurrence or progression during or after therapy. We have suggested that to overcome the tamoxifen drawbacks and to enhance its anticancer activity, iron-containing derivatives of this compound could be used. Numerous tumour cell strains produce large amounts of hydrogen peroxide (H 2 O 2 ). Ferrocene and other iron-containing compounds readily react with hydrogen peroxide producing free hydroxyl radicals. These radicals cause damage to DNA which, in turn, triggers the pathways leading to elimination of cancer cell via apoptosis. We used tamoxifen as a carrier for delivery of ferrocene to breast cancer cells, and synthesized a conjugate of ferrocene and tamoxifen (Tam-Fer). We have shown that the frequency of apoptotic events in MCF-7 breast cancer cells treated with Tam-Fer is significantly higher than in the control cells treated with 4-hydroxytamoxifen or ferrocene, and this increase well correlates with the increase in generation of free radicals in these cells. These results show that the accumulation of hydrogen peroxide in tumor cells can be used as a therapeutic advantage for treating cancer. Tetrahymena thermophila is a unicellular eukaryotic model organism for a variety of biochemical, molecular and biological studies. Its macronucleus genome sequence was annotated in 2006 and is approximately 104 Mb in length and composed of approximately 225 chromosomes. According to this it is expected to contain more than 27 000 protein-coding genes, a number comparable to that of human and other metazoan cells; however in recent databases only 43 proteins are integrated. In the present study, after removal of DNA and RNA molecules, total Tetrahymena's protein extracts were separated by twodimensional gel electrophoresis and analyzed by MALDI-MS and MALDI-MS-MS. Protein identification was carried out by peptide mass fingerprint (PMF) and peptide sequencing by post source decay (PSD). By that process we have been able to identify a total of 371 different proteins including 117 putative uncharacterised proteins with unknown functions. Thus, 33.67% of the identified proteins were hypothetical or predicted, 30.35% of the proteins were enzymes that participate in various metabolic processes, 12.24% were binding proteins such as ATP, GTP, RNA binding proteins and calcium-binding proteins, while 11.22% corresponded to structural proteins like actin, tropomyosin, centrin, dynein and GRL proteins. 3.06% were ribosomal proteins that take part in protein synthesis, 2.04% were translational proteins such as EF-1, and another 3.06% were chaperones like Heat shock proteins 90, 82 and DnaK. 3.10% were regulatory proteins like 14-3-3, AhpC/TSA and NSF and finally 1.02% of the identified proteins are known to participate in signal transduction as Ras and Mov34/MPN/PAD-1 proteins do.

Use of zero-length cross-linking to form lysozyme, horseradish peroxidase and lysozyme-peroxidase dimers Introduction: Zero-length cross-linking is defined when the peptide chains are covalently linked through existing functional groups without incorporation of a spacer group first crosslinking was performed for lysozyme, peroxidase and between lysozyme-peroxidase molecules in three separate experiments. Methods: The in vacuo cross-linking procedure was performed by the method of Simons et al. Lyophilized proteins were obtained from the supplier, reconstituted in distilled water to a concentration of 10 mg/ml for lysozyme and 5 mg/ml for peroxidase. Protein solutions were placed in glass tubes and lyophilized. These glass tubes were sealed under a vacuum of approximately 9 · 10 -5 mbar and after heating periods (24 hours) at 85°C.

Results: Approximately one-third of the total lysozyme becomes crosslinked and the dimer form was the major product for both enzymes. This modification induced some changes in the kinetic properties of the dimer peroxidase, as evident by two-fold increasing of V max compared to the monomer but the enzymatic activity of cross-linked lysozyme dimer was the same as monomer. The activity of lysozyme dimer remained constant up to 10 minutes at 80°C. The structural changes of the enzymes were investigated by circular dichroism and intrinsic fluorescence techniques. At the end formation of heterogeneous dimer, peroxidase-lysozyme, was achieved using this method.

Conclusion: In the present study we showed that in vacuum crosslinked proteins retained their biological activity when returned to an aqueous environment and even in the case of peroxidase dimer higher activity was obvious in comparison with the monomer form. In other cases, zero-length cross-linking of a protein or proteins may confer advantageous properties that are useful for applications in medicine and industry.

Diagnostic kit studies for hepatitis B viruses and application of a quick sandwich ELISA F. Yucel, E. Akcael, A. Manav, C. Saatcilar and A. Basalp Tubitak, Marmara Research Center, Institute for Genetic Engineering and Biotechnology, TURKEY Introduction: Hepatitis B virus is one of the major cause of acute and chronic hepatitis, cirrhosis and hepatocellular carcinoma, and it is a serious global public health problem. Worldwide, 2 billion people have been infected with hepatitis B and an estimated 1 million people die each year from hepatitis B. The diagnosis of acute and chronic hepatitis B infection is based on the detection of hepatitis B virus surface antigen (HBsAg). One of the most sensitive and specific methods for diagnosing HBV infections is ELISA and early detection of HBsAg may help for simplifying the treatment of HBV-associated dieases. In our country, the use of imported diagnostic kits causes important economical losses due to its high cost and dependency to supplier countries.

Methods: In this study, we developed diagnostic kit systems based on sandwich ELISA by using 2G3 monoclonal antibody which has high specificity for HBsAg. It was recently developed in a previous project in our lab. The monoclonal antibody was conjugated with horseradish peroxidase (HRP) and treated to accelerated stability test and then used in the quick sandwich ELISA system. Results and Conclusion: When our quick sandwich ELISA test was compared with the other conventional sandwich ELISA tests by using hepatitis B positive and negative infection serum, it was shown that our system gave reliable results. In our 'sandwich' system, the same 2G3 monoclonal antibody was used for both capturing and detecting. This system is quite practical and time-saving when compared with common and commercial sandwich ELISA. Introduction: Enzymes capable of hydrolyzing a-1,6 glycosidic bonds in pullulan are defined as pullulanases. Pullulanases have industrial applications in the starch industry like production of maltose syrups and highly pure glucose and fructose. Methods: A thermophilic strain L14, isolated from Iranian hot springs, produces pullulanase. 16S rDNA analysis was done. The enzyme was purified by ammonium sulfate precipitation, anion exchange and gel filtration chromatographies. The K m and V max for the enzyme activity on pullulan and starch was estimated by Lineweaver-Burk plot. Substrate specificity and end product analysis was done by thin layer chromatography.

Results: With regard to 16s rDNA analysis, it seems that L14 is a Geobacillus stearothermophilus strain. The enzyme was purified with a yield of 21%. Optimal conditions for the enzyme activity were pH 5.5 and T opt =65°C. It had good stability at 60-70°C. The apparent K m and V max for the activity on pullulan were 0.48 mg/ml and 0.044 mmol/minute and for soluble starch were 5 mg/ml and 0.08 mmol/minute, respectively. The products of enzymatic reaction on pullulan were glucose, maltose and maltotriose as well as conversion of starch to glucose and maltose. Also this enzyme can hydrolyze different types of sugars like maltotriose, maltotetraose, maltopentaos, maltohexaose and maltoheptaose. Conclusion: Our data establish that the strain L14 produces a new type of pullulanase that produces glucose, maltose, maltotriose from pullulan and attacks both a-1,4 and a-1,6 linkages in pullulan and starch. Therefore, this is the first report on the presence of a specific debranching enzyme which can produce glucose from pullulan. This unique enzyme can operate at temperature between 60 and 80°C and has a potential application for enhancement of the starch saccharification process in industry.

Protective effects of uric acid against experimental allergic encephalomyelitis-induced oxidative stress Although the pathogenic mechanisms involved in EAE/MS are not well understood, accumulating data suggest that oxidative stress plays a major role in lesion development, since modulation of oxygen free radi-cal production represents a new approach to the treatment of inflammatory and autoimmune diseases. Central nervous system tissue is particularly vulnerable to oxidative damage, suggesting that oxidation plays an important role in the pathogenesis of MS and EAE. To establish more conclusively whether uric acid (UA) is protective or therapeutic in EAE through inactivation of oxidant factors involved and sustain antioxidant capacity. Methods: We have assessed the effects of UA administration on several parameters relevant to the disease process and balance between oxidant and antioxidant system including Lipid peroxidation, superoxide dismutase and catalase enzyme activity, glutathione (GSH) and ferric reducing ability of plasma (FRAP) as well as Blood Brain barrier (BBB) permeability and also the clinical symptoms of EAE in the three interval (prior to, after onset and development) of disease.

The results of the present study demonstrate an ameliorating effect of UA on oxidative damage in actively induced EAE in C57/bl6 mice and a significant increases in antioxidant factors in the EAE + UA group versus the EAE group especially at prior to or after onset of EAE (first and second group of this study).

The impact of an ionizing radiation on fluoropyrimidine metabolism: a study in vitro Radiotherapy and fluoropyrimidine-based concurrent chemotherapy are commonly combined in effort to improve treatment efficacy of various cancers. The mechanism of interactions between fluoropyrimidines (5-fluorouracil, capecitabine) and ionizing radiation (IR) is, however, not fully clarified. An accelerated anabolism of fluoropyrimidines (FP) to the active forms due to induction of enzymes involved in their metabolism in irradiated cells might cause this effect. To verify this assumption, we measured the expression of enzymes involved in FP metabolism thymidine phosphorylase (TP), thymidine synthase (TS), thymidine kinase (TK) and dihydropyrimidine dehydrogenase (DPD) in HeLa cells after a single dose of 200 cGy in vitro. We used real-time RT-PCR and Western blot analysis to quantify the levels of mRNA and protein at several time intervals after irradiation (from 10 min. up to 120 h). Although a trend towards increased level of TP and TS was detected 6 h after irradiation, only a slight but insignificant change at protein levels was found. These preliminary results do not support the hypothesis that an accelerated FP metabolism to the active forms due to irradiation is responsible for the synergistic effect of radio-chemotherapy.

Signaling Molecules as Therapeutic Targets

Methods: Bothrops jararacussu venom was fractionated by gel filtration (Superdex 75) and ion exchange (HiTrap Q-Sepharose) chromatography in 50 mM Tris-HCl, pH 7.5-8.0, containing 1 mM MgCl. Cultured Balb/c 3T3 (10 4 cells/well) and HaCaT (7x10 3 cells/well) cells were incubated with venom and chromatographic fractions for 12 hours, after which the cell viability was assessed by MTT reduction. Results: Gel filtration yielded three fractions (GF1, GF2 and GF3)

Introduction: Carbon tetrachloride, a xenobiotic, using as experimental liver damage. Carbon tetrachloride metabolites that occur at the end of this stage cause DNA and lipid damages

Methods: Control (free diet), stobadine (three times a week

CCl 4 (three times a week, diluted in 1/10 olive oil, intraperitoneally), stobadine and CCl 4 groups (described above) each included 10 rats, were designed for 8 weeks. Enzyme activities of supernatants, 5¢-NT and ADA, were evaluated. Formalin-fixed, paraffinembedded sections were stained with Masson trichrome technique and then evaluated histologically

Institute for Cardiovascular Diseases

Nechifor 1 , C. Filipescu 2,3 , I. Gherasim 2

Victor Babes

Human and animal studies have demonstrated that procyanidin-rich diets reduce the risk of cardiovascular diseases and atherosclerosis. Some beneficial effects have been attributed to the wellknown antioxidant activity of procyanidins. In this study, we investigated another potential role of procyanidins in cholesterol influx and efflux and inflammation in macrophage-derived foam cells. Methods and Results: We cultured RAW 264.7 macrophages with highly oxidized LDL (oxLDL), moderately oxidized LDL (moxLDL) or with LPS (0.5 lg/ml) and oxLDL (LPS + oxLDL) to induce foam cell. Then, cells were treated with procyanidins derived from grape seed (PE, 45 lg/ml) for the last 12 hours of incubation with the different lipoproteins (25 lg/ml). After lipid extraction, we determined that total and esterified cholesterol and triglyceride accumulation in foam cells were increased by lipoprotein treatment but reduced by PE incubation in all the foam-cell models. In order to asses the effect of PE on gene expression we determined the relative mRNA levels of CD36, ABCA1, iNOS and IkBa by RT-PCR. We show that PE reduced the oxLDL scavenger receptor expression (CD36), and enhanced ABCA1 (ATP-binding cassette A1) expression, a key regulator of macrophage cholesterol efflux, thus reducing lipid influx and inducing its efflux. PE also downregulated inflammatory related genes such as iNOS (inducible nitric oxide synthase) and IkBa (kappa beta inhibitor-alpha). Conclusion: We provide evidence that procyanidins may attenuate the development of foam cell formation by reducing cholesterol accumulation through the modulation of key genes in cholesterol flux and inflammation. Introduction: According to the data of WHO, in 1990 the percentage of infertile families reached 20%, and in 50% the infertility is connected with men's reproductive ability. Decrease in concentration and motility of sperm is observed in the world. Therefore, investigation of factors affecting the sperm motility is relevant. Methods: Sperm motility was determined using Gorjaev's chamber and expressed as the percentage of the motile sperms relative to the total number of sperms. Results: It was shown that oxidants (hydrogen peroxide, hydroxyl radicals and superoxide anions) decreased significantly (by 50-100%) the sperm motility. The sperm-specific isoform of glyceraldehyde-3phosphate dehydrogenase (GAPDs) was inactivated in the sperms treated with oxidants. It is suggested that oxidants decrease the sperm motility due to the oxidation of the sulfhydryl groups of GAPDs, a key glycolytic enzyme that is necessary for sperm motility. Interestingly, sodium citrate (pH 7.5) above 30 mM decreased the sperm motility by 90-100% after 5 minutes of incubation. The mechanism remains unclear. Significant increase in the sperm motility (by 20-35%) was observed in the presence of calcium ions. Conclusion: The sperm motility was shown to decrease in the presence of oxidants and sodium citrate and to increase in the presence of calcium ions. Oxidants decrease the sperm motility due to the oxidation of the sulfhydryl groups of GAPDs, a key glycolytic enzyme. The role of citrate and calcium is discussed. Acknowledgements: The work was supported by the RFBR (grant no. 06-04-48240) and NATO (PDD(CP)-(CBP.NR.RIG 982779)).

We have extensively studied the biological metabolism of toxic compounds, nitriles (1). Recently, we reported an overall 'nitrile pathway' (aldoxime -> nitrile -> amide -> acid -> acyl-CoA) comprising aldoxime dehydratase, NHase, amidase and acyl-CoA synthetase (AcsA) of Pseudomonas chlororaphis B23 (2). AcsA forms a carbon-sulfur bond in the presence of ATP and Mg 2+ , yielding acyl-CoA thioesters from the corresponding free acids and CoA. Very recently, we discovered amide (peptide) bond synthetic activity of AcsA. When L-cysteine was used as a substrate instead of CoA, N-acyl-L-cysteine was detected as a reaction product. Results: Acyl-CoA synthetase belongs to the superfamily of adenylateforming enzymes, whose three-dimensional structures are analogous to one another. We confirmed that amide (peptide) bond synthetic activity was also observed for acetyl-CoA synthetase from Saccharomyces cerevisiae which belongs to the superfamily. These results indicate that acetyl-CoA synthetase and acyl-CoA synthetase from not only prokaryotes but also eukaryotes would exhibit N-acylation activity generally. Luciferase also belongs to the superfamily, although the enzyme catalyzes the oxidation of luciferin in the presence of ATP and O 2 to generate oxyluciferin and light (hm). We found that firefly (Photinus pyralis) luciferase catalyzes the N-acyl-L-cysteine synthetic reaction (amide bond synthesis). These findings indicate the generality of the new reaction in the superfamily of adenylate-forming enzymes. 

Antioxidant activity and effects on cell proliferation of (hydro)alcoholic extracts of Geranium robertianum Acknowledgement: Our study was possible by financial support from Romanian Academy.

Differentiation of Brucella melitensis field strains from the vaccine strain Rev- 

The effects of oxidative stress on the expression of PAI-1, tPA, uPA and uPAR in vascular endothelial cells Plasminogen activator inhibitor type-1 (PAI-1) is the primary inhibitor of tissue-type and urokinase-type plasminogen activators and play integral role in the control of the fibrinolytic system. High plasma PAI-1 level is a risk factor for thrombotic diseases such as myocardial infraction, stroke, and venous thromboembolism. PAI-1 is an early response gene known to be activated by hormones, cytokines, lipoprotein, angiotensin II, phorbol ester, and reactive oxygen species (ROS). The purpose of the present study was to establish whether or not an enhanced oxidative stress involving endothelial injury, activation of coagulation, and inflammatory reaction would induce an expression of PAI-1, t-PA, uPA or uPAR. Human endothelial cells were treated with hydrogen peroxide (100, 200 lM), sodium nitroprusside (1-100 lM) and oxidative stress were determined by lucigenin chemiluminescence and 2,7, dichlorofluorescein (DCF) fluorescence, respectively. PAI-1, tPA, uPA and uPAR transcription were determined by transfection of PAI-1, t-PA, uPA and uPAR promoter linked to a luciferase reporter. Ap1, Sp1 and NF and #954; B activity was determined by an electrophoresis mobility shift assay. Expression of PAI-1, t-PA, uPA and uPAR in vascular endothelial cells studied using microarray assay and confirmed by real time PCR.Introduction: Thromboxane A2 induces platelet aggregation and constriction of vascular and bronchial smooth muscle through the interaction with a G protein-coupled receptor that exists as two alternatively splice variants, termed TPa and TPb with different C-terminal cytoplasmic tails. TPa and TPb are usually co-expressed in native cells and form homo-and hetero-oligomers in transfected cells. Methods: Computational modelling techniques [1, 2] were first used to generate models of TPa monomer in free-and agonist-bound conformations, then subjected to docking simulations for modelling of TPa homo-dimers. Theoretical predictions were challenged by sitedirected mutagenesis of TPa and TPb. Wild type and mutant receptors fused to fluorescent proteins (GFP, CFP, YFP) were transiently expressed in HEK293 cells and their subcellular localization and dimerization studied by a combination of microscopy and fluorescence resonance energy transfer (FRET) techniques. Results: The most supported model of TPa homo-dimer was characterized by contacts between helices 1 of monomers, whereby cysteine 35 residues lie at a distance predictive of an inter-helical disulphide bridge. Upon substitution of cysteine 35 with alanine (C35A) homo-and hetero-dimerization of TPa and TPb as well as TPa trafficking was greatly impaired. However, C35A mutant TP receptors did not significantly differ from wild type in terms of [3H]-SQ29, 548 antagonist binding and accumulation of inositol phosphates induced by U46619 agonist stimulation. Conclusions: Mutation of cysteine 35 greatly impairs dimerization of TP receptors as anticipated by computational modelling but leaves unaffected the binding and signalling properties of monomeric receptors. 

Erbin is a novel SARA-interacting protein on early endosomes that negatively regulates TGF-b/ Activin signaling G. Sflomos 1,2 , E. Kostaras 1,2 , E. Panopoulou 2 , N. Pappas 1,2 , M. Hyvonen 3 , T. Fotsis 1,2 * and C. Murphy 2 * 1 Laboratory of Biological Chemistry, Medical School, University of Ioannina, Ioannina, GREECE, 2 Biomedical Research Institute, Foundation for Research and Technology-Hellas, Ioannina, GREECE, 3 Department of Biochemistry, University of Cambridge, Cambridge, UK Transforming growth factor beta (TGF-b) superfamily members are important regulators of many diverse developmental and homeostatic processes. SARA, an early endosomal FYVE finger protein, plays a leading role in this pathway by recruiting non-phosphorylated SMAD proteins to the receptor complex for phosphorylation. Furthermore, SARA also mediates receptor dephosphorylation by recruiting PP1 phosphatase to the activated receptor. Recently SARA has been shown to regulate the vesicular targeting of the photoreceptor protein Rhodopsin to newly synthesized discs. To further investigate the molecular mechanism of SARA action, we have undertaken the task of identifying additional SARA-interacting proteins. A yeast-two-hybrid screen identified the leucine-rich repeat (LRR) and PDZ domain-containing protein ERBIN as a binding partner of SARA. We have confirmed the interaction of ERBIN and SARA by pull-down assays and immunoprecipitation and have mapped the interaction domains on both partners. Furthermore, we have found that ERBIN is present on the early endocytic compartment and it also interacts with Activin Receptors. We have addressed the physiological role of this interaction using lossof-function approaches (siRNA) and we have observed that knockdown of the endogenous ERBIN enhances TGF-b/Activin-induced transcriptional responses, while gain-of-function reduces TGF-b/Activin-induced transcriptional responses. Thus, ERBIN appears to be a novel negative regulator of the TGF-b/Activin pathway. *These authors contributed equally.

The regulatory function of adenovirus E1A protein in JNK signaling pathway J. Shim Sejong University, Seoul, REPUBLIC OF KOREA The adenovirus E1A oncoproteins can induce resting human cells to enter the cell cycle providing an optimal environment for viral replication. On the other hand mitogen-activated protein kinase (MAPK) signaling pathways mediated intracellular signaling, which is initiated by a variety of extracelluar stimuli. This signaling pathway may be involved in diverse cellular activities that include cell proliferation, differentiation, and apoptosis. The persistent activation of the SAPK/ JNK(c-Jun N-terminal kinase) pathway often mediates intracellular signaling that leads to cell death. We demonstrated that E1A acts as an inhibitor for SAPK in a dose dependent manner. Besides this report show that E1A proteins are the putative substrate of SAPK/JNK. Taken together, these results suggest a possibility that E1A counteract SAPK activation to prevent apoptotic cell death of host cells.

Behaviour of the peptaibol trichogin in a mixed hybrid bilayer lipid membrane array (nanoBLMs) S. Smeazzetto 1 , T. Ferri 2 and M. R. Moncelli 1 1 Dipartimento di Chimica, Universita´di Firenze, Florence, ITALY 2 Dipartimento di Chimica, Universita´di Roma 'La Sapienza', Rome, ITALY Introduction: The peptaibols are a family of antibiotic peptides isolated from soil fungi that exhibit anti-bacterial and anti-fungal properties. The name peptaibol derives from their chemical composition: peptides containing Aib (a-amminoisobutyric acid or a-methyl alanine) residues and ending in a C-terminal alchol. Trichogin is one of the shortest peptaibols (11 residues) and it has a fatty acyl moiety linked to the N-terminal amino acid (n-octanoyl). Trichogin is too short to fully span a membrane, so its mechanism of action must be different from the long peptaibols, like alamethicin forming voltage-gated channel. Methods: We are studying these peptaibols using a new experimental model of biological membrane [1] . This model is constituted of a polycarbonate membrane with 1 lm diameter holes, covered by an octadecanthiol (ODT) self-assembled monolayer (SAM) on Au. The bilayer lipid membranes (BLMs) spontaneously self-assemble on the holes of the polycarbonate membrane. The characterization and stability of the nanoBLMs have been characterized by electrochemical measurements: cyclic voltammetry, AC voltammetry and Impedance Spectroscopy. Results: We identified the typical range of values of specific resistance, specific capacitance and conductance for well formed nanoBLMs: 0.1MX<R*cm 2 <10 MX 0.4microF<C/ cm 2 <1microF and 0<j<5 microS/cm respectively. By means of specific resistance data, we furthermore proved that nanoBLMs can last up to 24 hours. Conclusion: We set up the protocol to incorporate the trichogin in the phospholipidic bilayer and to study its selectivity. Gomel State Medical University, Gomel, BELARUS, 2 Belarusian State University, Minsk, BELARUS, 3 Gomel State Technical University, Gomel, BELARUS Peroxynitrite (ONOO ) , oxoperoxonitrate ()1)) is one from the reactive species generated by activated neutrophils. The work aimed at understanding the mechanisms of neutrophil interaction with exogenous ONOOusing chemiluminescence and atomic force microscopy (AFM) methods. We examined the conditions of the luminescence development (luminol-, lucegenin-dependent and spontaneous chemiluminescence) when the cells interacted with ONOO ) . With AFM methods we investigated the morphology and surface structure of neutrophils treated with ONOO -, adhered to glass slabs, fixed with glutaraldehyde and dried. The intensity of the chemiluminescence accompanying the neutrophil interaction with ONOO ) increased with increasing in pH level (in range of 5-9) and ONOO ) concentration (in range of 0.005-1.0 mM). The luminol-dependent chemiluminescence kinetics was characterized by the presence of some stages during which the generation of superoxide anion radicals, nitrogen monoxide and myeloperoxidase reaction products were observed. ONOO-induced spontaneous chemiluminescence of neutrophils was enhanced (5-15-fold) after the pre-incubation of the cells with such neutrophil stimulating agents as fMLP and latex. Atomic force microscopy analysis of neutrophil surface showed that 0.1 mM ONOO) induced neutrophil spreading on glass surface and neutrophil degranulation. The increase of the incubation time led to the increase in the spreading and degranulation levels of ONOO-treated neutrophils and to the damage of neutrophil through the NET (neutrophil extracellular traps) mechanism (after incubation for 3 hour). The findings are evidence of the modulation of some neutrophilic functions by exogenous ONOO ) .

Role of PLC-c in the control of the fertilization and mitotic calcium signals in sea urchin egg L. Tosca, R. Glass and B. Ciapa Universite Paris-Sud, Orsay, FRANCE Introduction: Egg activation after fertilization is mediated by a transient rise in intracellular free calcium (Ca i ) that is followed by other Ca i transients necessary for mitosis progression. In sea urchin egg, the cADPr (cyclic ADP ribose) and the IP 3 (inositol 1,4,5-trisphosphate) pathways are both necessary to generate the fertilization Ca i signal. In this study, we demonstrate that PLCc (phospholipase C c) control both fertilization and mitosis Ca i signals. Methods: PLCc contains two SH2 domains allowing interaction with phospho-tyrosine residues. Fusion proteins containing the N-ter and/or C-ter SH2 domain(s) and an histidine 'tag' were generated (His-SH2-PLCc). Proteins that are not functionnal carrying R253A (N-ter SH2) and R364A (C-ter SH2) mutations, which prevents interaction with phospho-tyrosine residues, were used as controls. All constructions were injected before or after fertilization and Ca i was mesured using a fura-2 fluorescent probe (Axon Inst. Image Analysis System). Results: Our results indicate that inhibiting the PLCc pathway did not prevent the fertilization Ca i signal. All control mutated constructs were without effect. However, we found that the C-ter SH2 domain only was capable of inhibiting mitosis. Conclusion: Our results clearly show that PLCc is not sufficient to trigger the fertilization Ca i signal, contrary to what is widely accepted in the litterature. However, PLCc is absolutely necessary to control progression through the first mitotic divisions.Introduction: In recent years, there is increasing interest towards the development of analytical methods for testing the authenticity of food products commanding a premium price. Coffee is an important food commodity worldwide. The commercial trade consists almost entirely of the Arabica and Robusta species. The former is considered to be of superior quality and thus attracts a premium price. We have developed a rapid, simple and robust method that allows detection of the two species based on the genotyping of a single nucleotide polymorphism (SNP) within the chloroplastic genome. Methods: DNA is isolated from coffee green beans and the segments that span the SNP of interest are amplified by PCR. Following purification, the products are subjected to two primer extension (PEXT) reactions in the presence of biotin-dUTP, using allele-specific primers that contain a (dA) 30 segment. The products are detected visually by a dry-reagent dipstick-type biosensor, in which the extension products are captured from immobilized streptavidin on the test zone of the sensor and detected by hybridization with oligo(dT)-functionalized gold nanoparticles forming a red line. A second red line is generated at the control zone of the biosensor by hybridization of the nanoparticles with immobilized oligo(dA). Results: Coffee amplicon mixtures were analyzed with this technique allowing clear discrimination of the two alleles. The dipstick test is complete within 10 minutes. Conclusions: The proposed dipstick assay is rapid and enables visual detection of either deliberate or accidental adulteration without the need for special instrumentation or highly qualified technical personnel.

Molecularly imprinted polymers that selectively recognize histamine F. Trikka 1 , K. Yoshimatsu 2 , L. Ye 2 and D. A. Kyriakides 1,3 1 Laboratory of Biochemistry, Department of Chemistry , Aristotle University of Thessaloniki, Thessaloniki, GREECE, 2 Pure and Applied Biochemistry, Chemical Center, Lund University, Lund, SWEDEN,   3 National Hellenic Research Foundation, Athens, GREECE Molecular imprinting is widely used for the preparation of polymeric materials containing recognition sites of predetermined specificity. Imprinted binding sites are generated by co-polymerization of a functional monomer with a cross-linker in the presence of a template molecule. Removal of the template molecule from the formed polymer results in cavities within the polymer that is complementary to the template. Consequently, the polymer is able to rebind the template molecule with high affinity and specificity, in a manner comparable to antibody-antigen or receptor-ligand binding. In this work, MIPs selective for histamine were synthesized by bulk polymerization using methacrylic acid as functional monomer and ethylene glycol dimethacrylate as cross-linker. Binding affinity of imprinted polymers for histamine and structurally related compounds was studied in both organic and aqueous medium. The specific binding sites of imprinted polymer selectively recognize histamine over L-Histidine under aqueous conditions. When polymers were tested against other biogenic amines in aqueous media, MIPs were capable of binding three times more histamine than putrescine. Binding analysis of histamine imprinted polymers with histamine receptor antagonists and compounds related to histamine action was studied as well. In organic media, MIPs selectively recognized histamine over dimentidene, ranitidine and fluoxetine. In aqueous media MIPs bound two times more histamine than ranitidine when 0.3% (v/v) Tween 20 was added. Selectivity of MIPs over structurally and functionally related compounds in aqueous environment makes feasible its potential bioanalytical applications. Acknowledgment: This work was supported by the European Union program: Nanoimprinting Technologies for Selective Recognition and Seperation, Contract no: NMP4-CT-2005-516981.

Monodispersed colloidal maghemite nanoparticles as conductors of magnetic hyperthermia for targeted dissolution of brain tumors 3 Institute of Biology National Center for Scientific Research 'Demokritos', Athens, GREECE, 4 Department of Radiology, University Hospital of Heraklion, Heraklion, GREECE Introduction: Ferrofluids are colloidal suspensions of single-domain magnetic nanoparticles, which are stabilized in water. Their long term colloidal stability is ensured by coating the nanoparticles with appropriate polymers, e.g. polysaccharides, to prevent aggregation and render these nanoparticles useful in medical diagnosis and therapy. Methods: Coated maghemite nanoparticles were prepared by mixing acidic iron solutions and added over in alkaline solution of NaOH. 1 wt% of dextran was added in the acidic media. Then 0.18 ml of ferrofluid (3.6 mg of maghemite) was injected in glioma C6 tumours grown in mice to 0.5 · 0.5 mm, in adult male Wistar rats. Following magnetic hyperthermia treatment for about 20 minutes the remaining glioma cell tumors were excised and processed for light microscopy. Results: Specifically in sites of ferrofluid injection but not in adjacent areas of tumour or normal tissue, nanoparticle-induced hyperthermia to 44°C via application of a magnetic field (temperature was monitored by an optical fibre thermometer), resulted in cell dissolution or cell damage: the remaining cells were deformed and appeared to be apoptotic with dense and fragmented nuclei, compared to the appearance of healthy cells in tumour areas free of ferrofluid. Apparently then the maghemite nanoparticles were able, by locally raising the temperature to interfere with cell viability and caused nanoparticle-targeted cell dissolution or cell death. Conclusions: The maghemite nanoparticles, because of their properties to conduct heat can be used as means to therapeutically intervene in massive tumours of the CNS by targeted tumour cell disruption and apoptosis. Introduction: Hepatitis B virus (HBV) is a small enveloped DNA virus, causing acute and chronic hepatitis, cirrhosis, hepatocellular carcinoma and considered a significant health problem worldwide. The proteins of HBV are the surface protein, the core protein and DNA polymerase. The HBV core genes codes for two different proteins; HBc antigen and HBe antigen (HBeAg). Presence of HBeAg in the sera of HBV infected individuals may indicate the high infectivity of the disease and poor prognosis of the disease treatment, so detection of HBeAg is important in clinical diganosis. Methods: One of the sensitive and commonly used methods to differentiate the type of viral infection and discriminate between acute and chronic hepatitis is ELISA, based on the use of monoclonal antibodies. In this study, BALB/c mice were immunized with HBeAg expressing M13 phage for the production of monoclonal antibodies specific for HBe antigen. It was expressed on the M13 phage by cloning of HBe gene into phagemid, transferring into E.coli TG1 and infection of positive clones with M13 helper phages. Results and Conclusion: After three subsequent immunization of BALB/c mice with HBe-phage, lymphocytes were isolated and fused with F0 myeloma cells. In order to maximize the probability of generating monoclonal antibodies, lymphocytes from spleen, lymph nodes and intraperitonal fluid were used. After selection of positive clones with ELISA, it was found that out of 690 hybrid clones, 10D12 was producing antibody recognizing both HBe antigen and human serum, whereas 5G12 was producing monoclonal antibody specific only for M13 phage.

A study for determining the direct effect of myeloperoxidase enzyme on doxorubicin Y. Basaran and H. Kuzu Yildiz Technical University, Bioengineering Department, Istanbul, TURKEY Introduction: Myeloperoxidase (MPO, EC 1.11.1.7) is an enzyme found in polymorphonuclear neutrophils and monocytes, and some populations of tissue macrophages. Myeloperoxidase plays an important role in the defense system against invading microorganisms and certain tumor killing actions via myeloperoxidase-H 2 O 2 -Clreaction system. Myeloperoxidase oxidizes various compounds such as phenols, anilines and their derivatives, and also b-diketons which may lead to the formation of free radicals. It has been hypothesized that peroxidase-catalyzed drug oxidation causes radical production, protein binding and oxidative stress cytotoxicity, and thereby contributes to reactive metabolite formation and cell death. This hypothesis includes the finding that peroxidase catalyzes the oxidation of drugs, carcinogens or xenobiotics to form radicals that cooxidize biomolecules and form reactive oxygen species [1] . From this standpoint, myeloperoxidase may cause free radical formation in drug metabolism. Methods: Myeloperoxidase was purified for the first time at sufficient amounts for laboratory scale experiments with good activity. The intensive effect of purified myeloperoxidase on Doxorubicin was studied for the first time. The reaction products which was formed at physiological temperature and pH (37°C; pH 7.2-7.3) were evaluated by using UV-Vis. spectrophotometer, FT-IR and LC-MS spectrometer systems. Results: The results obtained with UV-Vis spectrophotometer implied that the reaction took place. Occured reaction was clarified by evaluating FT-IR and LC-MS results for the products. Conclusions: The obtained data showed that Doxorubicin was fragmented with the formation of free radicals. Introduction: Dipeptidyl peptidase IV (DPP IV/CD26, EC 3.4.14.5) is a glycoprotein with multiple functions. As a serine protease, it could activate or inactivate biologically important peptides, involved in chronic diseases pathogenesis. Therefore, it could play an important role in the modulation of the immune response during inflammatory processes. The aim of this study was to evaluate the changes in serum DPP IV activity in adult patients with chronic diseases including inflammatory bowel diseases (IBD): Crohn's disease -CD and ulcerative colitis -UC; and rheumatoid diseases: rheumatoid arthritis -RA, psoriatic arthritis -PA, and patients with osteoporosis -OP. Methods: The diagnosis of CD, UC, RA, PA and OP were established on the basis of clinical history, laboratory, endoscopic and histological data. The control group included healthy blood donors. The serum DPP IV activity has been measured spectrofotometrically using Gly-Pro p-nitroanilide as substrate. Results: Serum DPP IV activities in patients with IBD (CD and UC) and RA were statistically significantly decreased compared with the levels in healthy controls (P < 0.001). In patients with PA and OP, serum DPP IV activities were not statistically significantly different from healthy controls. No statistically significant difference in the serum DPP IV activity between patients with CD and UC was found (P > 0.05). Conclusions: The results of this study sustained the statement that the serum DPP IV is involved in the pathogenesis of chronic diseases. The serum DPP IV activity appears to be useful as an available, non-invasive marker in the diagnosis of the disease activity. Introduction: Since embryonic stem (ES) cell-derived neural progenitors (NPs) have the potential to cure many diseases, knowledge of the signaling mechanisms that regulate their proliferation and differentiation is critical. We discovered the presence of functional mu opioid receptor (MOR) and kappa opioid receptor (KOR) in ES cells and NPs. To explore the relationship between opioids and ES cell-derived NP fate, we examined retinoic acid (RA) induced NP proliferation and differentiation upon opioid treatment. Method: An immunocytochemistry approach was taken that involved cell specific and BrdU antibodies to measure cell division and differentiation in NPs, treated with DAMGO (MOR agonist) or U69,593 (KOR agonist) for 1 or 5 days. Results: Both opioids inhibited NP proliferation and ES cell derived neurogenesis and astrogenesis. Some of these inhibitory effects were extracellular-signal regulated kinase (ERK) dependent. In contrast, mu and kappa opioids increased the number of proliferating ES-cell derived NG2 + oligodendrocyte progenitors (OPs). MOR and KOR immunoreactivity was detected in oligodendrocytes at three stages of their development. By using selective inhibitors, we found that the opioid induced proliferation of OPs was ERK but not p38 kinase mediated. Opioids did not affect proliferation of immature oligodendrocytes. Conclusion: Collectively, these results reveal that opioids can play a modulatory role in determining NP fate decisions. We have extensively studied the biological metabolism toxic compounds containing a triple bond between carbon and nitrogen such as nitrile (R-CN) (1) and isonitrile (R-NC). As for the latter compound, we discovered a novel enzyme that catalyzes the hydration isonitrile to the corresponding N-substituted formamide (R-NHCHO) in Pseudomonas putida N19-2 and named it isonitrile hydratase (2) . We recently discovered a novel enzyme, N-substituted formamide deformylase (NfdA) which catalyzes the hydrolysis N-substituted formamide to the corresponding amine and formate in Arthrobacter pascens F164 (3). Results: A. pascens F164 was able to degrade not only N-benzylformamide (which is an N-substituted formamide) but also the corresponding isonitrile, benzyl isocyanide. In the reaction the cell-free extracts prepared from the cells grown on media containing benzyl isocyanide as the sole nitrogen source, the isonitrile was confirmed to be hydrated to N-benzylformamide, which was further converted into benzylamine and formate by NfdA: isonitrile hydratase and NfdA activities were detected. These findings suggest that both enzymes are closely related in sequential isonitrile metabolism. We searched for an appropriate substrate whose reaction product was not degraded by NfdA and investigated the optimum culture conditions for the formation isonitrile hydratase. The highest enzyme activity was obtained when this strain was cultured in the presence N-benzylformamide as an inducer. This isonitrile hydratase was found to be an inducible enzyme depending on N-benzylformamide. Despite its importance in cell proliferation and tumorigenesis, very little is known about the molecular mechanism underlying the regulation of phospholipase D (PLD) expression. PLD isozymes are significantly co-overexpressed with cancer marker genes in colorectal carcinoma. Phorbol 12-myristate 13-acetate (PMA) treatment, as a mitogenic signal in colon cancer cells, selectively increases PLD1 expression in transcription and post-transcription. Moreover, experiments using intraperitoneal injection of PMA into mice showed selective PLD1 induction in the intestine and lung tissues, which suggests its physiological relevance in vivo. Therefore, we have undertaken a detailed analysis of the effects of PMA on the promoter activity of PLD genes. Protein kinase C inhibitors, but not a protein kinase A inhibitor, were found to suppress the up-regulation of PLD1 but not PLD2. Dominant-negative mutants of Ras, Raf, and MEK suppressed the induction and activity of PLD1. Moreover, depletion of the supposedly involved proteins reduced the endogenous PLD1 protein level. An important role for NFkappaB as a downstream target of ERK in PMA-induced PLD1 induction was also demonstrated using the inhibitor, small interfering RNA, chromatin immunoprecipitation assay, and site-specific mutagenesis. Furthermore, inhibitors of these signaling proteins and depletion of PLD1 suppressed PMA-induced matrix metalloproteinase-9 secretion and PLD1 induction. In conclusion, we demonstrate for the first time that induction of PLD1 through a protein kinase C/Ras/ ERK/NFkappaB-dependent pathway is involved in the secretion of matrix metalloproteinase-9 in colorectal cancer cells.

Endoplasmic reticulum stress and the role of T-cadherin Introduction: Endothelial dysfunction plays a key role in the pathogenesis of atherosclerosis. T-cadherin is an atypical GPI-anchored member of the cadherin superfamily. It is up-regulated in atherosclerotic lesion. Grp78, which co-associates with T-cadherin, is a key mediator of endoplasmic reticulum stress. This means that T-cadherin may possibly play a key role in ER stress. Methods: Endothelial cells are treated with ER stress inducing agents. Agents that are used are thapsigargin (1 lM), DTT (1 mM), Brefel-dinA (5 lg/ml), Tunicamycin (3 lg/ml), Calcium Ionophore A23187 (2 lM) and Homocysteine (15 mM). Immunoblotting and quantitative RT-PCR are used in order to determine the modulation of T-cadherin expression during endoplasmic reticulum stress, and for the changes in expression of UPR signaling molecules such as GRP78, ATF3 and phospho-eIF2a. Silencing (use of siRNA) and over-expression of Tcadherin (adenoviral vector system) is used to study the ability of Tcadherin to influence endothelial cell survival responses during ER stress.Results: T-cadherin is elevated in ER stress. Cultured EC after treatment with ER stress inducing agents show increased mRNA and protein levels of T-cad at early time points. GRP78 is gradually elevated through time.Conclusions: The role of these molecules in protection of cardiac and vascular cells from ER stress has to be evaluated. Also new insights into the impact of ER stress signaling in regulation of endothelial function would be given. T-cadherin seems to be a key player in ER stress. This study will shed more light on the mechanism of cardiovascular disorders. Cordyceps sinensis (C. sinensis) is a precious medicinal fungus, which has been used in China since ancient times. In modern scientific studies, C. sinensis possesses several bioativities, such as antitumour, immunomodulatory, anti-inflammation, anti-atherosclerosis and anti-diabetes. Due to the medicinal properties of C. sinensis, it has become increasingly scarce in recent years and the price for wild C. sinensis has increased sharply. Therefore, mycelial fermentation of Cordyceps fungal species is a feasible and sustainable method for producing the medicinal fungus and its active compounds. Methods: Cs-HK1, a Tolypocladium fungus, is a new species isolated from wild C. sinensis and cultivated by liquid fermentation which containing glucose, yeast extract, peptone and trace amount of mineral ions. The exo-polysaccharide (EPS) was isolated from the culture broth by centrifugation; the supernatant was collected and mixed with four volume of 95% (v/v) ethanol for the precipitation of EPS. The EPS precipitate was recovered by centrifugation and freeze-dried. The carbohydrate and protein content of EPS was measured by anthrone sulphuric acid and Lowry method. Results: EPS secreted by the Cs-HK1 mainly consisted of nearly 70% of carbohydrate and 30% of protein. Structural features of EPS were investigated by a GC and FT-IR, the results indicating that EPS has a backbone of b-D-glucose residues. The molecular weight of EPS was monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and showed that EPS is a wide molecular weight range of biopolymer, ranging from several to hundred of kilo-Dalton (kDa). During the in vitro assay, EPS shows a positive effect in both anti-oxidant and immunomodulatory activity. EPS showed the reducing activity with 50.2 lmol Fe (II) g/EPS during the ferric reducing ability of plasma (FRAP) assay. In radical scavenging activity by ABTS assay, 0.5 g/EPS ml could cause 55% of radical inhibition, which equivalence to 33.9 lmol Trolox g/EPS. In the in vitro cell model, EPS can also stimulate the immunocyte activity. 

In vitro anti-oxidant activities on phenolic and alkaloid extracts of two Algeria species Huge commercial demand for natural carotenoids has focused attention on developing of suitable biotechnological techniques for their production. A range of yeast species utilizing agroindustrial substrates have been tested for carotenoids formation. Yeast strains of Rhodotorula and Sporobolomyces formed b-carotene as the main pigment together with torulene and torularhodine as minor carotenoids. To increase the yield of these pigments, physiological regulation of the fermentation process has been employed. Molecular changes in yeast cells on genome, proteome and metabolome level were studied using PFGE, 2D-GE, LC/MS/MS and EPR techniques. These studies also revealed that activated biosynthesis of carotenoids by yeasts exposed to stress could be in a part explained by their scavenger characters as a protection against the harmful effect of the environment. During the last decades fast progress has been made within the field of biochemistry of carotenoid biosynthesis in bacteria, fungi, and plants. Although more than 600 different carotenoids have been identified in nature, only a few are used industrially. There are many yeast strains able to produce different carotenoids. However, the heterobasidiomycetous yeast Xanthophyllomyces dendrorhous, are the only microbial systems with commercial potentials for the production of astaxanthin. In this work three different carotenogenic strains (Sporobolomyces roseus., Rhodotorula gluitinis, Rhodotorula rubria) were enrolled into a comparative study. Each strain was cultivated at optimal growth conditions and in medium with modified carbon and nitrogen sources. Complex substrates as well as some waste materials (whey, rape pressing, potato extract) were used as nutrient source. Some types of exogenous stress were applied too. The production of carotene-enriched biomass was carried out in flasks as well as in laboratory fermentor. As the best pro-ducer of enriched biomass strain Rhodotorula glutinis CCY 20-2-26 was found. Production of biomass in R.glutinis was about 35 g/l. Total carotene enrichment of this biomass about 2.5 mg/g d.w. was obtained. Significant changes in biomass as well as carotenoid production were observed in individual strains according to cultivation conditions. Introduction: Ischemia or oxygen starvation of tissues is observed at cerebral stroke, brain edema, myocardial infarction, and various surgical operations. In mentioned conditions the tissue damage arise from apoptotic mechanism of cell death, which includes initiation of lipids peroxidation and activation of sphingomyelin cycle, comprising the secondary messengers ceramide, sphingosine, sphingosin-1-phosphate. The aim of our investigation was to study the effects of cysteine derivative on the lipid peroxidation processes, contents of sphingomyelin and glycosphingolipid and products of their hydrolytic dissociation, ceramide and sphingosine at hypoxic conditions. Methods: Brain hypoxia in white rats was induced by carotid ligation. Sphingomyelin cycle was studied by thin layer chromatography. Cysteine derivative (cysteine combined with nicotinic acid) was administered intraperitonealy in dose 5 mg/kg. Results: Brain hypoxia leads to the activation of lipid peroxidation, particularly to the level increase of diene conjugates, hydroperoxides and malonic dialdehyde, accompanied by the increase of the sphingosine level, decrease in sphingomyelines, galactosylceramides, and sulphogalactosylceramides content. The treatment by cysteine derivative leads to the normalization of studied parameters.Conclusions: Normalization effect of cysteine derivative is revealed in animals with hypoxia, which could be result of promotion of synthesis of glutathione, effective intracellular antioxidant and the main component of cell defense system in studied conditions.

Site-specific excision reaction in Anabaena PCC 7120 catalyzed by the integrase of coliphage HK022 The Integrase (Int) of coliphage HK022, a close relative of k catalyzes the site-specific integration and excision of the phage into and from the chromosome of its host, Escherichia coli. Int recognizes two different pairs of recombining sites attP x attB and attL x attR, thus enabling advanced site-specific recombination as a potent tool for gene manipulations. This system was introduced into the cyanobacterium Anabaena PCC7120. Two plasmids were consecutively co-introduced by conjugation into Anabaena PCC 7120. One plasmid carries the gene (int) encoding the HK022 Int recombinase, the other carries the excision substrate cassette pglnA-attL-Stop-attR-lacZ, where Stop is a transcription terminator that prevents expression of the lacZ reporter that encodes b-galactosidase. pglnA is the constitutive promoter of glutamine synthetase gene (glnA) in Anabaena. The Int-catalyzed site-specific recombination reaction was monitored by production of b-galactosidase that resulted from the eviction of the transcription terminator (Stop) as a result of the attL x attR excision reaction. This excision reaction was monitored when the substrate cassette was either on the plasmid or on the chromosome.Introduction: There has been growing interest in the development of fuel cell to power drug delivery devices for biomedical applications. However, there is a generally observed loss in fuel cell durability when enzymes are employed to catalyze these 'biofuel cell' redox reactions.The overall goal of this research project is to understand the factors responsible for the decrease in biofuel cell durability and how these can be prevented thus leading to the development of durable biofuel cell.In this work, we have non-covalently immobilized the enzyme alcohol dehydrogenase (ADH) on diamond nanoparticles (DNP) of 5 nm. Methods: Adsorption behavior and biophysical characterization of ADH at different loading densities and pH's were studied. Characterization of the absorbed protein on DNP surface was done by FT-IR, XPS, SEM and TEM. Furthermore, the activity of the immobilized enzyme at different loading densities was also determined and a release profile was performed.Results: The Langmuir adsorption isotherms revealed a pH dependence, dependant on the charges of both, ADH and DNP. The results of the enzymatic activity revealed that the activity is dependant on the surface coverage. The enzymatic activity increases as the surface coverage is higher. The release profile demonstrated that the absorption was not irreversible.Conclusions: This work demonstrated that DNP can be employed for non-covalent enzyme immobilization. It was found that the enzyme retains its activity after physical adsorption on DNP. Future experiments will include covalent immobilization of the enzyme on DNP and its biophysical characterization.

Aberrant phospholipase D expression enhances matrix metalloproteinase-2 expression and invasion of glioma cells via protein kinase C and protein kinase A/NFj B/SP1-mediated signaling pathwaysThe purpose of this study was to investigate the comparative levels of antibiotic resistance in different enterobacterial populations circulating in the hospital environment. Material and Methods: Two hundred Enterobacteriaceae strains belonging in majority to E. coli, followed by Klebsiella oxytoca, K. pneumoniae) and Enterobacter cloacae species isolated from surfaces in the hospital environment and respectively from different clinical sources were identified both by biochemical and serological tests. Both disc diffusion susceptibility test and microplate dilution technique were used to investigate the antibiotic resistance profile of these bacteria.The rapid test to nitrocephine and isoelectrofocusing techniques were used for the confirmation of the presence and type of beta-lactamases.Results: The enterobacterial strains isolated from surfacess in the hospital environment exhibited high resistance rates to cephuroxime (100%), cephtazidime (100%), ampicillin (98%), cefoxitin (98%), ticarcillin (62%), amoxicillin/ clavulanic acid (53%). 36% of these strains were confirmed for the production of ESBLs and 50% exhibited AMPc inducible beta-lactamases. All tested strains also exhibited high level resistance to tetracyclines (46-53%) and trimetoprim/sulphametoxazole. Curiously, the beta-lactam resistance levels were lower in strains isolated from clinical samples, as well as the incidence of ESBl, attesting the different origin of the clinical strains, which probably, is in a great number of cases endogenic. The clinical strains exhibited higher resistance levels to quinolones and aminoglycosides. Our study is demonstrating the polyclonal origin of the enterobacterial strains circulating in the hospital environment. The healthy effects of flavonoids are largely due to their antioxidant properties but also to their modulation of gene expression at the transcriptional level. The aim of this study was to determine proanthocyanidins' target genes in cholesterol and energy metabolisms in obese rats and in HepG2 cells. To this end, female Wistar rats were fed on a highfat diet (HFD) for 4 months. In the last 10 days some of the rats were treated with an oral dose of proanthocyanidins (25 mg/kg). The HFD increased total cholesterol and LDL-c plasma levels, and the rats' body weight rose by 40%. Moreover, in the cholesterol metabolism, liver mRNA levels of HMGCR reduced and CYP7A1 increased. In the energy metabolism, HFD decreased UCP-2, whereas IDH-3c and COX-5a remained unchanged. Proanthocyanidin administration, on the other hand, decreased LDL-c plasma levels and reduced body weight by 2%. In addition, HMGCR increased and CYP7A1 and COX-5a decreased. To gain further insight into proanthocyanidins' target genes, the Taqman Low Density Array was used with HepG2 cells. They were treated with a 50 mg/l dose for 9 hours. The most important results for cholesterol metabolism were the decrease in ACACA, ABCA1 and SOAT1, and the considerable increase in the LDL-receptor. Several nuclear receptors, including FXR, LXR and RXRa ??were also analysed, and a generalized repression was observed. In conclusion, HFD promotes obesity and dyslipidemia whereas proanthocyanidins act on the cholesterol metabolism both in vivo and in vitro. Several genes are regulated, which suggests an improvement in these pathologies. Acknowledgement: This work was supported by the Spanish Government (grant AGL2005-04889). *These authors contributed equally to this work. Background: Type I collagen plays an important role in maintaining and remodeling tissue architecture. Most of the collagen studies have been investigated using pepsin-hydrolyzed type I collagen (P-col). We found that actinidain-hydrolyzed type I collagen (A-col) shows a poor fibrillogenesis and nano-scale three-dimensional structure. It is not still understood how the superstructure of the collagen self-assembly forms. However, biological function of the collagen such as cell adhesion or migration should be altered. As it is well known that MMP-8 secreted by neutrophil cleaves type I collagen, we confirmed the cell behavior of neutrophil in the enzyme-hydrolyzed collagen assemblies. Methods: We prepared P-col and A-col from chicken skin. Neutrophil was collected from a mouse, and subsequently it was cultured on a plate coated with each collagen assembly. We observed the cell behavior of neutrophil using an ultra-resolution scanning electron microscopy (Hitachi, S-900). Also, we identified the epitope expression on the surfaces of the A-col and P-col using an anti-collagen antibody labeled with gold-particle. Results: We found that the neutrophil adhered and migrated to the Acol assembly, which was clearly different from the P-col. The binding of antibody showed to be significant difference in the epitope expression. Thus, several lines of evidence suggest that the A-col would be responsible for the activation of cell behaviors. Introduction: Atrophy of skeletal muscle is a caused by numerous diseases, including cancer and AIDS. Mechano growth factor (MGF) produced by active muscle appears to be the factor that controls local tissue repair, maintenance, remodeling and exhibits a neuroprotective effect. Therefore, the MGF has a potential to be used in medicines for regenerative capability of skeletal muscle, as well as prevention of neuronal damage. The Saccharomyces cerevisiae has been applied as host for production of recombinant human MGF. The yeast considered to be a perspective heterological protein producer due to its good secretion folding properties and biosafety. Results: MGF secreting expression cassette that contains synthetic gene under effective GAL1 promoter control has been developed. Two chaperons (BiP and PDi) were introduced in chromosome for folding improvement. Gene of protease B that catalyzes cleavage of MGF has been identified and disrupted. As a result strain synthesing and secreting soluble MGF has been obtained. For confirmation of MGF biological activity the assay on human postnatal myoblast has been carried out. These experiments proved that recombinant MGF showed the same level of biological activity as human MGF intensifying myoblast growth. For increasing MGF production, the conditions for yeast fermentation have been optimized. As a result the total yield of MGF production increased up to 50 mg/l of culture liquid. The Saccharomyces cerevisiae strain -producer of recombinant human MGF is shown to be a promising tool for enzymes production for bioactive peptides and proteins and therefore can be used for industrial application.

Fungal alpha-n-acetylgalactosaminidase from Aspergillus niger: cloning and expression in Escherichia coli This enzyme from the fungus Aspergillus niger has certain unique properties, and it was thus interesting to clone it for further structural investigations. Methods: a-N-acetylgalactosaminidase from Aspergillus niger CCIM K2 was partially sequenced by Edman degradation and MALDI MS. A gene fragment encoding a putative part of the a-N-acetylgalactosaminidase was amplified using cDNA prepared from Aspergillus niger K2 CCIM. Degenerated PCR primers were designed according to amino acids found in a-Nacetylgalactosaminidase from Aspergillus niger K2 CCIM The full-length coding sequence of a-N-acetylgalactosaminidase was cloned into pET 30a + , and the recombinant protein was expressed in Escherichia coli following induction by IPTG. Results: The cloned DNA consists of 1450 base pairs, and the deduced amino acid sequence (480 amino acid residues with molecular mass 54,814 kDa) is almost identical to that of the purified enzyme (as determinated by SDS-PAGE). Comparison of this amino acid sequence with the GenBank database revealed significant homology (96% identity) with sequence of a-galactosidase (EC 3. Introduction: Dengue fever is the most significant re-emerging disease in urban areas mediated through a mosquito vector, Aedes aegypti. The complex circulation of the dengue virus which mostly due to its existence as four serotypes of dengue-1 to 4, renders difficulty in producing a vaccine. Hence, many research groups are progressively searching for antiviral agent. Methods: The techniques involved were screening assay using fluorogenic peptides which targeted the NS2B/NS3 protease, in vitro antiviral activity employing virus inhibition assay and reverse-transcription polymerase chain reaction. Results: Through fluorogenic peptides complex assay, a competitive inhibitor, 4-hydroxypanduratin which is a cyclohexenyl chalcone derivative of the plant was identified. In vitro antiviral activity of crude extract and 4-hydroxypanduratin A assessed in HepG2 cells showed high inhibitory activity in a dose-dependent manner. At maximum non-toxic dose (MNTD) of 5 and 18 lg/ml respectively they exhibited complete inhibition of 100% in cells infected with DEN2 at TCID 50 10-1000 as indicated by the absence of cytophatic effects. Similar assay carried out in C6/36 cells showed low relative inhibition. At MNTD of 5 and 8 lg/ml respectively they inhibited DEN2 activity at a lower virus titer (TCID 50 1-10). Reverse-transcription polymerase chain reaction carried out for each sample harvested from in vitro inhibition assay showed decreasing intensity of the amplified DEN2 RNA transcript which correlates with increasing concentration of plant extracts and 4-hyroxypanduratin A given to infected cells.

The results strongly suggest that DEN2 viral activity is being affected by the bioactive compound at the viral replication phase.

Simultaneous determination of nucleotide sugars involved in protein glycosylation with high resolution ion-pair reversed-phase HPLC Nucleotide sugars are donor substrates of glycosyltransferases, and their availability and localization regulate glycosylation levels. Previous reports showed the levels of certain nucleotide sugars in tissue samples by using ion-pair reversed-phase or anion-exchange HPLC columns. However, the separation of nucleotide sugars and nucleotides was incomplete. Here we report a high resolution method using ion-pair reversed-phase HPLC to determine nucleotide sugars involved in glycosylation. A series of nucleotide sugars (CMP-NeuAc, UDP-Gal, UDP-Glc, UDP-GalNAc, UDP-GlcNAc, GDP-Man, GDP-Fuc, and UDP-GlcA) and nucleotides (CMP, CDP, CTP, GMP, GDP, GTP, UMP, UDP, UTP, AMP, ADP, and ATP) were perfectly separated on a C18 column and were detected by UV detector equipped with a semi-micro cell. The developed method gave a chromatogram with high sensitivity and reproducibility for cellular samples, which contained structurally similar nucleotide sugars and nucleotides. The limit of quantitation was 2 pmol per each injection, and coefficient variations for intra-and inter-day assay were smaller than 5.3 and 19%, respectively. Picomolar levels of nucleotide sugars could be determined in a single chromatographic run. We were able to determine levels of nucleotide sugars in Chinese hamster ovary (CHO) cells and their glycosylation-defective Lec mutants with this method. Interestingly, levels of particular nucleotide sugar in intracellular fractions of mutant cells were different from those of control CHO cells. Those levels were also changed both in the exponential and stationary phases of cellular conditions. Thus, this method provides useful tools for determination of nucleotide sugar levels in cytosol, Golgi apparatus and endoplasmic reticulum.Introduction: Geranium robertianum phytopreparations are currently in use, but little information exists regarding their effects at the cellular and molecular level. This study reports the effects of Geranium robertianum (hydro)alcoholic extracts on cultured cell proliferation. Methods: The extracts were obtained by mixing the herbal biomaterial for 8 hours at RT, 55°C, or by refluxing, with absolute or 70% ethanol, respectively. The extracts were analyzed for total polyphenols, by Folin-Ciocalteu colorimetric test and for antioxidant capacity, by DPPH assay determining radical scavenging activity. Various cell lines (HS27, HeLa and DOK) were tested for proliferation as a response on treatment with different extracts' concentrations, using MTS assay. Controls were done with medium supplemented with ethanol only. Results: The results showed that 70% ethanol extracts better polyphenols than absolute ethanol. RT extraction resulted in significantly lower polyphenol content than higher temperature extractions. The antioxidant activity does not significantly change as a result of extracting condition for 55°C, or by refluxing, but proved to be significantly lower for RT extracts. No significant difference for antioxidant activity was noted between 70% and absolute ethanol extracts. The extracts have increased cell proliferation, in a concentration dependent manner, for both HS27 and HeLa cells. Similar effects were observed for 70% or absolute ethanol extracts. The extracts showed no effects on DOK cell proliferation. BRCA1 and BRCA2 are major cancer predisposition genes, responsible for a large percentage of hereditary breast and ovarian cancer (HBOC) families. Screening for mutations in these genes is now standard practice for HBOC cases in Europe, and permits medical follow-up and genetic counselling adapted to the needs of individuals in such families. As very little information is available in Romania, we started the first characterization of hereditary breast and ovarian cancer risk in north-eastern Romania. Our study consists in HBOC families identification and recruitment, DNA collection achievement from these patients, implementation of molecular technology for mutations analysis and oncogenetic followup of mutations bearers. We managed to identify and recruit so far several HBOC families with unique features such as 5 ovarian plus 1 breast cancer, or 8 breast cancer cases within the same family line. All recruited families are now under systematic DNA sequencing for BRCA screening. We also investigated, within HBOC families and sporadic cases, the status of three known founder mutations (185delAG and 5382insC on BRCA1 and 6174delT on BRCA2), by optimization and comparison of several multiplex-PCR techniques. This part of our study showed a very important random factor within all multiplex-PCR methods. We demonstrate that although they are cheap, rapid and easy techniques, they often generate false results like primer dimmers, undesirable amplification products apparition or heteroduplexes. Therefore we always recommend combining multiplex-PCR with other pre-screening methods (SNP, heteroduplex analysis) for a good selection before DNA sequencing.Past efforts to differentiate the Brucella spp. have been hampered owing to the high genetic homogeneity among Brucella species. The availability of discriminatory molecular tools to inform and assist conventional epidemiological approaches is invaluable in controlling these infections. The hypervariable octameric oligonucleotide finger-printing method was implemented using microsatellite DNA to genotype Br. melitensis. An eight-base pair tandem repeat sequence was revealed in eight genomic loci of the Br. melitensis genome that existed in various, multiple and random repetitions. Specific primers that were previously described were used to amplify these particular loci. The composition of the omp2 locus from two gene copies with differences in their PstI restriction endonuclease sites was studied in 45 field strains of Br. melitensis and was compared with the vaccine strain Rev-1. Out of the nine loci studied for all samples, four exhibit differences and can be used for differentiating and biotyping of Br. melitensis pathogenic stains from the vaccine strain. In addition the digestion products of the omp2 amplicons by PstI enzyme yielded different fragments between vaccine and field strains of Br. melitensis. Hence contagious field strains can be differentiated from the vaccine strain both with HOOF-prints method and RFLP of omp2 gene.Background: B-cell epitope prediction facilitates the design and synthesis of short peptides for various immunological applications. Several algorithms have been developed to predict B-cell linear epitopes (LEs) from primary sequences of antigens, providing important information for immunobiological experiments and antibody design. This paper describes two improved methods, linear epitope prediction with/without local peak extraction (LEP-LP and LEP-NLP), based on antigenicity scale and mathematical morphology for the prediction of B-cell LEs. Results: Previous studies revealed that LEs occur in regions with lowto-moderate antigenicity but relatively high local antigenicity scales. Hence, we developed a method adopting mathematical morphology to extract local peaks from a linear combination of the propensity scales of physico-chemical characteristics at each antigen residue. Our methodology possessed sensitivity more than 60% of the confirmed epitopes in the AntiJen or Pellequer datasets. Comparison among LEP-LP/ LEP-NLP, BepiPred and BEPITOPE revealed that our algorithms performed better in retrieving epitopes with low-to-moderate antigenicity and achieved comparable performance according to receiver operation characteristics (ROC) curve analysis. of the identified LEs, over 30% were unable to be predicted by existing prediction methods employing an average threshold of antigenicity index. Conclusions: Our LEP-LP methods provide bioinformatic approaches for predicting B-cell LEs with low-to-moderate antigenicity. The webbased server was established at http://biotools.cs.ntou.edu.tw/lepd_antigenicity.php for free use.

Effect of different physico-mechanical factors upon the growth and expression of virulence in Escherichia coli strains isolated from drinking and sea water Aim: The purpose of the present study was to investigate the growth ability and expression of different virulence factors in aquatic Escherichia coli strains submitted to temperature and mechanical stress. Material and Methods: 100 strains of E. coli strains isolated in Romania from drinking and sea water were investigated for cell-associated (i.e. adhesion to the inert substrate and biofilm development) and soluble (i.e. sheep and rabbit erythrocytes haemolysins, CAMP-like factor, lecithinase, lipase, gelatinase, caseinase, amylase, mucinase, aesculin hydrolysis, DN-ase, indole-production) virulence factors. The experiments were performed before and after strains exposure to freezing temperature for 1 month and respectively, after microbial culture centrifugation at 10 000 rpm for 10 minutes. Results: As concerning the virulence hallmarks harbored by the tested strains, all tested strains produced haemolysins (spot and CAMP-like factors) and lipase and exhibited also high positively levels for the production of DN-ase, esculinase and gelatinase. These results pleading for the existence of a pathogenic potential in E. coli aquatic strains, which could colonize the cellular tissues and thereafter cause invasive infections, by the secretion of haemolysins and lipases (acting as poreforming toxins) and of tissue degrading enzymes, such as gelatinases. Our results point out the importance of detecting specific virulence factors before incriminating water as a source of human diseases. After the temperature and mechanical shock, 32% for the sea strains and only 2% of the drinking water strains were recovered demonstrating the role of the natural environment in the development of microbial resistance to stress factors. The virulence factors expression was drastically decreased after stress exposure. However, the maintenance of the growth ability and of some virulence properties in microbial strains exposed to different stress conditions are reflecting the survival and adaptation ability of these strains to variable environmental conditions and the role of aquatic medium in the constitution of the natural reservoir of virulence genes. An important mechanism which regulates mRNA translation is through the phosphorylation of eIF2a at serine 51. This event is mediated by a distinct family of kinases consisting of PERK, PKR, HRI, and GCN2 which are activated by endoplasmic reticulum (ER) stress, double stranded RNA, heme deficiency, and nutrient deprivation respectively. The tumor suppressor p53 is an important sensor of stress and a key regulator of proliferation, differentiation and apoptosis. Unlike the majority of stresses, which stabilize p53, our lab has demonstrated that stress in the ER leads to p53 degradation. More specifically, ER stress leads to the activation of the glycogen synthase kinase-3b (GSK-3b), which promotes the Mdm2 dependent nucleo-cytoplasmic export and degradation of p53. Interestingly, hypoxic tumor microenvironments elicit cytoprotective ER stress responses further validating a physiological significance for the GSK-3b/Mdm2/p53 pathway. Methods: Mouse and cancer cell lines bearing wildtype p53 treated with activators of eIF2a kinases including thapsigargin, tunicamycin, double stranded RNA, and hypoxia. Protein levels were monitored by immunoblot analysis and immunostaining. Activation of GSK-3b was further assessed using in vitro kinase assays. Results: We demonstrated that the degradation of p53 (through the induction of GSK-3b activity) is mediated by the activation of the eIF2a kinases. Moreover, we show that exposure to severe hypoxia results in p53 destabilization concomitantly with eIF2a kinase activation. Conclusions: Our findings reveal a novel cross-talk between the eIF2a kinases and p53 with important implications in hypoxic adaptation.

Inspired by the rich diversity of non-covalent interactions occurring in nature, researchers have pursued artificial receptors preparation by molecular imprinting technology. Recognition sites in molecularly imprinted polymers (MIPs), in case of non-covalent approach, are created by the polymerization of preorganized functional monomers around the template with weak interactions, leading to broad distribution of specific binding sites. The metal-coordination interactions are quite directional, therefore very promising to MIPs design. The well defined coordination sphere of many transition metals complexes allows control of the orientation of functionality in MIPs, offering a high degree of versatility in targeting different functional groups in many biologically molecules. The production of MIPs for the recognition of C-terminal cholecystokinin pentapeptide (CCK-5, NH 2 -Gly-Trp-Met-Asp-Phe-CONH 2 ) in the presence of metal ion is reported. The MIPs were produced under the same molar ratio of template to monomers (acrylamide, N, N¢-methylene bisacrylamide and 4-vinylpyridine) in the presence or absence of nitrilotriacetic acid-nickel (Ni-NTA) complex and their rebinding characteristics, net rebinding, imprinting factor and Lagmuir absorption constants, are reported. Introduction: Experimental transplantation in models of CNS injury has shown that Schwann cells (SCs), the myelinating glial cells of the PNS, are promissing candidates for cell therapy of CNS lesions. Overcoming SC limited integration within the CNS is critical for efficacious therapeutic intervention. As SC migration and myelination are mediated by extracellular matrix-cell surface interactions, our work has focused on genetic manipulation of SCs to alter their adhesive properties, by either expressing the polysialylated (PSA) form of NCAM on their surface or by over-expressing the cell adhesion molecule L1 and its secreted form L1-Fc. Methods: SCs from GFP-mice were transduced with retroviral vectors encoding either sialyl-transferase X (STX), the enzyme that transfers PSA on NCAM, or L1 in its transmembrane and in its soluble form L1-Fc, and they were transplanted in a mouse model of spinal cord injury. The animals were examined behaviorally and immunohistochemically for assessment of their recovery. Results: Transplantation of either STX-GFP-SCs or L1/L1-Fc-GFP-SCs in the injured mouse spinal cord promoted faster and greater locomotor recovery as compared to using control SCs or no cells. Morphological analysis revealed earlier and enhanced remyelination by grafted STX-GFP-SCs and L1/L1-Fc-GFP-SCs and also by endogenous SCs recruited from the periphery to the lesion. Moreover, enhanced sprouting of regenerating serotonergic nerve fibers was observed into and across the lesion site, in both cases. Additionally, STX-GFP-SC transplantation induced differentiation/remyelination by resident oligodendrocyte progenitors.

We provide proof-of-concept that grafts of SCs with altered adhesive properties may be important for therapeutic interventions. Recently we have published the purification and characterization of a PHA synthase from Thermus thermophilus HB8 [1] . Although the entire genome of T. thermophilus has been published [2] a large number of ORFs still remains as hypothetical proteins. In this study PCR-based methods were employed for the identification of pha biosynthesis genes in T. thermophilus genome. For pha loci amplification the LA Taq polymerase was used and genomic DNA isolated from the bacterial cells. The primers were designed based on the highly conserved nucleic acid sequences in regions of the class II pha loci of various Pseudomonas. PCR products were sucloned into PCRII and later into pUC19. Escherichia coli JM109 were transformed with the constructed plasmids and screened for PHA synthase activity. Three putative regions were identified and studied further. The amplification pattern indicates that T. thermophilus HB8 belongs to class II pha loci. Elucidation of the mechanism of PHA biosynthesis in a thermophilic microorganism may lead to polymers with improved properties of high biotechnological value. Blood coagulation factor IX is a plasma proenzyme which is activated during the coagulation cascade into an active serine protease. Among the detected polymorphisms of fIX gene, polymorphisms A21975G and A32881C cause the T194A and T461G mutations on the protein with a prevalence of 23% and 2% in the normal individuals, respectively. However, in our previous studies the A21975G polymorphism was detected in a higher percentage among a group of thrombophilic patients showing correction of their coagulation tests by in vitro adding of recombinant fIX. Hence, these two mutations were introduced into the truncated factor IX (rf9), and their effect on the expressed proteolytic activity was determined. The truncated fIX was overexpressed by pET22/rf9 lacking the first N-terminal domain which is essential for the protein expression in prokaryotic cells. In the fIX gene mutations at positions A21975G and A32881C were introduced. The native and the two mutated forms of rf9 were retrieved and purified from cytoplasm using a Q-Sepharose ff column. The purified proteins were activated by 'Russell's viper venom FX activating protein' (RVV-X). Both mutations significantly increased the proteolytic activity of the protein as shown by the K m and V max values, indicating that these polymorphisms might be responsible for the thrombophilic incidence in the above mentioned patients. Acknowledgements: Many thanks to Prof. Dr Brandstetter (University of Salzburg, Austria) for offering the pET22b vector carrying the truncated rf9 gene. Introduction: Mass Spectrometry (MS) has played an important role in study of phosphorylation which is occurred in cell signaling pathway and enrichment strategies such as Immobilized Metal affinity Chromatography(IMAC), Titanium dioxide(TiO 2 ) and Sequential elution from IMAC (SIMAC) are also important as much as MS. We compared three methods by evaluating the identification efficiency of the phosphorylation sites of a-casein with optimized methods in our group. Methods: After trypsin digestion on a-casein, tryptic peptides were enriched by each strategy and injected in our outstanding MS(nano-UPLC Q-TOF premier). Data analysis were performed by MS/MS expression system(MS e ) and the data were searched with MASCOT. Alkaline phosphatase was treated to confirm the new phosphorylated sites.

Results: It showed that IMAC prefers to multi-phosphorylated peptieds and TiO 2 prefers to mono-phosphorylated peptides as formerly reported. SIMAC enriched more peptides than the two strategies, because it uses three fractions, flow through, acid and base elution. Therefore, for whole phosphoproteome study, SIMAC could be an extraordinary tool. While we carry out our experiment, we identified new phosphorylated sites on Thr and Tyr residues of a-casein. New phosphorylated sites from a-casein were validated by alkaline phosphatase treatment which dephosphorylates the phosphate group of the protein.Conclusions: 1. SIMAC strategy was superior to other phosphoproteome enrichment methods. 2. We identified new phosphorylated sites on Thr and Tyr from a-casein. 3. The new sites were confirmed by using alkaline phosphatase. 4. This optimized method could be applied to cell system to elucidate novel signaling mechanism.

The mechanism of a local anaesthetic studied by Langmuir monolayer techniques and molecular modelling I. B. Pedersen and A. A. Skjevik Department of Biomedicine, University of Bergen, Bergen, NORWAY Introduction: It is well established and well documented that the nerve blocking effects of local anesthetics are secondary to their interaction with the Na+ channels thereby blocking nerve membrane excitability and the generation of action potentials. However, accumulating data suggest that local anesthetics also possess antimicrobial effects that may be due to their interaction with membranes. Articaine is a local anesthetic distributed under the name Septocaine ª and Septocaine forte ª . We have shown that this drug intercalates with biological membranes by Langmuir experiments and molecular dynamic simulations. Septocaine is currently administered as a racemic mixture and we have investigated the mechanism of action with respect to a potential stereo specific binding. Methods: Langmuir was utilized to obtain an indication of the membrane intercalations properties of Articaine. Molecular dynamics simulations were applied to study the molecular flexibility of Articaine and to select stable confirmations to be docked to the membrane. Docking of Articaine to biological membranes and docking to the pore domain of a human sodium channel was performed. Results: The results obtained from Langmuir confirm that Articaine intercalates with biological membranes. Molecular dynamics simulations give us insight into the molecular basis of this intercalation. Conclusions: The results give reason to believe that Articaine may exhibit its antimicrobial effect by intercalation into prokaryotic biological membranes. Introduction: Bacterial ice nucleation is a natural phenomenon responsible for frost damage of many crops, due to a multimeric protein complex formed on the outer membrane of certain Gram-negative ice nucleating bacteria of the genus Pseudomonas, Erwinia and Xanthomonas. The monomer of this complex is encoded by a single gene and undergoes post translational modifications before ancoring to the outer membrane. Methods: The ice nucleation gene of Pseudomonas syringae has been transfered and expressed heterologously in two Zymomonas mobilis strains by assisted conjugal transfer. Cell-free ice nuclei were quantified following separation by centrifugation and filtering through cellulose acetate membranes. Ice nucleation activity was quantified by the droplet-freezing test. Results: All recombinant strains proved to be efficient ice nucleators. Two of these were found to release ice nuclei in the enviroment. Release was not due to cell lysis, as cytosolic enzymes like pyruvate decarboxylase were not detected in the culture medium. Optimum release of cell-free ice nulei was obtained after growth on 2% glucose medium at 16°C. Freezing temperature threshold was below -7°C indicating class C nuclei. Conclusions: Cell-free ice nuclei from Z. mobilis could be a result of outer membrane blebbing and may prove useful for the production of cell-free active ice nucleation protein. Introduction: All quality parameters of wine depend in essence on microbial activities during fermentation and maturation. Thus, modern wine industries have focused worldwide on the development of starter cultures derived from wild yeast present in spontaneously fermentimg must. Methods: Isolation and molecular characterization of indigenous wine yeast strains. Sequential use of strains as starter cultures. GC-MS analysis for volatile compounds determination of the produced wines. Results: With regard to the total aromatic profile of the produced wine the interactions of non-Saccharomyces and Saccharomyces starters resulted to elevated concentrations of six measured higher alcohol esters (isoamyl acetate, ethyl caproate, ethyl caprylate, ethyl caprate, hexyl acetate and phenyl ethyl acetate) which are associated with pleasant fruity aromas. Additionally, it contained a more than 40-fold higher concentration of 2-phenyl ethanol, an alcohol contributing a delicate fragrance of rose petals to a wine's bouquet. Interestingly, three higher alcohols that produce an unpleasant fusel-oil taste and 1-hexanol that produces a grassy odour were found to be minimal. Conclusions: Interactions of indigenous non-Saccharomyces and Saccharomyces strains in a sequential manner as starter cultures, lead to the production of wine with a local character and richer flavour. 

Interaction of glutathione and ascorbate with hydrophobic antioxidants in protection against peroxidation of phosphatidylcholine liposomes induced by azo initiator AAPH L. Piotrowski and G. Bartosz Department of Molecular Biophysics, University of Lodz, Lodz, POLAND Biological membrane is an important cellular shield against exogenous oxidative stress thus action of radical chain-breaking antioxidants is crucial for cell protection. a-Tocopherol (a-TH) is a major lipid soluble antioxidant that protects biological membrane from peroxidation. It is known that water-soluble ascorbate can interact synergistically with a-TH prolonging its antioxidant capacity. Reduced glutathione (GSH) has also been shown to protect lipid layers in various ways including GSH-dependent recycling of a-TH but this mechanism is still unclear. Depending on antioxidants used, the effects of azo compound 2,2¢-azobis(2-amidinopropane) dihydrochloride (AAPH) on phosphatidylcholine (PC) liposomes peroxidation were tested. Egg yolk phosphatidylcholine liposomes were prepared using Mini extruder set (0.1 lm polycarbonate membranes). Hydrophilic antioxidants (GSH, ascorbate, melatonin) and hydrophobic antioxidants (butylated hydroxytoluene, a-TH, quercetin, bilirubin, lipoic acid, coenzyme Q 10 , menadione) were used. Hydrophilic antioxidants were encapsulated inside the liposomes containing selected hydrophobic antioxidants and optional interactions were checked. C-11 -BODIPY 581/591 fluorescence probe was used for detection of lipid peroxidation. The area under curve was calculated as a measure of antioxidants effect. Both ascorbate and GSH protected liposomes against AAPH but no significant antioxidants interactions were found. Quercetin exhibited antioxidative activity but no synergistic effect with ascorbate or GSH was observed. Other compounds had no significant protective effect at the concentrations used. Introduction: The value of polymorphisms in the Y chromosome has been largely recognized and used in studies of human evolution at the species level as well as in regional and population studies. A series of polymorphic Y-chromosomal short tandem repeats (Y-STRs) show high levels of heterogeneity within and between populations and thus provide a useful tool for local population structure and recent demographic history studies. The aim of the present study was to characterize 12 Y-STRs haplotype variation in the Latvian population. Methods: The population sample included 158 paternally unrelated healthy Latvians. One trinucleotide STR (DYS392) and 11 tetranucleotide STRs (DYS19, DYS385a, DYS385b, DYS389I, DYS389II, DYS390, DYS391, DYS393, DYS437, DYS438, and DYS439) were determined using PowerPlex Ò Y System (Promega, USA). Haplotype and gene diversity was calculated using the Arlequin 2.0 package. Results: A total of 115 haplotypes were identified, among which 95 were unique, and a haplotype 15-11-14-13-29-23-11-13-14-14-10-10 (defined by DYS19-385a-385b-389I-389II-390-391-392-393-437-438-439) of which occurred more frequently (8%). The haplotype diversity for 12 Y-STRs loci was observed 0.9923 demonstrating the usefulness and informative power of this Y-STR set. We have found four predominant alleles at loci DYS393 and DYS437 that make up 95.5% of variation within sampled population. The gene diversity values for the Y-STRs loci ranged from 0.4146 (DYS438) to 0.9631 (DYS393). Conclusion: The analysis of associated Y-STRs revealed differences between microsatellite diversity within the Latvian population. The results from the present study have showed genetic similarity to neighbouring populations from Eastern European region. Acknowledgement: This work has been supported by European Social Fund. Introduction: Our studies focused on hepcidin and the human RNase L Inhibitor (Rli). The first is a hormone, regulating iron homeostasis, the latter, a cytosolic iron-sulphur protein plays important role in antiviral protection. In order to identify in vivo protein-protein interactions, we screened a human liver cDNA library with our proteins using BacterioMatch two hybrid assay system. Methods: We used the BacterioMatch two-hybrid system to detect the protein-protein interactions. This system uses two plasmids, one of them contains the sequence of our protein of interest, while a cDNA library is expressed in the other plasmid. The protein-protein interaction is detected by the growth of carbenicillin-resistant colonies. After validation of the positive interactions, the participants were identified by sequencing. Results: The proteins which showed interactions with prohepcidin are the transthyretin, the a-1 acid protein, and the a-1 antitrypsin. We found that Rli binds to IFN induced transmembrane protein 3, ubiquilin 1 and to humanin 1. However, albumin gave interactions with Rli which is possibly a nonspecific binding. Conclusions: We found that the BacterioMatch system is useful for screening libraries for protein-protein interactions. One of our most important findings using this bacterial two-hybrid assay was, that a-1 antitrypsin, which is a serin protease inhibitor, binds prohepcidin. This binding may play an important role in the posttranslational regulation of prohepcidin by preventing its maturation. The proteins which interact with human Rli are important as well, because they may be involved in the action of interferon, or in accumulation of proteins in neurodegenerative disorders. Introduction: Parasitic diseases such as leishmaniasis have significant impacts on the world, specially in developing countries with infections spread over several hundred millions of people and it is one of the significant cause of morbidity and mortality. Conventional chemotherapies are often inadequate, toxic or are becoming less effective due to emergence of resistances as observed for antimonials; sodium stibogluconate (Pentostam) and meglumine antimoniate (Glucantime). In this study the leishmanicidal activities of a series of new synthetic 2,5-disubstituted-1,3,4-thiadiazole compounds like (5-chlorothiophen-2-yl) (4-(5-(1-methyl-5-nitro-1H-imidazol-2-yl)-1,3,4-thiadiazol-2-yl) piperazin-1-yl)methanone were investigated. Methods: In vitro activity of these compounds against Leishmania major and cytotoxic effects on the host cells were determined by MTT assay. Results: All of the agents presented more potent antileishmanial activity than did the standard pentavalent antimony, Glucantime. Also these compounds have not remarkable toxicity for murine macrophages rather than Glucantime. Conclusion: In general, nitrofuran and nitroimidazole derivatives of thiadiazoles or analogues with phenyl in piperazin ring presented potent activity against L. major. The most active compound was found to be 15a analogue, (5-chlorothiophen-2-yl)(4-(5-(1-methyl-5-nitro-1Himidazol-2-yl)-1,3,4-thiadiazol-2-yl) piperazin-1-yl)methanone with IC50 = 9.35 lM. Replacement of H with Cl or Br in thiophen moiety of nitrofuran and nitroimidazole derivatives, increase the leishmanicidal activity. The nitrothiophen components generally displayed less activity against both form of the parasite.

Investigation of some new synthetic thiadiazole components against Leishmania major F. Poorrajab Institute of Biochemistry and Biophysics, Department of Biochemistry University of Tehran, Tehran, IRAN Parasitic diseases such as leishmaniasis have significant impacts on the world, specially in developing countries with infections spread over several hundred millions of people and is one of the significant cause of morbidity and mortality. Conventional chemotherapies are often inadequate, toxic or are becoming less effective due to emergence of resistances as observed for antimonials; sodium stibogluconate (Pentostam) and meglumine antimoniate (Glucantime). In this study the effect of a series of new synthetic 2,5-disubstituted-1,3,4-thiadiazole compounds like (5-chlorothiophen-2-yl)(4-(5-(1-methyl-5-nitro-1H-imidazol-2-yl)-1,3,4-thiadiazol-2-yl) piperazin-1yl)methanone were investigated. In vitro activity of these compounds against Leishmania major and cytotoxic effects on the host cells were also investigated. These agents presented more potent an in vitro antileishmanial activity than did the standard pentavalent antimony, Glucantime. Also these compounds presented no toxicity to murine host cells rather than the reference drug, Glucantime. In general, nitrofuran and nitroimidazole derivatives of thiadiazoles or analogues with phenyl in piperazin ring presented potent activity against L. major. The most active compound was found to be 15a analogue, (5-chlorothiophen-2yl)(4-(5-(1-methyl-5-nitro-1H-imidazol-2-yl)-1,3,4-thiadiazol-2-yl) piperazin-1-yl)methanone with IC50=9.35lM. Replacement of H with Cl or Br in thiophen moiety of nitrofuran and nitroimidazole derivatives, increase the leishmanicidal activity. The nitrothiophen components generally displayed less activity against both form of the parasite. Introduction: Efficiency of conventional radiotherapy of melanoma is limited and shows only individual results. In attempt to realize more efficient killing of melanoma cells, in this in vitro study HTB140 cells were exposed to 12 C ion beam. Methods: Exponentially growing HTB140 melanoma cells were irradiated with 62 MeV/u 12 C ions produced by the superconducting cyclotrons at INFN-LNS, Italy. By interposing Perspex plates of different thickness in front of the cell monolayer three tissue equivalent irradiation positions along the Bragg curve were obtained. These positions belonged to: plateau region (A), maximum of the peak (B) and distal fall-off part of the peak (C), thus providing dose levels of: 22.04 ± 1.35%, 92.11 ± 4.76% and 21.42 ± 6.43%, respectively. Cell proliferation was evaluated using the 5-bromo-2-deoxyuridine (BrdU) assay. Cell cycle phase analysis and evaluation of apoptosis were performed using propidium iodide staining. Assays were performed 48 hours after irradiation. The study was performed on tissue and blood samples from patients undergoing colonoscopy for routine clinical indications, which were divided in three groups: health patients (GI, n = 14), polyp bearing patients (GII, n = 44) and cancer patients (GIII, n = 20). None of the patients had preoperative chemotherapy or radiotherapy. Results: None of the patients showed methylation of the promoter in blood samples for the genes hMLH1 and p16 while the methylation pattern for MGMT was the same in blood and tissue samples. Three patients showed the MSI-H phenotype (1/GII, 2/GIII) that was also accompanied by promoter methylation of the three genes (MGMT, p16 and hMLH1) as well as by changes on the number of the repeats of the target genes examined. Ten patients showed the MSI-L phenotype (4/GII, 6/GIII) but in this case only the GIII patients showed simultaneous promoter methylation of the three genes and only two GII patients an alteration of TGFbRII coding repeats. Conclusions: Our results suggests that there is a link between methylation and MSI phenotype which is caused by defective DNA mismatch repair (MMR) system which drive on the adenoma to carcinoma transformation through alterations on repeats number of the 'target genes'.Marine pollution produces multiple consequences at the organism, population and ecosystem level, detected as biochemical, cellular and physiological alterations that are defined as biomarkers. Protein synthesis depression is a life-saving mechanism in response to various cellular stresses. Therefore, an experimental approach to reveal responses to pollution stress could be to measure the ribosomal subpopulation existing as polysomes which serve as an indicator of the translation efficiency status. To test this approach, specimens of Mytilus galloprovincialis were immersed in a clean coastal region, and two marine stations differently polluted by heavy metals. After 1 month exposure, digestive gland cells were isolated from the caged mussels and their content in polysomes and efficiency of ribosomes to initiate protein synthesis were estimated. In parallel, a suite of standard biomarkers, including metallothionein content, lysosomal membrane stability, superoxide radical concentration and lipid peroxidation were assessed in the same samples. Metals in the surrounding waters and mussel tissues were also measured. Alterations in protein synthesis were found to be strongly correlated with the other biomarkers and the results of metal analysis. Our data support the notion that downregulation of the global protein synthesis is an important component of the cellular response to pollution stress and can be exploited as a biomarker of marine pollution.

Inorganic polyphosphate -induced proinflammatory cytokine profiles on human macrophage -derived THP-1 cells Inorganic polyphosphate (polyP) is chain of tens or many hundreds of phosphate residues linked by high-energy phosphoanhydride bonds, which is ubiquitous in both prokaryotic and eukaryotic cells. However, the polyP regulated genes necessary and/or sufficient for the biological effects remain undefined. Since polyP is known as highly negative charged polymer, it can be a possible stimulator like any other known molecules such as LPS a well defined endotoxin. To test this hypothesis we compared polyP against LPS on human monocyte derived macrophage cell line THP-1. Our cDNA microarray analysis showed that polyP was able to induce IL-6, IL-8, TNF-a, TGF-b, IL-10 and IL-1b to that of control, which was similar to LPS, treated THP-1 cells. These results were confirmed by expression of appropriate cytokine at protein levels. Based on these results we hypothesizes that polyP posses an LPS like activity on human monocyte macrophage cell line THP-1.

Genetic fusion of CTB with Sm14: biochemical characterization and its use as an antischistosomiasis vaccine H. Ramos 1,2 , P. A. Miyasato 2 , C. R. R. Ramos 3 , A. P. Areas 4 and P. L. Ho 1,2 1 Instituto de Quıḿica, University of Sa˜o Paulo, Sa˜o Paulo, BRAZIL, 2 Instituto Butantan, Sa˜o Paulo, BRAZIL, 3 FIOCRUZ, Rio de Janeiro, BRAZIL, 4 UFABC, Santo Andre´, BRAZIL Introduction: Developing a vaccine against schistosomiasis would be an important advance on the control of this chronic and debilitating disease which afflicts millions of people worldwide. Herein we describe the use of the non-toxic B subunit of cholera toxin (CTB) genetically fused to Sm14 -a fatty-acid binding protein from Schistosoma mansoni -as an attempt to elicit a better immune response for a vaccine antigen candidate. Methods: Recombinant proteins were expressed on a prokaryotic system, purified by different chromatographic methods and both immunochemical and spectroscopically characterized. Intranasal immunization experiments were performed on BALB/c mice and vaccine's efficacy was assessed by analyzing the worm-burden after challenge infection with S. mansoni cercariae. Results: When intranasally administered, Sm14 was not able to reduce the worm burden on vaccinated animals, however either when coadministered with or genetically fused to CTB, Sm14 reduced about 20% the worm burden on vaccinated animals. Furthermore, coadministration of CTB and Sm14 reduces in 33.87% the area of hepatic granulomas around trapped eggs. Conclusion: Despite subcutaneous vaccination of mice with Sm14 was described to induce 40-60% of protection on vaccinated animals, when intranasally administered, this antigen did not elicit the same protection levels. Acknowledgements: Support: FAPESP, CNPq, Fundac¸a˜o Butantan. Introduction: Cellulases are a group of enzymes with the capability of hydrolysing the beta 1,4 glycosidic linkage of cellulose fibers to various products with wide range of industrial applications. Trichoderma reesei because of having complete cellulase complex is an efficient and well studied cellulose-degrading microorganisms. Methods: In this respect we immobilized the cellulolytic broth of T.reseei on microcrystalline cellulose. The enzymatic systems were then eluted with citrate buffer (method I). In other approach the cellulolytic enzymes which adsorbed to microcrystalline cellulose, were desorbed with in glycerol and diethylether and then concentrated using n-buthanol (method II). In addition we concentrated directly broth of T.reseei using lyophylization (method III). Results: Based on sequential ion-exchange chromatography (DEAE-Sepharose CL-6B) and gel filtration on obtained cellulase compositions in three methods, we found out the chromatographams are different regarding the Avicelase and CMCase activities. Method I was more specific for having particular cellulase enzymes. In method II the obtained cellulolytic system was more complete, but the chromatogram in this method is the result of further fractions (because of more aggregation of enzyme proteins in n-buthanol). We have the most complete cellulolytic complex in method III. Conclusions: We can use method I for different industry when the goal is using a particular cellulase enzymes, but more efficient method for obtaining the whole cellulolytic complex with more purity of these enzymes, is method II. Method III has a complete cellulase complex too, however the purity of enzymes is less than the other methods. Introduction: Lateral roots are essential to form a dense root network allowing plants to take up water and minerals effectively from the soil. Laterals are initiated from a specific organogenic cell layer within the differentiated root known as the pericycle. The establishment and maintenance of the pericycle and the subsequent process of lateral root formation is an interesting model to study how stem-cells can be maintained within an otherwise differentiated and endoreduplicated tissue, and how these cells are re-activated into division in response to mitogenic signals. Activation and progression through the major phases of the cell cycle (G1, S, G2 and M) are regulated by cyclin-dependent kinases, composed of catalytic (CDK) and regulatory (cyclin) subunits. Modulation of CDK activity is not primarily regulated by the abundance of the CDK subunit, but by controlled transcription, translation, intracellular localization, or regulated destruction of the cyclin or by stimulatory and inhibitory phosphorylation of the CDK. In addition, activity can also be inhibited by the binding of inhibitor proteins (KRPs). Results and Conclusions: In this work, we present that the interaction of KRP2 with the D-type cyclin CYCD2;1 could be relevant in this process. KRP2 drives D-type cyclin CYCD2;1 and kinase CDKA to the nucleus of these cells, stabilizing the complex and retaining the capability of entering in the mitotic cell cycle and we propose that auxin could control CDKA-CYCD2;1 activity by modulating the levels of KRP2.

Comparative enzymatic parameters of peroxidase models of hemin and MP-11 in reverse SDS micellar medium There is great interest in Fe (III) protoporphyrin (IX) (hemin) and microperoxidase-11(MP-11) as designable artificial heme enzymes for analytical, biotransformation and biotechnological purposes. In order to examine their catalytic activity, peroxidase activity of microperoxidase-11 and hemin in reverse micellar media were investigated. The reverse microemulsion composed of dodecane, aqueous phosphate buffer, SDS and 1-hexanol was prepared and hemin and MP-11 were directly encapsulated into the aqueous cores of reverse microemulsions through the self-assembly formation mechanism. The capability of peroxidase models in catalyzing the oxidation 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) in the presence of hydrogen peroxide was followed spectrophotometrically. Kinetic parameters including initial reaction rates, V max , Michaelis constant (K m ), turnover number (k cat ) and catalytic efficiencies were obtained. The activatory effect of histidine on the catalytic parameters of the enzyme models was also taken into account to improve the peroxidatic activity and functional stabilities of the prepared mini-enzymes. The table indicates the comparative kinetic parameters for the various prepared peroxidase models. The catalytic parameters illustrates that these heme biocatalysts represent acceptable peroxidatic activity. As it can be concluded from the table the most potent peroxidase models are histidine-hemin(in organic phase) and MP-11(in aqueous phase). High values of catalytic efficiencies probably arise from high solubility of the biocatalyst in the corresponding reaction medium. Introduction: In food and beverage industries, yeast cells are exposed to various stress conditions. Thus, obtaining robust yeast strains and understanding the genetic mechanisms of stress resistance as a complex phenotype is important to improve yeast fermentation performance. In this study, to increase acid resistance of the yeast Saccharomyces cerevisiae, we have applied 'evolutionary engineering' approach based on random mutation and selection. Methods: We started with chemically mutagenized yeast cells to increase initial genetic diversity and applied citric acid stress continuously throughout batch cultivations with increasing acid concentrations for 15 successive generations. Starting from 50 mM citric acid concentration as the initial stress level, we obtained a population that resisted up to 240 mM citric acid. Individual mutants were selected randomly from this final population. To determine relative survival level compared to wild type S. cerevisiae, a high throughput procedure in 96-well plates based on the most probable number (MPN) method was applied to the mutants and wild type.Results: Resistance of mutant strains against citric acid, and some other stresses particularly relevant for food industry such as heat, osmotic, ethanol and oxidative stress were investigated using MPN methodology. Resistances of these strains were compared with those of the wild type strain in terms of percent survival. Conclusions: We have successfully employed an evolutionary engineering approach to obtain acid-resistant yeast mutants. Further genetic analysis of the mutants will reveal the genetic basis of acid resistance in yeast, which can be exploited to improve industrial bioprocess efficiency. 'Lipofuscin-loaded-RPE-model' was either maintained in the dark or exposed to 390-550 nm light. The gene copy numbers of lysosomal and antioxidant enzymes were measured by real-time quantitative PCR. Results: Significant upregulation was observed in genes of acid phosphatase (AP-70%), cathepsin D (CAD-35%), catalase (CAT-71%), superoxide dismutase (SOD-63%) and glutathione-peroxidase (GPX-52%) in lipofuscin-fed-RPE compared to unfed-ones just before the start of blue-light exposure. In lipofuscin-ingested RPE exposed to bluelight increase in gene expression of AP, CAT and SOD observed until 3-hours decreased subsequently; however, CAD and GPX genes showed upregulation during similar time. Dark-treated lipofuscin-ingested RPE showed similar expression as light treated ones for AP, CAD, and GPX genes, although the absolute gene copy numbers were much lower. Blue light alone exposed RPE showed gene expressions to various degrees.Conclusions: Lipofuscin has a potential to regulate expression of the lysosomal and antioxidant genes. The upregulation of enzyme expression possibly occur to compensate for the previously observed lipofuscin-coupled loss in enzyme activity. The data consolidates previous observations on liposuction toxicity, highlighting its role at molecular level and implicating lipofuscin as an important component in cellular ageing and in age-related pathogenesis of eye diseases. We attempted to construct a new recombinant protein characterized by fibrin-specific properties of plasminogen activation combined with antithrombin and antiplatelet activities. To the C-terminal part of recombinant staphylokinase (r-SAK), which is a promising profibrinolytic agent, we assembled (i) the Kringle two domain (K2) of tissue type-plasminogen activator (t-PA), containing a fibrin-specific binding site, (ii) the RGD sequence (Arg-Gly-Asp) for the prevention of platelet aggregation and (iii) the antithrombotic agent -hirudin. The cDNA for hybrid protein SAK-RGD-K2-Hir was cloned into pESP-3 yeast protein expression vector. The introduction of K2 t-PA, RGD sequence and hirudin into r-SAK molecule did not alter the staphylokinase activity. The plasminogen activation rate (determined by KM and Kcat) of SAK-RGD-K2-Hir was not significantly different from that of r-SAK. Affinity and binding strength of the recombinant protein to fibrin immobilized on the biosensor were higher than to r-SAK. We observed a higher clot lysis potency of SAK-RGD-K2-Hir as evidenced by a faster and more profound lysis of 125I-labeled human fibrin clots. The potency of thrombin inhibition by the hirudin part of the recombinant fusion protein SAK-RGD-K2-Hir was the same as that of r-Hir alone. In conclusion, presented results from in vitro study suggest that the SAK-RGD-K2-Hir construct can be a more potent and faster acting thrombolytic agent with antithrombin and antiplatelet properties compared to standard r-SAK. In eukaryotes, genomic DNA is packaged into the chromatin structure. Nucleosome, which is the fundamental unit of chromatin, is composed of a histone octamer and a 146 bp DNA. The histone octamer contains two each of the core histones, H2A, H2B, H3, and H4. Five nonallelic histone H3 variants, H3.1, H3.2, H3.3, H3t, and CENP-A, have been identified in mammals. Histones H3.1 and H3.2 are produced at S phase, while histone H3.3 is constitutively expressed through the cell cycle. Consistently, histones H3.1 and H3.2 are incorporated into chromatin in a replication dependent fashion, while, histone H3.3 is incorporated into transcriptionally active chromatin region in a replication independent fashion. CENP-A is the centromere-specific H3 variant, which specifically associated with the centromeric region of chromosomes. Histone H3t is a testis-specific H3 variant, and is robustly expressed in the testis. Recent proteomics and tissue-specific RT-PCR experiments suggested that histone H3t may function in somatic tissues, in addition to testis. In the present study, we purified human histone H3t as a recombinant protein, and tested its nucleosome formation ability in vitro. We found that H3t/H4 was proficient in the nucleosome formation by the salt-dialysis method, but it was not efficiently incorporated into the nucleosome by human Nap1, due to its defective H3t/H4 deposition on DNA. Intriguingly, human Nap2, a paralog of hNap1, efficiently promoted nucleosome assembly with H3t/ H4. Mutational analyses revealed that the Ala111 residue, which is conserved among H3.1, H3.2, and H3.3, but not in H3t, is responsible for the hNap1-mediated nucleosome assembly.PP8-183 Translocation of two candidate T3SS effectors from Bradyrhizobium japonicum USDA 110 into plant cells through the HRP pathway C. Fotiadis and A. Tampakaki Department of Agricultural Biotechnology, Agricultural University of Athens, Athens, GREECE Introduction: The type III secretion systems are widely conserved in plant and animal pathogens but are also present in rhizobia, in some non-pathogenic plant-associated bacteria and in certain endosymbionts. These systems mediate the transfer of diverse bacterial proteins (termed 'effectors') directly into the host cell cytosol. To explore the T3SS effector repertoire of Bradyrhizobium japonicum USDA110, we initially isolated two genes encoding proteins with weak similarity to AvrPphB of the bean pathogen Pseudomonas syringae pv. phaseolicola. Methods: The two annotated genes (BLR2058 and BLR2140) were PCR amplified and cloned in-frame to the coding region of the C-terminal 177 aa of the effector protein AvrRpt2, a well-documented in vivo reporter for type III secretion. AvrRpt2 fusion plasmids were electroporated into P. fluorescens 55 (pLN18) carrying the entire hrp cluster from strain Pss-61. The P. fluorescens strains were infiltrated into Nicotiana spp. The genes under study were also cloned in a Ti vector for Agrobacterium-mediated transient expression in plants.Results: Infiltration of P. fluorescens (pLN18) expressing AvrRpt2 fusions of the two rhizobial proteins in leaves of various Nicotiana spp. elicited a typical hypersensitive reaction (HR). However, only the BLR2140 protein elicited HR when it is expressed through Agrobacterium.

The results suggest that both rhizobial proteins have the appropriate type III secretion signals for their delivery inside plant cells via a heterologous type III secretion system. The lack of HR activity of BLR2058 may be due to lack of accessibility of functionally complimentary receptors in the plants tested.

The protective effect of Mucuna pruriens seed against cobra venom poisoning: immunological studies Seed of Mucuna pruriens (Velvet beans) has been prescribed by traditional medicine practitioners in Africa as antisnake remedy. Recent studies indicated that pre-treatment with Mucuna pruriens seed extract (MPE) in mice did confer protection against the lethal effects of Echis carinatus and cobra venom. In the present studies, survival experiments using rats showed that the MPE pre-treatment did confer effective protection against lethality of Naja naja sputatrix (NNS) venom and some protection against Calloselasma rhodostoma venom. In vitro neutralization experiments using mice showed that antibody raised against MPE effectively neutralized the lethalities of Naja naja sputatrix, Naja kaouthia and Naja nivea venoms Both immunobloting and in vitro neutralization experiments showed that the anti-MPE cross-reacted with the two major toxins of NNS venom: phospholipase A 2 and neurotoxin, but was not reactive against the venom cardiotoxin. The results indicated that anti-MPE could be used in the antiserum therapy of Asiatic cobra (Naja) bites, and that the protective mechanism of MPE pre-treatment against Asiatic cobra venom poisoning involves immunological neutralization of cobra venom phospholipase A 2 and neurotoxin by antibodies elicited by MPE pre-treatment.PP8-189 A model system for gene knockout in chinese hamster ovary (CHO) cells M. Tsukahara, Y. Maseki, A. Shimanuki and I. Kitazumi Kirin Pharma Co., Ltd., Takasaki, JAPAN Introduction: CHO cells are extensively employed for the production of recombinant protein therapeutics. Genetic modification of CHO cells provides a useful tool for an addition of extra values to recombinant therapeutic proteins and/or improvement of cell growth and viability. Targeted disruption for gene of interest has proven useful as a method not only for study of gene function but also for permanent modification of cell character. In our previous studies, six genes including caspase-3 and a-1,6-fucosyltransferase were knocked-out in CHO-K1 cells. However, KO efficiency is low and we need to improve the knockout system. Methods: Hygromycin resistance gene with intron; Hyg-gene was divided into two exons by insertion of 4.6 kb non-sense sequences as artificial intron. KO host cells; Host cells were established by transfection with the following DNA construct, CMV promoter/hygromycinartificial exon1/artificial intron/polyA terminator. KO vectors; KO vectors consisted of various length of artificial intron as 5¢-homologous region/hygromycin-artificial exon2/polyA terminator as 3¢-homologous region. Measurement of KO efficiency; 3 weeks later after transfection of KO vector, hyg-resistant colonies were picked and KO was confirmed by DNA sequencing of PCR products with KO specific primers.Results: Hyg-resistant clones were obtained by transfection of KO vectors. These cells had the functional hyg-gene in host cells. KO efficiency was dependent on the length of homologous arms. Conclusion: We established the assay system for gene targeting using hyg-resistance. This simple system will be available to find the factors affecting the gene targeting efficiency.

Synthesis and evaluation of novel benzothiazole complexes as potential biochemical, cellular and solid tumour radiotracers 2-(4-Amino-3-methylphenyl) benzothiazole (DF 203) has emerged from the NCI screening as an agent with markedly differential activity in distinct cancer cell types, among which MCF-7 and T-47D breast carcinoma and ovarian IGROV1 cells are exquisitely sensitive. Consequently, this agent is an outstanding candidate for the development of a radiotracer for differential diagnosis of ovarian and breast cancer. Furthermore, it may equally serve as a biochemical and cellular tool of high sensitivity for the investigation of the mode of action of DF-203 and other similarly performing anticancer agents. This work describes the synthesis of the DF 203 rhenium and technetium complexes and their preliminary evaluation in MCF-7 and IGROV1 cell lines. The complexes were efficiently synthesised in high chemical and radiochemical yield and purity. The DF-Re complex was evaluated for its cell uptake in both sensitive and insensitive cell lines, as well as in healthy fibroblasts. Uptake detection was based on the fluorescence properties of the complex, which can be clearly visualised under epifluorescence microscope equipped with DAPI filter. Several concentrations were investigated to appreciate the potential of a dose-dependent uptake.The nuclear uptake was also investigated with fluorescence observation. The corresponding technetium complexes is evaluated by means of autoradiography. Cellular localisation in different compartments and their affinity for the arylhydrocarbon receptor, are currently in progress. The results so far are encouraging for the application of these radiotracers for the elucidation DF-203 mode of action and for the development of a promising tumour radiodiagnostic agent.

Effect of rice germ on plasma adiponectin level in C57BL/6j mice Rice is one of the most important crops in the world but unfortunately most of rice germ produced is wasted. The goal of this study is to evaluate physiological function of dietary rice germ extract on type 2 diabetes of mice. C57BL/6j mice were fed with a high dextrin diet and a high dextrin diet containing rice germ extract. High dextrin diet remarkably increased body weight, compared with dextrin diet containing germ. Rice germ containing diet repressed total plasma cholesterol and LDL cholesterol, and increased plasma adiponectin level that has been known to stimulate fatty acid oxidation and enhance insulin sensitivity. Single oral administration of rice germ extract also increased plasma adiponectin level. Gamma aminobutyric acid (GABA), one of main components of rice germ extract, increased adiponectin level quickly as well as rice germ extract. These results suggest that GABA would upregulate adiponectin secretion. The paper will discuss about the GABA function for modulating adiponectin secretion from adipocyte and the promising functional foods against type 2 diabetes.

Prevention of Duchenne muscular dystrophy by inhibition of hematopoietic prostaglandin D synthase Introduction: Duchenne muscular dystrophy is a X-linked muscular abnormality caused by the loss of dystrophin and is one of the most gravely genetic disorders. We have recently found that hematopoietic prostaglandin D synthase (H-PGDS) induced in grouped necrotic muscle fibers in Duchenne's muscular dystrophy (DMD). Cyclooxygenase-2 and phospholipase A 2 , synchronously induced in the H-PGDSexpressing muscle fibers. In this study, we developed a novel therapy for DMD by inhibition of H-PGDS. Results and Methods: H-PGDS was induced in the necrotic muscle fiber in a bupivacaine (BPVC)-induced rat model, and in a dystrophin null mdx mouse model. Expressions of H-PGDS-mRNA and -protein peaked at 12-24 hours after the BPVC injection into the muscle. The necrotic muscle in the mdx mice constitutively expressed H-PGDS. We developed novel H-PGDS inhibitors based on the X-ray crystallographic analysis of human H-PGDS complexed with its prototype inhibitor, HQL-79. We also developed a non-invasive, continuous visualization method of necrotic muscle by using X-ray computed tomography (CT) imaging. Oral administration of a novel H-PGDS inhibitor prevented the wound expansion in both a BPVC-induced muscular necrosis model and a mdx mouse model, as evaluated by CT imaging, demonstrating that PGD 2 produced by H-PGDS plays important pathological roles on the expansion of muscle necrosis. H-PGDS inhibitor also accelerated the accumulation and activation of macrophages in the necrotic area.These results indicate that PGD 2 produced by H-PGDS is involved in the wound expansion in DMD and that inhibition of H-PGDS is a novel therapy for DMD.