key: cord-0006386-xta38e7j
authors: nan
title: Deutsche Gesellschaft für Experimentelle und Klinische Pharmakologie und Toxikologie e.V.
date: 2012-02-22
journal: Naunyn Schmiedebergs Arch Pharmacol
DOI: 10.1007/s00210-012-0736-0
sha: ae9adf2e31d4b2c2e2b1577adbd1c3a68ee4ee35
doc_id: 6386
cord_uid: xta38e7j

nan

Nucleoside diphosphate kinases (NDPKs) are multifunctional enzymes involved in a variety of cellular processes including cancer metastasis and heart diseases. The plasma membrane content of the three major NDPK isoforms NDPK A, B and C is increased in human heart failure. We have previously shown that the NDPK B isoform regulates cAMP levels and cardiac contractility through a receptor-independent Gprotein activation involving direct G protein β subunit phosphorylation. The precise role of NDPK C in the heart is unknown and was the object of this study. NDPK C function was assessed in neonatal (NRCM) and adult (ARCM) rat cardiomyocytes with real-time PCR, immunoblotting, and quantification of cAMP content. Heart failure was induced by chronic treatment with isoproterenol (ISO, 2.4 mg/kg/d 4 days) via minipumps. Chronic ISO increased mRNA levels of NDPK C by 9.4±1.9-fold and its protein levels by 2.1±0.11-fold. Immunoprecipitation of the G protein β subunit resulted in coimmunoprecipitation of NDPK C and the stimulatory Gαs subunit: ISO enhanced this interaction. Upon ISO stimulation, NDPK C translocated from the cytosol to the plasma membrane within 3 hours in both NRCMs and ARCMs. Adenoviral overexpression of NDPK C in NRCMs caused a 1.5-fold increase in basal and ISO induced cAMP synthesis, whereas siRNA mediated knockdown of endogenous NDPK C decreased cAMP levels by ~50%. Our results establish NDPK C as a novel and critical regulator of cAMP synthesis and Gs signaling in the heart. The up-regulation of NDPK C and the increased responsiveness to ISO in failing hearts point to NDPK C as a potential counterregulatory factor in the onset of heart failure.

Uptake and metabolism of methylated myricetin derivatives: Studies in cell culture and C. elegans Ackermann D. 1, 2 , Büchter C. Secondary plant compounds like flavonoids that are ubiquitary present in fruits and vegetables are believed to exert health protective effects in terms of lowering the incidence of widespread diseases such as cardiovascular diseases and cancer. Besides their antioxidative effects, flavonoids may also modulate cell signaling pathways and thereby performing their disease-protective actions. Though this class of dietary polyphenols has become increasingly popular as dietary supplements, only little is known about their metabolic fate in vivo. Therefore, we investigated the absorption and metabolism of several flavonoids such as myricetin and its methylated derivatives laricitrin, syringetin, and myricetin-3',4',5'-trimethylether in the human colon carcinoma cell line Hct116 and the human hepatoma cell line HepG2 as well as the model organism Caenorhabditis elegans. All flavonoids were rapidly taken up by both cell lines as shown by HPLC analyses. The intracellular amount of myricetin and laricitrin did not increase with time and was only half of that of syringetin and myricetin-3',4',5'-trimethylether. Interestingly, no metabolites of these flavonoids could be detected which might at least in part be due to their low intracellular amounts. Absorption as well as intracellular distribution was also evidenced by using the fluorescent dye "Naturstoff reagent A" NSRA) . Fluorescence microscopy indicated a predominant cytosolic distribution of the employed flavonoids. In the model organism Caenorhabditis elegans, the flavonoids were exclusively distributed in the intestine as visualized by NSRA. The antioxidative capacity of the four flavonoids (measured by using the cell free TEAC assay and the H 2DCF-DA assay in Hct116 cells) decreased with increasing methyl groups in the B-ring. Myricetin-3',4',5'-trimethylether (three methyl groups) was the least effective radical scavenging flavonoid in both the cell free system and in Hct116 cells compared to myricetin (no methyl groups).

In conclusion, for exerting their biological effects, uptake and distribution as well as metabolism of flavonoids in certain organs such as liver and gut are important and more research in that field is warranted.

Munich Heart Alliance, München, Germany

Signaling through G protein-coupled receptors is affected by receptor polymorphisms, yet the molecular basis for the functional differences of individual receptor variants is unclear.

To investigate the impact of the frequent Gly389Arg variant of the β1-adrenergic receptor (β1AR) on receptor conformation we used β1AR-sensors capable of fluorescence resonance energy transfer (FRET). These sensors retained the pharmacological and functional characteristics of the native receptors. Upon stimulation of the sensors we determined the activation characteristics of the polymorphic receptors in real time and in living cells. We found the β1AR variants to behave similar upon a single stimulation with an agonist, but to differentially respond with a change of their activation kinetics during subsequent stimulations. While the Arg389-β1AR did not show altered activation kinetics after prestimulation, the Gly389-β1AR became slower compared to the initial stimulation suggesting that β1ARs possess a memory of previous activation. We then permeabilized β1AR-sensor-expressing cells with saponin to remove soluble cytosolic factors. Upon permeabilization the β1AR variants did not display receptor memory, suggesting that the β1AR memory depended on the interaction of the receptors with soluble cytosolic factors upon their initial activation including the phosphorylation of agonist-bound receptors by protein kinase A or G protein-coupled receptor kinases. Our findings suggest an intrinsic, polymorphism-specific property of βARs that alters activation kinetics upon continued stimulation and that might account for individual drug responses.

Micro-RNA replacement therapy: Nanoparticle-mediated in vivo delivery of miRNA-145 or miRNA-33a exerts antitumor effects in colon carcinoma xenograft mouse models Weirauch U. 1 Micro-RNAs (miRNAs) control the expression of various genes, and under pathological conditions several miRNAs are up-or downregulated. Previous in vitro studies have established a pro-apoptotic and anti-proliferative role of miR-145, which shows decreased levels in colon carcinoma. In contrast, while miR-33a is only weakly expressed in several tumors as well, its role in cancer has not been analysed so far. In this study, we demonstrate the tumor-relevance of miR-33a and identify the protooncogenic kinase Pim-1 as a target of miR-33a. Pim-1 harbours a highly conserved miR-33a binding site within its 3'-UTR, and seed mutagenesis of this target sequence abolishes the miR-33a-mediated downregulation of Pim-1. The knockdown of Pim-1 by RNAi or miRNA transfection inhibits proliferation in leukemia and in colon carcinoma cells by decelerating cell cycle progression, thus establishing a tumor inhibitory function of miR-33a. We furthermore introduce polyethylenimines (PEIs) for the therapeutic application of miRNAs in vivo, which is critically dependent on the development of appropriate delivery tools. PEIs are able to form non-covalent complexes with miRNAs, leading to miRNA protection after systemic application in combination with an attractive biodistribution profile and the efficient uptake in target organs/cells. Therapeutic effects of PEI-mediated miRNA delivery were demonstrated in subcutaneous colon carcinoma xenograft mouse models. The in vivo application of miRNA-145 through systemic or local injection of PEI/miRNA complexes resulted in efficient miRNA delivery and in antitumor effects, based on the concomitant repression of ERK5. Likewise, tumor growth inhibition was observed upon treatment of tumor-bearing mice with PEI-complexed miR-33a. This is due to the miR-33a-mediated downregulation of Pim-1 expression and resembles the Pim-1 knockdown through RNAi / Pim-1 siRNAs. Taken together, in tumor xenograft mouse models we establish miRNA replacement therapy through the PEI-complexation of miRNAs as a novel therapeutic strategy and demonstrate that miR-145 and miR-33a may be promising miRNAs in colon carcinoma therapy. MD 288, a hybrid of chloroquine and primaquine, is a potential drug against infectious diseases such as malaria. Since one moiety of the hybrid, the known antimalarial drug chloroquine, is a known intercalator, the potential of MD 288 to intercalate into DNA was determined. Due to the ability of intercalators to cause frame shift mutations, the mutagenic potential of MD 288 was also investigated. The potential of MD 288 to intercalate into DNA was investigated by means of fluorescence based micro plate assay using ethidium bromide (EB) and isolated calf thymus double stranded DNA. As a positive control chloroquine was used. The potential of MD 288 to cause gene mutations was determined using the hypoxanthine-guanine phosphoribosyltransferase (HPRT) test in Chinese hamster V79 lung fibroblasts (V79 cells). V79 cells were treated with 0.4 µM, 0.8 µM and 1.5 µM MD 288 or the positive control, the direct mutagen 4-nitroquinoline-N-oxide (NQO, 1 µM) for 24 h. On day 6, mutants exhibiting loss of hprt function were selected with 6-thioguanine . Whereas at 1 µM chloroquine, a 38% decrease in fluorescence intensity of EB (indicating DNA intercalation) was observed, 10 µM MD 288 were needed to observe a similar decrease (28%) in fluorescence intensity of EB. Therefore, MD 288 is 10fold less potent to intercalate into DNA than its moiety chloroquine. The frequency of spontaneous 6-TG resistant mutants per 10 6 colony-forming cells was 9 ± 2. As expected, 1 µM NQO caused a significant increase in the mutant frequency (MF, 168 ± 9) . In contrast, MF was not significantly affected by treatment with MD 288 at both noncytotoxic (0.4 µM: 5 ± 4) and cytotoxic (0.8 µM: 9 ± 7) concentrations.

In conclusion, MD 288 is a less potent intercalator than the known antimalarial drug chloroquine. Furthermore, MD 288 does not cause gene mutations in the HPRT test.

Since current studies show that various metabolites of MD 288 are formed in vitro, their mutagenic potential is currently under investigation as well. -conducting channels but depends critically on the membrane potential. TRP channels form cation entry channels thereby either contributing to Ca 2+ entry or depolarisation. Recently, we showed that TRPM4 acts as a Ca 2+ -activated non-selective cation channel and critically determines the driving force for Ca 2+ influx in mast cells following FcεRI-stimulation (1) . In addition to TRPM4 we also identified the expression of other TRP transcripts in bone marrow derived mast cells (BMMC) including those encoding TRPC2, TRPC3, TRPC5, TRPC6 and TRPM7. In peritoneal mast cells (PMC), RT-PCR indicated expression of TRPC1, TRPC4, TRPC5, TRPC6, TRPM2, TRPM4 and TRPM5. To identify the functional role of those TRP channel proteins for mast cell activation we analysed Ca 2+ signaling using microfluorimetry in BMMCs and PMCs after stimulation with substances known to activate TRPC6 channels in other cell systems such as the diacylglycerol analogue OAG, the hyperforin analogue HYP-9 and flufenamic acid (FFA), but could not evoke a rise in the [Ca 2+ ]i in both PMC and BMMC. Sphingosine 1phosphate and lysophosphatidylcholine, which were reported to activate TRPC5 channels, induced only minor rise in [Ca 2+ ]i in BMMCs, respectively. Here, we will present our analysis of Ca 2+ signaling following stimulation of the FcεRI receptor and application of secretagogues that are supposed to affect Ca 2+ -dependent mast cell activation such as adenosine, endothelin-1, substance P and compound 48/80 in BMMCs and PMCs derived from mouse lines with inactivation of TRPC1, TRPC3, TRPC4, TRPC5 or TRPC6 since specific antagonists are still lacking for these TRP channels.

The α2A-AR is the main AR in the central nervous system and it plays a crucial role in regulating norepinephrine (NE) release from nerve terminals via presynaptic feedback inhibition. It is also associated with a number of physiological effects, including hypotension, pain perception, sedation and modulation of mood. NE, once released in the synaptic space, binds to α2A-ARs and induces a rearrangement of the receptors from the inactive state into an active conformation. This allows the binding and activation of the cognate Gi-protein and, hence, the transduction of the transmembrane signal to the downstream effectors. Generally, α2A-AR activation has been deduced from the stimulation of a receptor-mediated biological response that could be easily followed experimentally. However, most of these approaches do not employ living cells and are normally applied under equilibrium conditions that need prolonged incubation periods incompatible with the physiological temporal dynamics of NE. Here, we monitored the NE-mediated α2A-AR and Gi-protein activation by using a fluorescence resonance energy transfer (FRET)-based approach in living cells. To examine the effects of increasing concentrations of NE on the speed and extent of α2A-AR activation with very high temporal resolution, we took advantage of the previously described α2A-AR FlAsH/CFP sensor [1] . The results indicate that in our system the efficacy of NE in eliciting α2A-AR FlAsH/CFP activation increases in a time-dependent way and reaches the maximum with a half-life of ~ 70 ms. The EC50 values decrease in an exponential manner and arrive at ~ 2 µM with a half-life of ~ 330 ms. Next, we analyzed the ability of increasing concentrations of NE to trigger a downstream intracellular response after α2A-AR stimulation by monitoring the kinetics and amplitude of Gi activation in living cells. We applied the previously well characterized Gi CFP/YFP sensor [2] . The results show that both the efficacy and the potency of NE in inducing Gi activation reach the steady state slower compared to receptor activation (half-life ~ 700 ms and ~ 3,000 ms respectively). In conclusion, we were able to monitor NE-mediated events occurring in the millisecond time scale and reaching the equilibrium in a time interval compatible with physiological conditions. Sphingosine-1-phosphate (S1P) is an immune modulator produced by sphingosine kinase 1 (SphK1) and sphingosine kinase 2 (SphK2) and de-phosphorylated or degraded irreversibly by S1P phosphatases and a lyase, respectively. We recently showed that TLR4-induced IL-12p70 is selectively counter regulated by SphK1, S1PR1 and its extracellular ligand S1P. On the other hand, Spiegel et al. have demonstrated that specific, SphK1-dependent, binding of S1P to TRAF2 enhances the TNF-alpha signaling. Therefore we were interested whether the TLR/TIR and TNF-alpha-signaling pathways are interfered with each other and are modulated by S1P. In a first approach we focused our investigations on SphK1 effects on both TRAF2 and TRAF6 stimulatory signals and cytokines produced downstream. Experimentally, with GM-CSF expanded, bone marrow-derived DCs we first desensitized the LPS-TLR4 or CpG-TLR9 signal by a defined time period of costimulation with TNF-alpha. The initial results showed a partial decrease of IL-12p70 secretion in TNF-alpha-co-stimulated DCs in contrast to LPS stimulation alone. This might indicate that TRAF2 activated via TNF-alpha interacted with the TRAF6 pathway to reduce IL-12p70. Further series with DCs derived from SphK1-deficient mice confirmed our former results that IL-12p70 in contrast to other cytokines is specifically sensitive to SphK1-S1P feedback, but did not change the effects of TNF-alpha on WT DCs IL-12p70 release. In comparison, CpG-TLR9-induced IL-12p70 release reached only 20% of LPS-induced IL-12p70 levels and was less sensitive to TNF-alpha costimulation. However, SphK1-deficiency strongly augmented CpG-dependent IL-12p70 production.

In ongoing experiments we started to analyze the details of TRAF2/RIP1 and TRAF6/TAK1 activation by ubiquitination blots in WT, SphK1-and S1Plyase-deficient DCs. In conclusion, we hope to unravel possible mechanisms of the observed differential effects of S1P and its enzymes on inflammation and cancer-relevant cytokines.

Identification of the KH type splicing regulatory protein (KSRP) as a new important mediator of the anti-inflammatory effects of resveratrol Art J. 1 , Besche V. 2 , Bros M. 2 , Li H. 1 , Handler N. 3 , Bauer F. 3 , Erker T. 3 , Behnke F. 4 , Mönch B. 5 , Förstermann U. 1 , Dirsch V. M. 6 , Werz O. 5 , Kleinert H. 1 , Pautz A.

University of Vienna Department of Pharmacognosy, Althanstr. 14, 1090 Wien, Austria Resveratrol, a polyphenol derived from different plants, possesses multiple pharmacological functions such as anti-oxidative, anti-diabetic, cardioprotective, anticancer, neuroprotective and anti-inflammatory properties. Many of these effects have been attributed to its anti-oxidative activity but resveratrol also modulates signal transduction pathways like the p38 MAPK pathway or the activity of different transcription factors like NF-κB redox-independently. Moreover, the histone deacetylase sirtuin 1 (SIRT1) is an important mediator of resveratrol effects. Nevertheless the direct molecular target of resveratrol remains unclear. In target fishing experiments we identified the RNA-binding protein KSRP as direct resveratrol binding partner. KSRP is an RNA-binding protein that controls proinflammatory gene expression on the post-transcriptional level by modulation of mRNA stability. Moreover, it is involved in the biogenesis of miRNAs. Resveratrol treatment of human DLD-1 cells resulted in a decreased mRNA expression of a number of well known KSRP target mRNAs and enhanced miRNA-155 function. Downregulation of KSRP expression by siRNA prevented the mRNA destabilizing effect of resveratrol. As the activity of KSRP is mainly regulated on the post-translational level by phosphorylation of different serine and threonine residues we analyzed whether resveratrol changes KSRP activity by altering the phosphorylation of the protein. Indeed, our immunoprecipitation experiments demonstrated that resveratrol reduces the p38 MAPK-mediated phosphorylation of threonine residues in the KSRP protein and thus leads to an increase of KSRP activity. Interestingly, resveratrol does not block p38 MAPK activation or activity. In addition we have evidence that SIRT1 is not involved in the resveratrol mediated activation of KSRP. So we believe that activation of KSRP by resveratrol is the major mechanism mediating the anti-inflammatory effects of resveratrol.

In vitro testing of OECD reference nanomaterials (NM-series) in rat precision cut lung slices Aumann A. 1 The OECD has defined reference nanomaterials (NM) to be tested in different endpoints concerning human health and environmental safety (1) in order to evaluate if the toxicity of nanomaterials can be linked to their physico-chemical properties. For nanomaterials, inhalation presents the major exposure route of concern and can be assessed using acute inhalation toxicity studies in rodents. However, these in vivo studies are resource intensive and animal consuming. The OECD working party on nanomaterials has named several alternative methods as being of particular interest for testing of nanomaterials; among them is the precision-cut lung slices model (PCLS) to estimate respiratory toxicity. We have tested all 16 NM in PCLS measuring cytotoxicity, apoptosis, oxidative stress and inflammatory response of the tissues as well as observing them histological. For in vitro exposure of PCLS the test material was dispersed in medium. Since it is the nature of these materials to change their surface characteristics and agglomeration state in different environments, a standardized dispersion method (nanoCare) using bovine serum albumin as a stabilizing agent, was used. Particle size-distributions of the nanomaterial dispersions were characterized via analytical ultracentrifugation and found the nanomaterials well dispersed. Silver and Zinc oxide but none of the other NM showed cytotoxicity to the lung tissue in the tested concentrations. However, differences in cytokine profiles among the NM were observed and showed several correlations to the results obtained in in vivo inhalation or instillation studies.

Universität des Saarlandes Institut für Molekulare Zellbiologie, Gebäude 61, 66421 Homburg, Germany TMEM2 proteins show similarities in their primary sequence to motifs that are conserved amongst various members of the TRP protein family. Based on hydropathy analysis these proteins exhibit 6 to 10 membrane spanning domains. In contrast to TRP channels there is no evidence that these proteins form ion channels in the plasma membrane following overexpression of their cDNA in HEK293 cells. TMEM2 -/mice are viable and show no obvious signs of disease, but exhibit increased pancreatic amylase and lipase plasma levels. Microfluorimetric measurements using Fura-2 revealed that the elevation of the cytosolic Ca 2+ concentration after stimulation with carbachol and the cholecystokinin analogue caerulein is unchanged in TMEM2-deficient acinar cells. TMEM2 is expressed in several cell types including pancreatic acinar cells, cardiac myocytes, cardiac fibroblasts, but their subcellular localization is still unkown. We generated several constructs encoding TMEM2 fusion proteins with fluorescence protein tags by fusing eYFP, mcherry and tagRFP-T to the N-and C-terminus of the protein, respectively. Based on Western blot experiments and expression in HEK293 cells the TMEM2-eYFP construct was most suitable for further colocalisation analysis and generation of viral vectors including Adenovirus and Semliki forrest virus. In contrast to the prediction by the PSORT II algorithm TMEM2-eYFP could not yet be identified in the plasma membrane of fibroblasts, cardiac myocytes or acinar cells but showed a vesicular subcellular localization pattern. We localized TMEM2-eYFP in acidic compartments and predominantly in lysosomes (Pearson coefficient (PCC) 0,79 ± 0,03, n= 4 using LysoTracker ® dye). Analysis of subcellular localization with independent TMEM2 fusion constructs and additional vesicular markers will be presented as a framework to get insights towards the cellular function of TMEM2 and to reveal the mechanisms underlying increased amylase release from acinar cells of TMEM2 -/mice.

The small molecule Bcl-2/Mcl-1 inhibitor TW-37 shows single-agent cytotoxicity in neuroblastoma cell lines Bachmann H. S. 1 , Akdeli N. High-risk neuroblastoma (NB) remains a therapeutic challenge in paediatric oncology. Pro-survival Bcl-2 family proteins critically regulate apoptosis, and may represent important therapeutic targets in NB. Primary NB tumours heterogeneously express Mcl-1 or Bcl-2, with high expression correlating to high risk phenotype. Co-expression can be detected in approximately 10% and is correlated to reduced survival. Recent studies with two inhibitors that predominantly target Bcl-2 and other proteins, but not or to a lesser extend Mcl-1, elucidated the importance of Mcl-1 inhibition for cytotoxicity in NB. TW-37 is a small molecule inhibitor that showed almost equal affinity to Bcl-2 and Mcl-1.

To explore the effect of combined Bcl-2/Mcl-1 inhibition on neuroblastoma cells, four cell lines (SK-N-AS, IMR-5, SY5Y and Kelly) were treated with TW-37 and changes in growth properties were determined. Furthermore, nude mice with Kelly (human neuroblastoma cell line) xenografts were treated with TW-37. Using siRNA, we investigated the functional relevance of Mcl-1 and Bcl-2 in Kelly cells. For in vitro cell viability we observed IC50 values of 0.59 ± 0.39 µmol/l. On treatment with 1 µmol/l dose of TW-37, all neuroblastoma cell lines analyzed showed significantly reduced proliferation and increased apoptosis rates. Bcl-2 as well as Mcl-1 knockdown induced apoptosis in Kelly cells. Interestingly, TW-37 was able to reduce, but not to abrogate growth of Kelly neuroblastoma xenografts in nude mice. In conclusion, combined inhibition of Bcl-2 and Mcl-1 using TW-37 exhibits strong single-agent antitumor activity on human neuroblastoma cells in vitro, but limited single-agent activity in vivo. Therefore, inhibition of Bcl-2/Mcl-1 may represent an interesting therapeutic strategy, most likely in combination with conventional chemotherapy and other specific inhibitors.

Localization and functional characterization of membrane transporters for sulfated steroid hormones in the human testis Bakhaus K. 1 , Wapelhorst B. Circulating sulfated steroid hormones like estrone sulfate (E1S) or dehydroepiandrosterone sulfate (DHEAS) are delivered to the testis via membrane uptake carriers such as the sodium-dependent organic anion transporter (SOAT). Inside the cell these sulfated steroids can be metabolized to active steroid hormones by the catalytic activity of the steroid sulfatase (StS), which shows high enzymatic activity in the testis ("sulfatase pathway"). In addition to SOAT, other candidate carriers like the organic solute carrier protein 1 (OSCP1) and the organic anion transporting polypeptides OATP6A1 and OATP1C1 are predominantly expressed in the human testis and demonstrate transport activity for sulfated steroids. We aimed to evaluate the cellular expression of SOAT and the other steroid sulfate carriers and their co-localization with the steroid sulfatase (StS) in human testis. Furthermore we want to perform functional transport studies with the steroid sulfate carriers in stably transfected HEK293 cells. We detected SOAT by RT-PCR and Western Blot analysis in the human testis. Single cell analysis and in situ hybridization revealed pachytene primary spermatocytes to express the SOAT mRNA. SOAT expression in specimens showing maturation arrest at the level of early round spermatids seems to be severely reduced or absent. StS mRNA was detected by RT-PCR in testis homogenates. Preliminary immunohistochemical data showed that StS may be expressed in germ cells and interstitial Leydig cells. HEK293 cells stably expressing the SOAT carrier protein showed significant transport activity for DHEAS. This was demonstrated by using a radiolabeled [ 3 The hepato-intestinal induction of the detoxifying enzymes CYP3A4 and CYP3A5 by the xenosensing pregnane X receptor (PXR) constitutes a key adaptive response to oral drugs and dietary xenobiotics. In contrast to CYP3A4, CYP3A5 is additionally expressed in several, mostly steroidogenic organs, which creates potential for induction-driven disturbances of the steroid homeostasis. Using cell lines and mice transgenic for a CYP3A5 promoter we demonstrate that the CYP3A5 expression in these organs is noninducible and independent from PXR. Instead, it is enabled by the loss of a suppressing yin yang 1 (YY1)-binding site from the CYP3A5 promoter which occurred in haplorrhine primates. This YY1 site is conserved in CYP3A4, but its inhibitory effect can be offset by PXR acting on response elements such as XREM. Taken together, the loss of YY1 binding site from promoters of the CYP3A5 gene lineage during primate evolution may have enabled the utilization of CYP3A5 both in the adaptive hepato-intestinal response to xenobiotics and as a constitutively expressed gene in other organs. Our results thus constitute a first description of uncoupling induction from constitutive expression for a major detoxifying enzyme. They also suggest an explanation for the considerable tissue expression differences between CYP3A5 and CYP3A4.

Serum albumin adducts as biomarkers for systemic bioavailability of active metabolites of various glucosinolates in animal models and humans Barknowitz G. 1 , Engst W. Glucosinolates (GLS) are natural pesticides of Brassicales, which comprise many important food and feed plants. Upon physical damage to the plant, the enzyme myrosinase can convert GLS to reactive metabolites (e.g. isothiocyanates). The same reaction can also be catalyzed by enzymes of the intestinal microbiota. Modification of sensor proteins (e.g. Keap-1) by some GLS metabolites leads to adaptive responses, resulting in enhanced detoxification of reactive metabolites and other protective reactions. At least in experimental models, this mechanism can be exploited for chemoprevention of carcinogenesis induced by various chemical carcinogens. However, own research revealed that certain reactive GLS metabolites can covalently bind to DNA in vitro and in vivo, involving possible genotoxic and carcinogenic risks. Animal models are useful for studying beneficial and adverse effects of GLS. However, it has to be taken into account that the toxicokinetics of GLS may differ between rodent and humans and that the active metabolites are short lived and thus difficult to detect and quantify. Likewise, exposure of humans to GLS and their breakdown products may enormously vary depending on (i) food preferences, (ii) cultivars, growth conditions and preparation of plants consumed and (iii) variations in human xenobiotic metabolizing system and composition of intestinal microbiota. In order to estimate individual levels of systemic exposure to reactive GLS metabolites, blood protein adducts may be useful. We have developed LC-MS/MS methods for quantifying serum albumin adducts formed by glucoraphanin, glucotropaeolin and neoglucobrassicin. The method involves digestion of the protein to amino acids and the usage of isotope-labelled amino acid adducts as internal standards. Serum albumin adducts were detected in mouse models after feeding broccoli and pak choi respectively, as well as after administration of purified GLS and breakdown products. Likewise, GLS adducts were detected in human blood plasma after consumption of broccoli or cress. This work was financially supported by the Bundesministerium für Bildung und Forschung (Grant 0315370D) . Barlow S. Harrington House, 8 Harrington Road, Brighton, BN1 6RE, Great Britain Values for cancer endpoints for the application of the TTC approach have been derived by linear extrapolation of results from animal carcinogenicity studies to calculate "virtually safe doses" (VSDs). A VSD is defined as an exposure that represents an estimated upper bound increase in risk of 1 in a million of developing cancer during a lifetime. In 1995, from consideration of a range of VSDs for carcinogens, the US Food and Drug Administration (FDA) proposed and adopted a value of 0.5 micrograms/kg of diet (0.5 ppb) as a Threshold of Regulation to be used for substances present in food contact materials. The FDA considered that if exposure to a substance in the diet was below this value, consumers would be protected "with reasonable certainty of no harm" and no toxicological data on the substance need be submitted. The value of 0.5 ppb is equivalent to 1.5 micrograms/person per day, assuming that 1500 g of food and 1500 g of fluids diet might be consumed daily. This value was subsequently incorporated into the TTC approach to be used for assessment of substances without a structural alert for genotoxicity. In 2004, Kroes and colleagues further explored cancer as an endpoint and recommended a lower TTC value of 0.15 micrograms/person per day for substances with a structural alert for genotoxicity and exclusion from the TTC approach of certain groups of high potency carcinogens (with VSDs below this value). In this presentation, the data underpinning these TTC values will be discussed from the perspective of their reliability for risk assessment of substances with low exposures, for which there are no toxicity data, but which may in fact be genotoxic or non-genotoxic carcinogens.

stable conjugates which are recognized by the immune system. In the current investigations, the ability of chemical pre-treatment to interfere with antibody-protein binding has been investigated using ovalbumin (OVA) as the model protein and naturally occurring anti-OVA IgG from healthy human donors. Preparation of conjugates: OVA (1 mg/mL) was dissolved in sodium borate buffer (0.1M, pH 9.4) . For chemical treatment various amounts (50-200 mg) of 1-fluoro-2,4dinitrobenzene (DNFB; sensitizer) or 2,4-dichloro-1-nitrobenzene(DCNB; non-sensitizer) was added and stirred for 2 hrs at room temperature. Unbound compound was removed by consecutive dialysis against phosphate buffered saline (PBS) and distilled water. Inhibition ELISA: Plates were coated with 100 µg/mL OVA and blocked with 10% FCS in PBS. OVA samples (native or conjugated; 0.6 -10 µg/mL) were pre-incubated with polyclonal antibodies (Ab) from pooled normal human serum for 30min and then added to the plates. Human anti-OVA IgG Abs were detected by colorimetric analysis using ortho-phenylendiamine as a substrate. The concentration of soluble native OVA or conjugate required to displace 50% of Ab to plate-bound OVA (IC50) was calculated (minimum of n=3 independent experiments). Treatment of OVA with the chemical sensitizer and protein reactive DNFB resulted in increased IC50 value, whereas mock treatment resulted in comparable IC50 values to native OVA. Treatment with DCNB showed that the presence of a chemical per se (and possible denaturation) was not sufficient to alter the IC50 value; conjugation of the compound to the protein was required. The analysis is not test chemical specific (specific Ab against compound-protein conjugates are not required) and the colorimetric analysis is unaffected by absorbance of the compound itself. Thus, this method may have utility for the identification of chemical sensitizers which are directly protein reactive.

2´-deoxy-cAMP in human cell lines: Another second messenger? Beckert C. 1 , Hinz C. we have shown that 2´-deoxy-cAMP (dcAMP) is synthesized by recombinant human soluble adenylyl cyclase (sAC). Here, we report that dcAMP can be detected and quantified by liquid chromatography coupled to mass spectrometry (LC-MS) in various human cell lines. In most cells a ratio of cAMP : dcAMP of ~10 was observed. As a remarkable exception, in HL-60 promyelocytic leukemia cells, the dcAMP concentration exceeded the cAMP concentration more than 3-fold. A differential regulation of cAMP versus dcAMP was determined upon replacement of the incubation medium (proliferating condition with serum / serum-free resting condition). For example, cAMP was dramatically reduced in HEK293 cells after 24 hours under resting conditions whereas dcAMP was significantly increased. In cellular subfractions of HEK293 cells AC assays (Mn 2+ /forskolin-or Mn 2+ /bicarbonate-stimulated) with either ATP or dATP as substrate revealed that comparable amounts of cAMP and dcAMP accumulated. In addition to sAC, membranous ACs such as AC V were capable of forming dcAMP with vmax and KM values for dATP comparable to those for ATP. We also analyzed the substrate-specificity for several human phosphodiesterases. PDE3 and PDE4 hydrolyzed dcAMP more effectively than cAMP. Taken together, these data point to a putative second messenger role of dcAMP in human cells. We are currently investigating the regulatory role(s) of cAMP and dcAMP in apoptosis of HEK293 cells. As a novel tool for these studies, we will use the cellpermeant dcAMP-acetoxymethylester which penetrates the plasma membrane and releases dcAMP intracellularly.

The biogenic amine histamine is recognized by target cells via four different histamine receptors subtypes (H1R -H4R), which all belong to the family of G-protein-coupled seven-transmembrane receptors. Histamine plays a crucial role in allergic reactions such as rhinitis or conjunctivitis and also in allergic asthma. Previously, we showed an interaction of the effects of antagonists at the H1R and H4R in a mouse model of allergic asthma. However, not much is known about the signaling pathway activated by murine H1R and H4R. In order to analyze these signaling pathways, we established a cellular model using transfected HEK 293 cells which stably express recombinant mH1R or mH4R. Proper expression of the receptors was verified by western blot analysis and flow cytometry. In functional assays we demonstrated that histamine stimulation results in the increase of intracellular Ca 2+ concentration ([Ca 2+ ]i) in cells expressing either of both, mH1R (pEC50 = 8.2) or mH4R (pEC50 = 6.9). As a second readout, we analyzed the modulation of forskolin-induced cAMP-accumulation. In mH1R-expressing cells the intracellular cAMP concentration was increased by stimulation with histamine, while in mH4R-expressing cells forskolin-induced cAMP accumulation was reduced. The histamine-induced effects in H1R-expressing cells were blocked by the H1R antagonist mepyramine ([Ca 2+ ]i: pKb = 8.6) and those in the H4R-expressing cells by the H4R antagonist JNJ7777120 ([Ca 2+ ]i: pKb = 8.8) or by pertussis toxin, which selectively blocks receptor Gi-protein coupling. JNJ7777120, which behaves as a partial mH4R agonist in the steady-state GTPase assay using membranes of infected SF9 cells, was without effect on [Ca 2+ ]i and forskolin-induced cAMP-accumulation in the mH4R-expressing cells. Currently, we are investigating mitogen-activated protein (MAP)-kinase pathways activated by the H1R and the H4R. Using a phospho-MAP-kinase array, histamine dependent phosphorylation of Erk1/2, p38, JNK, CREB, PKB (Akt), and MKK 3/6 were detected in cells expressing either of both, mH1R and mH4R. In summary, the HEK 293 cell lines stably expressing selective histamine receptors are very useful tools to investigate HxR signaling pathways in-vitro.

Enhanced fibroblast motility in the absence of the β3 regulatory subunit of voltage-activated calcium channels Belkacemi A. 1 CaVβ subunits of voltage-activated Ca 2+ channels are required for trafficking the poreforming CaVα1 subunit to the plasma membrane and modulate the kinetics of its current. Mouse embryonic fibroblasts (MEFs), acutely isolated cardiac fibroblasts (CFs) and NIH 3T3 fibroblasts do express CaVβ2 and CaVβ3 subunits, but we could not detect any voltage-activated Ca 2+ influx. Whereas in mouse cardiomyocytes or HEK 293 cells coexpressing CaVβ3 and CaVα1 subunits a dihydropyridin-sensitive voltage-activated Ca 2+ influx was readily detectable. Apparently, CaVβ subunits serve functions in fibroblasts unrelated to voltage-activated Ca 2+ influx. Among the proteins potentially interacting with CaVβ3 are the inositol 1,4,5-trisphosphate receptors (IP3Rs) [1, 2] . We therefore coexpressed mouse CaVβ3 and mouse IP3R type 1, 2 or 3 in COS-7 cells and found coimmunoprecipitation of IP3Rs using an antibody for CaVβ3 and vice versa. To study the release of Ca 2+ from IP3-sensitive stores we performed FURA-2 measurements on fibroblasts isolated from wild type and CaVβ3-deficient mice either in the presence of thapsigargin or after stimulation of Gq-coupled receptors by PAR-1, LPA or Bradykinin. Receptor-activated Ca 2+ release was more pronounced in β3-deficient MEFs and CFs, whereas thapsigargin-induced Ca 2+ release was the same in cells from both genotypes. In addition, IP3 production measured by a radioreceptor assay was already increased in β3-deficient cells under basal conditions. Fibroblasts are migrating cells and involved in various physiological and pathophysiological processes. We therefore started in vitro assays for proliferation, migration and angiogenesis as well as in vivo assays for skin wound healing. Angiogenesis and proliferation were apparently not different in both genotypes but migration (measured as transwell migration and in scratch assays) and wound healing were affected in different ways. Fluorescent staining of cytoskeleton and quantification of the F-actin/G-actin ratio show similar results in both genotypes, suggesting that the increased migration rates and wound repair in β3 knockout may result, in part, from the increased amount of IP3-releasable Ca 2+ . [1] Berggren, Yang, Murakami, et al., Removal of Ca channel β3 subunit enhances Caoscillation frequency and insulin exocytosis. Cell 119, (2004) 273-284. [2] Müller, Haupt, Bildl, et al.,  Quantitative proteomics of the Cav2 channel nanoenvironments in the mammalian brain. PNAS 107, (2010) 14950-14957.

Bender-Sigel J., Closs E. I. Universitätsmedizin Mainz Institut für Pharmakologie, Obere Zahlbacher Straße 67, 55101 Mainz, Germany Human cationic amino acid transporters (hCAT) are a family of multimembrane spanning proteins that mediate the transport of cationic amino acids through the plasma membrane. Our earlier results have demonstrated that activation of either protein kinase C (PKC) by PMA or Cdc42 by EGF leads to an internalization of these transporters. In addition, in a recent collaboration with the group of Alexander Sorkin (University of Colorado Denver) we found that ubiquitination and clathrin-dependent endocytosis are necessary for the down regulation of hCAT-1-mediated arginine transport by PMA (Vina-Vilaseca et al, J Biol Chem 2011 286:8697) . This mechanism requires Nedd4 E3 ligases, but hCATs do not contain a PPXY motif to bind the ligases, suggesting that an adaptor protein takes part in this process. However, an involvement of the adaptor protein beta-arrestin in this mechanism could be excluded. Using siRNA against PKC alpha we now show that PKC alpha is the major isoform that induces the reduction of arginine transport in human U373 glioblastoma cells overexpressing hCAT-2A-EGFP. In addition, siRNA-mediated knock down of Cdc42 prevented the decrease of hCAT-2Amediated transport induced by PMA. Taken together PKC seems to negatively influence the constitutive cycling of CATs by activation the ubiquitination machinery and clathrinmediated endocytosis. Cdc42 is part of this pathway.

Converging of the classical mitochondria-related pathway in Parkinson and nuclear DNA-repair signaling? Scherr A. -L. 1 Parkinson disease is the second most neurological disorder worldwide. Despite the fact that most cases are idiopathic and only few can be traced back to specific genes, general progression between both tracks of the disease is comparable. The variety of clinical symptoms in motor control like tremor, rigor and postural problems all originate from loss of dopaminergic neurons in the substantia nigra pars compacta of the brain. Several proteins mutated in PD are involved in surveillance pathways, monitoring functionality and integrity of proteins and organelles either by the proteasome degradation machinery or by clearance of mitochondria via autophagy (mitophagy). Disturbed calcium-and redox-homeostasis seems to play a major role in susceptibility to cell death signals in dopaminergic neurons, but if this is a preceding or successive event in cell death related to PD progression is not known. On the other hand, experimentally elicited Parkinsonism by oxidative stress inducers like paraquat or rotenone and MPTP (inhibitors of mitochondrial complex I) lead to damage in nuclear DNA and activation of the DNA repair protein poly(ADP-ribose) polymerase 1. By consuming substantial amounts of its substrate NAD + , this enzyme can drastically decrease energy levels and disturb the redox balance within a cell, also sensitizing it to stress induced cell death. Inhibition of poly(ADP-ribose) polymerase 1 has been proven to be beneficial to some extent in cell culture models as well as in experimental PD in mice. Our research focuses on a putative crosstalk mechanism we recently discovered between the two pathways of experimentally induced cell death in culture models, i.e. mitochondrial signaling and PARP1-dependent poly(ADP-ribosyl)ation. Both converge on two mitochondrial chaperones, Mortalin and TRAP1. Whereas mutations in Mortalin have been reported recently to be responsible for some Parkinson disease cases in humans, TRAP1 is a specific target of the kinase Pink1 (PTEN induced putative kinase), which is often mutated in autosomal-recessive forms of the disorder. Pink1 is a central regulator of the mitophagy process, tagging mitochondria with dissipated membrane potential for destruction. We could show now that both chaperones bind to short-chain poly(ADP-ribose) specifically synthesized by poly(ADP-ribose) polymerase 1. We will present our most recent findings about regulation of these two chaperones after application of Parkinson-inducing toxins.

Aldrich). The effect was reversible within ten minutes when cells were re-incubated in regular cell culture medium. Stimulatory effects were not due to osmolarity or cell stress due to medium exchange. Analysis of different components of both media (Table 1) revealed that bicarbonate stimulates accumulation of cCMP and cUMP besides cGMP and cAMP in a time-and concentration-dependent manner. Bicarbonate is known to activate soluble adenylyl cyclase (sAC) and particulate guanylyl cyclase G (pGC-G), regulating sugar metabolism, sperm motility and olfaction by synthesis of cAMP and cGMP, respectively. In order to identify a responsible cyclase for cCMP and cUMP generation after bicarbonate treatment, we are currently analyzing transiently and stably transfected HEK293 cells overexpressing various known adenylyl and guanylyl cyclases (sAC, mAC1, mAC3, mAC9, soluble guanylyl cyclase, pGC-G, pGC-A, and pGC-D) for their pyrimidinyl and purinyl cyclase activity in vivo and their regulation by bicarbonate. In addition, cell fractions will be analyzed for the detection of specific cyclase compartments. Question: Long term ventricular pacing, especially at the right ventricle (RV), results in left ventricular (LV) failure. There are several lines of evidence that disturbed Ca 2+ homeostasis is involved in the pathophysiology of human heart failure. In this study we examined if ventricular pacing affects the Na + -and Ca 2+ -channels and the expression of Ca 2+ -handling proteins and investigated if there is a differential effect between right ventricular free wall (RVFW) pacing and left ventricular apex (LVA) pacing. Methods: After AV-node ablation 14 minipigs underwent ventricular pacing at 120 beats/min (DDD mode) for one year. 7 minipigs were paced from the RVFW and 7 minipigs from the LVA, respectively. 7 minipigs with normal sinus-rhythm served as control group. Patch-clamp-experiments were studied to measure Na + -and Ca 2+ currents. Western-Blots were carried out to investigate the expression of the Ca 2+handling proteins L-type Ca 2+ -channel, Serca2 and phospholamban. Results: Both RVFW-and LVA-pacing led to significant decreased Ca 2+ -currentdensities in cardiomyocytes of the LV compared to the control group. The plateau phase of the action potential was significantly shortened after ventricular pacing in relation to control minipigs. Furthermore cardiomyocytes of RVFW-and LVA-paced minipigs had significant lower Na + -current-densities than control minipigs. The action potential amplitude was significantly decreased after RVFW-and LVA-pacing whereas the diastolic potential remained unchanged. The expression of the L-type Ca 2+ -channel was significantly reduced after ventricular pacing, regardless of the pacing site. In contrast RVFW-and LVA-paced minipigs showed significant increased Serca2-expression. The expression of phospholamban remained unchanged after RVFW-and LVA-pacing compared to control minipigs. Conclusion: In a chronic animal model ventricular pacing leads to remodeling of ionchannels and Ca 2+ -handling-protein-expression, regardless of the pacing site.

Investigation on metabolic competence of dermal systems: native human skin, in vitro skin models and keratinocytes Blatz V. 1 The implementation of reconstructed human skin equivalents (RHEs) as an alternative method for dermal toxicity testing became very prominent in the last decades. Their advantages are e. g. the human cell origin and an organ-like 3D structure. Already regulatory accepted methodologies are widely in use for testing the skin corrosion (OECD 431) and irritation (OECD 439) within RHEs. But there are still some questions open, one of them the metabolic competence of such dermal systems.

In this context, enzyme activities of oxidizing (CYP; FMO; ADH; ALDH) and conjugating enzymes (NAT; UGT) were investigated in subcellular fractions of in vitro systems such as keratinocytes and RHEs (epidermis model EpiDerm TM (MatTek), full-thickness skin models EpiDerm TM FT (MatTek) and Phenion ® FT (Henkel AG)) and compared to those of native human skin. Activities of CYP 1A, 2B and 3A isoenzymes were measured fluorometrically by oxidative desalkylation of alkoxyresorufines. FMO 1/3 activities were evaluated by HPLC/FLD detection of N-oxygenated product of Benzydamine [1] . ADH and ALDH activities were investigated by photometrical detection of NADH generation during ethanol (ADH) [2] or propanal (ALDH) oxidation [3] . NAT1 activity was followed by HPLC/UV detection of acetylated p-aminobenzoic acid. UGT1 activity was quantified fluorimetrically by glucuronidation of methylumbelliferone [1] .

During the course of this study the following results were observed: (LOQ = limit of quantification) Since the metabolic competence of RHEs is confirmed, these in vitro systems are estimated as suitable for further toxicity tests (e. g. genotoxicity by Comet Assay), where metabolic activation of substances may play a crucial role. However, for the data assessment, the determined metabolic profiles should be taken into account. We acknowledge BMBF funding this project (0315226D).

signalling pathway indicates that in B104 cells the adenine receptor couples to a Gqprotein followed by activation of phospholipase C pathway. These findings represent a new signalling pathway of the adenine receptor and allow the assumption that different adenine receptor subtypes exist in the rat brain. In the scope of the project LExUKon ("Foodborne exposure to environmental contaminants -Data analysis to support and standardise exposure assessments based on NVS II") exposure to the heavy metals cadmium (Cd), lead (Pb) and mercury (Hg) via food consumption has been assessed for the German adult population based on the National Nutrition Study II ( The updated intake assessments show that especially foods regularly consumed such as vegetables and grain contribute mainly to exposure of Cd that is about 1.5 µg/kg body weight (bw) per week for average consumers over all food groups. This corresponds to 58% of the tolerable weekly intake (TWI) of 2.5 µg/kg bw for Cd defined by the European Food Safety Authority (EFSA) in 2009. Beverages and vegetables are the food groups most relevant for exposure to Pb. About 3.7 µg Pb/kg bw is taken up by average consumers that is below the benchmark dose for renal toxic effects (4.41 µg/kg bw) defined by EFSA for the weekly Pb intake. For Hg the intake amounts for all population groups examined were significantly below the toxicological reference values. For average consumers the weekly intake of Hg is 0.49 µg/kg bw that is primarily taken up by eating fish and fishery products. However, individual population groups and high consumers reach and/or exceed the toxicological reference values for the daily intake amounts for Cd and Pb. High consumers almost reach the TWI for the Cd with 94%. For Pb a weekly intake of 5 µg/kg bw was estimated for high consumers that exceeds the benchmark dose for renal toxic effects for the weekly Pb intake.

The results show that data collection should also focus on highly consumed and not only on highly contaminated foods. Further, uncertainties in concentration levels should be reduced e.g. by lowering and standardizing the analytical limits. It's recommended to consider further measures in view of the reduction of contents of environmental contaminants in foods. However, other sources can also contribute to the intake of the mentioned heavy metals (e.g. smoking). Major cell biological processes are regulated by Rho-GTPases, actin-mediated processes in particular. Amongst others, Rho-GTPases are stimulated by the receptormediated activation of Gα12/13 and Gαq via specific RhoGEFs. The p63RhoGEF is activated by Gαq and plays a major role in the acute response of vascular smooth muscle cells to angiotensin II treatment. The aim of the present study was to establish a FRET-assay between Gαq-CFP and Venus-p63RhoGEF and characterize the dynamics of p63RhoGEF-Gαq-interaction in single living cells. The fusion of p63RhoGEF with Venus resulted in a functional Gαq-regulated p63RhoGEF-protein as determined by means of Rho-Luziferase-Assays. Whereas no specific FRET signal was observed between the two interaction partners in the absence of receptor stimulation, a robust and rapid FRET signal developed in response to stimulation of histaminergic H1and cholinergic M3-recptors. The onset of this signal after rapid application of agonist paralleled Gαq activation kinetics. Similar to the kinetics of Gαq-protein deactivation the dissociation of p63RhoGEF and Gαq after withdrawal of agonist was slow (tens of seconds). The specificity of the FRET signal between Gαq-CFP and Venus-p63RhoGEF was verified by introducing point mutations rendering p63RhoGEF unable to bind to active Gαq. Furtehrmore we observed a robust acceleration of the dissociation of p63RhoGEF and Gαq upon cotransfection of RGS2, suggesting a very short lifetime of the p63RhoGEF-Gαq-complex or the ability of RGS2 to bind to p63RhoGEF-associated Gαq. Taken together, FRET-based imaging of the interactions between p63RhoGEF and Gαq revealed fast interaction kinetics closely resembling G-protein activation kinetics, both of which can be regulated by RGS2.

Toxicity of silver nanoparticles in intestinal cells Boehmert L. 1 The rapid development of nanotechnology has been accompanied by an increased concern for the safety of nanomaterials. Especially silver nanoparticles are used in many manufacturer identified consumer products including silver coated food contact materials or hydrosol silver supplements. These products lead to an intentional or unintentional oral uptake of silver nanoparticles and hence to a contact with the intestinal barrier. The human cell line Caco-2 is a well established model system in studying effects on human enterocytes. Although these cells are colon carcinoma cells and exhibit typical features of cancer cells when they are kept sub confluent, these cells have the capability to differentiate into polarized cells with morphological and biochemical properties of small enterocyte cells.

We investigated the effects of silver nanoparticles on these colon carcinoma (proliferating) and small intestinal epithelium like (differentiated) Caco-2 cells. The silver nanoparticles AgPURE were commercially available from rent-a-scientist GmbH. The behaviour of these silver nanoparticles in cell culture medium were characterised using asymmetric flow-fild flow fractionation (A4F), small ankle x-ray scattering (SAXS) and dynamic light scattering (DLS). We investigated the particle toxicity on both cell states using Cell Titer Blue Assay, xCELLigence impedance measurements, Annexin-V and Caspase measurements, diclorofluorescein assay and antioxidant pre incubated cells.

The AgPURE stock solution is an aqueous suspension of silver particles with a metal core radius of 7.2 nm stabilised with tween-20 und polyoxyethylenglycerol trioleate. AgPURE silver nanoparticles were toxic for proliferating as well as differentiated Caco-2 cells in a time and concentration depending manner. The presence of foetal calf serum in the incubation medium has a minor influence on the toxicity. Prior to cell death, morphological abnormal adherence characteristics and morphological changes in the cells were observed using microscopy and quantified by xCELLigence impedance measurement. It is concluded that cell death is caused by an oxidative stress related mechanism rather than apoptosis.

The release of sphingosine-1-phosphate from human platelets during acute coronary syndrome is attenuated by aspirin Böhm A. 1 , Polzin A. In addition to atherosclerosis TTP knock out mice develop more cardiovascular dysfunctions. In tail vein bleeding assays we monitored a significant difference in the bleeding times of TTP deficient mice in comparison to wildtype mice, triggered by a stronger granulopoeisis. Our results leed us to the assumption that the chonic inflammation seems to be more improtant for the development of cardiovascular diseases in RA patients than the traditional risk factors.

Differentially expressed cardiac genes in a mouse model with heart-specific overexpression of PP2A Bollmann P., Makarova E. A., Gergs U., Neumann J. Institute for Pharmacology and Toxicology Medical Faculty, Magdeburger Str. 4, 06112 Halle (Saale) , Germany

In transgenic (TG) mice with cardiac myocyte-specific overexpression of the catalytic subunit of protein phosphatase 2A (PP2A) reduced cardiac protein phosphorylation, cardiac hypertrophy and impaired cardiac contractility were noted compared to wild type (WT) littermates. The hearts of TG mice also suffered from ventricular dilatation and a diminished response to β-adrenergic stimulation. Analyses of mRNAs expressed in TG and WT hearts (n=3) using Affimetrix mouse genome microarray chips resulted in several candidate genes possibly differentially regulated. In this study, we focussed on verifying the mRNA data of selected genes important for stress response and signal transduction on protein level in cardiac homogenates by Western blotting. Hearts from WT littermates were used as control. Compared to WT heat shock protein 25 (HSP25) and calcium calmodulin dependent protein kinase type II (CaMKII) mRNAs were upregulated in TG but only HSP25 protein was increased (p<0.05, n=7-8) but not CaMKII (p>0.05). Protein phosphatase type 5 (PP5) and superoxide dismutase (SOD) were downregulated on mRNA level in TG but on protein level this could be found only for SOD (p<0.05, . In contrast, PP5 protein was upregulated (p<0.05, in TG compared to WT. For comparison the regulatory A-subunit of PP2A and HSP90 were studied. Both genes were unchanged on mRNA level in TG: Western blotting revealed the same results for the corresponding proteins. In summary, mRNA expression data could only partially be confirmed on protein level elucidating the importance of Western blotting studies. These data indicate that increased PP2A activity is associated with modified gene expression in TG hearts possibly affecting stress response and regulation of cell signalling. (Supported by the Deutsche Forschungsgemeinschaft)

Center for Regenerative Therapies, Technische Universtität, Dresden, Germany Cell therapy in the form of beta cell replacement to cure diabetes has been practiced for decades without become a routine clinical therapy. More widespread clinical application is hindered by the scarcity of suitable organ donors, a dramatic loss of transplanted cells within the first days post-transplant, the requirement of long term immunosuppression to maintain graft survival, and despite this, a loss of graft function from a recurrence of autoimmunity in some patients. Research is currently dedicated to overcome each of these limitations. Additional beta cell sources investigated include embryonic stem cell derived insulin producing cells, human insulin producing cells lines, and xenogeneic beta cells. Parallel to these efforts are the development of encapsulation devices to protect these sources from immune and inflammation mediated destruction, and transplantation into new sites such as the muscle and bone marrow to infuse beta cells. Additional therapy to reduce immune suppression includes the infusion of T regulatory cells to control autoimmune and alloimmune response, and cytokine and chemokine receptor directed compounds aimed at blocking early inflammation or autoimmunity. These efforts are likely to lead to an expansion of clinical activity to replace beta cells in diabetes, and to novel pharmaceutical therapies that may be more generally applicable in patients with diabetes.

PDGF-BB induces the H2S producing enzyme Cystathionine-γ-lyase via a ROSdependent mechanism in rat renal mesangial cells Boosen M. 1 , Hassan M. There is increasing evidence that hydrogen sulfide (H2S) that is endogenously produced in several cell types serves as a potent gasotransmitter in a wide variety of physiological processes involving vascular homeostasis and inflammation. In the present study we investigate the expression and the regulation of the hydrogen sulfide synthesizing enzyme cystathionine γ-lyase (CSE) in cultured rat renal mesangial cells. As demonstrated by qPCR and Western blot experiments, mesangial cells showed a marked time-and dose-dependent upregulation of CSE mRNA and protein levels after treatment with platelet-derived growth factor (PDGF-BB). The CSE upregulation by PDGF-BB is accompanied by a marked increase in reactive oxygen species (ROS) formation. Interestingly, co-administration of the ROS scavenger N-acetylcysteine, the glutathione peroxidase mimetic ebselen and the NADPH oxidase inhibitor diphenylen iodonium chloride (DPI) drastically reduced PDGF-induced CSE expression, indicating a role for endogenously produced ROS in mediating regulation of CSE. As demonstrated by electrophoretic mobility shift (EMSA) experiments PDGF-BB induces binding of the redox-sensitive transcription factor NF-E2-related factor 2 (Nrf2) to a consensus antioxidant response element and this effect was also diminished by co-administration of antioxidants (DPI, NAC, ebselen) . Furthermore, LPS/IFNγ-as well as PDGF-BB-induced CSE upregulation was nearly completely abolished in Nrf2 -/spleen macrophages and mesangial cells, respectively. As a consequence of the elevated CSE levels we could demonstrate increased H2S levels and a higher CSE enzyme activity in mesangial cells after stimulation with PDGF-BB by using the colorimetric methylenblue method and a CSE activity assay. Importantly, in a rat model of anti-Thy-1-induced proliferative glomerulonephritis we observed a marked upregulation of CSE protein during the course of the disease paralleled by a stabilization of Nrf2 protein. From our data, we hypothesize that PDGF-BB-mediated regulation of CSE via a redox-mediated activation of Nrf2 may constitute a protective mechanism during glomerular inflammatory disease.

Rac1 knockout protects from acute hepatic damage following doxorubicin treatment Bopp A., Wartlick F., Fritz G. Heinrich-Heine-Universität Institut für Toxikologie, Universitätsstr. 1, 40225 Düsseldorf, Germany Rac1 belongs to the best characterized members of the Ras-homologous (Rho) family of small GTPases, which are key regulators of the actin cytoskeleton. Furthermore, Rac1 is part of the activation of the NADPH oxidase, which produces reactive oxygen species and regulates the activity of stress kinases (e.g. SAPK/JNK) and transcription factors such as NF-κB and AP1. Anticancer drugs cause DNA damage, which in turn stimulates the DNA damage response (DDR) regulating DNA repair, cell cycle progression and, in case of non-repairable DNA damage, triggers apoptosis. So far, a role of Rac1 in the DDR has not been reported. Based on its exceptional function as a regulator of transcription and because of its recently found ability to translocate to the nucleus, we hypothesize that Rac1 may be involved in the DDR. To study the in vivo function of Rac1 we used an inducible Cre-based knockout mouse model (Rac1 flox/flox/MxCre ). Mice were treated with different doses of doxorubicin for different periods of time. We monitored gH2AX foci formation as a marker of DNA strand breaks, used the Masson-Goldner staining for the detection of collagen accumulation, analyzed phosphorylated Histone 3 as a marker of mitotic events and performed a Tunel assay to detect apoptotic cells. In the absence of Rac1 the basal mRNA expression of pro-fibrotic CTGF was decreased. Collagen levels were increased and MMP1 mRNA expression was reduced in the liver of Rac -/animals as compared with Rac1 proficient animals. In addition we found more apoptotic cells in Rac1 -/mice. 96 hours after treatment with the anthracycline derivative doxorubicin the number of gH2AX foci in Rac1 -/animals was reduced in comparison to Rac1 +/+ animals. We also found lower level of CTGF mRNA expression and reduced amount of collagen in Rac1 -/mice. None of these protective effects resulting from Rac1 deficiency could be detected after administration of three consecutive doxorubicin injections over a time period of 21 days. There were no significant differences in the number of gH2AX foci or collagen accumulation. The mRNA expression of CTGF was even higher in Rac1 -/animals. Furthermore the number of mitotic events was almost two times higher in the Rac1 -/mice compared to the Rac1 +/+ mice. Summarizing, our findings show that impaired hepatic expression of Rac1 protein is hepatoprotective against acute damage following doxorubicin exposure, but does not protect against doxorubicin-induced subacute toxicity.

In vitro cytotoxicity of tBHQ (tert-butyl-hydroquinone) Braeuning A., Vetter S., Schwarz M. Institut für Experimentelle und Klinische Pharmakologie und Toxikologie Toxikologie, Wilhelmstrasse 56, 72074 Tübingen, Germany At high concentrations, tert.-butyl-hydroquinone (tBHQ), a phenolic antioxidant frequently used as a food preservative, exerts cytotoxic effects, which are closely linked to its ability to form reactive oxygen species as a consequence of redox cycling processes. Here we describe that treatment of murine 3T3 cells with tBHQ in 96-well culture plates induces the death of untreated cells in neighboring wells on the same plate. The mechanisms underlying that effect were investigated. Death of the seemingly untreated neighboring cells was caused by a more toxic and volatile tBHQ oxidation product which was formed in a non-enzymatic process involving metal ions and oxygen. The unexpected perturbation of cytotoxicity testing by the volatile tBHQ metabolite shows that not only metabolic processes, but also non-enzymatic mechanisms have to be considered as important parameters for in vitro assays. Furthermore, our data show that even cells several wells distant from the site of treatment do not necessarily constitute proper "untreated" controls when cells are treated with tBHQ, e.g. in assays aimed to analyze the activity of the tBHQ-inducible Nrf2 pathway. S14 056 Angiotensin II causes oxidative stress and DNA damage in mouse kidneys via the angiotensin II type 1 receptor Brand S. 1 , Amann K. 2 , Schupp N. 1 1 Universität Würzburg Institut für Pharmakologie und Toxikologie, Versbacherstr. 9, 97078 Würzburg, Germany 2 Universität Erlangen-Nürnberg Institut für Pathologie, Krankenhausstraße 8, 91054 Erlangen, Germany

Angiotensin II (Ang II), the reactive peptide of the renin-angiotensin-system, causes vasoconstriction and, in higher levels hypertension, which is connected with an increased cancer risk in the kidney. Treatment of male C57BL/6 mice with Ang II results in the formation of superoxide radicals and DNA damage in the kidney as well as in the heart. To answer the question if the DNA damage is caused by hypertension or by elevated Ang II concentrations, mice were treated with different compounds: the angiotensin-converting-enzyme blocker ramipril, the Ang II receptor blocker ramipril, the Ang II receptor candesartan, the antioxidant tempol and the vasodilator hydralazine. The effect on blood pressure and renal function of Ang II-treated C57Bl/6 mice was examined. Treatment with Ang II led to a significant increase in blood pressure. Candesartan and hydralazine led to a decrease, whereas intervention with ramipril and tempol had no effect. Equal conditions could be found by examining renal function regarding the excretion of urinary albumin, which was ameliorated by candesartan and hydralazine. In addition, histopathological changes were investigated. There was significant glomerular damage and tubulointerstitial damage in Ang II-treated animals compared to control animals, which was significantly improved by candesartan and tempol. Hydralazine and ramipril mitigated the observed renal damage but were less effective than candesartan. Furthermore, the Ang II-induced formation of superoxide radicals in the kidney and the heart was slightly affected by all interventions. Genomic damage, in the form of double strand breaks was prevented by the Ang II receptor antagonist candesartan and the antioxidant tempol.

To sum up, the results from this study show that Ang II induces the elevation of markers of kidney failure and DNA damage, which is prevented by substances lowering blood pressure like candesartan, showing the receptor responsibility for the induction of DNA damage. Actually by substances not lowering blood pressure like tempol, the oxidative stress and DNA damage was ameliorated, showing the involvement of reactive oxygen species.

Optimization of the BALB/c-3T3 cell transformation assay by coupling a drug metabolizing system Brauneis M. D., Steinberg P. Stiftung Tierärztliche Hochschule Hannover Institut für Lebensmitteltoxikologie und Chemische Analytik, Bischofsholer Damm 15, 30173 Hannover, Germany

The analysis of the carcinogenic potential of chemicals plays an important role in toxicology. Up to now the acquisition of such data requires a large amount of animal experiments. The aim of this study is to reduce the number of experimental animals being used by further optimizing the BALB/c-3T3 cell transformation assay, an already well-established in vitro method. This method, which is also well suited for high throughput screening applications, allows a quantitative analysis of the aforementioned carcinogenic potential. The incubation of BALB/c-3T3 cells (murine embryonic fibroblasts) with mutagenic compounds leads to a loss of contact inhibition between these cells, which results in the development of so-called foci. These foci can be distinguished by characteristic changes in cell growth behaviour, a result of the treatment with carcinogenic compounds, and their number is therefore directly related to the genotoxic potential of the latter. A major disadvantage of the "classic" BALB/c-3T3 cell transformation assay is that a number of compounds initially require a metabolic transformation to gain their full genotoxic potential. Hence, without prior metabolic transformation many chemicals are not detected as carcinogenic in the abovementioned test system. To overcome this drawback the BALB/c-3T3 cell transformation assay has been coupled to a drug metabolizing system, in this case the so-called liver S9. In a first step the well-known genotoxic agents benzo[a]pyrene, aflatoxin B 1 and Nnitrosodimethylamine were tested in this assay. All three compounds led to a concentration-dependent increase in the number of foci formed, whereby this concentration-dependent increase was observed in a non-cytotoxic concentration range. In a next step the BALB/c-3T3 cell transformation assay will be coupled to further drug metabolizing systems as well as to the soft agar assay.

This study is being financially supported by the Stiftung SET and the Doerenkamp-Zbinden Foundation. Adenylyl cyclases (ACs) synthesize the second messenger cAMP. The family of ACs consists of nine membranous and one soluble isoforms with AC5 and AC6 being the predominantly expressed isoforms in the heart. In the heart, ACs integrate β-adrenergic (β-AR) signaling as the main physiological mechanism to improve cardiac performance. Although AC5 and AC6 share high sequence homology, opposing effects on cardioprotection have been reported, where disruption of AC5, as well as overexpression of AC6 both exerting beneficial effects in heart failure. Prospective pharmacological treatment of heart failure on the level of AC is under investigation. Our study explored the impact of AC5 KO on AC-activities in the heart at a functional level.

Complementary, mRNA expression studies of the β-AR-G-protein-AC signaling cascade were performed to detect possible compensatory alterations. Hearts from 16-20 week old homozygote AC5 knockout and wild-type male littermates were examined in this study. AC activities where measured in cardiac membrane preparations from left ventricles. AC activities were assessed under β-AR and G-protein (G s) stimulation by isoproterenol, guanosine 5'-triphosphate (GTP) and 5'-O-(3thiotriphosphate) (GTPγS) as well as for direct activation by forskolin. Relative mRNA expressions for AC1-9, Gs-, Gi-a and β1-, β2-AR where measured by quantitative realtime PCR. Surprisingly, assessment of basal, β-AR and G-protein-mediated AC-stimulation as well as direct activation by forskolin revealed no changes in AC activities. Besides from detection of the AC5 knockout, mRNA expressions analysis of AC1-9, Gs-, Gi-a and β1-, β2-AR did not detect any compensatory alteration. These findings suggest that proximal adrenergic signaling in the heart does not necessarily require AC5. Whether physiological integration of beta adrenergic signaling in the heart is mediated by both isoforms AC5 and AC6, or can be attributed to one main isoform remains to be elucidated.

Melanocortin-promoted PKA activation decreases AMPK activity via ERK-1/2 and LKB-1 in hypothalamic GT1-7 cells Breit A., Ellen D., Gudermann T. Goethestrasse 33, 80336 München, Germany α-melanocyte stimulating hormone (α-MSH)-induced activation of the melanocortin-4 receptor (MC4R) in hypothalamic neurons increases energy expenditure and inhibits food intake. Intrahypothalamic injection of melanocortins decreased food intake due to the inhibition of AMP-activated protein kinase (AMPK) that has recently been reported to enhance food intake in rodents. Until now, it is not clear if α-MSH affects AMPK via direct intracellular signaling cascades or if the release of paracrine factors is involved. Herein, we used a murine, hypothalamic cell line (GT1-7 cells) and monitored AMPK phosphorylation at Thr 172 which has been suggested to increase AMPK activity. We found that α-MSH dephosphorylated AMPK at Thr 172 and consequently decreased phosphorylation of the established AMPK substrate acetyl-CoA-carboxylase at Ser 79 . Inhibitory effects of α-MSH on AMPK were blocked by specific inhibitors of protein kinase A (PKA) or extracellular-regulated kinases-1/2 (ERK-1/2), pointing to an important role of both kinases in this process. Since α-MSH-induced activation of ERK-1/2 was blunted by PKA inhibitors, we propose that ERK-1/2 serves as a link between PKA and AMPK in GT1-7 cells. Furthermore, down-regulation of liver kinase B-1 (LKB-1), but not inhibition of calcium-calmodulin-dependent kinase kinase-β or transforming growth-factor-beta-activated kinase-1 decreased basal phosphorylation of AMPK and its dephosphorylation induced by α-MSH. Thus, we propose that α-MSH inhibits AMPK activity via a linear pathway including PKA, ERK-1/2 and LKB-1 in GT1-7 cells. Given the importance of the melanocortin system in the formation of adipositas detailed knowledge about this pathway might help to develop drugs targeting obesity.

Autism Spectrum Disorder (ASD) is a complex neurodevelopmental disorder with dysfunction of social interaction and communication. A hitherto unknown complexgenetic principle of origin probably underlies ASD. So far, more than 100 candidate genes were identified in literature. The patients affected with the monogenic Timothy syndrome show multiorgan dysfunction including lethal arrhythmias, immune deficiency, skeleton-dysplasia, syndactylia and autism. This single gene disorder serves as a model disease for ASD, giving insights in a possible pathophysiology. Here, a point mutation in a highly-conserved region of the pore-forming subunit of the voltage-dependent calcium channel (Ca V) CaV1.2 gene (CACNA1C) results in incomplete inactivation of the L-type calcium currents (Splawski et al., Cell 2004; 119:19-31) . Functionally similar biophysical effects can be induced by structural variation β1-and β2-subunits of the voltagedependent calcium channels (Herzig et al., FASEB J. 2007; 21:1527-38; Jangsangthong et al., Pflugers Arch. 2010; 459:399-411) . Supported by findings in a meta-analysis of linkage data of ASD patients (Trikalinos et al., Mol Psychiatry. 2006; 11:29-36) , we are investigating a function-based candidate gene hypothesis linking the β2 subunit gene (CACNB2) with ASD. We performed a case control study sequencing all exons and flanking intronic regions of CACNB2 in 155 patients with ASD. We found three rare missense mutations in ASD patients, but not in 259 unaffected controls. All three mutations occur at highly conserved positions and might alter protein function; additionally results one amino acid substitution highly probable in a post-translational modification by phosphorylation. So far, we characterized two of these mutations and also a phosphorylation-mimicking mutant in electrophysiological studies. All variants show a decelerated and incomplete time-dependent inactivation of the co-transfected CaV1.2 subunit. Furthermore, two variants exhibit a significant increased slope factor of voltage-dependent steady-state inactivation. We here present mutations in the β2 subunit gene of ASD patients that result in a retardation of inactivation behavior, thus phenocopying the monogenic Timothy syndrome mutations of CaV1.2. β2 subunit mutations may influence neuronal function or development in some ASD patients.

Jangsangthong, W., Kuzmenkina, E., Khan, I.F., Matthes, J., Hullin, R., and Herzig, S. (2010) . Inactivation of L-type calcium channels is determined by the length of the N terminus of mutant beta (1) subunits. Pflugers Arch 459, 399-411. Herzig, S., Khan, I.F., Grundemann, D., Matthes, J., Ludwig, A., Michels, G., Hoppe, U.C., Chaudhuri, D., Schwartz, A., Yue, D.T., et al. (2007) . Mechanism of Ca(v)1.2 channel modulation by the amino terminus of cardiac beta2-subunits. FASEB J 21, 1527-1538. Splawski, I., Timothy, K.W., Sharpe, L.M., Decher, N., Kumar, P., Bloise, R., Napolitano, C., Schwartz, P.J., Joseph, R.M., Condouris, K., et al. (2004) . Ca(V)1.2 calcium channel dysfunction causes a multisystem disorder including arrhythmia and autism. Cell 119, 19-31. Trikalinos, T.A., Karvouni, A., Zintzaras, E., Ylisaukko-oja, T., Peltonen, L., Jarvela, I., and Ioannidis, J.P. (2006) . A heterogeneity-based genome search meta-analysis for autism-spectrum disorders. Mol Psychiatry 11, [29] [30] [31] [32] [33] [34] [35] [36] Background: Brain serotonin (5-HT) has been implicated in the regulation of food-intake. The ingestive effects of 5-HT are mediated by a number of different receptor subtypes under which the 5-HT1A-receptor plays a central role. Former in vivo studies have shown an increased intake of food, elicted by 5-HT-receptor agonists. The aim of this behavioural pharmacologic project was to determine if the hyperphagic effect is mediated by presynaptic 5-HT1A autoreceptors in the raphe nuclei or by postsynaptic 5-HT1A heteroreceptors in serotonergic terminal structures. Methods: The effect of the 5-HT1A receptor agonist 8-OH-DPAT (0.1, 0.5 or 1.0mg/kg) was investigated on feeding behaviour in non-food-deprived young-adult and adult NMRI and transgenic L35 mice. L35 mice are characterized by an overexpression of postsynaptic 5-HT1A receptors. Results: The administration of the 5-HT1A receptor agonist induced hyperphagia in all groups of mice, except for the adult transgenic mice which showed no drug effect. Conclusion: The results confirm a key role of the 5-HT1A receptor in food intake. Further, we make the assumption that the hyperphagic effect of 8-OH-DPAT is mediated by presynaptic 5-HT1A autoreceptors in the raphe nuclei which decreases 5-HT function in the central nervous system. It can be speculated that the aberrant feeding behaviour of the adult transgenic mice refers to a possible opposite role of the postsynaptic 5-HT1A receptors. These receptors might affect the release of neuropeptides in the hypothalamus. The efflux transporter ABCC2 (MRP2) expressed at different compartment barriers is important for the elimination of various endogenous and exogenous compounds. With some evidence inflammatory processes regulate ABCC2 expression and cause changes of absorption, distribution and clearance of a number of xenobiotics. The investigation of the influence of interleukin (IL) 1β on ABCC2 mRNA and protein expression in various cell lines representing specific tissues is the aim of our study. A further aim is to characterize the signaling pathways regulating ABCC2 expression while inflammation. Three different cell lines a) HepG2 cells (liver tissue) and b) CaCo2 (colon tissue) both without naïve IL1β expression and c) SkHep1 cells representing physiological liver tissue with naive IL1β expression, were stimulated with different concentrations of IL1β (range 10 pg/ml to 10 ng/ml). Over a period of 48h samples were taken at defined time points. ABCC2 mRNA and protein expression were quantified by qRT-PCR and Western blot analysis, respectively. By using small molecule kinase inhibitors for signal transduction proteins (p38 MAPK, AKT, ERK1 and JNK) we analysed the signal transduction pathways associated with IL1β-mediated transcriptional ABCC2 regulation. On ABCC2 mRNA level an up-regulation in CaCo2 cells (1, 35 fold) and HepG2 cells (3, 6 fold) within the first hour after stimulation with 1 ng/ml IL1β was shown. In contrast SkHep1 cells demonstrated a decreased ABCC2 mRNA expression (0,59-0,62 fold) in comparison to unstimulated controls. The ABCC2 protein expression exhibited a time and IL1β dependent regulation as well. The analysis for the signal transduction showed for p38MAPK a moderat time dependet down regulated phosphorylation (15%) in HepG2 cells whereas it showed no effect in CaCo2 cells. Concluding, the expression of ABCC2 is regulated moderately by IL1b in a concentration and time-dependent manner. Interestingly, the effects are strongly tissue-dependent concerning ABCC2 expression and signal transduction pathways and show partly contradictory results. The regulation of the different signaling pathways is currently subject of ongoing investigations. Introduction: Despite the remarkable success of imatinib treatment of chronic myeloid leukemia (CML), therapy resistance emerged as a major clinical problem. The aim of this study was to identify microRNAs, which may serve as biomarkers for therapy response or predict pathways involved in pharmacoresistance of imatinib treatment.

Methods: Blood was collected from 21 CML-patients, ten of whom responded to imatinib therapy. After RNA extraction from leukocytes, we performed a TaqMan Low-Density Array screen to determine the expression of 667 microRNAs. Statistical analysis using the 2 -∆Ct method was performed. MicroRNAs showing a p-value<0.01 and a fold change>2 were considered to be significantly differently expressed. In addition, by using microRNA target prediction databases (TargetScan 1 , mirDB 2 , PicTar 3 , MicroCosm 4 , Diana microT 5 ), selected putative target genes were further functionally investigated by the DAVID bioinformatics database 6 .

Results: Comparing treatment-naïve responders and non-responders four microRNAs were identified to be deregulated that were predicted to target 97 genes, especially transcription regulators (21%). Pathway analysis showed that six of the predicted genes are relevant in cancer pathways, four of which play a role in CML (SMAD4, NRAS, RB1, RAF1). When comparing patients' expression profiles before and under treatment, seven microRNAs were identified to be deregulated in responders and five microRNAs in nonresponders. Ninety-nine targets of the latter include transcription regulators (19%), but also cellular transporters (18%, especially uptake transporters of the SLC-family). Most target genes are involved in MAPK signalling or endocytosis pathways. Conclusion: Analysis of microRNA expression profiles revealed four microRNAs involved in imatinib-response and 12 microRNAs deregulated during imatinib treatment. Predicted target genes code mainly for transcription factors as well as oncogenes relevant for CML and are involved in transporter expression and endocytotic processes.

Dissociations in the effects of beta2-adrenergic receptor agonists on cAMP formation and superoxide production in human neutrophils Brunskole I. 1 , Buschauer A. Activation of the β2-adrenergic receptor (β2AR), a classically Gs-coupled receptor, in neutrophil granulocytes results in an inhibition of inflammatory responses [1] , which could be further therapeutically exploited. The aim of the present study was to evaluate the effects of various β2AR ligands on cyclic adenosine 3',5'-monophosphate accumulation (cAMP assay) and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)induced superoxide anion production (O2 •assay) in isolated human neutrophils, which are a physiologically relevant native test system. cAMP concentration in neutrophils was determined by HPLC/tandem mass spectrometry, and O2 •formation was assessed by monitoring the superoxide dismutase-inhibitable reduction of ferricytochrome c.

(-)-Isoproterenol, (-)-adrenaline, salbutamol and dobutamine were more potent in inhibiting fMLP-induced O2 •production than in stimulating cAMP accumulation. (-)-Ephedrine and dichloroisoproterenol were devoid of any agonistic activity in the cAMP assay, but could partially inhibit fMLP-induced O2 •production at higher concentrations. Moreover, (-)-adrenaline and dobutamine were equi-efficacious in both assays whereas the efficacy of salbutamol was more than two fold higher in the O2 •assay. This suggests that salbutamol is able to stabilize a different receptor conformation than the other two ligands. Thus, ligand-directed signaling via β2AR can also occur in human neutrophils. In addition, differences between the data from neutrophils and recombinant test systems [2, 3] were noticed, pointing to the problem of insufficient comparability of effects in recombinant and native test systems. The investigation of β2AR antagonists on neutrophil granulocytes is subject of ongoing work, in order to find out whether pKB values of β2AR antagonists in the cAMP assay and the O2 •assay are different. Such differences were previously reported for β2AR antagonists in other test systems [4] . Moreover, studies with protein kinase A inhibitors should give deeper insight into the signaling events in neutrophils that result in inhibition of fMLP-stimulated O2 •production and clarify how cAMP increase interferes with this events.

Agonist-selective internalization of the human 5-HT2A receptor Buchborn T., Kahl E., Höllt V., Koch T. Otto-von-Guericke-Universität Magdeburg Institut für Pharmakologie und Toxikologie, Leipziger Straße 44, 39120 Magdeburg, Germany

The serotonin 2A (5-HT2A) receptor is a G protein coupled receptor and the molecular target of LSD-like hallucinogens. Downregulation of 5-HT2A receptors is an adaptive process considered relevant for the therapeutic action of diverse serotonergic antidepressants, such as SSRIs. Since the antidepressant targeting of 5-HT2A receptors, however, is largely restricted to indirect agonists and/or antagonists, little is known about the mechanisms and implications of their regulation by direct agonists. In the present study we, therefore, investigated the capacity of various agonists to regulate the human HA-tagged 5-HT2A receptor by internalization. Using immunocytochemical techniques in stably transfected HEK293 cells, we show that agonists differ in their capacity to internalize the receptor. Serotonin, quipazine and DOI are the agonists most efficaciously internalizing the receptor, DMT and methysergide, on the other hand, hardly internalize; other agonists like psilocin, ergotamine and LSD induce low to intermediate internalization. The specificity of the agonistic effect was demonstrated by the 5-HT2A selective antagonist ketanserin, which blocked the agonist-induced internalization. In additional experiments, we show that the internalized 5-HT2A receptors colocalize with A488-labelled transferrin receptors, and that the internalization can be blocked by high molar sucrose; these results are indicative of a clathrin associated sequestration of 5-HT2A receptors in recycling endosomes. Also, we demonstrate that the proteinkinase C activator PMA efficaciously induces 5-HT2A internalization in the absence of an agonist, and that the DOI-induced internalization can be blocked by the proteinkinase inhibitor staurosporine. We, thus, confirm previous findings that the activation of proteinkinases seems to be necessitated for the 5-HT2A internalization to occur. Overall, we conclude that the internalization of the human 5-HT2A receptor is agonist-selective, and employs a proteinkinase (possibly PKC) dependent, clathrincoated endosome associated pathway. As there is recent evidence that the regulation of 5-HT2(A) receptors by agonists might have antidepressant (-like) properties, knowledge about the agonist-selective processing of 5-HT2A receptors could help to identify agonists most promising for future (pre-)clinical research.

Non-clinical safety assessment of homeopathic medicinal products: criteria for establishing a first safe dilution Buchholzer M. -L., Werner C., Knoess W. BfArM Bundesinstitut für Arzneimittel und Medizinprodukte Zulassung 4, Kurt-Georg-Kiesinger-Allee 3, 53175 Bonn, Germany Like all human medicinal products the homeopathic medicinal products for human use must demonstrate adequate safety. In general, they are regulated according to the analogue non-clinical safety principles (Points to Consider on Non-Clinical Safety of Homeopathic Medicinal Products of Botanical, Mineral and Chemical Origin, adoption by HMA 2007). One particular approach is the recently introduced concept of a first safe dilution (FSD; INTRODUCTION TO THE LIST OF FIRST SAFE DILUTIONS, adoption  by HMA 2010) . This contribution summarizes the first experiences in establishing FSDs of a selection of given homeopathic preparations by BfArM. For a given preparation the major toxicological concern and available data set is identified. This determines the safety assessment route: food regulation, permitted daily exposure (PDE), threshold of toxicological concern (TTC) or lowest human recommended dose (LHRD/100). Finally the acceptable amount/tolerable daily intake is derived and the respective FSD is calculated. For example the draft evaluation for Reserpinum (Ph. Eur. method 4.1.1) and for Atropine (Ph. Eur. method 3.1.1 or 4.1.1) based on LHRD leads in each case to a suggested FSD of D7 related to 10 g of preparation. Furthermore, the draft evaluation for Potassium iodide (Ph. Eur. method 3.1.1 or 4.1.1) based on food legislation emerged a proposed FSD of D6 related to 10 g of preparation. The concept of FSD combines a scientific and at the same time pragmatic approach in differentiated risk assessment of homeopathic medicinal products.

Impact of myricetin and its methylated derivatives laricitrin, syringetin and myricetin-3`,4`,5`-trimethylether in C. elegans Büchter C. 1 , Ackermann D. Polyphenolic compounds ubiquitously present in herbal food are discussed to contribute to the health beneficial effects of a diet rich in vegetables and fruits. Additional to a strong antioxidative activity of various flavonoids, most of these substances display a variety of other pharmacological properties. We investigated the flavonoid myricetin found in several species of berries, as well as the methylated derivatives laricitrin, syringetin and myricetin-3`,4`,5`-trimethylether. In this study Caenorhabditis elegans was used as a model to explore the impact of myricetin and its methylated derivatives in vivo and to investigate molecular modes of action. Myricetin (100 µM) caused an increase in mean and median adult lifespan of C. elegans. This longevity effect was associated with a decrease of the aging marker lipofuscin as well as a decrease in ROS induction, measured by using the H 2DCF-DA assay.

However, myrictin failed to improve heat stress resistance, an attribute often associated with longevity in C. elegans. The methylated myricetin derivatives (100 µM) showed a decrease in lipofuscin accumulation and ROS induction and they further improved the heat stress resistance. In order to elucidate the basis of the life prolonging action of myricetin, we investigated its influence on factors known to have important functions in stress response and the regulation of aging, namely the FoxO homologue DAF-16, the NAD + -dependent protein deacetylase SIR-2.1 and the heat-shock transcription factor HSF-1, respectively. Lifespan extension by myricetin disappeared in daf-16 and sir-2.1 loss of function mutant strains, showing the effect is at least partially dependent on these signaling molecules. By using a hsf-1 loss of function mutant strain of C. elegans, it was further shown that the life prolonging effect of myricetin is independent of hsf-1.

In conclusion, our results indicate that the life prolonging effect of myricetin is at least in part dependent on daf-16 and sir-2.1, probably due to a modified expression of target genes.

Stimulatory and inhibitory control of phospholipase C-gamma 2 Bühler A., Walliser C., Becker L., Gierschik P. Universität Ulm Institut für Pharmakologie und Toxikologie, Albert-Einstein-Allee 11, 89081 Ulm, Germany

Activation of phospholipase C-γ2 (PLCγ2) upon B cell antigen receptor (BCR) stimulation has been implicated to be a critical step in the BCR-mediated calcium signaling. Therefore it is important to understand the mechanisms of how the activity of PLCγ2 is stimulated and inhibited. The mammalian PLCs are divided into six subfamilies, designated β, γ, δ, ε, ζ, and η. Within the PLCγ subfamily, the two PLCγ isoforms share a number of features that are distinct from those of the other PLC subfamilies. The most striking difference is the insertion of additional domains between the catalytic subdomains X and Y. This specific array (SA) contains a second, split pleckstrin homology (spPH) domain, consisting of two halves separated by two Src homology 2 (SH2) domains and one Src homology 3 (SH3) domain. There is abundant evidence in the literature that PLCs are autoinhibited in their basal state by structural elements within their X/Y linker, pointing to a conserved role of the X/Y linker in autoinhibitory regulation of PLC isozymes. Data from our group show that PLCγ 2 is also regulated by autoinhibitory elements within its specific array (Walliser et al., 2008; Everett et al., 2011) . Our recent data demonstrated that PLCγ2 is negatively regulated by its SA. Specifically, within the SH domain tandem, the C-terminal SH2 (SH2C) and the SH3 domain in combination, but not either one alone, cause the strongest autoinhibitory control of PLCγ2. PLCγ2 has been shown to be phosphorylated at tyrosine residues 753 and 759 upon BCR stimulation (Kim et al., 2004) . Both tyrosines are located in the linker between the SH2C and the SH3 domain, which we have shown to be the major elements involved in autoinhibitory regulation of PLCγ2. Interestingly, a novel phosphorylation site in PLCγ2 was found in non-small cell lung cancer (NSCLC) tissue which is located at tyrosine residue 733 (Rikova et al., 2007) . In this work, we demonstrate, for the first time, the activation of PLCγ2 by phosphomimetic mutations in these three positions and the functional interplay of the three tyrosine phosphorylation targets. Most interestingly, mimicking phosphorylation of Tyr733 is critical to fully activate the enzyme. The results not only point to a crucial role of PLCγ2 in pulmonary tumorigenesis, but also prompt and stimulate the search for the protein kinase involved in phosphorylating PLCγ2 at Tyr733.

Molecular characterization of hepatotoxic effects of perfluorooctanoic acid (PFOA) Buhrke T., Scharmach E., Lampen A. Bundesinstitut für Risikobewertung (BfR) Lebensmittelsicherheit, Max-Dohrn-Str. 8-10, 10589 Berlin, Germany Perfluorooctanoic acid (PFOA) is an industrial chemical that is used for the fabrication of numerous products with oil-, dirt-and water-repellent properties. PFOA is resistant to chemical, thermal and biological degradation and has become a global contaminant of soil, water, air and food in the meantime. The toxicological data of PFOA give cause for concern as the substance was shown to damage the liver of rodents and to impair embryo development. Currently, the hazard potential of PFOA for humans is controversially discussed. In this study the human liver cell line HepG2 was employed to analyse the hepatotoxic effects of PFOA on the cellular and on the molecular level. PFOA was shown to stimulate cellular proliferation at concentrations in a range between 5 µM and 25 µM. At concentrations higher than 25 µM the substance was cytotoxic to the cells (IC 50 47µM). Cytotoxicity was not due to apoptotic mechanisms as no increase of caspase activity was detected up to a level of 100 µM PFOA. On the molecular level PFOA is known to act as an agonist of the peroxisome proliferator-activated receptor alpha (PPARα), and the observed hepatotoxic effects in rodents are associated with PFOA-mediated PPARα activation. Here we show that PFOA has the capacity also to activate the human isoform of PPARα. Additional human nuclear receptors were tested for activation by PFOA, and PPARγ as well as the pregnane X receptor (PXR) were shown to be activated at high concentrations of PFOA whereas PPARδ and the liver X receptor alpha (LXRα) were insensitive to activation by PFOA. Notably, we observed a significant inhibition of the activity of the hepatocyte nuclear factor 4α (HNF4α) by incubating the cells already with moderate concentrations of PFOA at a level of about 1 µM. These findings indicate that additional, PPARα-independent mechanisms may contribute to the observed hepatotoxicity of PFOA. The elucidation of novel modes of action of PFOA is relevant for the ongoing risk assessment of the substance. S17 070 Human breast stem cells as a toxicological model for endocrine disruptors, such as soy isoflavones Stempin S. 1 , Bumke Scheer M. (Kao et al., 1995) . Two daughter cell lines were developed from M13SV1 after X-ray irradiation (M13SV1 R2) and an additional transfection with a mutated ERBB2 oncogene (M13SV1 R2-N1), resulting in high and low tumorigenicity respectively and showing a change in estrogen response after growth in minimal media (Wang et al., 2010) . Isolated isoflavones are currently widely used in the treatment of postmenopausal symptoms of women. According to the stem cell theory of carcinogenesis, breast stem cells are the ideal target for the proposed research. However, the epidemiological data to the effect of isoflavone intake on breast cancer is contradictory. Therefore, we want to develop a toxicological model using these human breast stem cell lines to test the effect of endocrine disruptors, such as soy isoflavones in a human relevant model. In the present study we analyzed the effect of the phytoestrogen genistein, the most intensively studied soy protein, on the differentiation of the 3 HBEC lines. The expression of different luminal epithelial cell markers, estrogen receptors and stem cell markers was measured on the mRNA level by quantitative real time PCR. The analysis of several of these markers was also performed on the protein level using western blot. Additionally, a broad number of genes related to breast cancer and estrogen receptordependent signal transduction were studied using a commercial PCR-Array. In parallel we are also analyzing the changes on the protein level using 2D gel electrophoresis. We want to use this panel of different markers to establish a toxicological model that can be used in the future to analyze a wide range of different endocrine disruptors.

Kao CY, Nomata K, Oakley CS, Welsch CW, Chang CC. (1995) Bronchial asthma is a common inflammatory disease of the airways whose occurrence has increased dramatically over the past decades. Histamine plays an important role in mediating the inflammatory response leading to characteristic symptoms like wheezing, coughing, chest tightness, and shortness of breath. Since antagonizing the histamine H1-receptor (H1R) shows no ameliorating effects on asthmatic symptoms, H4R antagonists may be new drugs for asthma therapy. In addition to the H3R-and H4Rselective antagonist thioperamide, the selective H4R antagonist 1-[(5-chloro-1H-indol-2yl)carbonyl]-4-methylperazine (JNJ7777120) is used in pharmacodynamic studies. A correct interpretation of the collected data requires the detailed knowledge of the pharmacokinetics of the applied substances. For this reason, we developed a fast and robust method based on high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) which allows the simultaneous quantitation of thioperamide and JNJ7777120 as well as the selective H3R antagonist 1-({4- [3-(piperidin-1-yl) propoxy]phenyl}methyl)piperidine (JNJ5207852) in murine plasma and lung tissue. The treatment of plasma samples based on protein precipitation performed with a mixture of methanol and 0.2 M ZnSO4 using 30 µL of plasma. Analyte extraction from lung tissue was achieved by treating 150 -200 mg of tissue with a mixture of ethanol and water followed by rigorous mixing using a FastPrep-System. Ten µL of the extracted samples were transferred to a Synergy Polar-RP 80A Mercury column (10 x 2mm; 4µm) connected to a Polar-RP Security Guard. Chromatographic separation was performed via a gradient using an acetate buffer (pH 5) and methanol at a flow rate of 0.4 mL/min. The analytical run-time was 5 minutes. For plasma samples the assay was linear over a concentration range of 0.078 -40 µg/mL for JNJ7777120 and JNJ5207852, and 0.313 -40 µg/mL for thioperamide, respectively. In tissues, thioperamide could be quantified in a concentration range of 0.008 -2 µg/sample, JNJ7777120 and JNJ5207852 in a range of 0.004 -2 µg/sample. Our results show that the developed HPLC-MS/MS method is suitable for the quantitation of all tested histamine-receptor ligands in murine plasma and lung tissue. The functionality of the heart greatly depends on strict homeostasis and interplay of a range of signalling cascades. Deregulation of either one is always harmful and eventually detrimental for life. Some of the most relevant signals in the adult heart are triggered by the stimulation of G protein-coupled receptors such as adrenergic or Angiotensin receptors. Those in turn are modulated by a small subset of kinases, the G protein-coupled receptor kinases (GRKs). Interestingly, GRKs, which for the longest time were believed to regulate only G protein-coupled receptors were shown to modulate also non-receptor-mediated signalling pathways. By now it is well documented that GRK5 plays important roles in both the physiological as well pathological setting of the adult heart. In spite of the important functions of GRKs in the adult heart, it must be assumed that GRK5, one of the two main cardiac GRKs, may also be involved in signal modulation in the course of heart development. Deregulation of GRK5-dependent pathways may very well be causal for impaired cardiac development up to congenital heart disease. In fact, GRK5 is already expressed during embryogenesis and we can detect it in the developing heart. However, GRK5 has not been studied yet for a potential function during embryonic development in general or heart formation in particular. We have established zebrafish as a very time and cost efficient vertebrate model to investigate the role of GRK5 on cardiac signalling and development. Tools for GRK5 specific loss-as well as gain-of-function analyses have been developed in our lab. We revealed an unexpected role of GRK5 in the development of left-right asymmetry in zebrafish. Clinically, this has been associated with disorders such as heterotaxy and other syndromes linked to ciliary dysfunction. Many of those disorders are known to affect proper heart development resulting for example in septum defects or in detrimental translocation of the outflow tract. Precisely, depletion of the close homolog of human GRK5 in zebrafish mirrors the human syndrome called heterotaxy by displaying randomized placement of inner organs, aberrant heart looping and disrupted valve formation in the heart. In addition, loss of zebrafish GRK5 results in a lower heart rate as well as dilatation of the embryonic heart at later stages of development. Therefore, we believe that GRK5 may potentially serve as a candidate gene for congenital heart disease.

Identification of a HCN3 interacting protein in mouse brain Cao-Ehlker X., Hammelmann V., Zong X., Fenske S., Biel M. Department of Pharmacy, Center for Drug Research Ludwig-Maximilians-Universität, Munich Center for Integrated Protein Science CIPSM, Butenandtstr. [5] [6] [7] [8] [9] [10] [11] [12] [13] 81377 München, Germany

Hyperpolarization activated cyclic nucleotide-gated cation channels (HCN) pass a depolarizing current (Ih) that is involved in cardiac pacemaking and the control of numerous basic functions in neuronal circuits. The four HCN channel types (HCN1-4) display specific expression pattern in brain suggesting that each channel fulfills a distinct physiological function. While HCN1, HCN2 and HCN4 channels have been studied in quite some detail there is only little information on the particular role of the HCN3 channel. As an important step towards achieving a better understanding of HCN3 function we set out in this study to identify proteins that are assembled with HCN3 in brain tissue. To this end we performed a yeast two hybrid screen with a mouse cDNA library using the HCN3 C-terminal domain as bait. Several proteins were obtained and confirmed for interaction with HCN3 using heterologous coexpression in HEK293 cells.

Here, we provide an in-depth analysis of the functional interaction between HCN3 and one of the identified interacting proteins (HIP3.1). We show that HIP3.1 physically binds to HCN3 channels in vitro and in vivo. Still several open issues remain to be clarified i.e. the precise function of TPC1 and its tissue-specific and subcellular distribution. Therefore we established a mouse model with a general deletion of TPCN1 and generated a series of TPC1 antibodies. Using these tools we investigate the closer molecular and vesicular environment by different biochemical approaches i.e. affinity purification from native tissue derived from wild-type and as a control from knock-out mice, density gradient based vesicle separation, fluorescence activated organelle sorting (FAOS), total internal reflection fluorescence (TIRF) and confocal microscopy. So far, we confirmed the TPC1 knock-out model by our self-generated TPC1 antibodies. TPC1 knockout mice are viable and do not show any obvious deficits. To isolate TPC1 containing vesicles or protein complexes, tissue or cell culture derived material was prepurified by sucrose density gradient centrifugation. For a subsequent mass spectrometric analysis this preparation was taken as a source material for coimmunoprecipitation or FAOS respectively. In another approach the migration pattern of TPC1 containing endosomes on linear density gradients was compared with a series of endolysosomal markers i.e. different Rab-, ER-, Golgi-and lysosomal antibodies. Potentially interesting markers were then in turn analyzed for their co-localization with TPC1 by confocal microscopy/TIRF. By combining these results with that from mass spectrometric analysis of FAOS samples we collect data to get detailed information on the precise endolysosomal distribution pattern of TPC1. FOXOs are involved in a wide spectrum of cellular functions, including cell proliferation, apoptosis and regulation of oxidative stress. In order to identify novel target genes of FOXO transcription factors and to achieve further insight into their role in cancer cells, DNA microarray analysis was performed using wild type MCF-7 breast cancer cells and MCF-7 cells overexpressing FOXO. We found that several genes involved in the TNF receptor/NF-κB pathway were differentially regulated. One of the genes that was identified to be up-regulated in FoxO4 overexpressing cells was A20 a negative regulator of NF-κB signaling pathway. At both mRNA and protein level FOXO4-dependent up-regulation of this ubiquitin modifying enzyme was confirmed. To determine whether A20 is a direct target of FOXO4, a luciferase reporter containing a 1.2 kb of the A20 promoter was co-transfected with different amounts of FOXO4 wild-type expression construct. FOXO4 induced a dosedependent increase in A20 promoter activity, supporting the assumption that A20 is a direct transcriptional target of FOXO4. Overexpression of FOXO4 led to decreased activity of NF-kB signaling pathway as confirmed by reporter gene and NF-kB specific ELISA assays. In addition, MCF-7 cells can be sentisized to TNF-α mediated cytotoxicity which is assocciated with a dimineshed activation of NF-κB. Altogether, we identified A20, an ubiquitin modifying enzyme, as a novel FoxO4 target gene. Our data implicate that sustained FoxO4 expression may be involved in regulation of TNF receptor/NF-κB pathway and leading to reduced cell survival. TRPM7 is a bi-functional protein consisting of a transient receptor potential ion channel segment linked to an α-type protein kinase domain. TRPM7 is essential for motility, proliferation and cell growth. Up-regulation of TRPM7 function is involved in anoxic neuronal death, cardiac fibrosis and tumor cell proliferation. Recently, we have demonstrated that the recombinant TRPM7 channel is inhibited by the known modulators of SK1-3 channels such as antimalarial plant alkaloid quinine, CyPPA, dequalinium, NS8593, SKA31, UCL 1684. The most potent of these compounds, NS8593 (IC50 1.6 µM), interferes with the regulation of TRPM7 by cytosolic Mg 2+ . Here we show that NS8593 (10 µM) fully and reversibly inhibits native TRPM7-like currents in HEK 293 cells, freshly isolated smooth muscle cells, primary podocytes and ventricular myocytes. Furthermore, we examined whether targeting of the native TRPM7 currents by NS8593 would impact cellular processes known to be affected by a genetic inactivation of TRPM7. We found that NS8593 (10-30 µM) suppressed motility of HEK 293 cells without a detectable effect on cell viability. Taken together, our findings indicate that NS8593 is a potent and reversible inhibitor of endogenous TRPM7 currents and may be a good candidate drug for pharmacological targeting of TRPM7.

Sulfur mustard (2,2'-dichlorodiethylsulfide; SM) is a highly toxic and mutagenic warfare agent classified as a weapon of mass destruction. As soon as SM was first used as a warfare agent, research aimed at the development of an effective antidote was launched. Early studies with first-generation inhibitors of poly(ADP-ribose) polymerases (PARP) have revealed promising therapeutic potential in SM-induced skin injury, but the underlying mechanism remains elusive. The current renaissance of PARP inhibitors in cancer chemotherapy has revived the discussion on their use for treatment of SM injury. Thus we established a comprehensive study aiming the elucidation of the role of PARP in SM pathology based on model substance 2-chloroethyl ethyl sulfide (CEES), which is not classified as warfare agent.

We have recently demonstrated that PARP becomes rapidly activated in living human keratinocytes (HaCaT) after treatment with CEES. The maximal PARP activity was observed 10 minutes after treatment with 3 mM CEES. The activation was transient and dose dependent. To our knowledge this is the first demonstration of PARP activation after treatment with mustards in the context of live cells. An important question is how PARP-1 becomes activated upon treatment with mustards. PARP-1 is a first-line protein involved in the cellular response to DNA strand breaks. However, mustards do not directly induce large numbers of such lesions. One possibility is that PARP-1 is activated by DNA breaks incorporated as base excision repair (BER) or nucleotide excision repair (NER) intermediates. Thus, we performed knockdown experiments of APE1 and ERCC1, i.e. endonucleases involved in BER and NER, respectively. The reduction of APE1 expression had no effect on PARP activity. Surprisingly, the knockdown of ERCC1 almost completely abolished the cellular PAR production after CEES treatment. The functional consequence of the ERRC1-PARP cross-talk with regards to adduct removal is under investigation. However, our present data indicate that PARP activity is not obligatory for the survival of cells upon CEES treatment, as revealed by the lack of effect of the potent PARP inhibitor ABT-888. Expression and activity of G protein coupled receptor kinase 2 (GRK2) are elevated in several conditions of compromised heart function. Although GRK2 inhibition has been characterized as a promising therapeutic strategy in heart failure, a specific GRK2inhibitor is not available. Raf kinase inhibitor protein (RKIP) inhibits Raf1 but it also acts as a physiological inhibitor of GRK2 upon phosphorylation by PKC at serine153. A detailed understanding of the RKIP/GRK2 interaction may help to identify inhibitory compounds for GRK2. Since phosphorylation often induces homo-oligomerization of proteins, we investigated whether this could be implicated in switching RKIP from a Raf1-into a GRK2-inhibitor. Co-immunoprecipitation assays showed that RKIP self-association was substantially increased after PKC-mediated phosphorylation of RKIP. RKIP mutants either lacking or mimicking S153 phosphorylation confirmed that this phosphorylation is indeed a prerequisite for RKIP/RKIP association. Cross-linking experiments with myc-tagged RKIP in living cells or with purified RKIP revealed that RKIP phosphorylation by PKC promotes RKIP dimers -not oligomers.

To test whether dimerization is a critical step for the association of RKIP with GRK2, we generated a peptide to inhibit RKIP dimerization. Intriguingly, the peptide did not only prevent RKIP dimerization but also attenuated RKIP/GRK2 association. This implicates, that dimerization of RKIP is essential to bind GRK2. To determine whether RKIP dimers consequently inhibit GRK2 activity, we established RKIP mutants with high tendency to form dimers. Subsequent functional analyses demonstrated that enhanced dimerization of RKIP indeed translates into increased GRK2 inhibition. We conclude that PKC-mediated phosphorylation of RKIP is important for dimerization and that these dimers are essential for GRK2 binding and inhibition. Our results reveal new insights in the molecular mechanism of RKIP/GRK2 interaction and will help to develop specific GRK2 inhibitors.

Expression and function of TRPM3 ion channels in epithelial MDCK2 cells Dembla S., Meiser J., Philipp S. University of Saarland Institute for experimental and clinical pharmacology and toxicology, Kirrberger Str. 1, 66421 Homburg, Germany TRPM3 proteins build Ca 2+ permeable cation channels [1] activated by steroids [2] and sensitive to increased temperatures [3] . TRPM3 channels are expressed in pancreatic ßcells as well as neurons of the dorsal root ganglion, where they act as mediators of insulin release [2] or as nociceptors of noxious heat, respectively [3] . However, Northern blots and in situ hybridization experiments revealed that TRPM3 is also expressed in epithelial cells of the choroid plexus and the ciliary body [1] as well as in the kidney. PCR analysis of different epithelial cell lines indicated that TRPM3 is also expressed in Madin-Darby canine kidney2 (MDCK) cells. Quantitative analysis of TRPM3 expression by qRT-PCR revealed a ~ 5 fold upregulation in MDCK2 cells grown in confluency compared to well separated, proliferating cells. In contrast the level of expression of TRPM7, a related ion channel described as regulator of proliferation in other cell types, remained constant. HEK293 cells overexpressing TRPM3 channels did not proliferate in the presence of the TRPM3 agonist pregnenolone sulphate. However, as indicated by impedance analysis, the proliferation of MDCK2 cells in the presence PregS was only slightly affected. When we analysed the transepithelial resistance (TER) of MDCK2 epithelial cells in transwells as a measure for the formation of tight junctions, we found that the TER of cells grown in the presence of PregS was reduced. Interestingly, Ca 2+ imaging experiments using Fura2 revealed that pregnenolone sulphate induces Ca 2+ entry in well separated MDCK2 cells but not in cells growing in confluency. We hypothesize that TRPM3 might act as a regulator of cell proliferation and/or the formation of tight junctions in MDCK2 cells. 

Inhibition of GRK2 by RKIP improves cardiac contractility and structure in a transgenic mouse model of heart failure Denzinger S., Schmitt J. P., Lohse M. J., Lorenz K. Institut für Pharmakologie und Toxikologie Pharmakologie, Versbacher Str. 9, 97078 Würzburg, Germany

The Raf kinase inhibitor protein (RKIP) has been identified as a physiological inhibitor of G-protein coupled receptor kinase 2 (GRK2). GRK2 initiates G protein coupled receptor (GPCR) desensitization. Since expression and activity of GRK2 are upregulated in human heart failure, it has been proposed that GRK2 inhibition may resensitize badrenergic receptor activity in heart failure patients. In this study, we evaluated chronic GRK2 inhibition by RKIP as a potential strategy to improve cardiac function in heart failure.

To analyse the effect of RKIP on heart failure, RKIP transgenic mice were crossed with mice carrying a mutation in phospholamban (PLN R9C ). PLN R9C causes severe heart failure and premature death in humans and transgenic mice. Cardiac function was significantly improved in the presence of RKIP as shown by left ventricular catheterization and echocardiography. Expression of heart failure marker genes ANF and BNP was indistinguishable between wild-type mice and mice co-expressing RKIP and PLN R9C . In line with these findings, the life span of double transgenic mice was significantly prolonged compared to PLN R9C transgenic mice. Slow calcium transport into the sarcoplasmatic reticulum was characterised as cause for dilatated cardiomyopathy of PLN R9C transgenic mice. Since Western blot analyses of RKIP transgenic heart lysates showed increased phosphorylation of important regulators of cardiomyocyte relaxation, we analysed calcium transients and contractility of isolated cardiomyocytes as possible mechanism of the RKIP mediated rescue. In the presence of RKIP, calcium reuptake into the sarcoplasmatic reticulum was accelerated and cardiomyocyte relaxation improved. Furthermore, coexpression of RKIP significantly attenuated pathological cardiac remodelling. Interstitual fibrosis and apoptotic cells were quantified in histological sections after Sirius Red-and TUNEL-staining. This study revealed a protective function of RKIP in a genetic mouse model of human dilated cardiomyopathy by improving cardiac contractility and attenuating interstitial fibrosis and apoptosis. A detailed understanding of this rescue may help to find a new therapeutic strategy to improve cardiac contractility in heart failure. Gαi-proteins comprise a group of three highly related members characterized by specific expression patterns. Based on previous work of Gi-mediated signaling pathways in cardiomyocytes and platelets, we checked Gαi expression in mouse heart and platelets. The analysis revealed the presence of Gαi2 and Gαi3 with Gαi2 as the predominant isoform. Gene-targeted mice lacking either Gαi2 or Gαi3 were analyzed to unravel the physiological role of Gαi-proteins in the cardiovascular system. Extraordinarily prolonged bleeding times in Gαi2-deficient animals were an obvious phenomenon. Detailed analysis using isolated platelets Gαi2-deficient mice exhibited reduced platelet activation and attenuated aggregation in response to stimulation by various agonists accompanied by reduced thrombus formation and diminished stability on a collagen-coated surface. Employing in vivo injury/thrombosis models revealed abrogated thrombus formation selectively in Gαi2-deficient mice. Comparable results were obtained in experiments using mice with megakaryocyte/platelet-specific Gαi2deficiency. To assess the pathophysiological consequences of platelet Gαi2 function, we challenged these mice in experimental models of myocardial and cerebral ischemia. The results clearly show that platelet-Gαi2-deficient mice were protected from both, myocardial and cerebral ischemia. In contrast, conventional Gαi2-deficient mice subjected to the heart ischemia/reperfusion model exhibited a significantly increased susceptibility to ischemic injury as compared to wild type controls. In contrast, Gαi3deficient mice were strongly protected from injury. Thus we suggest that Gαi2 and Gαi3 play distinct roles in major cardiovascular disorders pointing to specific, non-redundant functions of these two highly related Gαi isoforms. The cGMP signaling pathway is activated by nitric oxide (NO), natriuretic peptides (ANP, BNP & CNP), and cGMP-elevating drugs. It regulates several physiological functions such as smooth muscle relaxation, platelet inhibition, and cell growth and differentiation.

Recent studies indicate that cGMP signaling might also play a role in tumorigenesis, but the cellular and molecular mechanisms of cGMP's potential pro-and/or anti-tumor activities are not well understood. This study has examined the expression and function of components of the cGMP pathway in melanoma cells of murine and human origin. We have found that mouse B16 melanoma cells specifically express the alpha isoform of the cGMP-dependent protein kinase type I (cGKIalpha) but not the beta isoform. Treatment of intact cells with the membrane-permeable cGMP analog 8-Br-cGMP induced the phosphorylation of cGKI substrates, vasodilator stimulated phosphoprotein and phosphodiesterase 5. ANP and CNP, ligands of the membrane-bound guanylyl cyclase GC-A and GC-B, respectively, did also activate the endogenous cGMP/cGKI pathway. However, B16 melanoma cells did not respond to DEA-NO, which stimulates NO-sensitive soluble guanylyl cyclases. Interestingly, activation of cGMP/cGKIalpha signal transduction was associated with an increase in ERK1/2 and p38 phosphorylation, growth and migration of B16 melanoma cells. Similar results were obtained with WM1205 human melanoma cells. In conclusion, we have identified a GC-A/GC-B/cGMP/cGKIalpha pathway in melanoma cells, which stimulates tumor cell growth and migration in vitro. Pharmacologic inhibition of cGMP signaling may offer a promising strategy for the treatment of melanoma. An increasing body of evidence supports important roles for voltage-gated calcium channels in idiopathic generalized epilepsies (IGEs), however which calcium channels participate in IGE pathogenesis and how has yet to be fully understood. Recently, it has been proposed that Cav2.3 (R-type) and T-type calcium channels jointly contribute to oscillatory bursting in the reticular thalamus (RT) 1 , which is associated with absence epilepsy. Cav3.2 is one of the two T-type calcium channels known to be expressed in the RT 2 . It has been demonstrated that ablation of either Cav2.3 or Cav3.2 reduces susceptibility to experimentally induced epilepsy 3;4 and in addition that both channels share several pharmacological properties [5] [6] [7] . To gain further insight into interacting mechanisms of these two channels in epilepsy, we tested Cav2.3(-|-), Cav3.2(-|-) and Cav3.2(-|-)xCav2.3(+|-) mice side-by-side in the kainic acid model of epilepsy. We provide first in vivo data supporting a synergistic mode of action for Cav2.3 and Cav3.2 calcium channels in epileptogenesis.

The deubiquitinase CYLD regulates mechanisms of RIP1/RIP3-dependent necroptosis in neuronal cells Diemert S. 1 , Krieg S. 

vivo model of cerebral ischemia, we found, that CYLD -/-mice exhibit significantly reduced infarction volume compared to control littermates. Overall, these data reveal a role for CYLD in RIP1/3-dependent mechanisms of necroptosis in a model of glutamate toxicity in neuronal cells and further suggests CYLD-mediated mechanisms of neuronal cell death as a potential therapeutic target after acute brain injury in vivo.

Cyanamide-mediated inhibition of N-acetyltransferase 1 Dierolf D., Bonifas J., Blömeke B. University Trier Department of Environmental Toxicology, Universitätsring 15, Bldg. N, 54286 Trier, Germany Cyanamide has been used for decades for medical purposes in the treatment of alcoholism and for agricultural purposes as a plant growth regulator and bud-breaking agent. Its therapeutic effect is mediated by reversible inhibition of aldehyde dehydrogenase and it was reported to be metabolised in vivo mainly via coenzyme A dependent N-acetylation by N-acetyltransferases.

Reported to be a substrate for N-acetyltransferases, cyanamide has a different molecular structure to arylamines and hydrazines, the preferred substrates for Nacetyltransferases. Therefore a more detailed investigation of its interrelations with Nacetyltransferases was performed. We analysed NAT1 enzyme activities after incubation of THP-1 cells with cyanamide for 24h, and found that the metabolic conversion of the classic substrate para-aminobenzoic acid was significantly reduced at physiologically relevant concentrations. In detail a significant dose-and time-dependent NAT1 protein inhibition was observed for 100 and 1000 µM cyanamide using over-expressed human recombinant NAT1 (insect cell cytosol containing recombinant human NAT1*4). However, no inhibition was found in the presence of recombinant NAT2*4. As we also provide evidence that cyanamide is not metabolised via coenzyme A dependent Nacetylation in vitro by human NAT1 or NAT2 cytosol, by THP-1 cells or by human liver cytosol, we can conclude that this inhibition is not based on substrate-dependent downregulation of NAT1. Further mechanistic and kinetic studies indicated that cyanamide reacts with the active site cysteine residue of NAT1, leading to its rapid inhibition. Since the presence of the reduction agent dithiothreitol did not reverse the results it could be that it is possibly not caused by oxidative processes. In sum these data indicate that cyanamide is able to interact with cysteine residues of human NAT1, which causes its inhibition but not by a substrate-dependent mode of action. Taken together our results show, that cyanamide is not N-acetylated by human NATs, but might modulate NAT1 dependent detoxification and activation of arylamines.

Dissecting the signal transduction pathway of acute hypoxic vasoconstriction (HPV) in precapillary pulmonary arterial smooth muscle cells ( Low levels of oxygen in the pulmonary airways induce acute hypoxic pulmonary arterial vasoconstriction (acute HPV) redirecting blood flow to normoxic areas of the lung to assure optimal uptake of oxygen during ventilation. Acute HPV lasting several minutes occurs predominantly in the precapillary region of the pulmonary vascular tree [1] . Therefore, precapillary pulmonary arterial smooth muscle cells (PASMC) have been suggested as sensor as well as effector cells and TRPC6 a member of the classical transient receptor potential (TRPC) ion channel family was identified to be essential for the initiation of Ca 2+ influx and the subsequent contraction of PASMC [2] . However, the underlying oxygen sensor and the exact signal transduction pathway(s) in PASMC have not been fully elucidated yet. By using gene-deficient mouse models as well as downregulation of potential candidate proteins by specific small interfering RNAs (siRNAs), we aim to dissect signaling cascades of TRPC6 channel activation in acute HPV. For PASMC isolation and culture from mice we use a technique based on magnetic separation of intrapulmonary arteries originally developed in rats [3] . TRPC-expression in freshly isolated and passaged PASMC cultured in low (5%) and high fetal bovine serum (25%) was analyzed. Interestingly higher passage numbers resulted in a significant down-regulation of TRPC1 and TRPC6 the most predominantly expressed channel monomers in PASMC, while different serum concentrations resulted in no significant changes in their expression rates. SiRNAs were designed, transfected and successfully tested in HEK293 cells and PASMC. Initial results of the dissection of the signal transduction pathway activating TRPC6 and inducing acute HPV in PASMC will be presented.

References [1] Staub, N. C. (1985) . Site of hypoxic pulmonary vasoconstriction, Chest 88, 240S-245S. [2] Weissmann, N. et al. (2006) . Classical transient receptor potential channel 6 (TRPC6) is essential for hypoxic pulmonary vasoconstriction and alveolar gas exchange, Proc. Natl. Acad. Sci. U.S.A. 103, 19093-19098 TRPV5 is a highly selective calcium channel expressed in various tissues amongst others in placenta. The channel may be involved in transcellular calcium transport in epithelial tissues thereby playing some role in calcium homeostasis of the body. In the placenta TRPV5 is assumed to contribute to the maternal-fetal calcium transport. Most probably TRPV5 is part of a multiprotein channel complex but most of the components of this complex are unknown so far. Our aim is to find interaction partners of the TRPV5 protein in the placenta that might contribute to the regulation of the TRPV5 protein function. Therefore we expressed the intracellularly located N-and C-terminal parts of TRPV5 (aa 1-330 and 571-723) as TRPV5-GST (glutathione-S-transferase)-fusion proteins and used the purified recombinant proteins for pulling down proteins from human placenta cell extracts. The proteins pulled down by this approach were analysed by mass spectrometry. We identified several potential TRPV5 interacting proteins which were not associated with the GST protein only. One of the proteins which was highly enriched with the N-terminal part of the TRPV5 protein is Calpain 6. In contrast to the classical calpains (calcium activated cystein proteases), Calpain 6 is unique in that it lacks the cysteine residue in the active site. Calpain 6 is mainly expressed during embryogenesis and is reported to be involved in cytoskeleton stabilisation but with unknown function in placenta. The interaction between TRPV5 and Calpain 6 was confirmed in reciprocal pulldown experiments and the TRPV5 binding region for Calpain 6 was narrowed down by using overlapping N-terminal TRPV5-GST fusion proteins. After injection of TRPV5 cRNA into Xenopus laevis oocytes calcium uptake into the oocytes was measured; this uptake was largely reduced after co-injection of the Calpain 6 cRNA. In further experiments we want to study potential regulatory effects of the TRPV5 protein on the Calpain 6 function in cell culture models. 

Comparative studies on the effects of the human carcinogen inorganic arsenite and its recently identified thiolated metabolite thio-DMA V on human urothelial cells Ebert F., Leffers L., Unterberg M., Schwerdtle T. Universität Münster Institut für Lebensmittelchemie, Corrensstr. 45, 48149 Münster, Germany It has been demonstrated that chronic ingestion of 50-200 µg/day inorganic arsenic is associated with an increased risk for cancers of the skin, the lung and the bladder, but until now the underlying toxic modes of action are still under debate. In this context, in the last five years one main focus has been given to the role of human inorganic arsenic metabolism and nowadays it is generally accepted that human biomethylation contributes to inorganic arsenic induced carcinogenicity. Due to further improvements in arsenic speciation techniques recently a new thiolated arsenite metabolite, the thio analogue of the well known metabolite dimethylarsinic acid (DMA V ), the so called thiodimethylarsinic acid (thio-DMA V ), has been discovered in human biological samples. After synthesizing and analytically characterizing this metabolite (Bartel et al. 2011 , J Toxicol. 2011 we investigated its toxic effects in direct comparison with iAs III in human urothelial cells. Thereby cell cycle studies revealed a G2/M-and S-phase arrest as well as subG1 peak formation in case of thio-DMA V . Moreover, thio-DMA V induced apoptosis (subG1, caspase 3 activity) at lower concentrations and earlier time points as compared to iAs III . Most likely this is partly due to the higher cellular bioavailability of thio-DMA V (AAS/ICP-MS). Regarding genotoxicity, a generation of DNA single strand breaks (alkaline unwinding technique) as well as an increased formation of reactive oxygen species (ROS, DCFH-DA-fluorescence) occurred only at high cytotoxic concentrations. However, thio-DMA V strongly increased H2O2 induced ROS formation at very low nanomolar concentrations, which might result in cogenotoxic effects. Since our earlier studies have shown a strong inhibition of H2O2 induced poly(ADP-ribosyl)ation especially by trivalent methylated arsenic metabolites, actual studies investigate the impact of thio-DMA V on cellular poly(ADP-ribosyl)ation, PARP-1 gene expression, protein level and cellular cleavage, which might hopefully give further hints regarding the mode of action behind thio-DMA V induced apoptosis.

Mitochondrial dysfunction in models of Alzheimer´s disease Eckert A. Universitäre Psychiatrische Kliniken Basel Neurobiology Laboratory, Wilhelm Klein Strasse 27, 4012 Basel, Switzerland The histopathological characteristics of Alzheimer's disease (AD) are amyloid-ß (Aß) containing plaques and neurofibrillary tangles (NFTs) as well as neuronal and synaptic loss. Until today, the underlying mechanisms of the interplay of plaques and tangles remained unresolved. There is increasing evidence that mitochondrial dysfunction might be a possible link, as revealed by studies in several APP and tau transgenic mouse models. Recently, we examined mitochondrial function in a novel triple transgenic mouse model (pR5/APP/PS2) -tripleAD mice -that combines both pathologic features of the disease in brain. Using comparative, quantitative proteomics (iTRAQ) and mass spectroscopy we found a massive deregulation of 24 proteins, of which one-third were mitochondrial proteins mainly related to complexes I and IV of the oxidative phosphorylation system (OXPHOS). Remarkably, deregulation of complex I was related to tau, whereas deregulation of complex IV was Aß dependent, both at the protein and activity levels. The tripleAD mice showed synergistic effects of Aß and tau already at the age of 8 months, resulting in a depolarized mitochondrial membrane potential. At 12 months, the strongest defects on OXPHOS, synthesis of ATP and reactive oxygen species were exhibited in the tripleAD mice, again emphasizing synergistic, ageassociated effects of Aß and tau in impairing mitochondria. Evidences from AD post-mortem brain as well as cellular and animal AD models indicate that Aß and tau protein trigger mitochondrial dysfunction through a number of pathways, such as impairment of oxidative phosphorylation, elevation of reactive oxygen species production, alteration of mitochondrial dynamics, and interaction with mitochondrial proteins. Moreover, recent reports indicate that Aß may also interact directly with intracellular proteins such as the mitochondrial enzyme ABAD (Aß binding alcohol dehydrogenase) in executing its toxic effects. Mitochondrial dysfunction occurs early in AD, and Aß's toxicity seems to be in part mediated by inhibition of ABAD. In total, a vicious cycle as well as several vicious circles within the cycle, each accelerating the other, can be drawn emphasizing the synergistic deterioration of mitochondria by tau and Aß. Olesoxime is a novel mitochondrial-targeted compound that is orally active and crosses the blood brain barrier. The cholesterol-oxime targets proteins of the outer mitochondrial membrane and represents a promising drug candidate for neurodegenerative diseases 1 . We evaluated Olexoxime's neuroprotective effects against mitochondrial dysfunction in an animal model for Alzheimer`s disease (AD). Dissociated brain cells (DBC) and mitochondria were isolated from brains of C57/BJ6-Thy1-APPSL (AD-mice) mice that were fed with 600 mg Olesoxime/kg feed for 3 months. Drug plasma levels reached approx. 600 ng/ml. Respiration of isolated mitochondria were significantly diminished in AD-mice due to reduced complex I, I+II and COX activities. Consequently, mitochondrial membrane potential (MMP) was significantly reduced in DBC from AD-mice. Olesoxime normalized respiration chain complex activities and the MPP. To further evaluate the beneficial effects of Olesoxime on complex I activity, we challenged DBC with rotenone ex vivo and observed that Olesoxime treatment was protective. To further clarify the mode of action, we analyzed the ability of Olesoxime to prevent opening of the mitochondrial permeability transition pore (mPTP) in vitro using energized brain mitochondria by measuring Ca 2+ -and atractyloside (ATR) induced swelling. The opening of mPTP precedes apoptosis and can be induced by mitochondrial dysfunction due to calcium overload, oxidative stress, elevated phosphate concentrations or adenine nucleotide depletion. Olesoxime prevented Ca 2+ -as well as ATR induced mitochondrial swelling. ATR inhibits the adenine nucleotide translocase (ANT) that requires appropriate membrane properties to mediate mitochondrial permeability transition (MPT). Since cholesterol (CHO) and its derivates represent potent modulators of membrane viscosity, we related the effects of CHO and Olesoxime on mPTP opening to membrane properties. Both, CHO and Olesoxime reduced the flexibility of membrane acyl-chains in energized mitochondria and prevented ATR induced mPTP opening. However, CHO didn`t prevent Ca 2+ -induced mPTP opening, indicating a different mode of action for Olesoxime. Our data confirm Olesoxime as drug candidate against mitochondrial dysfunction, which is considered to play a pivotal role in neurodegenerative diseases. The work was supported by the European Union under the 7th Framework Program for RTD -Project MitoTarget -Grant Agreement HEALTH-F2-2008-223388.

Several inflammatory glomerular kidney diseases are accompanied with a massive production of reactive oxygen species (ROS) that may attack the glomerular filtration barrier by affecting podocyte function and may contribute to apoptotic or necrotic cell death of mesangial cells. Otherwise, ROS also trigger fine-tuned signaling processes that may result in cell proliferation or cell migration. To define such redox-driven signaling devices, we performed a non hypothesis-driven proteomic approach, to identify homo-or heteromeric protein complexes induced by ROS. To this end, protein lysates of human podocytes were treated with or without hydrogen peroxide (250 µM) for 10 min. Thereafter, the cell lysates were subjected to diagonal 2D gel electrophoresis and putative redox-affected proteins were analyzed by MS/MS-analysis. By this approach, we could identify a series of proteins that form interprotein-disulfide bonds in a redoxdependent manner. One of those proteins could be characterized as the regulatory subunit of protein kinase A (R-subunit of PKA), which belongs to the family of serine/threonine kinases. To evaluate whether ROS is capable to activate PKA also in a more physiological setting, we treated rat mesangial cells with PDGF-BB to induce ROS formation and we could demonstrate that PDGF-BB induces dimerization of R-subunits in a redox-dependent manner. To demonstrate whether PDGF-BB induces PKAdependent pathways, we analyzed the effects of PDGF-BB on phosphorylation of serine 157 of vasodilater stimulated protein (VASP) a classical target of PKA. In fact, PDGF-BB induced VASP phosphorylation independently of intracellular cAMP levels. Moreover, elevating cAMP levels via activation of adenylate cyclase with forskolin did not change the dimerization state of R-subunits. PDGF-BB-induced dimerization of the R-subunits and subsequent phosphorylation of VASP was blocked by diphenyljodonium (DPI), indicating activation of a NADPH oxidase is essential for PKA activity. Taken together, we demonstrate a redox-dependent activation of PKA by PDGF-BB and this may hint also for a probably protective role of ROS in rat mesangial cells. Testing the potential sensitizing capacity of chemicals is currently done by using the murine local lymph node assay (LLNA). Animal welfare and EU cosmetics directive demands alternative methods to animal tests. The purpose of this study was to establish an in vitro assay for the prediction of skin sensitizers. Based on the finding that the majority of skin sensitizers are electrophilic or have the potential to be metabolized to electrophilic substances, it is assumed that they can activate the Nrf2-Keap1-antioxidant response element (ARE) regulatory pathway. Here, we report the results obtained from the LuSens assay that detects electrophilic chemicals using the Nrf2 pathway. The cell line LuSens was derived from immortalized keratinocyte HaCaT cells and carries a luciferase reporter gene under the control of an ARE-element from the rat NADPH quinone reductase NQ1. The LuSens assay was in house validated with a panel of 54 chemicals and cosmetic ingredients including the 22 performance standard substances of the local lymph node assay. The predictivity of this assay was compared to the predictivity of the murine LLNA and to human patch test data and can be considered as reliable screening approach (accuracy of 83% compared to human data). However, in order to cope with the complex multi-step mechanism of skin sensitization, an integrated approach of in vitro assays mimicking several steps was designed; thereof, the ARE-dependent gene activation represents one module.

Time-resolved fluorescence ligand-binding assays for parathyroid hormone receptors Emami-Nemini A. 1 Ligand-binding studies represent essential tools for pharmacological research on G protein-coupled receptors. In recent years, time-resolved fluorescence gained significant relevance as readout for ligand binding studies. However, ligand-binding assays for parathyroid hormone receptors (PTHRs) utilizing fluorescent parathyroid hormone (PTH) were missing. Therefore, we generated various fluorescent PTH analogues which exhibit properties of native PTH in terms of affinity, potency and internalization. For the purposes of academic and commercial research, we utilized labeled PTH to set up three time-resolved fluorescence assay formats: (I) Classical separation binding assay, based on time-resolved fluorescence and suitable for native receptors; (II) Homogeneous timeresolved fluorescence resonance energy transfer (HTRF) based on tag-lite technology for high through-put screening; (III) HTRF based on antibodies, a synergistic approach using HTRF with minimized receptor modification. This work will facilitate the development of new drugs directed to the PTHR as well as fundamental research on the PTHR.

Anandamide Production in Eosinophilic Granulocytes is Independent of IL-5 and Eotaxin Stimulation Engeli S., Reinke J., Zörner A., Tsikas D., Jordan J. Medizinische Hochschule Hannover Institut für Klinische Pharmakologie, Carl-Neuberg-Straße 1, 30625 Hannover, Germany Introduction: Some animal and in vitro studies suggest that endocannabinoids exert anti-inflammatory effects. Specifically, inhaled anandamide reduced the obstructive effect of leukotrien D4 in airways, and a specific cannabinoid receptor agonist significantly reduced pulmonary inflammation in guinea pigs. We have recently shown that segmental bronchial allergen challenge is associated with significant increases of anandamide concentrations in bronchoalveolar fluid of patients with asthma. The concomitant increase in eosinophilic counts, eotaxin and IL-5 concentrations in bronchoalveolar fluid led us to hypothesize that anandamide is produced by eosinophilic granulocytes in response to chemotactic stimuli.

Peripheral eosinophilic granulocytes were isolated from whole blood by means of Percoll gradient centrifugation and magnetic separation employing CD16antibodies conjugated to magnetic beads. Isolated cells were counted and anandamide measurements were typically performed in whole cell lysates of 1.5x10 6 eosinophils. We stimulated eosinophils with varying concentrations of IL-5, eotaxin-1 (CCL11), eotaxin-2 (CCL24), and eotaxin-3 (CCL26). To prevent anandamide degradation, a specific fatty acid amide hydrolase (FAAH) inhibitor (oloxa) was employed. Anandamide concentrations and FAAH activity were determined by stable isotope dilution using LC-MS/MS protocols.

Results: First, we confirmed the ability of eosinophilc granulocytes to synthesize anandamide. However, cellular anandamide content could only be measured when FAAH was effectively blocked with oloxa, and strong FAAH activity was demonstrated in eosinophils. With oloxa, typical anandamide concentrations were in the range of 2-4 pM/1.5x10 6 eosinophils. Neither IL5 (50-500 pg/ml), nor any of the eotaxins (50 ng/ml either alone or in varying combinations) did stimulate anandamide production after 30min of incubation.

Our results suggest that chemotactic molecules like eotaxin and IL-5 are not responsible for increased anandamide formation in eosinophils during allergen challenge. In a next step, the effects of anandamide on eosinophils and bronchial epithelial cells need to be determined. The suprisingly high FAAH activity in eosinophils may point to an alternative pathway facilitating prostaglandin and leukotriene synthesis by production of arachidonic acid.

Screening Methodology for estimatation of dermal absorption in vitro Fabian E., Goth C., Guth K., Mehling A., van Ravenzwaay B., Landsiedel R. BASF SE Experimentelle Toxikologie, Carl-Bosch-Strasse 38, 67056 Ludwigshafen, Germany Dermal absorption is used in the evaluation of the effectiveness of pharmaceutical or cosmetic formulations, but often dermal absorption is a critical parameter in risk assessment of pesticides or chemicals. Therefore, knowledge of dermal absorption is e.g. helpful in formulation development. Skin absorption is routinely measured in vivo or in vitro following OECD TG 427 or 428. However, these tests are complex, time consuming and expensive. Therefore, a method was developed to allow simple and rapid screening. The experiment uses dermatomized skin in modified Franz type diffusion cells. 10 µl of test substance preparation are applied to the skin preparation. After 6h, the skin is washed and the amount of penetrated substance is quantified. The receptor fluid and the washing solutions are optimized for subsequent analyses by LC-MS. We performed dermal absorption screenings in parallel to our routine guideline studies and demonstrated a good correlation of the results of both study types: the total recovery found in the screening studies is somewhat lower than in the corresponding guideline studies but is always in an acceptable range above 80%. The efficacy of the skin washing procedure is lower than under routine conditions, most probably due to the change to an LC-MS-compatible washing solution.

Overall, the dermal absorption screening is an easy, fast and cost-effective screening method for the estimation of dermal absorption of a wide variety of test substances and formulations. The p53 tumor suppressor protein is frequently inactivated in human cancers by diverse mechanisms. Owing to its fundamental role in the maintenance of genomic stability and cancer prevention, p53 is an attractive target in cancer therapy and several approaches were pursued to restore p53 function in tumor cells. Polyamidoamine (PAMAM) dendrons are positively charged molecules with a systematically branched structure that interact with the negatively charged cell membrane, inducing cellular uptake via endocytosis. In the present study, biotin-labeled p53 protein was attached to a dendronized streptavidin nanocarrier to facilitate its internalization into different tumor cell lines. First, biotin-substituted PAMAM dendrons were conjugated with streptavidin to allow formation of the dendronized streptavidin nanocarrier. The nanocarrier displayed uptake into HeLa cervix carcinoma and A549 lung adenocarcinoma cells without detectable cytotoxicity. Biotin-labeled p53 was then conjugated to the dendronized streptavidin, preserving its specific DNA-binding in vitro. Immunoblot analysis revealed efficient internalization of biotin-p53 into HeLa cells in the presence of dendronized streptavidin. In line with this finding, specific cellular uptake of biotin-p53 was observed by confocal microscopy, which showed a cytoplasmic and peri-nuclear localization in HeLa, A549 and SaOS osteosarcoma cells. The internalized biotin-p53 also partially co-localized with early endosomes. Importantly, the delivery of biotin-p53 into p53-deficient SaOS cells resulted in impaired cell viability and upregulation of caspase 3/7 activity, demonstrating its biological functionality. This study intriguingly demonstrate the efficient delivery of functional biotin-p53 into different tumor cell lines using the novel streptavidin nanocarrier, which can be further modified to allow cell-type specific targeting and combined with cytotoxic drugs such as doxorubicin.

Identification of novel AhR target genes in rat liver oval cells Faust D. 1 , Vondracek J. The aryl hydrocarbon receptor (AhR) is a transcription factor involved in physiological processes, but also mediates most, if not all, toxic responses to 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD), some polycyclic aromatic hydrocarbons (PAHs) and dioxin-like polychlorinated biphenyls (PCBs), such as PCB126. Activation of the AhR by these ligands leads to its dimerization with ARNT and transcriptional activation of several phase I and II metabolising enzymes. While it is generally accepted that many PAHs are thereby transformed to genotoxic metabolites, this classical signalling pathway so far failed to explain the tumour promoting effects of the nongenotoxic compounds TCDD and PCB126. Thus, there is an urgent need to define genetic programmes orchestrated by AhR to unravel its role in physiology and toxicology. We have recently shown that treatment of rat liver oval cells with TCDD or PCB126 leads to a release from contact-inhibition involving activation of the AhR, elevation of JunD protein levels and transcriptional activation of cyclin A (1, 2) . Loss of contact-inhibition is one hallmark of tumour promotion. To better understand AhR-driven pathways we identified the transcriptional programme using high density microarrays in response to PCB126. Already 6 h after treatment, 69 genes were found to be upregulated and 76 genes downregulated indicating that these are direct AhR-dependent target genes. DAVID analysis revealed that these genes are involved, for instance, in drug and lipid metabolism, cancer pathways, TGF-b signalling and cell-cell communication. Ten of the 69 genes were selected for confirmation by semi-quantitative RT-PCR. Using the AhR inhibitor CH-223191 and siRNA directed against AhR and ARNT, we further demonstrated that AhR-and ARNT-function is required for transcriptional activation of the selected genes. Finally, we identified the transcription factor Foxq1as a novel AhR target protein in rat liver oval cells. Although the function of Foxq1 is poorly understood, it has been shown very recently that Foxq1 is overexpressed in colorectal cancer and is involved in epithelial-mesenchymal transition in breast cancer cells. Its function in AhRmediated tumour promotion, however, remains to be determined. The practical relevance of histamine H1 and H3 receptors in the brain can be easily deduced since H1 receptor antagonists of the first generation have a sedative effect and an inverse H3 agonist, pitolisant (close to its introduction to the market), is active against excessive daytime sleepiness associated with narcolepsy. In this context the question arises whether also H2 and H4 receptors possess a practical relevance in the brain. To this end, we examined whether the electrically evoked 3 H-noradrenaline release in superfused human cerebral cortex slices is affected by agonists at the above receptors. The H2 agonist impromidine 10 µM failed to affect noradrenaline release in human cortex slices although it facilitated noradrenaline release in guinea-pig cortex slices; the maximum extent of facilitation was 50 %, the pEC50 was 6.9 and the pA2 of the H2 antagonist ranitidine against impromidine was 7.2. With respect to H4 receptors there is some controversy in the literature whether they occur in the brain at all. However, we were able to detect H4 receptor mRNA in the human and mouse cortex by the reverse transcriptase polymerase chain reaction. In cortex slices of either species, noradrenaline release was not affected by the H4 agonist 4-methylhistamine 10 -30 µM but inhibited by histamine 10 µM via H3 receptors by 28 and 55 %, respectively. In mouse cortex membranes, 4-methylhistamine 30 µM also failed to affect 35 S-GTPγS binding although R-α-methylhistamine 3 µM, acting via H3 receptors, increased it by 20 %. In conclusion, H2 receptors facilitating noradrenaline release are detectable in the isolated guinea-pig but not human cortex. Despite the presence of H4 mRNA in the brain, functional readouts of this receptor, i.e. modulation of noradrenaline release (humans, mice) and modulation of 35 S-GTPγS binding (mice), could not be shown.

Murine Cx40 promoter activity is dependent on the transcription factor CREB Fels B., Nunes F., Schmitz W., Müller F. U. Westfälische Wilhelms-Universität Münster Institut für Pharmakologie und Toxikologie, Domagkstraße 12, 48149 Münster, Germany

Connexin 40 (Cx40) is a gap junction protein expressed in atrial myocytes and the ventricular conduction system, mediating the electrical intercellular communication in the myocardium. Alterations in Cx40 function were linked to the pathophysiology of atrial fibrillation and heart failure. In the heart, cAMP dependent gene transcription is regulated by members of the CREB/CREM/ATF family which bind to cAMP responsive elements (CREs). Similar to the human Cx40 gene promoter, the murine promoter contains one CRE. Cardiomyocyte-specific overexpression of a CREM-isoform (CREM-Ib∆C-X) led to Cx40 down-regulation, suggesting that CREB related transcription factors are involved in Cx40 gene regulation. In order to study the functional role of CREB in the regulation of the Cx40 promoter we have generated a luciferase based murine Cx40 promoter reporter gene construct and monitored its activity in a permanent cell line upon overexpression of constitutive-active CREB (caCREB) and a non-phosphorylatable dominant-negative CREB (dnCREB) isoform. Surprisingly, overexpression of caCREB and dnCREB both lead to a reduction of Cx40 promoter activity (caCREB 46% ± 5% vs control 100% ± 3%; p<0.05 vs control , n=15), dnCREB 45% ± 2% vs control 100% ± 3%; p<0.05 vs control, n=18).

The activity of the murine connexin 40 promoter is modulated by CREB. Both caCREB and dnCREB led to Cx40 down-regulation, which could be explained by induction of inhibitory transcription factors CREB/CREM/ATF1 transcription factor family, which in turn could suppress Cx40 promoter activity. (Supported by the DFG)

Results: FXa increased PAR-2 mRNA, protein and cell-surface expression and augmented PAR-2-mediated mitogenesis. PAR-1 expression was not influenced. The regulatory action of FXa on PAR-2 was concentration-dependent and mimicked by a PAR-2 selective activating peptide. The thrombin inhibitor argatroban or PAR-1 gene silencing did not influence FXa-stimulated PAR-2 expression. FXa increased oxidative stress and expression of the NADPH oxidase subunit NOX-1 in SMC. NOX-1 gene silencing prevented FXa-stimulated PAR-2 regulation, as did ebselen and catalase. Exogenous hydrogen peroxide increased PAR-2 expression and mitogenic activity. FXa induced nuclear translocation and PAR-2 DNA binding of nuclear factor kB (NFkB). Inhibition of NFkB prevented FXa-stimulated PAR-2 expression. In separate studies, FXa promoted PAR-2 mRNA stabilisation through increased human antigen R (HuR)/PAR-2 mRNA binding and cytoplasmic shuttling. HuR gene silencing abolished FXa-stimulated PAR-2 expression. Conclusion: Expression and mitogenic activity of vascular PAR-2, but not PAR-1, is upregulated by FXa. This action involves transcriptional and post-transcriptional mechanisms mediated through NOX-1-containing NADPH oxidase and its downstream effectors hydrogen peroxide, NFkB and the mRNA stabilising protein HuR. Continued generation of FXa by the mural thrombus, and the autoregulatory feedback control of PAR-2 may maintain the inflammatory and proliferative state of the injured vessel, thereby promoting vascular remodeling.

The mRNA stabilising factor HuR is a critical regulator of human proteaseactivated receptor 4 Aim: We recently reported that functional expression of PAR-4 thrombin receptors is induced in human saphenous vein SMC exposed to high glucose. This effect could be attributed in part to transcriptional mechanisms mediated through NFkB but the contribution of post-transcriptional effects such as mRNA stabilisation is not known. This study explored the potential role of the mRNA stabilising factor human antigen R (HuR) in the regulation of PAR-4. Methods: Human saphenous vein SMC were serum-deprived prior to study. Gene expression was determined by realtime PCR, protein expression by western blotting. Gene silencing utilized commercially available siRNA. HuR binding to PAR-4 mRNA was determined by immunoprecipitation ("pull-down") PCR. Results: High glucose (25 mM vs 5.5 mM) slowed PAR-4 mRNA degradation in the presence of actinomycin D. PAR-1 mRNA decay was not affected. HuR binding to PAR-4 mRNA and nucleo-cytosolic shuttling was enhanced by high glucose, total HuR protein expression was not affected. HuR siRNA abolished the high glucose-stimulated induction of PAR-4 mRNA. Hydrogen peroxide (H 2O2) also induced cytosolic HuR shuttling and increased PAR-4 mRNA and total protein expression. The role of endogenously generated H2O2 in the regulatory effect of high glucose on PAR-4 expression was investigated with the NADPH oxidase inhibitors apocynin/diphenyliodonium (to prevent H2O2 generation) and cell-permeant catalase (to degrade cellular H2O2). Both approaches prevented the stimulatory effect of high glucose on PAR-4 expression. Cyclic AMP has been reported to suppress HuR activation and in the present study, the cyclic AMP stimuli forskolin and cicaprost (prostacyclin analog) suppressed basal HuR shuttling and PAR-4 transcript stability. Cicaprost also attenuated high glucose-induced HuR binding to PAR-4 mRNA and as a consequence normalised PAR-4 expression and inflammatory signalling in high glucosetreated cell. Conclusion: The regulation of PAR-4 thrombin receptors in human vascular SMC is critically dependent on the mRNA stabilising actions of HuR. Through activation of HuR, high glucose and other HuR stimuli such as Ang II and exogenous H2O2, increase PAR-4 expression, while cyclic AMP agonists such as prostacyclin oppose this effect. Such interactions could potentially represent a fine-tuning mechanism to control PAR-4 expression and ultimately also the mitogenic and inflammatory actions of thrombin in the vessel wall. Nucleoside diphosphate kinase B (NDPK B) is a member of a family of ubiquitously expressed enzymes required for nucleoside triphosphate synthesis. Thus, they are involved in the regulation of a variety of cellular processes, e. g. G protein mediated signal transduction. However, whether NDPK B has a specific role in the regulation of angiogenic processes in endothelial cells is unknown. Therefore, we studied the function of NDPK B in the vasculature in a developmental, an ischemia-induced and an in vitro model of angiogenesis. Firstly, depletion of NDPK B expression was achieved by morpholino-mediated knockdown in zebrafish embryos in which the developing vasculature can be visualized by EGFP expression in the endothelium. 72h post fertilization, NDPK B knockdown larvae showed a dramatic inhibition of intersegmental and dorsal longitudinal anastomatic vessel formation compared to control injected fish. This phenotype could be rescued by early re-expression of NDPK B. Secondly, ischemia driven angiogenesis was studied in NDPK B-depleted mice and wildtype littermates after excision of the left femoral artery. Hind limb blood flow was assessed by laser Doppler perfusion imaging immediately before and after ligation (day 0) and on postoperative days 3, 7, 14, 21, 28, and 35 . A significant reduction of recovery was observed in the NDPK B depleted mice at days 3 and 7. Thirdly, in vitro-sprouting angiogenesis was analyzed in human umbilical vein endothelial cell (HUVEC) spheroids with and without siRNA-mediated NDPK B knockdown. Vascular endothelial growth factor (VEGF)induced sprouting was significantly attenuated by NDPK B knock down by more than 50% in comparison with control transfected HUVEC. We conclude from these results that NDPK B is an essential regulator of angiogenesis. The loss of NDPK B may specifically interfere with the VEGF-induced migration and proliferation during endothelial sprouting.

Ethylene oxide in blood of ethylene-exposed volunteers Ethylene (ET) is a commercially important high volume industrial chemical. Inhaled and endogenous ET is metabolized in mammals to ethylene oxide (EO), which is carcinogenic in rats and mice. Until now, no data on the oxidation of ET in ET-exposed humans has been published.

In the present study, we investigated the formation of EO in four male adult volunteers exposed for 4 hours to constant atmospheric ET concentrations of 5, 20, or 50 ppm by means of a breathing mask. During exposure, ET concentrations were measured in inhaled and exhaled air by GC/FID and EO concentrations in venous blood by GC/MS. Rates of ET metabolism were obtained from the product of the differences in the ET concentrations with the pulmonary ventilation. In each subject, linear correlations were found between the ET exposure concentration and the rate of ET metabolism or the EO concentration in blood. Mean rate of ET metabolism was 5.5 ± 1.4 nmol/h/ppm/kg body weight. Steady-state concentrations of EO in blood differed by a factor of 2 between the volunteers. These inter-individual differences likely reflect the polymorphism of glutathione S-transferase theta, the main EO metabolizing enzyme in human liver. Mean EO concentration in blood at steady state was 1.5 ± 0.08 nmol/l blood per ppm of ET. The data will be used for validating a physiological toxicokinetic model which will describe the ET related EO tissue burdens in rodents and humans. The model predictions will support risk evaluations of ET. Financially supported by the Lower Olefins Sector Group of Cefic.

In vitro effect of STW11 on human dendritic cells Fink C. 1 , Bonaterra G. A. Extracts of Echinacea (purple coneflower) are used in the prevention and therapy of infectious diseases. The medicinal product STW 11 contains the extract from purple coneflower, and in addition, extracts of monkshood, venom of honey bee and bushmaster snake in homeopathic dilutions. Previous studies showed a stimulation of the cellular and humoral immune response. Dendritic cells (DCs) are antigen presenting cells that act at the interface of the innate and adaptive branches of the immune system. During stages of DC differentiation, the ability to internalize antigens varies and decreases during maturation. In this study, we determined the influence of STW11 on the expression of maturation related genes (CD1a, CD83), cytokines (TNFα, Il-4, IL-12), chemokines (CCR7), major histocompatibility complex II(MHC-II) and Toll-like receptors (TLR2, TLR4). In mature (mDC) and immature DC (iDC) using real-time RT-PCR. Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats of human volunteers by densitygradient (Ficoll ® ) and seeded in 6 well plates. Non-adherent cells were eliminated. To induce iDC development, 50 ng/ml IL-4 and 50 ng/ml granulocyte macrophagecolony stimulating factor (GM-CSF) were added. At day 6, maturation was induced by addition of lipopolysaccharide (LPS) at a final concentration of 1µg/ml and cultured for additional 3 days.

After incubation with different concentrations (0. In iDC, compared to control, we found a significantly increased expression of CD83 (2.1-2.6 fold) and TNFα (2.6-3.3-fold) genes after treatment with 0.1-5% STW11, respectively, but no effect was found on the expression of CD1a, IL-4, IL12, ADAM19, CD11c, CD40,TLR2, TLR4, MHC-II and CCR7. In summary, these data demonstrate a stimulatory effect of STW 11 in iDC and especially in mDC, concerning an increase of various genes related to maturation (CD83), immunomodulation (TNFα, CD40), adhesion (CCR7) and antigen presentation (MHC-II) and are in accordance with the therapeutic use in infectious diseases. Waterproofing sprays are widely used consumer products containing for example fluorinated polymers or silicon based compounds dissolved in alcohols or volatile petroleum distillates. There have been repeated reports on cases of severe acute respiratory disorders especially when using products that newly entered the market. It is hypothesized that impairment of the pulmonary surfactant by deposition of inhaled respirable particles of the active compound is one of the main causes of the acute lung injury. Since the inhalation toxicity cannot be predicted a priori based on the physical and chemical properties of the formulation, proper test strategies are required to ensure consumer safety. We propose to combine screening tests addressing both, exposure and acute lung toxicity. The exposure potential of the spray product is characterized by determining the release fraction of the active compound in the respirable particle size range under conditions relevant for the product application. This is carried out by spraying defined quantities of the product into a control volume and measuring the concentration of health related size fractions. This procedure takes into account spray ageing, especially size reduction of the droplets due to solvent evaporation. The isolated perfused lung is used as a model for testing acute toxicity. Ventilated rat lungs are exposed to aged aerosols with proper particle size of approximately 1 µm MMAD generated from the liquid spray formulation. Lung compliance and lung resistance are continuously monitored during exposure. Dose dependent deviations from the normal values (without exposure) are used as read-out parameters. Using the combined procedure, different sprays could be ranked according to their realistic exposure risk and, most importantly, sprays with known lung toxicity could be uniquely distinguished from those that have been shown to be safe. In its current stage of development the simple test method is recommended for screening of substances only.

Induction of oxidative damage in calf thymus DNA by the Fusarium mycotoxin zearalenone after metabolic activation with liver microsomes Fleck S. C., Pfeiffer E., Metzler M. KIT -Institute of Applied Biosciences Chair of Food Chemistry, Adenauerring 20a, 76131 Karlsruhe, Germany Zearalenone (ZEN) is an estrogenic mycotoxin produced by Fusarium species. The adverse effects of ZEN and its reductive metabolite zearalenol (ZEL) are often compared to those of 17-beta-estradiol (E2) and estrone (E1). These endogenous estrogens are associated with an increased risk for cancer, which may be mediated by two mechanisms, i.e. (i) hormonal activity and (ii) genotoxic effects by P450-catalyzed metabolic activation to catechols (Wang et al., Chem Res Toxicol 23, 1365 . Like E2 and E1, ZEN and ZEL exhibit marked estrogenicity and also undergo aromatic hydroxylation to catechol metabolites (Pfeiffer et al., Mol Nutr Food Res 53, 1123 . The aim of the present study was to examine the formation of catechol metabolites of ZEN by liver microsomes of various species and their potential for redox cycling. Catechol metabolites are frequently associated with the generation of reactive oxygen species and subsequent oxidative damage of DNA, for which 8-oxo-7,8-dihydro-2'deoxyguanosine (8-oxo-dG) is a common biomarker. The propensity of the catechol metabolites of ZEN and ZEL to cause the formation of 8-oxo-dG in isolated calf thymus DNA was determined using a LC-ESI-MS/MS method. To this end, ZEN was incubated with microsomes from human, rat, mouse, bovine and porcine liver as well as with human CYP1A2, and the incubations were extracted with ethyl acetate. The extract was analyzed with LC-MS and then added to a solution of calf thymus DNA in the presence of copper(II) ions and NADPH. The formation of 8-oxo-dG could be demonstrated with each extract. The levels of 8-oxo-dG correlated directly with the extent of catechol formation, which increased from steer to swine to human to mouse to rat microsomes. 15-Hydroxylated ZEN/ZEL, which is the major catechol, was more reactive than 13-hydroxylated ZEN/ZEL to form 8-oxo-dG.

In conclusion, our study has shown that the catechol metabolites of ZEN are highly reactive and give rise to oxidative DNA damage in vitro. In addition, recent research from our laboratory revealed that the catechols of ZEN are less efficiently inactivated by catechol-O-methyl transferase than the catechols of E2 and E1. The genotoxic potential of ZEN may constitute another biological activity in addition to the well-known estrogenicity.

Supported by Deutsche Forschungsgemeinschaft (Grant ME 574/32-1) and "Food and Health" of KIT.

Thrombin regulates expression of sphingosine kinase-1 (SPHK-1) in human vascular smooth muscle cells -inhibition by dabigatran reduces vascular SPHK-1 expression and atherosclerotic burden in vivo Flößer A. 1 Results: Thrombin induced a time-and concentration-dependent (1-100 nmol/L) increase in SPHK-1 mRNA and protein expression in human saphenous vein SMC, n=6-7. This was mimicked by a synthetic PAR-1 ligand. Inhibition of SPHK-1 attenuated thrombin-induced SMC proliferation but not SMC migration (n=5). The regulatory action of thrombin on SPHK-1 expression was suppressed by siRNA against the mRNA stabilisiserHuR. In thrombin-stimulated SMC, HuR binding to SPHK-1 mRNA and subsequent nucleo-cytosolic shuttling was enhanced. Accordingly, thrombin induced SPHK-1 mRNA stabilisation in SMC in the presence of actinomycin D. In ApoE-deficient mice, long-term treatment with the direct thrombin inhibitor dabigatran significantly reduced aortic SPHK-1 expression by 50% (n=5) and plaque size by 35% compared to control animals (n=10). Conclusions: Thrombin induces SPHK-1 expression and S1P synthesis in vascular SMC via the mRNA stabilising protein HuR. This leads to increased SMC proliferation. Inhibition of thrombin by dabigatran treatment in vivo attenuates progression of plaques possibly by reducing SPHK-1 expression.

Mycotoxin contamination and cytotoxicity of grain mill products Typical grain mill products from North-Rhine Westphalia, i.e. grains, flour, wholemeal flour and bran (from wheat, rye and spelt) as well as typical by-products (outsourced fractions) from the milling process were analysed for their mycotoxin content by LC-MS/MS. The cytotoxicity of sample extracts with known mycotoxin composition was then assessed in V79 cell cultures by means of the neutral red uptake assay, in parallel with pure reference mycotoxin mixtures. Extracts from flour and wholemeal flour samples with low levels of deoxynivalenol and enniatin B (from not detectable to 0.4 µg/g DON and 0.5 µg/g EnnB) induced no measurable cytotoxicity. On the other hand, although mycotoxin contamination levels were also rather low in bran, these samples induced strong cytotoxicity: Extracts of bran derived from rye and spelt were more cytotoxic than those of wheat bran. The cytotoxic effects of the bran samples cannot be related to their mycotoxin content as comparable concentrations of pure mycotoxins and mycotoxin combinations tested in parallel were not cytotoxic. By-products from certain stages of the milling process (sorting and waste fractions) were found to contain mycotoxins at rather high levels: Enniatin B was detected in nearly all samples, and also T-2 toxin, HT-2 toxin, ergotamine, ergocornin and deoxynivalenol were present. Waste sample extracts with notable mycotoxin levels (up to 6 µg/g DON, 7 µg/g EnnB, 16 µg/g ergotamine, 50 ng/g HT-2 toxin) exert pronounced cytotoxicity in V79 cells. The cytotoxicity of these samples was somewhat stronger than expected when compared with mixtures of reference mycotoxins tested in parallel. In summary, the tested flour and wholemeal flour extracts contained only low levels of mycotoxins and were not cytotoxic. In contrast, bran samples showed cytotoxicity which cannot be explained solely by their mycotoxin content. This unexpected observation in real samples and combination effects of mycotoxin mixtures require further studies. The AhR is a ligand-activated transcription factor that mediates the toxicity of dioxins and related compounds. Upon ligand binding the AhR translocates into the nucleus and dimerizes with ARNT to modulate gene expression, e.g. of CYP1A1. Recently, we have shown that UVB irradiation of human keratinocytes results in activation of the AhR and associated EGFR signaling leading to an enhanced expression of CYP1A1 and proinflammatory COX-2, respectively. The initial step is the UVB induced intracellular formation of the tryptophan photoproduct 6-formylindolo [3,2b] carbazole (FICZ), a high affinity AhR ligand. Thus, the FICZ activated AhR is an important mediator of the DNA damage independent part of the UVB response. Our current study aims to identify further aspects of AhR mediated UVB responses. Therefore, we analysed changes in protein expression, proliferation and apoptosis in AhR+/+ and AhR-/-keratinocytes (NCTC 2544) by western blot, flow cytometry and BrdU incorporation.

UVB exposure of NCTC cells led to a dose-dependent increase in apoptosis. Compared to AhR+/+ cells, AhR-/-cultures exhibited an increased amount of apoptotic cells. This finding was confirmed in irradiated AhR+/+ cells, pretreated with the AhR antagonist 3'methoxy-4'-nitroflavone. Moreover, the proliferation of sham as well as UVB irradiated AhR-/-cells was significantly decreased. In AhR-/-cells we found a reduced expression of checkpoint kinase 1 (Chk1), an important cell cycle regulator that arrests the cell in G2/M upon DNA damage. Interestingly, UVB exposure led to a higher net phosphorylation of Chk1 in AhR-/-cells, indicating that this pathway is responsible for the observed AhR-dependent differences in proliferation and apoptosis. Further expression analyses of Chk1 client proteins emphasize our hypothesis. In conclusion our study identifies the AhR as an anti-apoptotic player in UVB irradiated human NCTC cells. Therefore, we propose that the AhR is a suitable target to prevent UVB induced skin diseases.

Synthetic progestins exert divergent effects on thrombosis in a murine model of atherothrombosis Background: Medroxyprogesterone acetate (MPA), a synthetic progestin often used in postmenopausal hormone replacement therapy, has previously been described to be pro-thrombotic in a murine model of accelerated atherosclerosis. However, nothing is so far known about effects of progestins with receptor profiles different from MPA (i.e. agonism or antagonism of mineralocorticoid-or androgen-receptors), such as drospirenone, levonorgestrel and norethisterone acetate. Methods: Apo -/mice were bilaterally ovariectomized (OVX) and substituted subcutaneously with MPA, drospirenone, levonorgestrel and norethisterone acetate as well as the respective placebo pellets for 90 days on a Western-type diet. Subsequently, thrombosis was induced by photochemical injury to the right carotid artery using Rose Bengal and a green light laser. Results: Compared to placebo, animals substituted with MPA showed significantly shortened times to occlusion of the right carotid artery (placebo MPA: 50.0 ± 5.8 min. vs. MPA: 33.4 ± 4.2 min., n = 6 -9, p < 0.05). In contrast, drospirenone, levonorgestrel or norethisterone acetate did not alter thrombotic responses. However, at least drospirenone (placebo drospirenone: 53.7 ± 5.4 min. vs. drospirenone: 47.5 ± 4.8 min., n = 7 -8) and levonorgestrel (placebo levonorgestrel: 47.0 ± 3.2 min. vs. levonorgestrel: 41.8 ± 2.1 min., n = 6) showed a trend towards shorter times to stable occlusion. Furthermore, analysis of aortic gene expression revealed that in aortas of MPA-treated mice expression of matrix-metalloproteinase 9 (MMP-9) was induced as compared to placebo-treated mice. Conclusion: MPA, a progestin with glucocorticoid effects, exerts a pro-thrombotic effect that is either progesterone-or glucocorticoidreceptor-dependent while progestins with receptor profiles different from MPA do not show a significant pro-thrombotic effect. Furthermore, the pro-thrombotic effect exerted by MPA may be associated with increased expression of MMP-9, a metalloproteinase being known to destabilize atherosclerotic plaques and make them more prone to rupture.

Rapid Screening for Mitochondrial Toxicity In Vitro Using an Oxygen-sensitive Phosphorescent Probe Freyberger A. Bayer HealthCare BPH GDD GED Toxikologie -P & CP, Aprather Weg 18, 42096 Wuppertal, Germany Impaired mitochondrial function has been implicated with disease, aging, and druginduced toxicities. Analyzing mitochondrial respiration (MR) rates is one of the most informative ways to assess mitochondrial function as it provides information on the the bioenergetic capacity of a tissue, however, previously measurements using polarography (Clark electrode) were cumbersome with only low throughput. In this work we explored Luxcel's water-soluble phosphorescent oxygen-sensitive probe MitoXpress TM for the assessment of mitochondrial toxicity in freshly isolated male rat liver mitochondria (RLM) in a 96-well plate format using glutamate/malate (20 mM/0.5 mM) and succinate (25 mM) as respiratory substrates. Inhibition of mitochondrial complexes I to IV, adenosine triphosphate synthetase and the adenosine diphosphate (ADP) / adenosine triphosphate (ATP) antiporter by rotenone, thenoyltrifluoracetone (TTFA), antimycin A, potassium cyanide, oligomycin, and atractyloside was readily detected in ADP-stimulated RLM. Use of the two different substrates in parallel allowed to discriminate complex I inhibition by rotenone from complex II inhibition by TTFA, whereas downstream of these complexes inhibition by the other model inhibitors occurred independent of the substrate used. Decoupling of MR from oxidative phosphorylation by carbonylcyanid-p-trifluormethoxyphenylhydrazone (FCCP) was detected best in the absence of ADP. Compared to polarographic measurement, the use of an oxygen-sensitive probe is superior with regard to assay cycle time and sample throughput and offers new opportunities to characterize and screen for mitochondrial toxicity, but also to support studies on mitochondria-mediated modes of action of new chemical entities. The murine local lymph node assay (LLNA) and the guinea pig maximization test (GPMT) have been used to study the sensitization potential of a series of unsaturated compounds by Kreiling et al. (2008) . We have examined the same substances in the loose-fit coculture-based sensitization assay (LCSA), developed by our working group (Schreiner et al., 2007) . Eight unsaturated compounds [oleic acid (OA), linoleic acid (LA), linolenic acid (LnA), undecylenic acid (UA), fumaric acid (FA), maleic acid (MA), squalene (SQ), 1-octyn-3-ol (OC)] and succinic acid (SuA) were investigated using a coculture of keratinocytes and dendritic cell-related cells (DCrc). Sensitization potential was quantified by flow cytometry measuring the increase of CD86 expressed on DCrc (EC50 = half maximal effective concentration). A pronounced induction of CD86 at low concentrations was seen with LA, LnA and OA (EC50: 3, 5 and 5 µmol/L, respectively). UA exhibited an intermediate response (EC50: 307 µmol/L). With OC and MA, we observed effects at higher concentrations only (EC50: 1340 and 2293 µmol/L). No significant increase of CD86 was observed with FA, SuA and SQ. Because of poor solubility, SQ could not be studied adequately. Induction of CD86 was generally observed at concentrations which did not cause a major impairment of cell viability. Our results show a high degree of concordance with those obtained by the GPMT, except for OA. In comparison with the results of the LLNA, those compounds which showed a strong effect in the LLNA (OA, LA, LnA) also induced an increase of CD86 at low concentrations, whereas those with low stimulation indices in the LLNA induced no significant increase of CD86 (FA, SuA) or only at higher concentrations (UA). We observed a discrepancy between the tests with MA and OC, causing a strong stimulation of the murine lymph nodes, while the expression of CD86 was increased at high concentrations only. We assume that MA and OC might be false-positives in the LLNA, because they were also negative in the GPMT. Background: The first step in elimination of many cationic drugs is their uptake from the blood into hepatocytes and renal proximal tubular cells by the organic cation transporter 1 (OCT1) and OCT2, respectively. The pivotal role of OCTs in the excretion of cationic drugs raises the possibility of drug-drug interactions in which one drug reduces OCTmediated elimination of a second drug. Although many psychoactive drugs are cationic at pH 7.4 and some of these have already been recognized as OCT inhibitors, a systematic screen of this class of compounds is missing. Methods: We screened a drug library of 50 most frequently prescribed psychoactive drugs (inpatient prescriptions in Germany, at least 4 million DDD each) for their inhibitory interaction with OCT1 and OCT2. Human embryonic kidney (HEK) cells stably overexpressing OCT1 or OCT2 and the prototypical OCT substrate 1-methyl-4phenylpyridinium (MPP+) were used as a test system. Cells transfected with the empty vector were used as controls.

Results: At 20 µM, 59% and 51%, respectively, of the tested compounds significantly decreased OCT1-and OCT2-mediated uptake of MPP+. The most potent inhibitors (inhibition >75%) of OCT1 were chlorprothixen and clomipramine, whereas olanzapine, clomipramine and doxepin were the most potent inhibitors of OCT2. In contrast, neither at 20 µM nor at 200 µM carbamazepin, haloperidol, lithium, moclobemide and valproic acid did significantly inhibit MPP+ uptake into HEK-OCT1 or HEK-OCT2 cells. There was a significant correlation between the degree of OCT1 and OCT2 inhibition (p Conclusions: Our results demonstrate that inhibition of OCT function by psychoactive drugs has to be considered as a potential mechanism underlying drug-drug interactions.

Considering estimated peak sinusoidal concentrations e.g., of chlorprothixen and clomipramine between 30 and 80 µM in humans, inhibitory interactions of these compounds with hepatic OCT1 have to be taken in account. Our data will help to create a chemoinformatic model to predict potential OCT-dependent interactions of psychoactive drugs with the hepatic or renal elimination of coadministered drugs. This project is supported by the German Federal Ministry of Education and Research (BMBF), project grant No. 01 EX1015B.

Cardiac gene expression is altered during the development of hypertrophy and heart failure compared to the healthy heart. The molecular mechanisms controlling gene expression in cardiac failure are only partially known. DNA methylation is one epigenetic mechanism that regulates long-term changes in gene-expression. To elucidate whether DNA methylation is altered during the development and progression of chronic heart failure, genome-wide DNA methylation profiles were determined in myocardial biopsies from control patients and patients with cardiac hypertrophy or failure.

Cardiac biopsies were obtained from patients with aortic aneurysm who served as control and did not show clinical signs of chronic heart disease (EF: 58±3 %, n=3) and from patients with aortic stenosis. The latter group was subdivided according to the ejection fraction into hypertrophic (EF: 63±7 %, n=3) and failing patients (EF: 28±1 %, n=3). After bisulfite conversion of extracted DNA, the methylation status of genomic DNA was quantified using the Infinium® HumanMethylation450 BeadChip (Illumina). This microarray allows analysis of more than 485,000 methylation-sites throughout the whole genome at single-base-pair resolution. These experiments identified 1280 CpG sites in hypertrophic samples and 1365 CpG sites in failing samples which were differentially methylated compared to control specimens (delta >15%; p<0.05). 523 CpG sites were significantly altered in both aortic stenosis groups compared with control hearts. From these CpGs, 496 sites were altered concordantly in hypertrophic and failing samples. Analysis of regions harbouring distinct CpG densities revealed that most changes occured in shelf regions of CpG islands whereas the methylation status in the CpG islands was more stable. Further analysis showed that differences in methylation were most frequent in gene body, enhancer and 3`UTR regions. Specifically 9 probes spanning a CpG-island at the promotor region of the muscle-specific serine kinase 1 (SRPK3) showed diminished CpG-methylation in hypertrophic (-11.5±0.02%) and failing (-11±0.02%) as compared to control biopsies.

Remarkably, no alterations of DNA-methylation were observed in loci of classic marker genes of chronic heart failure like NPPA, SERCA, CTGF, MYH6 or MYH7.

These results indicate that DNA methylation is specifically altered in chronic heart disease but does not affect classic marker genes of chronic heart failure. Gliomas are the most abundant type of primary brain tumor in the central nervous system in adults. The current standard of Glioblastoma multiforme (GBM) therapy is surgery followed by radiotherapy and chemotherapy. However the morbidity and mortality of GBM remain very high and the median survival period is only 15 months even with treatment. Therefore it is important to identify novel drugs to reduce GBM cell proliferation.

Purine-analogues (PA) are well known for their anti-proliferative effects on eukaryotic cells. In this study novel PA were synthesized and the library of substance-derivatives was tested using different GBM cell lines namely LN18, U87-MG and GL261. The effect on proliferation and viability was assessed by using BrdU and Resazurin assays. Using these in vitro methods we were able to identify several compounds with cytotoxic and anti-proliferative effects in vitro showing IC50 values in the deeper µM range. Cytotoxicity of selected compounds was further analyzed by assessment of caspase 3 and propidium iodide based cell cycle FACS analysis to discriminate between apoptosis and cell cycle arrest.

Based on these data purine-derivatives might inhibit proliferation and induce apoptosis in glioma cells. As a result we hypothesize that these compounds could be potentially interesting for the drug-development of GBM therapy and therefore a clue for chemical modifications. Further studies are required to identify the exact underlying mechanism of action of the tested purine-analogues.

The Biological Role of Adenosine Receptors in Brown Adipose Tissue Gnad T. 1 Brown adipose tissue (BAT) is responsible for basal and inducible energy expenditure in mammals. BAT contains large amounts of mitochondria and is highly vascularized. BAT lipolysis and thermogenesis are stimulated by sympathetic neurons. Importantly, recent findings indicate that adult humans possess metabolically active BAT 1 . Here, we analyzed the expression and function of adenosine receptors in BAT. Adenosine receptors (AdoR) are members of the superfamily of G protein-coupled receptors. There are four subtypes of AdoRs in humans referred to as AdoRA1, A2A, A2B and A3. They are widely expressed in tissues and mediate a variety of cellular functions, mostly due to their regulation of cAMP levels within cells. Interestingly, it has been shown that adenosine can either inhibit or stimulate lipolysis in white adipocytes through AdoRA1 or A2A, respectively 2 . However, the role of adenosine in the differentiation of brown preadipocytes to adipocytes and in BAT function is not clear. To analyze the role of AdoRs in BAT, we use preadipocytes isolated from BAT of newborn mice and subjected them to a differentiation protocol. 3 Abundance of AdoRA1, A2A, A2B and A3 mRNA was measured using qPCR. All four receptor subtypes are present in preadipocytes with AdoRA2B being the most abundant. AdoRA1, AdoRA2A and AdoRA3 are significantly transcriptionally upregulated -albeit at varying degree -during differentiation. AdoRA1 is upregulated 4.4 fold (+/-0.3 fold) and 11 fold (+/-0.49 fold) at day 4 and at day 7, respectively, as compared to preadipocytes (n=5). AdoRA2A is 23 fold (+/-1.96 fold) upregulated at day 4 and 57 fold upregulated (+/-2.48 fold) at day 7, respectively (n=4). AdoRA3 was found upregulated 3.6 fold (+/-0.46 fold) at day 7 (n=4). In contrast to this, AdoR2B was downregulated to 0.87 fold (+/-0.09 fold) at day 4 and to 0.69 fold (+/-0.04) day 7 compared to preadipocytes (n=5).

To investigate the functional role of AdoR in BATi cells, we analyzed lipolysis in mature cells after acute treatment with specific agonists and antagonists. We observed that AdoRA2A activation by CGS21680 significantly increased lipolysis by 88% (+/-0.33%) compared to untreated control. Moreover, AdoRA1 antagonist PSB36 increased lipolysis by 35% (+/-0.05%) (n=4). In conclusion, AdoR are highly regulated during brown fat cell differentiation. Lipolysis of mature brown fat cells is significantly increased by AdoRA2A agonist or AdoRA1 antagonist, respectively. Munich Heart Alliance, München, Germany Activation of the sympathetic nervous system and the subsequent activation of βadrenergic receptors (βARs) through catecholamines represents the strongest mechanism to increase cardiac function. However, long-term activation of cardiac βARs is clearly detrimental and β-blockers have been introduced as an effective treatment modality in cardiac failure. Despite their central role in cardiac physiology and disease, our knowledge about the intracellular mechanism of βAR stimulation is confined to a few targets and is likely incomplete.

Here, we report a functional proteomics approach to directly assess the entire phosphoproteome of βAR-stimulated mouse hearts in vivo.

To identify proteins that are phosphorylated in response to β-adrenergic stimulation in vivo, we treated mice with isoproterenol or, as a control, with propranolol. After lysis of hearts and tryptic digest, phosphopeptides were enriched by TiO 2 or immobilized metal ion affinity chromatography (IMAC). Subsequent analysis of eluated peptides by tandem mass spectrometry (MS/MS) mapped several phosphopeptides to cardiac proteins, among which known mediators of βAR signaling such as phospholamban, troponin I and myosin binding protein C. We then employed multiple reaction monitoring (MRM) as a quantitative approach to assess changes of phosphorylation after βAR stimulation. Using this combination of MS approaches, we identified 39 peptides with PKA consensus phosphosites that were more abundantly detected under βAR stimulation. Among those, we found myozenin-2 (MYOZ2) and G protein signaling modulator 1 (GPSM1, also termed AGS3) as proteins previously not related to βAR signaling. We validated the βAR-dependence of phosphorylation at these sites in isolated cardiomyocytes by in vivo labelling or phosphoepitope-specific antibodies. Current efforts aim at the functional characterization of these novel candidate mediators of βAR signaling in the heart. Taken together, we report the β-adrenergic phosphoproteome of the mammalian heart in vivo. We have identified several new targets of βAR signaling that may represent essential factors in cardiac physiology and disease. Background: Drug measurement in autopsy material is normally used to investigate the cause of death. In our study it was possible to measure concentrations of drugs that were part of a regular treatment without connection to the cause of death. Metamizole is used as an analgetic and spasmolytic agent. The active metabolite MAA (4-Methyl-Aminoantipyrin) is metabolized by the liver and eliminated by the kidney. Hepatic and renal dysfunction can therefore influence MAA clearance. Methods: MAA concentrations were measured in different samples of the autopsy material (heart blood, venous blood, urine, liver, kidney and brain) using an HPLC-MS/MS method. Information about the dosage and time of drug application as well as information about existing renal or hepatic disorders were taken from the corresponding patient records. Because of the low number of cases an explorative single-case study was necessary. Results: 10 cases with oral intake of Metamizole in a customary continuous dosage could be indentified. The MAA distribution into body liquids and organs depended on the time between last oral intake and death. In two cases without renal or hepatic diseases MAA blood levels were below 10 µg/ml. Five cases with combined renal and hepatic disorders showed either increased blood levels of 40-50 µg/ml or prolonged MAA elimination half-life of up to 12 hours. In one case with manifest hepatic insufficiency an MAA concentration of more than 200 µg/ml was measured in venous blood. Two cases with renal insufficiency alone had MAA venous blood levels of less than 10 µg/ml. (PET) . PET detects the positron emission of neutron-deficient radioactive nuclides and allows their external localization in vivo. FET, a modified amino acid, is not incorporated in proteins but accumulates in glioblastomas. One pathway responsible for its accumulation is the preferential transport into the tumor cells, probably via amino acid transporters. We investigated in more detail (a) which individual, cloned amino acid transporters accept FET as substrate and (b) which transporter is responsible for the major FET transport into glioblastoma cells. Studies with Xenopus laevis oocytes, expressing individual human amino acid transporters, revealed that system L, y + L and b 0+ amino acid transporters recognize FET as substrate (LAT1 and 2, y + LAT2, and b 0+ AT, respectively). In contrast, y + LAT1 and ATB 0,+ did not transport FET. RNA expression studies using qRT/PCR revealed that LAT1 is the dominant amino acid transporter in all glioblastoma cells investigated (LN229/U373/U87MG/U251/A172/T98G). A strong LAT1 expression was also shown on the protein level. To find out whether LAT1 is the main transporter responsible for FET accumulation, we first studied transport of the parent amino acid L-tyrosine in LN229 glioblastoma cells. [ 3 H] Tyr uptake was completely Na + -independent and inhibited by Leu, Phe and Trp, but not by Arg, Pro or Ser. siRNA-mediated down-regulation of LAT1 in LN229 cells led to a concomitant decrease of LAT1 mRNA and Tyr transport (down to 3% and 20%, respectively). These results indicate that Tyr is exclusively transported by LAT1 in LN229 cells. We are currently performing transport studies using [ 18 F]FET to investigate whether FET transport is also exclusively mediated by LAT1 in glioblastoma cells. A further question is if LAT1, a sodium-independent transporter, can be responsible for the accumulation of FET observed in glioblastoma cells. If true, other amino acid derivatives that are LAT1 substrates might also proof useful in cancer diagnosis.

Telmisartan reduces adipose tissue inflammation and biglycan accumulation in diabetogenic LDL-receptor knockout mice Grandoch M., Nagy N., Fischer J. W. Institut für Pharmakologie und Klinische Pharmakologie, Universitätsklinikum der Heinrich-Heine-Universität Düsseldorf, Moorenstraße 5, 40225 Düsseldorf, Germany In addition to lowering blood pressure some of the angiotensin II AT1 receptor antagonists (ARB) such as telmisartan have additional beneficial effects on the onset of type 2 diabetes mellitus and obesity. This was contributed mainly to peroxisome proliferator activated receptor (PPAR)γ modulating activity. Hyaluronan (HA), a high molecular weight polysaccharide and the small leucine rich proteoglycans, decorin and biglycan, are known to be involved in atheroprogression. Mechanistically these matrix components contribute to inflammatory processes via toll-like receptor-signalling and are supposed to modulate lipid retention. The aim of this study was to elucidate the effects of telmisartan in comparison to valsartan, an ARB without PPARγ activity, on extracellular matrix remodelling and inflammation in atherosclerosis and the interrelationship with adipose tissue inflammation using the LDLR-/-model of accelerated atherosclerosis. Male LDLR-/-mice were fed either a diabetogenic diet alone or in combination with telmisartan (10 mg/kg), valsartan (25 mg/kg) or valsartan (50 mg/kg) from 8 weeks of age for 17 weeks. All treatment groups except of the lower valsartan dose showed significant effects on reducing the aortic plaque score. The content of HA, collagen and decorin in the aortic root were not changed. However, telmisartan reduced the content of biglycan in the aortic root significantly in contrast to valsartan. In addition, a trend towards decreased mac2-positive macrophages in abdominal adipose tissue was detectable after telmisartan treatment as well as a strong reduction in the adipose tissue mRNA expression of biglycan. Finally, telmisartan reduced the expression of hyaluronan catabolizing enzymes potentially leading to an increase of high molecular weight HA in the adipose tissue, which is thought to be homeostatic and antiinflammatory. In summary, the results of this study underline the pronounced anti-inflammatory capacity of telmisartan on atherosclerosis and adipose tissue inflammation in comparison to valsartan and strongly suggest that biglycan might be an additional target of telmisartan not only concerning matrix composition of atherosclerotic lesions but also concerning the structure of adipose tissue and metabolic effects of the compound. human primary malignant cancer cells derived from peritoneal effusions of a patient with colorectal carcinoma, as assessed by comet assay. The primary cancer cells were more efficient in DSB repair than HT-29 cells, and their doxorubicin IC50 was four times higher. Comparative protein expression levels showed that the primary cells had less Rad 51 and 52 as well as less topoIIα, while Ku70 and 80 levels were similar. Another very interesting protein is the MRN (Mre11-Rad50-Nbs1) complex that initializes the phosphorylation of ATM and thereby starts the signalling cascade. The newly described MRN-ATM pathway inhibitor mirin interrupts MRN activity by inhibiting the exonuclease activity of Mre11. The toxicity of mirin in HT-29 cells was measured using a luminescence-based assay detecting the amount of ATP, which is correlated with cellular viability. Mirin did not show any toxic effects up to a concentration of 100 µM and incubation times of 24 hours, indicating that mirin can be used under these conditions without detrimental effects. We are currently investigating the effect of mirin on the toxicity of topoIIα inhibitors. The inhibition of DNA repair may be a valuable strategy to enhance the effect of DNAdamaging anticancer drugs. Since tumours (even of the same entity) are not only heterogeneous, but also polyclonal, a broad selection of response modifiers of anticancer drugs would be helpful to individually enhance chemotherapeutic effectiveness.

Evidence has been provided that diet and environmental factors directly influence epigenetic mechanisms associated with cancer development in humans. Epigenetics play an important role in the control of gene expression. Epigenetic mechanisms comprise modulation in DNA methylation, histone modification and non-coding RNA. Several polyphenols have been reported to possess histondeacetylase (HDAC) inhibitory properties [1] . Histone deacetylation is generally linked to transcription repression. Furthermore, HDAC belongs to the group of small ubiquitin-related modifier (SUMO) substrate proteins. SUMOylation of HDAC is associated with a modulation of its biological activity [2] . Little is known so far about the mechanism by which HDAC SUMOylation mediates inhibition of gene transcription. We addressed the question whether SUMO E1 and HDAC 1 expression and whether potential HDAC-SUMOylation will be affected by polyphenols such as chlorogenic acid, genistein and (-)epigallocatechin-3-gallate (EGCG).

Chlorogenic acid, genistein and EGCG decreased SUMO E1 protein level in the human colon carcinoma cell line HT29 after 24h of incubation measured with Western Blot analysis. EGCG exhibited the most pronounced effect at concentrations ≥ 50 µM. HDAC 1 expression was also affected by these polyphenols. The direct impact of polyphenols on the HDAC SUMOylation is detected by co-immunoprecipitation experiments with the respective antibodies against HDAC-1 and SUMO E1. These experiments are still under investigation.

In conclusion, chlorogenic acid, genistein and (-)-epigallocatechin-3-gallate influenced the SUMO and HDAC expression in vitro. In further studies the direct impact on subtract-SUMOylation will be investigated. These studies contribute to a better understanding of potential chemopreventive effects of dietary polyphenols on specific epigenetic alterations may provide chemopreventive strategies for reducing cancer risk. The NO/cGMP cascade is thought to be essential for penile erection. Within the smooth muscle of corpus cavernosum, nitric oxide activates the NO-sensitive guanylyl cyclase (NO-GC) which raises the intracellular concentration of cGMP. This second messenger activates the cGMP-dependent protein kinase I (PKGI) and subsequent phosphorylation of target proteins leads to relaxation of cavernosal smooth muscle. Knock out of key enzymes of the NO/cGMP cascade has led to discrepant results: The deletion of PKGI in the mouse has been shown to lead to erectile dysfunction whereas mice lacking neuronal NO synthase are fertile.

To investigate the role of the NO receptor in fertility we have generated mice lacking NO-GC (GCKO), a bottleneck enzyme of the NO/cGMP cascade. We have shown that lack of NO-GC resulted in arterial hypertension concomitant with a totally abolished NO responsiveness of vascular and gastrointestinal smooth muscle. In addition, we generated a mouse line in which NO-GC was specifically deleted in smooth muscle cells (SM-GCKO).

Using these KO strains we here examined the role of NO/cGMP signaling with regards to the smooth muscle relaxation of corpus cavernosum. NO failed to affect corpus cavernosum from GCKO in organ bath experiments: neither exogenously produced NO by NO donors nor endogenous NO release from neurons induced by electrical field stimulation led to relaxation. Similar results were observed in the corpus cavernosum of SM-GCKO mice. To our surprise, the GCKO animals were fertile and produced offspring albeit at a reduced rate compared to WT animals. Our data show that interruption of NO/cGMP signaling results in complete absence of NO-induced relaxation of penile corpus cavernosum in mice and reduces the ability to produce offspring but does not abolish fertility.

Novel modes of invasive cell motility regulated by the formin class of actin nucleators Khan J., Grosse R. Philipps-Universität Marburg, Pharmakologisches Institut, Karl-von-Frisch-Str. 1, 35034 Marburg, Germany

Pathological invasive cell migration essentially reqires actin polymerization. Formins are the largest group of Rho-GTPase effectors involved in actin nucleation and assembly as well as microtubule dynamics. Here we studied the role of formins in cytoskeletal regulation during homotypic cancer cell invasion. We identified the actin-dependent steps and structures involved for this process. Using live cell analysis we characterize the distinct actin dynamics controlled by formin-like 2 and Rho function. The specific involvement of this signaling module will be discussed.

Formin-driven Nuclear Actin Assembly Controls MAL/SRF Activity Baarlink C., Wang H. Polymerization of actin in the cytoplasm is tightly linked to transcriptional activation of the SRF cofactor MAL (also known as MRTF-A) through release of actin/MAL interactions and subsequent nuclear accumulation of MAL. Formins directly promote assembly of actin filaments thereby efficiently regulating MAL-dependent transcription for cell shape, adhesion and motility. Here we show that formins assemble F-actin and promote MAL activation inside the mammalian nucleus. The Rho-GTPase effector mDia2 rapidly enters the nucleus in a signal-dependent fashion and an active mDia confined to the nucleus potently promotes release of G-actin from MAL to specifically activate SRF. Live cell imaging reveals formin-mediated nuclear actin dynamics. Moreover, using actin assembly assays we find that inhibition of endogenous mDia formins controls F-actin turnover in isolated nuclear extracts. Thus, formin activity is dynamically compartmentalized to the mammalian nucleus to potently regulate actindependent MRTF function. In women the placenta becomes the main source of maternal estrogens during pregnancy. Placental estrogen biosynthesis is located in the syncytiotrophoblast, a syncytium that builds the main part of the placental barrier and limits the transfer of substances between the fetal and maternal compartment. Since the human placenta is unable to convert cholesterol into 17-OH-pregnenolone, the placenta tissue highly depends on the supply of C-19 steroids for their conversion into C-18 estrogens. In contrast to lipophilic unconjugated steroids that penetrate the cell membrane passively via diffusion, circulating sulfated steroid hormones are delivered to the placenta via carrier-mediated transport, followed by their reactivation via the catalytic activity of the steroid sulfatase (StS). DHEAS of maternal and fetal origin contributes about equally to the placental formation of estrone (E 1) and estradiol (E2), while 16αOH-DHEAS supplied by the fetus contributes to over 90% of placental estriol (E3) synthesis. SOAT, a member of the SLC10 family with highest expression in hormone-responsive tissues such as testis, placenta, and mammary gland has been shown to transport the sulfoconjugated steroid hormones dehydroepiandrosterone sulfate (DHEAS), estrone sulfate (E1S), and pregnenolone sulfate (PREGS) [1] . Aim of this project is to investigate the role of SOAT for placental estrogen synthesis by means of the choriocarcinoma cell line JEG-3 as in vitro model for the human syncytiotrophoblast. Therefore, we characterized a JEG-3 cell line that transformed DHEA into E2 and 16αOH-DHEA into E3. By qRT-PCR we found expression of StS and aromatase, both essential for estrogen synthesis in these cells. Upon transient transfection of SOAT the carrier was located in the cell membrane of transfected JEG-3 cells. Currently we investigate the transformation of DHEAS of these SOAT-JEG-3 cells by LC-MS-MS. We could demonstrate transport of 16αOH-DHEAS for stably transfected SOAT-HEK293 cells. In situ hybridization and immunohistochemistry showed coloured syncytiotrophoblasts and vascular endothelial cells in late term placenta.

In conclusion, SOAT-mediated transport of sulfated steroids could play a pivotal role for placental estrogen synthesis from sulfated steroid hormones. Developing non-animal test systems for evaluation of toxicity was important in the past and will remain essential in the future. Here we present a toxicity test using the chicken yolk sac area vasculosa (CAV) of fertilized white leghorn chicken eggs [1, 2] and compare it to Hen's egg test on chorioallantoic membrane (HET-CAM) [3] for polymer toxicity testing. Fertilized chicken eggs were incubated and after 72 h explanted shell less into sterile petri dishes. Test substances were applied on the CAV and the appearance of different effects (vascular lysis, haemorrhage, aggregation of blood components, lethality) was determined by light microscopy after 1 -48 h (Fig.1 ). These effects were combined to a CAV test score based on the irritation score calculation used for HET-CAM evaluation. Different polymers like poly(ethylene glycol) (PEG; neutral), poly(ethylene imine) (PEI; cationic) and dextran sulphate (DS; anionic), as well as guideline-conform (recommended HET-CAM protocol from the Interagency Coordination Committee on the Validation of Alternative Methods) negative (0.9 % NaCl) and positive controls (1 % sodium dodecyl sulphate (SDS) and 0.1 N NaOH) were investigated. Additionally LD 50 values for different cationic polymers have been determined. Within the selected incubation times (1 -48 h) , effects such as vessel lysis and blood component aggregation could be detected. Additionally to HET-CAM, lethality as well as recovery of the CAV could be observed. Differences between neutral, positively and negatively charged polymers were obtained. PEI showed strong vessel lysis and aggregation of blood components whereas DS and PEG showed none of these effects. Lethality was found to increase from PEG < DS < PEI and is concentration and time dependent. The results demonstrate that differences, regarding the toxicity of the used polymers, can be shown with this test. These findings in the CAV test can be well correlated with already existing data. In summary, the CAV test provides same data (testing control substances) and more information (recovery and lethality) compared to HET-CAM and could be a suitable model for toxicity testing of polymers. Risk characterisation of chemicals consists of three steps (1) hazard identification and characterisation, based on substance-specific toxicological hazard data, (2) estimates of the level of exposure toward the substance and (3) the comparison between the toxicologically safe level and the exposure level. In contrast to the classical risk assessment approach, the threshold of toxicological concern (TTC) approach is developed as a tool to assess the risk of substances without toxicity data. Its application requires (1) information on human exposure, for which it is essential that exposure is fully captured and (2) knowledge of the chemical structure to assess whether the chemical is not excluded from the application of the TTC concept. Instead of chemical specific no observed (adverse) effect levels (NOELs/NOAELs), the TTC approach utilises knowledge on the empirical distribution of several hundreds of NOELs/NOAELs, originally 613, based on toxicological testing in animals (Munroe et al., 1996) . With the basis on NOAELs, the TTC concept builds on the fundamental principle of toxicology, that toxicity is a function of dose and that a dose exists, below which no adverse effects of the substance can be detected. It is assumed that exposures below this level will not result in health risks. Three separate TTCs were derived (Munroe et al., 1996) by classifying the chemicals into three toxicity classes using a decision tree based on a series of 33 questions related to chemical structure, and on natural occurrence in food and in the body (Cramer et al., 1978) . The TTC values are derived from empirical distribution of the NOELs/NOAELs in the class taking the 95th percentiles and dividing them by the default uncertainty factor of 100. It is assumed that the probability is very low that the unknown NOAEL of a not tested chemical will be lower than the value of the 95th percentile in the distribution of the known NOELs/NOAELs. Hence, at exposures below the TTC values, the probability of adverse effects on human health is considered to be very low. Introduction: KIBRA, mainly expressed in kidney and brain tissue, is involved in brain development and memory formation as a postsynaptic scaffold protein. In podocytes, KIBRA is proposed to regulate cell motility as a linker between components of the cytoskeleton and polarity protein complexes (Duning et al, JASN 2008) . Furthermore, KIBRA has been identified as key regulator of the Hippo pathway, which is involved in organ size control and tumorigenesis. In the current study, we focused on the identification of KIBRA gene expression regulation and functional promoter characterization.

Methods: Serial promoter deletion constructs were generated by cloning 3627 bp of the 5'flanking region of KIBRA into the pGL3-vector system. Deletion constructs were transiently transfected into human neuroblastoma cells (SH-SY5Y) and immortalized human kidney epithelial (IHKE) cells. Potential transcription factors (TFs) were investigated in cotransfection experiments. Transcriptional start sites (TSS) were determined by rapid amplification of 5'cDNA ends (5'RACE) . TSS utilization between cell lines was assessed by semiquantitative PCR.

Transcriptional activity (TA) of the KIBRA promoter P1 was separated by ~1060 bp into two distinct regions, promoter P1a and P1b. Deletion constructs harbouring promoter P1b were transcriptionally active only in IHKE cells. 5'RACE revealed two alternative TSS in both cell lines upstream of the annotated TSS (NM_015238). Exclusively in IHKE cells, two additional TSS were detected in intron 1, resulting in two alternative exons. Deletion constructs harbouring the putative regulatory regions (P2 and P3) of both exons were transcriptionally active only in IHKE cells.

Overexpression of full length TCF7L2 (transcription factor 7-like 2 [T-cell specific, HMGbox]) resulted in a ~3-fold increase of promoter P1a and intron promoter P2 TA.

KIBRA gene expression is driven by a complex alternative promoter system comprising the constitutional promoter P1 and three alternative promoters P1b, P2 and P3. The TSS utilization is cell type-specific. Subsequent usage of an alternative translation start site within exon 3 could result in truncated KIBRA protein isoforms. TCF7L2 is involved in the differential KIBRA gene expression regulation. Resulting KIBRA protein isoform and their cellular function will be assessed in further studies. Skin Absorption in vitro based on the study of human/animal skin ex vivo or reconstructed human epidermis, respectively, is an alternative method which is accepted by the OECD. Guideline TG 428 and a corresponding technical guidance document (GD 28) give technical guidance how to perform valid experiments 1, 2 . The requirements include integrity evaluation tests for the skin samples. Different tests are proposed to ensure an exclusively use of undamaged skin. To decide which test suites best to our routine test strategy, we investigated the correlation between integrity test results and absorption profiles of various penetrants (logP range: -0.07 -6.2).

Finite dose experiments using rat and human skin were performed with 14 C-labeled testosterone, caffeine, MCPA (4-chloro-2-methylphenoxyacetic acid) and its 2-ethylhexyl-ester MCPA-2EHE. For each experiment at least three of the five following integrity tests were conducted: transepidermal electrical resistance (TEER), transepidermal water loss (TEWL), transepidermal tritiated water flux (³H2O), transepidermal absorption of methylene blue (BLUE) , transepidermal absorption and flux of a ³H-labeled internal standard (ISTD); ³H-Testosterone or ³H-Mannitol was used as ISTD. The applied radioactivity of the ³H-ISTD was selected to show no analytical interference with the 14 C-penetrants. TEER, TEWL and ³H2O represent pre-study, ISTD concurrent and BLUE post-study tests. Calculated maximal permeability constants (Kp) and absorbed doses (AD) of the penetrants were compared to the results of the integrity tests. Individual linear regression analysis was used to evaluate the correlation The correlations varied over a wide range for all five methods and four penetrants. The best correlations in average were achieved with the ISTD. No inverse correlations were obtained for the ISTD, but partly for TEWL, TEER, ³H2O and BLUE.

In conclusion, the ISTD represents best the achieved absorption profiles of the test compounds and is based on that the most suitable integrity test for our dermal absorption studies. We will further confirm its effectiveness and generate a sufficient historical database in order to include the ISTD in our routine test protocol. 

Investigation of miRNA expression and DNA Methylation in focal and non-focal brain tissue of therapy-resistant epilepsy patients Haenisch S. 1 Background: Resistance to anticonvulsants affects one third of all epilepsy patients. Limited bioavailability of the drug at the target site caused by increased expression of efflux transporters on the blood brain barrier or alterations of target genes are potential mechanisms for therapy resistance. However, these mechanisms alone cannot completely explain the observed resistance and it is likely that multifactorial alterations lead to pharmacoresistance. There is increasing evidence that expression of microRNAs probably caused by DNA modifications is deregulated in many neuronal diseases. We hypothesize that miRNA regulation of target genes is involved in drug resistance in epilepsy. Methods: Hippocampal focal and cortical non-focal brain tissue samples from 13 patients diagnosed with MTS (mesial temporal sclerosis) who underwent neurosurgery have been screened for miRNA expression using TaqMan low density arrays. In silico approaches for both a hypothesis-based (efflux-transporter and target gene) as well as a hypothesis-free approach were used to identify potential phenotype-relevant target genes. Using the program R (Bioconductor) a Mann-Whitney-U test was performed to compare miRNA expression between brain regions. Pyrosequencing was performed to investigate methylation status 5'-upstream of DNA regions encoding for selected candidate miRNAs. Results: Out of 754 miRNAs, 150 were detected in both tissue types. The expression of one miRNA was 7.2 fold higher (q=0.01) and another was 3.8 fold lower (q=0.01) in the hippocampus relative to the cortex. Evidence could be found that down-regulation of the latter is possibly caused by hypermethylation of 5'-flanking region of its encoding DNA locus. Bioinformatic analysis has identified eight genes important for neuronal regulation and signal transmission (e.g. SOX11, MECP2, BSN), as well as one ABC effluxtransporter, as potential targets for these differentially regulated miRNAs. Conclusion: Differential regulation of two miRNAs could contribute to an altered function of several genes resulting in an imbalance between neuronal excitation and inhibition that is independent from mechanisms presently targeted by anticonvulsants. This work was supported by a fellowship from DFG and NIH grant GM61390. Recently, it has been reported that human B cells express and secrete the cytotoxic protease Granzyme B (GrB) after the combined stimulation of the IL-21-and the B cell receptors. GrB produced by B cells is enzymatically active and B cells deliver GrB to sensitive cancer cell lines, thereby inducing apoptosis. To date, there is little experimental evidence on the mechanisms involved in GrB expression, or its function in B cell biology. As experimental transgenic murine systems should enable us insights into these issues, we assayed for GrB in C57BL/6 B cells using an extensive array of physiologically relevant stimuli, but were unable to detect either GrB expression or its proteolytic activity, even when antigen specific transgenic B cell receptors were cross-linked. Similar results were also obtained with B cells from DBA/2, CBA or BALB/c mice. In vivo, infection with either influenza virus or murine γ-herpesvirus induced the expected expression of GrB in cytotoxic T lymphocytes, but not in B cell populations. We also investigated a possible role of GrB on the humoral immune response to NP-KLH, but GrB-deficient mice produced normal amounts of antibody with typical affinity maturation and heightened secondary response, demonstrating conclusively the redundancy of GrB for antibody responses. Our results highlight the complex evolutionary differences that have shaped the immune systems of mice and humans and demonstrate the need to develop novel in vivo systems to study human humoral immune responses.

Investigations of the cholinergic neurotransmitter system in DYT1 mice Hamann M. 1 Early-onset torsion dystonia is an autosomal dominant inherited movement disorder associated with the DYT1 gene defect with deletion of a glutamic acid residue in the protein torsinA. Despite the gene defect, the pathophysiology is poorly understood. Animal models can help to understand the underlying mechanisms and thereby to develop new therapeutic strategies. Sharma et al. (2005, J. Neurosci. 25 [22] , 5351-5355) initially described a transgenic mouse model (DYT1 mice) with overexpression of mutant torsinA. Previous studies in these mice pointed to alterations in the cholinergic system. To investigate the functional relevance of these in-vitro findings, we carried out pharmacological in-vivo experiments and determined the density of striatal cholinergic interneurons as well as the expression of choline acetyltransferase in different brain regions. The acute intraperitoneal administration of the cholinomimetic drug pilocarpine (75, 100 and 125 mg/kg) as well as a long-term treatment over 21 days (100 mg/kg/d) did not induce pronounced effects in DYT1 mice compared to wildtype controls. The higher incidence of epileptic seizures in DYT1 mice compared to controls after repeated local striatal applications of pilocarpine (25 and 50 µg/0.5 µl/hemisphere) let presume an altered synaptic plasticity in DYT1 mice. The immunohistochemical investigations revealed a moderately reduced density of striatal cholinergic interneurons in the dorsomedial subregion of DYT1 mice compared to wildtype controls, while significant differences in other striatal subregions were not detected. Western Blot analysis did not show clear differences in the expression of choline acetyltransferase between DYT1 and wildtype control mice. These results indicate that the cholinergic system seems not to play a key role in this line of DYT1 mice. Ongoing receptor autoradiographic analysis of binding to different muscarinic receptors subtypes have to further clarify the existence of possible alterations within the cholinergic system of these DYT1 mice. Inhibitors direct against cell cycle-regulatory kinases are being tested in clinical trials as anti-proliferative agents. Thus, the ATP-competitive kinase inhibitor PD332991 which inhibits CDK4 and CDK6 is currently tested in patients with solid tumors such as glioma. We found that PD332991 suppressed IL-1-induced expression of IL-8 suggesting that CDK4 or CDK6 may have unknown anti-inflammatory properties. To study the effects of CDK6 on the IL-1-signaling network, we established a bidirectional doxycyline-inducible system to express a constitutively active mutant of CDK6, CDK6 S178P, in asynchronized HeLa cells. CDK6-expressing cells were identified by GFP which was expressed from the same promoter, isolated by laser-microdissection and analysed for mRNA expression using a down-scaled RT-qPCR assay. Compared to the uninduced state, CDK6 S178P enhanced IL-1-induced IL-8 and IL-6 mRNA expression. Moreover, shRNA-mediated suppression of endogenous CDK6 confirmed a role of this kinase in regulation of maximal IL-1-induced gene expression of IL-8. These data also revealed that the contribution of CDK6 to inflammatory gene expression is highest in G1, when activity of endogenous CDK6 is activated by D-type cyclins. These findings were corroborated in HeLa cells expressing fluorescent ubiquitin-dependent cell cycle indicator (Fucci) proteins. HeLa-Fucci cells from G1, G1/S, G2 or mitotic states were isolated by laser-microdissection and analyzed by RT-qPCR for TNF-inducible gene expression. Stable knockdown of CDK6 in HeLa Fucci or inhibition by PD332991 suppressed inducible IL-8 expression. Microarray experiments identified many additional genes that required active CDK6 for maximal IL-1-or TNF-inducible gene expression. We also found that CDK6 co-immunoprecipitated with p65 NF-κB, colocalized with p65 in the nucleus and was recruited together with the p65 subunit to the proximal IL-8 promoter as assessed by ChIP and Re-ChIP experiments. Collectively, these results suggest an unexpected control of inflammatory gene expression through a classical cell cycle regulatory pathway. These results also imply that pharmacological targeting of CDKs may have effects and side-effects on the immune system in addition to inhibition of cell cycle progression. TRP channels form a heterogeneous family of calcium-permeable channels, which play major roles in physiological functions ranging from sensory reception to cellular signal transduction. Members of the TRPC subfamily (classic transient receptor potential channels) are downstream targets of hormone receptors. Of particular interest is the biological role of TRPC6 channels. They are directly activated by diacylglycerol due to phospholipase C-driven signalling pathways which are involved in smooth muscle contractility, neuronal plasticity, keratinocyte differentiation and renal function. Their impact in renal function became evident from analyzing patients suffering from familial forms of focal segmental glomerolusclerosis (FSGS) which could be linked to TRPC6 mutations. Since the first descriptions at least 17 different pathogenic mutations have been identified in humans to cause FSGS. In order to study the underlying pathophysiological mechanisms of TRPC6 mutations, we have analysed all mutated TRPC6 channels known to date heterologously expressed cells. One set of mutations showed a gain-of-function phenotype which has been previously suggested to cause an increased intracellular calcium load and subsequent cell death. Hence, gain of function mutations fit to the current paradigm of FSGS pathophysiology. However, another set of mutations found in the patients showed a loss-of-function phenotype. Our results enable a change in the current paradigm for the role of TRPC6 in renal pathophysiology and may provide a basis for our understanding of the pathophysiology of loss-of-function mutations in familial focal segmental glomerolusclerosis.

Karlsruher Institut für Technologie (KIT) Institut für Angewandte Biowissenschaften, Abteilung Lebensmittelchemie und Toxikologie, Adenauerring 20a, 76131 Karlsruhe, Germany Risk assessment for genotoxic carcinogens is an important challenge in toxicology. Even though manifold attempts have been made to substitute carcinogens and to reduce exposures, their complete elimination appears to be not possible. Thus, low concentrations of known or suspected genotoxic carcinogens are present at workplaces, in the environment and in food. In order to deal with this situation and to set priorities for risk management, different concepts have been established such as the ALARA principle (As Low As Reasonably Achievable) and the Margin Of Exposure (MOE), based on the ratio between concentrations being carcinogenic in experimental animals and the actual exposure of humans for example via foodstuff. While usually linear doseresponse-relationships have been used as default assumption, analytical methods are now available to assess the induction and repair of DNA lesions on low exposure conditions, including environmental background exposure, and to relate the extent of exposure-induced DNA lesions to endogenous DNA damage. This may be an important prerequisite to establish health-based limit values for selected genotoxic carcinogens. Within this workshop, different examples will be discussed and research need will be identified.

Dendritic cells from H4R-deficient mice lose their ability to properly stimulate T lymphocytes Hartwig C., Seifert R., Neumann D. MHH Pharmakologie, Carl-Neuberg Str. 1, 30625 Hannover, Germany

The incidence of allergic airway diseases is increasing throughout the world, especially in Western countries. Although histamine (HA) is found at high concentrations in asthmatic lungs, a role for HA in bronchial asthma is still a neglected topic in clinical research. In particular, the capacity of HA to modulate the underlying immune reaction is far from being understood. The histamine H4-receptor (H4R) is involved in acute inflammation and Th2 cytokine production. Consequently, we intended to analyze the role of H4R in a murine Th2 lymphocyte transfer-based model of asthma. Specifically the ability of H4R expressed on dendritic cells (DCs) to modulate T cell function was analyzed. OVA-specific CD4 + T cells were polarized in vitro under Th2-favoring conditions with OVA peptide-pulsed DCs, obtained either from wild-type or H4R -/mice. Analysis of the polarized T cells after in vitro restimulation revealed a marked decrease of IL-4 production in T cells polarized in the presence of H4R -/-DCs compared to those polarized in the presence of wild-type DCs. Thus, on DCs, the H4R is essential for proper stimulation of spleen T cells and for directing their polarization towards a Th2 phenotype. The transfer of in vitro polarized T cells into recipient mice and subsequent provocation elicited an asthma-like disease. The H4R on DCs not only affects in vitro polarization of T cells, but also the in vivo function of the obtained polarized T cells. A parameter indicating allergic inflammation is the enhanced influx of inflammatory immune cells into the lung tissue, mainly driven by eosinophils, which are virtually absent in non-asthmatics. When analyzing the number of eosinophils, a dramatic difference due to the polarizing conditions of T cells occurs. In BAL fluids of mice that received T cells polarized in the presence of wild-type DCs, about 40% eosinophils were detected. In contrast, the transfer of T cells polarized in the presence of H4R -/-DCs yielded only about 10-20% eosinophils in BAL fluids. In summery, the H4R on DCs plays an important role for T cell polarization and consequently affects the allergic reaction during sensitization. Since the lack of the H4R on DCs reduced their ability to stimulate proper Th2 polarization of CD4 + T cells, we conclude that HA via the H4R significantly affects the manifestation of asthmatic inflammation.

Antioxidant polyphenols and their effects on Nrf2 (SKN-1) signalling in a cell culture system and the model organism C. elegans Havermann S., Wätjen W. Heinrich-Heine-Universität Düsseldorf Institut für Toxikologie, P.O. Box 101007, 40225 Düsseldorf, Germany Oxidative stress has been connected with a variety of diseases, (e.g. Alzheimer`s and Parkinson´s disease), cancer and ageing over the last years. Certain polyphenols were shown to have an antioxidant capacity as well as being able to activate the protective Nrf2 signalling pathway. Compared to direct radical scavengers modulators have the advantage of building up a permanent defense against oxidative insults whereas scavengers do not protect any more after consumption or may even cause stress due to redox cycling. We have employed cell culture based assays (DCF, Western Blot, GFP reporters) to analyse the effects of polyphenols. Further we tested the coumpounds in vivo in the nematode C. elegans where SKN-1 is the Nrf2 homologue. Baicalein and caffeic acid phenethylester (CAPE) protected cells and the nematode from ROS accumulation after application of stress (shown by DCF assay). Activation of Nrf2 signalling is correlated with translocation of the transcription factor into the nucleus. In both systems Nrf2::GFP accumulation in the nuclei could be observed after incubation with baicalein (fluorescence microscopy). But while CAPE is a potent activator of Nrf2 in cells, it has no effect on SKN-1 localisation. Further the effect on the Nrf2 protein amount was investigated by western blot analysis. The expression of target genes can be investigated by differing means: While PCR methods and western blotting are standard for in vitro studies, the vast number of available GFP reporter strains offers opportunities for research using C. elegans. We have performed congruent assays in a cell culture system and the model organism C. elegans to compare antioxidative capacity and effects of polyphenols on Nrf2 signalling. Therefore, depending on the substance tested, C. elegans is a suitable model system to investigate effects of natural compounds in an organism. Being associated with adverse health effects, the human exposure to DEHP is subject to concern. Quantifying the population's exposure and determining the contributions of different exposure routes is a key task of environmental health risk assessment.

The study presented comprises a review of the available data on DEHP levels in foods, consumer products, and house dust. Extensive survey data, e.g. from the current National Nutrition Survey II and the EU RAPEX system were processed for modeling the exposure by the oral, inhalative and dermal path of the population in Germany. The study also included analytical analyses of DEHP levels in selected foods and consumer goods (incl. migration rates for mouthing). Probabilistic techniques allowed elucidating the exposure's variation and the relevance of different routes.

Mean exposure estimates for German children and adults to DEHP are 36 and 26 µg/(kg d), resp. For children, food accounts for 36% of the total exposure, followed by mouthing (30%) and house dust (19%). The adult exposure is almost entirely (82%) due to food. As dietary exposure is a result from concentration and consumption, foods exhibiting high contamination e.g. Butter (8%) and dressings (mayonnaise) (14 %) as well as highly consumed foods e.g. bread and bakery (16%) and vegetables (6,8 %) contributed significantly. The mean estimate of children's DEHP exposure via mouthing revealed 0,9 µg/(kg d).

High exposures were estimated (95th percentile) up to 10,8 µg/(kg d).

On average people in Germany are exposed to DEHP below the current TDI of 50 µg/(kg d). However, individual exposures exceeding the TDI still cannot be excluded. Current data on DEHP and other plasticizers in foods are scarce, which warrants broader monitoring. Our findings highly facilitate further exposure modeling focusing on DEHP substitutes and risks of combined exposure.

This study was funded by the Federal Ministry for the Environment, Nature Conservation and Nuclear Safety in the frame of the environmental research plan (Umweltforschungsplan, Förderkennzeichen (Ufoplan) 3707 61 201). On the basis of the available measurements of DEHP, the exposure assessment has been focused on 37 food categories characterising a selection of the most important food groups covering all major food classes of the German population. Based on an extensive literature survey, the analysis considered the available data of DEHP measurements in food, as well as the official German food control data taken from the National Food Consumption Survey. The high amount of considered data allowed the consideration of several exposure assessment tiers (deterministic and probabilistic by using Monte Carlo simulation).

A quantitative evaluation of the uncertainties of the estimate of the 37 food categories groups has been performed by means of a sensitivity analysis by using the methodology proposed within the 2008 WHO IPCS guidance document of characterising and communication uncertainty in exposure analysis. Qualitative uncertainty analysis (tier 1) was applied to determine the most important sources of uncertainty, i.e. concentration of DEHP in all food categories. The probabilistic Monte Carlo simulation (tier 2) was then used to rank the cumulative probability distributions of the exposure assessments of 37 food categories on the basis of the food categories that appear to dominate. Sensitivity analyses were applied to prove the impact correlation of food groups for uncertainties. By a scenario based concept, the aggregation of the 37 food groups to 10 groups has been evaluated, as well as the sensitivities by characterising particular scenarios. For this purpose, particular "meals" have been described as fixed combined scenarios and.

The aggregation leads to a considerable higher exposure estimate which can be explained by the combination of high contaminated foods with others of high consumption. The evaluation confirms the considerable role of possibly high contaminated foods e.g. fats, or mayonnaise. The evaluation shows that quantitative probabilistic sensitivity analysis is a suitable and pragmatic tool for uncertainty analysis in exposure assessment. The transcription factor cAMP response element (CRE)-binding protein (CREB) plays a critical role in regulating gene expression in response to activation of the cAMPdependent signaling pathway, which is implicated in the pathophysiology of heart failure. We observed CREB knock-out cardiomyocytes to be larger than wildtype cardiomyocytes (cell area in µm 2 ; mean±SEM; CREB-KO 4871±258 vs. WT 3396±171; n=68/69 cells, N=4 mice, p<0.01 vs. ctr.). The Nuclear factor of activated T-cells c3, NFATc3, is another transcription factor involved in the development of heart failure and also a known positive regulator of hypertrophy. Hence, we investigated whether inhibition of the CRE-dependent transcriptional activation has an impact on the NFATc3 signaling pathway. We first studied the effects of an overexpression of a dominant negative CREB mutant (dnCREB) or of NFATc3 on the activity of a NFAT-dependent model promoter in a permanent cell line. Overexpression of dnCREB evoked an 8.0±1.5 fold increase of the NFAT model promoter activity (n=36; N=6 transfections), but had no impact on Kv4.2 promoter activity which is known to be regulated by NFATc3 (1.2±0.1 fold; n=18; N=3; p<0.05 vs. ctr.). NFATc3 overexpression led to a 4.5±0.7 fold increase of the NFAT-dependent model promoter activity (n=12; N=2) and to an inhibition of Kv4.2 promoter activity (0.8±0.1 fold; n=18; N=3; p<0.05 vs. ctr.). We conclude that CREB is a negative regulator of NFAT-mediated gene transcription and that activation of NFATc3 might contribute to the observed hypertrophy of CREB-KO cardiomyocytes.

Neuroleptika der Perazin-Klasse sind potente Modulatoren des P2X7-Rezeptors -Perazine-type neuroleptic drugs are potent modulators of P2X7 receptors Hempel C., Nörenberg W., Urban N., Sobottka H., Schaefer M. Universität Leipzig Rudolf-Boehm-Institut für Pharmakologie und Toxikologie, Härtelstraße 16-18, 04107 Leipzig, Germany P2X7 receptors belong to a family of ATP-gated, non-selective cation channels, which play an important role in immune cell activation, inflammatory hyperalgesia and neuropathic pain. They differ from other P2X family members by the low ATP affinity, and by the ability to form or recruit dilated pores in the sustained presence of ATP. Owing to its involvement in many diseased states, P2X7 is a promising target for pharmacological intervention. Accordingly, P2X7 blockers are currently tested in phase II clinical trials. In an attempt to identify P2X7-modulating properties of approved drugs or natural compounds, we performed a medium-throughput screen, using an appropriate compound library (Spectrum Collection) and a stably transfected HEK293hP2X7 cell line.

With IC50 values of 1-2 µM, the tricyclic antipsychotics prochlorperazine and trifluoperazine showed a high potency and efficacy to block the ATP (1 mM)-triggered increases in the intracellular Ca 2+ concentration ([Ca 2+ ]i) that was mediated by human P2X7 (hP2X7). The closely related phenothiazine-class neuroleptic drugs, such as chlorpromazine or triflupromazine did not have an appreciable effect on hP2X7mediated Ca 2+ influx. Whole-cell inward currents, measured at -60 mV, were blocked by more than 60% by 3-10 µM prochlorperazine. The inhibitory effects of perazines developed within about 200 ms, hinting to a direct mode of action by binding to the P2X7 protein. Prochlorperazine added intracellularly via the patch pipette did not substitute for the extracellularly applied drug, indicating that its binding site is accessible from the extracellular side. In addition, both compounds blocked Yo-Pro-1 uptake when preincubated before P2X7 stimulation with 1 mM ATP or when applied subsequent to the agonist. Interestingly, when added to a HEK293 cell line expressing the rat P2X7, perazines potentiated the ATP-induced increase in [Ca 2+ ]i. Measurements in human monocyte-derived macrophages confirmed the ability of prochlorperazine and trifluoperazine to inhibit ATP-evoked increases in [Ca 2+ ]i, changes in Yo-Pro-1 permeability and whole cell currents. Taken together, we conclude that perazine-type neuroleptics impede on P2X7 activity in a species-specific manner, presumably by binding to an extracellularly accessible binding site of recombinant or natively expressed P2X7. Similarly, pre-treatment with LOV also lowered DOX-induced stabilisation of p53 and phosphorylation of CHEK1 and SAPK/JNK. While LOV had no influence on IR-induced initial DNA damage formation in HUVEC and rat cardiomyoblasts (H9c2), it decreased DOX-and ETO-induced phosphorylation of histone H2AX, which is a surrogate marker of DNA-double strand breaks. This indicates that LOV specifically protects against the genotoxicity of topoisomerase type II poisons.

In an acute and subacute Balb/c mouse model LOV protected from IR-induced toxicity. This effect rested on inhibition of pro-inflammatory and pro-fibrotic processes as measured via quantification of mRNA levels of Il6, Ctgf and Tnfα. The same was true for DOX-induced toxicity, i.e. heart and liver damage. Similar to the in vitro experiments, DOX-induced hepatic DNA-damage was attenuated by LOV treatment. Overall, liver and heart toxicity were reduced by LOV as mirrored by the serum levels of Gldh/Gpt and cTn-I, respectively. Both in liver and in heart we observed collagen rich perivascular areas following DOX treatment. Under situation of LOV-co-treatment these areas occurred more rarely and were less pronounced, pointing to a lowered level of fibrosis. PCR-array-based mRNA analyses showed inhibitory effects of LOV on DOX-triggered expression of genes involved in oxidative stress response, drug transport, DNA repair, cell cycle progression and cell death. For instance, up-regulation of p21, Wee1, cJun/Fos and Hmox-1 following DOX administration was attenuated by LOV. Altogether, we suggest that including LOV in current cancer therapeutic regimen might widen the therapeutic window of anticancer therapeutics by lowering normal tissue damage.

the p values. In addition, array data underwent cluster analysis for identification of substantial differences of gene regulation among the three different types of biopsies. Results: Of particular interest in our study was the expression of genes coding for metabolism and transport proteins. Therefore 42 genes from the 156 significant differentially regulated genes, were selected for the qRT-PCR analysis. Genes coding for ABCB1 and ABCG8 transport proteins showed higher expression in the jejunal tissue one year after surgery compared to the duodenal tissue (fold change 1.80 and 1.73). Moreover, CYP7A1 mRNA involved in metabolic processes is higher expressed in postoperative jejunum than in the jejunum tissue taken during the surgery (fold change 1.9). In conclusion Roux-en-Y gastric bypass operation leeds a change of mucosal gene expression profile in the jejunum during one year. There was also a significant differential gene expression between the original duodenum and jejunum one year after surgery. These results give strong evidence that jejunum not exposed to pancreatic but only to gastric fluids may change its gene regulation. Background. Numerous genome-wide association studies (GWAS) identified polymorphisms located in transporter genes such as SLC2A9, ABCG2, NPT1, and URAT1 as predicitive for the serum levels of urate 1 . These genes encode membrane proteins expressed in the apical membrane of human kidney proximal tubule cells and are assumed to facilitate tubular exchange of urate 2, 3, 4, 5 . Importantly several single nucleotide polymorphisms (SNP) located in vicinity of SLC2A9 have been identified as highly associated with serum urate levels.

Little is known about the transcriptional regulation of SLC2A9. Therefore, the aim of our study was to investigate which sequences in the SLC2A9 gene harbour ciselements and regulate its gene expression. We also asked whether intronic SNPs influence gene expression at the transcriptional level. Methods and Results. Performing dual luciferase reporter gene assays we found gene regulating modules in the SLC2A9 gene. DNA from human kidney samples was then genotyped for rs6449237 being part of this region. Next total SLC2A9 mRNA-expression levels of the samples were determined using real-time quantitative RT-PCR assay. Male samples with two minor alleles of SNP rs6449237 showed lower SLC2A9 mRNA levels than samples with the wild type alleles. The effect was not seen in females. Reporter gene constructs with either minor or major allele of rs6449237 were then used in luciferase assays, however showing no significant difference in activity. Furthermore mRNA-expression levels of other urate transporter genes were determined in kidney samples. After linear regression a positive correlation of mRNA-expression of SLC2A9, URAT1, NPT1, and OAT10, respectively was observed. Conclusion. Our data suggest that the SLC2A9 SNPs rs6449237 and rs74794351 might influence SLC2A9 mRNA-level without controlling the transcriptional activity. It needs to be elucidated whether those SNPs alter mRNA stability. However, the mRNA coexpression of SLC2A9 and other urate transporter might be attributed to a common gene regulating pathway of an "transportosome" controlling urate homeostasis. 

Sulfotransferases mediate the bioactivation of methyleugenol to a reactive sulfate ester binding to DNA in vitro and in vivo Herrmann K. 1 Methyleugenol (ME) is a secondary metabolite occurring in many herbs and spices. Although ME is hepatocarcinogenic in rodents, standard genotoxicity tests were negative. This may be due to the lack of critical activating enzymes responsible for the terminal bioactivation of ME to a genotoxicant. ME is initially hydroxylated by cytochrome P450 enzymes yielding 1´-hydroxymethyleugenol (1´-OHME). This alcohol can be further activated by sulfotransferases (SULTs) to an electrophilic sulfate ester that can be easily attacked by DNA. The DNA adducts formed could lead to mutation and further carcinogenicity observed in animals. The aim of the present study was to clarify whether individual human (h) and murine SULT forms are involved in the activation of ME to a genotoxicant. In order to identify critical SULTs, mutagenicity tests including bacteria expressing different SULT forms were conducted. (±)-1´-OHME (separated into its enantiomers) served as test compound. We could show that hSULT1A1, standing out due to its high expression level in many tissues, can efficiently activate both enantiomers even at low concentrations. Furthermore, DNA adduct formation in hSULT1A1-proficient and SULT-deficient bacteria was examined after incubation with 7 µM of (+)-or (-)-1´-OHME. For selective detection and quantification of ME-derived 2´-deoxyadenosine (dA) and 2´-deoxyguanosine (dG) adducts we developed a sensitive tandem mass spectrometry method including stable isotope dilution analysis. Adduct formation was only observed in bacteria expressing hSULT1A1. The concentration dependence of adduct formation in hSULT1A1-proficient bacteria was examined for (+)-1´-OHME. Both adducts turned out to be concentrationdependent. To check the extent and organ specificity of adduct formation in vivo we administered 54 mg/kg bw (±)-1´-OHME (i.p.) to mice carrying the hSULT1A1/1A2 gene cluster. Mice getting only the vehicle served as controls. Animals were sacrificed and DNA from eight organs was extracted. By means of tandem mass spectrometry adducts were measured and quantified. dA and dG adduct formation was observed in all tissues studied, but not in untreated animals. Furthermore, adduct levels were higher than in experiments using wild-type mice. Altogether, we herein could show that sulfo conjugation leads to bioactivation of ME to a DNA-binding intermediate in vitro and in vivo. This work was financially supported by Bundesinstitut für Risikobewertung. Objective: The soluble adenylyl cyclase (sAC) activates the Na + /K + -ATPase in renal epithelial collecting duct cells. Nuclear sAC constitutes a functional complex with cAMP response element binding protein (CREB), suggesting a more general role of sAC in overall gene regulation. We determined the chromatin binding capacities of sAC at CRE sequences and its influence on genes, which play a role in aldosterone signalling. Furthermore, we functionally characterised expression relevant promoter portions of sAC and the influence of aldosterone and cAMP mediated signalling pathways on sAC gene regulation. Design and Methods: In vascular endothelial cells (EA.hy926) and in human kidney cell lines (HEK293T; IHKE), we performed Chromation Immunoprecipitation (ChIP) assay with antibodies against sAC and CREB. We conducted transfection with a CRE luciferase reporter vector and sAC promoter constructs, following treatment with sAC inhibitors and aldosterone. Total RNA of EA.hy926 cells, which were treated with sAC inhibitors and aldosterone, was isolated and subsequently analysed by real-time PCR for expression of genes involved in aldosterone signalling.

In vivo binding of sAC at CRE motifs was shown using CRE consensus sequences in ChIP experiments. Specific pharmacological inhibition of sAC led to a significant decrease of transcriptional activity of the CRE control vector in endothelial and kidney cell lines. Furthermore, we were able to show the different effects of sAC on the expression of downstream targets of aldosterone signalling, e.g. mineralocorticoid receptor and Na + /K + -ATPase alpha1 and beta1 and sAC itself. Regulation of sAC itself is mediated by two different promoter portions, which are influenced by aldosterone and inhibition of sAC and differentially accessed in kidney and endothelial cells.

sAC has transcriptional trans-acting properties as it interacts with CRE sites and potentially influences the expression of genes, which play a role in aldosterone signalling. Transcription of sAC is regulated via aldosterone and cAMP. The location of promoter TA is cell type-and stimulation-specific.

The role of the sodium-calcium exchanger (NCX1) in cardiac pacemaking Herrmann S., Stieber J., Ludwig A. Friedrich-Alexander-Universität Erlangen-Nürnberg Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Fahrstrasse 17, 91054 Erlangen, Germany

The mammalian heart is driven by the sinoatrial node, the primary cardiac pacemaker. The unique feature of sinoatrial node (SN) cells is the ability to generate a spontaneous diastolic depolarization that periodically initiates action potentials which set the heart rhythm. The molecular origin of this cardiac pacemaker activity is still a matter of debate. Recent findings point to a coordinated interplay between intracellular Ca 2+ -cycling processes and plasma membrane-localized ion channels which determines the origin, periodicity and rate modulation of pacemaker potentials. In this study, we investigated the contribution of the cardiac sodium-calcium exchanger (NCX1) to pacemaking. NCX1 is a key sarcolemmal protein for the maintenance of calcium homeostasis in the heart. It was speculated that the membrane depolarizing current INCX, whose activity is dependent on intracellular Ca 2+ -fluctuations, represents a main determinant of the spontaneous diastolic depolarization. We used an inducible and sinoatrial node-specific Cre-transgene to delete NCX1 in the murine pacemaker system. The successful creation of a cardiac pacemaking and conduction system specific NCX1 knockout (cpNCX1KO) was demonstrated by transcript quantification as well as immunofluorescence experiments. Telemetric ECG recordings of cpNCX1KO displayed a distinct cardiac phenotype. Mutant animals were deeply bradycardic and lost their capability of maintaining a stable heart beat as demonstrated by various ECG abnormalities like SN arrhythmia, SN pauses, AV block and ventricular tachycardia. Analysis of the spontaneous activity of isolated SN preparations showed a slower and arrhythmic contraction rate of the mutant tissues strips confirming that the bradycardia and arrhythmia induced by deletion of NCX1 results from a slower and arrhythmic intrinsic pacemaker activity. A battery of experiments using different heart rate lowering as well as increasing drugs revealed an altered heart rate modulation in cpNCX1KO animals as compared to controls. In conclusion, these initial results establish NCX1 as a major contributor to cardiac pacemaking. A wide variety of contaminants are ingested through food, among them the procarcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene (BP) which is resorbed and partially metabolized in the enterocytes of the small intestine. Previous in vitro studies revealed that BP phenols are excreted as phase II metabolites including BP glucuronides and BP sulfates. This export is mediated by the breast cancer resistance protein (BCRP/ABCG2). The ultimate carcinogenic phase I BP metabolite anti-BP-7,8dihydrodiol-9,10-epoxide (BPDE) can be detoxified by glutathione conjugate formation catalyzed by various glutathione S-transferases. In the present study, differentiated human intestinal Caco-2 cells were used as a model for the human small intestine to investigate the detoxification of BPDE and the subsequent transport of the stereoisomeric glutathione conjugates in the presence of an inhibitor (acivicin) of the glutathione-cleaving enzyme gamma-glutamyl transpeptidase (gGT) at the surface of the cells. The results indicate that the glutathione conjugates of BPDE are formed and excreted mainly to the apical and to a minor extent to the basolateral side of the polarized Caco-2 monolayer. To stimulate the transport rate several inducers known to enhance gene expression of xenobiotic-metabolizing enzymes as well as transport proteins were used (quercetin, oltipraz, butyrate). However, solely oltipraz substantially increased the efflux of BPDE glutathione conjugates after inhibition of gGT. Inhibition studies revealed that the multidrug resistance-associated proteins (MRPs/ABCCs) are involved in the transport of the BPDE glutathione conjugates. Stable ABCC1, ABCC2 and ABCC3 knockdown cell lines were generated allowing to demonstrate that ABCC1 mediates the basolateral, ABCC2 the apical excretion of the BPDE glutathione conjugates. In conclusion, the ultimate carcinogen BPDE is detoxified via glutathione conjugation and subsequently excreted by Caco-2 cells in both apical and basolateral directions. .This finding is equivalent to a transport into the feces as well as blood system in the in vivo situation.

Signaling via IRAG regulates Store operated Calcium Entry (SOCE) in aortic VSMC Hieke B., Hüttner J., Schlossmann J. University of Regensburg Department of Pharmacology and Toxicology, Universitätsstr. 31, 93053 Regensburg, Germany

The mechanisms involved in the activation of store operated calcium entry (SOCE) through depletion of intracellular stores and their regulation are not yet fully understood. We examined the effect of inositoltriphosphate-receptor associated cGMP-kinase substrate (IRAG) on SOCE. Aortic vascular smooth muscle cells (VSMC) from wild type (WT) and IRAG-Knock-out (KO) mice were loaded with the calcium indicator Fura 2-AM and SOCE was measured as a change in the intracellular calcium concentration. In experiments with VSMC from WT mice SOCE was attenuated by the application of 8-Br-cGMP. This effect was not observed in VSMC isolated from IRAG-KO mice. These differences in the strength of the SOCE-signal were abolished by the replacement of extracellular sodium with N-Methyl-D-Glucamine. The observed sodium dependence of the SOCE regulation via IRAG suggests, that an alternated sodium conductance might be responsible to some extent for the differences detected in WT and IRAG-KO VSMC. As a change in sodium conductance might result in a changed membrane potential we tried to track these changes with the FLIPR membrane potential assay kit while executing the SOCE protocol with and without 8-Br-cGMP. No significant differences in membrane potential could be detected in the various stages of SOCE. In conclusion, our results indicate that IRAG exhibits a dual action on calcium regulation as it inhibits not only the intracellular calcium release but also the extracellular calcium influx through SOCE.

Induction of the ICER promoter in vascular smooth muscle cells Hildebrandt I. 1 Tokyo Metropolitan Institute of Gerontology, Tokyo Japan Several transcription factor isoforms are encoded by the Crem (cAMP response element modulator) gene. One prominent isoform is the inducible cAMP early repressor (ICER), which acts as a transcriptional repressor on so called cAMP responsive elements (CREs) in its target gene promoters. The Icer mRNA expression is regulated by an intronic promoter of the Crem gene. In vascular smooth muscle cells (VSMCs) CREM/ICER is involved in the regulation of cell proliferation and apoptosis with physiological consequences in vivo. For instance CREM-knockout mice, in which none of the known isoforms can be expressed, exhibit an increased neointima formation after carotid ligation as well as an increased atherosclerotic plaque formation after high fat diet on an ApoE background. These observations were associated with an increased proliferation rate in isolated CREM deficient VSMCs. On this background we wanted to clarify the specific role of ICER isoforms in the vasculature. In first experiments we examined the inducibility of Icer in primary VSMCs and smooth muscle cell lines. Reporter luciferase assays showed that the activity of the Icer promoter is induced at the maximum of fourteen fold after 4 hours of stimulation with forskolin (FSK) in immortalized rat VSMCs (13.7 ± 0.93; n=12 from 3 isolations). In A7r5 rat smooth muscle cells the Icer promoter showed a maximum stimulation of 3.2 ± 0.16 fold after two hours of FSK stimulation (n=20 from 4 isolations). These pilot experiments showed that the Icer promoter is inducible in VSMCs by cAMP dependent pathways. Further experiments have to be carried out to elucidate the role of ICER in the vascular system for example by stimulation of primary VSMCs and analysis of ICER knockout mice. (Supported by the DFG)

Overexpression of transmembrane channel-like proteins (TMCs) uncouples receptor-mediated calcium mobilisation Hill K., Urban N., Straub I., Schaefer M. Universität Leipzig -Universitätsmedizin Rudolf Boehm-Institut für Pharmakologie und Toxikologie, Härtelstr. 16-18, 04107 Leipzig, Germany

The family of transmembrane channel-like proteins (TMCs) consist of 8 members (TMC1-TMC8) All TMC genes are predicted to encode transmembrane proteins with at least six membrane-spanning helices. Mutations of TMC1 cause deafness in human and mice whereas TMC6 and TMC8 (also referred to as EVER 1 and 2) are linked to epidermodysplasia verruciformis (EV), a skin disorder, which is characterised by an enhanced susceptibility towards cutanous infections by human papillomaviruses. The cell biological and physiological functions of TMC proteins still remain elusive. We have overexpressed TMC6 and TMC8 in HEK293 cells to get insights into their physiological function. All TMCs were located within the endoplasmic reticulum (ER) after overexpression of YFP or CFP-tagged constructs. Ratiometric calcium imaging revealed that after overexpression of TMC6 or TMC8, stimulation of Gq-coupled receptors with carbachol and ATP resulted in a greatly reduced amount of calcium release from the ER. Moreover, challenging TMC6-or TMC8-expressing cells with the SERCA pump inhibitor thapsigargin was also not followed by a release of ER-based calcium within the cell. To test whether the lack of calcium release was caused by a reduced calcium content within the ER, we investigated calcium dynamics within the ER using an ER-targeted FRET-based calcium indicator (D1ER Cameleon). The experiments revealed that the amount of calcium within the ER was reduced upon overexpression of TMC6 or TMC8. Recently, it has been reported that TMC6 and TMC8 might influence intracellular zinc distribution in human keratinocytes. We could confirm the presence of TMC6 and TMC8 mRNA in a human keratinocytes cell line (HaCat). Upon overexpression of TMC8, HaCat cells revealed the same phenotype as described above for the HEK293 cells with an uncoupling of the receptor-mediated calcium mobilisation due to a depletion of the ER calcium store. The mechanism by which overexpression of TMC proteins causes a reduced calcium concentration within the ER remains unclear. Considering that the presumed topology of the TMC proteins distantly resembles those of other ion channel superfamilies such as anoctamins, one might speculate that a conductance through the TMC protein itself leads to a calcium leak from the ER. Terahertz radiation is defined as radiation between 0.1 THz and 10 THz. A number of applications are currently being developed using radiation in this frequency range. These applications will lead to exposure of the general public, making it very important to study potential effects on biological systems. Historically, only a few studies on effects caused by terahertz radiation have been conducted because of the lack of suitable generators and detectors. During the last decade, a number of studies on effects caused by radiation with frequencies around 100 GHz have been published. The present study investigated the genotoxic potential of terahertz radiation at three different frequencies, 0.106 THz, 0.380 THz and 2.520 THz. Two skin cell types were used, primary human dermal fibroblasts (HDF) and a keratinocyte cell line (HaCaT). The cells were irradiated applying different exposure times and different power intensities. Two genotoxicity tests were applied: the comet assay quantifies DNA strand breaks as well as alkali-labile sites whereas the micronucleus test quantifies chromosomal damage. All experiments were performed and evaluated under blinded conditions as three independent replicate experiments. Positive (MMS-treated) and negative (untreated, sham-exposed) controls were included. In the comet assay no DNA damage was observed as a consequence of the exposure under all experimental conditions. The same was true for the chromosomal damage investigated with the micronucleus test. The latter finding was particularly interesting for the experiments at 0.106 THz, because this type of radiation had been reported to cause mitotic disturbances. Therefore these experiments were extended, applying higher power intensities and longer exposure periods. Also with these modifications, no genomic damage was observed in the form of micronucleus formation. All in all, terahertz radiation did not induce genomic damage under the applied experimental conditions. This result is in line with published findings on genotoxicity of low-frequency terahertz radiation around 0.1 THz. The question, why the reported mitotic disturbances do not lead to manifest genomic damage remains open and requires further research. Introduction: Tea flavonoids derived from camomile and green tea such as apigenin and epigallocatechin gallate (EGCG) can inhibit intestinal neoplasia. Recurrences of adenomas and cancers were reduced in patients with resected colorectal cancer by treatment with tea bioflavonoids after tumor operation [1] . To clarify the biomolecular pathway for suppression of neoplasia we investigated the anti-inflammatory effect of a nutritional supplement Flavo Natin® (FN) which had been used in the clinical study on tertiary tumor prevention and of EGCG in a colon tumor cell line. The aim of our study was to investigate if tea flavonoids are capable to suppress the inflammatory markers produced by tumor cells after cytokine stimulation. Method: We studied the cytotoxicity of FN in the colon cancer cell line T-84 by resazurin fluorescence and compared it with the placebo supplement. Additionally, the T-84 cells were incubated with FN, EGCG or placebo and stimulated with TNF-alpha, IF-gamma and IL-1-beta. After the cytokine stimulation the mRNA expression of IP-10, IL-8 and TNF-alpha was measured by quantitative Real-Time PCR (qRT-PCR). Results: Stimulation of T-84 cells increased the expression of IP-10 (gamma-interferon inducible protein 10), TNF-alpha and IL-8. By preincubation with FN at 10 µM the mRNA expression of IP-10 was strongly reduced (log2-ratio -14). The TNF-alpha mRNA was also but less decreased by FN. EGCG displayed an inhibition pattern similar to FN. Placebo did not influence the mRNA expression of the chemokines and TNF-alpha. Discussion & Conclusion: Clinically useful dietary tea bioflavonoids inhibit the expression of inflammatory genes in a colon cancer cell line. Down-regulation of inflammatory gene products could be achieved in vivo by botanicals without clinically relevant side effects.

[1] H. Hoensch, B. Groh, L. Edler, W. Kirch (2008) . Prospective cohort comparison of flavonoid treatment in patients with resected colorectal cancer to prevent recurrence. World J Gastroenterol, 14, 2187-2193.

The CXCR7 C-terminal domain mediates efficient CXCL12 uptake and degradation Hoffmann F., Müller W., Schütz D., Schulz S., Stumm R. Universitätsklinikum Jena Institut für Pharmakologie und Toxikologie, Drackendorfer Str. 1, 07747 Jena, Germany CXCL12-signaling mediated by the G protein-coupled CXCR4 receptor plays a key role during embryonic development and disease states including cancer and inflammation. The second CXCL12-receptor CXCR7 modulates CXCL12/CXCR4-signaling by acting as a CXCL12-scavenger. Given the distinct functions of CXCR4 and CXCR7, we hypothesized that trafficking and receptor stability are differently regulated by the distinct C-terminal domains. Here, we examined epitope-tagged wild type and C-terminal mutant receptors expressed in human embryonic kidney cells (HEK293) with respect to trafficking, stability, 125 I-CXCL12 radioligand degradation, and G protein-coupling. We found that the 24 C-terminal residues of CXCR7 were sufficient for CXCR7 to undergo rapid spontaneous internalization. Replacement of the CXCR7 C-terminal domain with that of CXCR4 (CXCR7-4tail mutant) abolished spontaneous internalization but permitted ligand-induced internalization in conjunction with C-terminal phosphorylation. Conversely, replacement of the CXCR4 C-terminal domain by that of CXCR7 caused ligand-independent internalization of CXCR4. Receptor-mediated 125 I-CXCL12-uptake, release of 125 I-CXCL12-degradation products, and degradation of the receptor protein itself were accelerated with receptors bearing the CXCR7 C-terminus. While the CXCR7 C-terminus was sufficient to abolish G protein coupling in the CXCR4-7tail mutant, replacement of the CXCR7 C-terminus, CXCR7 second intracellular loop or both domains with the corresponding CXCR4 domain did not generate a G protein-coupled CXCR7 chimera. Taken together, we provide evidence that the CXCR7 C-terminal domain influences the ligand-uptake/degradation rate, G protein-coupling, and stability of the receptor. This suggests that heterologous regulatory pathways targeting the CXCR7-C-terminal domain may effectively control CXCR7 functions.

Soluble guanylyl cyclase is a key mediator of brown adipocyte differentiation Hoffmann L. S. 1 Brown adipose tissue (BAT) uses energy to produce heat by inducible thermogenesis. Recent studies show that active BAT is present in adults and involved in human energy balance, suggesting that the energy consuming property of BAT might be exploited to fight obesity and related diseases. The NO/cGMP signaling pathway is a key player in diverse physiological processes. Recently, we have shown in BAT that cGMP signaling is connected with insulin signaling and abrogation of cGMP signaling leads to impaired BAT differentiation and function (Haas, B. et al., Sci Signal, 2009 ). Here we investigated the role of the cGMP generating enzyme soluble guanylate cyclase (sGC) in BAT differentiation in vitro. Mesenchymal stem cells isolated from BAT of newborn sGCβ 1 -/mice and wt littermates were differentiated in vitro into brown adipocytes in the presence or absence of cGMP. Abundance of sGC isoforms was determined by qRT-PCR and Western Blotting. BAT differentiation was assessed by RedO staining of accumulated intracellular lipids, measurement of triglyceride (TG) content, determination of expression of BAT marker proteins PPARγ, C/EBPα, aP2 and BAT marker genes UCP1, PGC1α, Cidea. The α2 and β1 isoforms of sGC were highly expressed in BAT whereas α1 sGC could not be detected. RedO staining of wt brown adipocytes showed basal differentiation which was increased upon addition of 8-pCPT-cGMP. In contrast, staining was lower in sGCβ1 -/cells compared to wt under control conditions and increased in the presence of cGMP. TG measurement showed that sGCβ1 -/brown adipocytes contain approximately 50% less lipids than wt cells under basal conditions. Addition of cGMP doubled TG content in both genotypes. Similar results were observed for marker protein expression. Deletion of sGC resulted in 56-40% decrease in C/EBPα, PPARγ and aP2 expression compared to wt. Again, cGMP roughly doubled protein expression in sGCβ1 -/and wt cells compared to control. Under basal conditions, BAT marker gene expression was decreased by approximately 80% in sGCβ1 -/cells compared to wt cells. This decrease was prevented by addition of cGMP. These results show that sGC deletion leads to dysfunctional BAT differentiation and emphasize the central role of cGMP signaling in BAT differentiation. Further investigation of sGC/cGMP signaling in BAT might reveal new drugable targets bringing BAT-dependent pharmacological therapy to treat obesity and related disease into closer reach. 

Comparative Inhalation toxicity of Carbon-Nanomaterials (multi-wall carbon nanotubes, graphene and carbon black) Hofmann T. 1 Carbon black is a spherical carbon anomaterial whereas multi-wall Carbon nanotubes (MWCNT) are cylindrical and graphene is a laminar allotrope of carbon. Processing and handling as well as abrasion processes can set free inhalable CNT particles. Results of rodent studies collectively show that regardless of the process by which CNTs were synthesized and the types and amounts of metals they contained, CNTs were capable of producing inflammation, epithelioid granulomas, fibrosis, biochemical and or toxicological changes in the lungs (Lam et al. 2004 , Muller et al. 2005 , Ma-Hock 2009 , Pauluhn 2010 . Graphene possess similar physical properties as CNT but may different toxicological property. We performed short-term inhalation studies in rats to compare the toxic potency of four different CNT, two graphenes and one carbon black. The materials are characterized thoroughly according to the OECD list. The four MWCNT caused morphological changes as descriped above. Several biochemical and cytological parameters in the broncho-alveolar lavage fluid were strongly increased consistent with the histological findings. Two MWCNT exhibited a higher toxic potency than two other MWCNTS and findings caused by one Graphene typ were even less severe. The graphene with lower surface area as well as low surface area carbon black did not cause any adverse effects up to 10 mg/m 3 . The short-term inhalation studies were able to descriminate different toxic potencies of carbon-based nanomaterials and is hence used for the selection of less toxic materials for further product development as well as to define and prioritize higher-tier toxicological testing of nanomaterials.

165 Synthesis and analytical assessment of possible DNA adducts formed after activation of the tobacco alkaloid myosmine Högg C., Zwickenpflug W., Gudermann T. Walther-Straub-Institut Abt.: Toxikologie, Nußbaumstraße 26, 80336 München, Germany Myosmine represents one of the minor tobacco alkaloids and its effective uptake from smokeless tobacco or tobacco smoke, as well as by consumption of food is not understood in detail. Myosmine can be activated by peroxidation and N-nitrosation yielding 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) which is well known as reactive intermediate during activation of a variety of tobacco specific N-nitrosamines (TSNA). Therefore, myosmine may be a potential candidate for possible mutagenic or carcinogenic risk to human health. Furthermore, myosmine N-nitrosation yields the tobacco specific nitrosamine N-nitrosonornicotine (NNN), which is classified as carcinogenic to humans. Considerable efforts have been undertaken, especially in organic synthesis, to verify and elucidate the significance of the HPB precursor the pyridyloxobutyl (POB) intermediate and its DNA-adducts, which were analysed only in animal experiments till now. The formation of 3-pyridylmethanol was observed under myosmine peroxidation and identified as a metabolite in rat urine after application of myosmine to rats. The formation of the 3-pyridylmethanol intermediate, might provide for the reactive electrophilic picolinium ion which could interact with DNA. These possible adducts might be of special interest to elucidate the role of myosmine concerning its uptake by smokers and passive-smokers in contrast to non smokers ingesting the substance by consumption of food. DNA adducts may help to obtain more information about possible risk assessment of myosmine and to differentiate between the activation from the other nicotinoids and TSNA. The specific DNA adducts, 5-methyl-1,3-bispyridin-3-ylmethyl-1H-pyrimidin-2,4-dion and 3-(3''-picolyl)thymidine have been synthesised as reference substances. The former was prepared by addition of diisopropylazodicarboxylate (DIAD) to a mixture of 3-pyridylmethanol, thymine and triphenylphosphine. The reaction product was identified using NMR ( 1 H, 13 C, COSY, HMQC, HMBC). For synthesis of 3-(3''-picolyl)thymidine the hydroxyl groups of thymidine were initially acetylated using acetic acid anhydride and 4dimethylaminopyridine (DMAP). The existence of this thymidine adduct was confirmed using NMR. This adduct was labelled with 5-([4,6,-dichlorotriazin-2-yl]amino)fluorescin (DTAF) to enhance the sensitivity using HPLC-fluorescence chromatography and used as reference substances for analysis of DNA samples from biological tissue. Supported by DFG grant (TY 81/1-1). Cancer and cardiovascular diseases such as atherosclerosis are the most important causes of death in western societies. Common to both diseases is a deregulation of cell death, with significant contribution of inflammatory processes. Enhanced oxidative stress plays a dominant role in such events as it forms a vicious cycle with inflammation and controls multiple forms of cell demise. Therefore, anti-oxidative enzyme systems gained considerable interest since control of reactive oxygen species (ROS) has the capacity to regulate cell death in either direction. The human enzyme family of paraoxonases consists of three members, known as PON1, PON2 and PON3. While PON1 is found predominantly in the circulation, PON2 and PON3 are intracellular enzymes with established anti-oxidative functions. It has been shown that both PON2 and PON3 are protective against atherosclerosis. Underlying mechanisms of their protective and antioxidative functions however remained elusive. Here we demonstrate that both enzymes locate to the endoplasmic reticulum (ER) and mitochondria where they fulfill vital functions in the control of ROS generation. In particular, PON2 and PON3 were shown to interact with coenzyme Q10 which diminishes mitochondrial ROS formation. As a consequence, these enzymes reduce execution of mitochondrial apoptosis, such as cardiolipin peroxidation, cytochrome C release and caspase activation. Moreover, PON2 and PON3 reduced ER stress-triggered cell death, i.e. by diminishing JNK signaling and CHOP expression. While these results elucidate their protective role in cardiovascular diseases, it also establishes a relevant function in survival of tumor cells. In accordance, we demonstrate that both enzymes are frequently found overexpressed in various tumors. In cancer cell culture studies, overexpression of both enzymes granted considerable resistance against chemotherapeutics. In turn, knock-down of PON2 caused spontaneous apoptosis of several cancer cell lines. Finally, our analyses also revealed that PON2-knockout mice show severe alterations of the hematopoetic stem cell compartment, which implies a significant role in leukemias where these enzymes are frequently found overexpressed. Together, our results propose PON2 and PON3 as new putative anti-tumor candidates and demonstrate the efficacy of interventions targeting cellular redox-balance. Steigerwald Arzneimittelwerk GmbH Wissenschaftliche Abteilung, Havelstr. 5, 64295 Darmstadt, Germany STW 5 (Iberogast ® ), a multi-component herbal drug, is successfully used in the therapy of functional dyspepsia and irritable bowel syndrome (IBS). Previous studies revealed effects of STW 5 on disturbed motility and inflammatory processes. Although the antiinflammatory properties of STW 5 are well examined, the contribution each of the individual herbal constituents to the anti-inflammatory effect remains unclear. Therefore, we studied the effects of STW 5 and its components on inflammation-induced cell death and on the release of the pro-inflammatory cytokine TNF-α. The aim of these investigations was to analyse additive or synergistic effects of the components. The experiments were carried out on CaCo-2 cells after stimulation with LPS (10 ng/ml) for 2 hours. Cytotoxicity was evaluated using a commercially available LDH (lactate dehydrogenase)-assay. Furthermore, the release of TNF-α after LPS (100ng/ml) stimulation of differentiated THP-1 cells was measured using a commercially available ELISA. STW 5 (62.6 -500.5 µg/ml) reduced LPS (10 ng/ml)-induced cell death in a concentration-dependent manner with a maximum inhibition of 50.0 %. The herbal components in equivalent concentrations contributed to the inhibitory effect of STW 5 to different extents. The maximum inhibition differed in a wide range between the components. STW 5 (500.5 µg/ml) reduced significantly the release of TNF-α by 87 % in LPS (100 ng/ml)-stimulated differentiated THP-1 cells while having no effect in untreated cells. In concentrations equivalent to STW 5 caraway, milk thistle, lemon balm and greater celandine had no effect on the LPS-induced increase in TNF-α release. Bitter candytuft, peppermint, chamomile, liquorice and angelica reduced the TNF-α release, though less pronounced as compared to STW 5, indicating a possible synergistic effect.

Our results indicate a multi-target effect of STW 5. The anti-inflammatory effect may be due to a reduction of the cytotoxic effect on intestinal mucosa cells and to an inhibition of the release of the pro-inflammatory cytokine TNF-α from immune cells. The individual herbal components seem to contribute by different mechanisms of action to the overall effect of STW 5.

Immune cell-induced local steroidogenesis in the lung: implications for asthmatic disease and therapeutic intervention Hostettler N. 1 , Brunner T. Glucocorticoids are steroid hormones with potent anti-inflammatory properties. Synthetic glucocorticoids are frequently used for the therapeutic treatment of inflammatory disorders, such as asthma. Endogenous glucocorticoids are predominantly produced in the adrenal glands in response to emotional, physical and immunological stress. Recent years, however, revealed several alternative sources of these immunoregulatory steroid hormones. Thus, we have found that the intestinal epithelium is a rich source of glucocorticoids and intestinal glucocorticoids contribute to the maintenance of local immune homeostasis (1) . As the intestinal and the pulmonary epithelium have much in common, i.e. barrier functions and transport of nutrients, resp. gases, we wondered whether the lung mucosa is also capable of synthesizing immunoregulatory glucocorticoids in response to immune cell activation. The murine lung was found to expresses all enzymes required for the synthesis of corticosterone from cholesterol. While most enzymes where expressed in a constitutive manner, Cyp11a1, encoding P450ssc, was strongly induced in response to immunological tress. Treatment of mice with T cell-activating anti-CD3 antibody of macrophage-activating lipopolysaccharides induced strong local glucocorticoid synthesis, which was effectively blocked by the corticosterone synthesis inhibitor metyrapone, indicating that glucocorticoids measured were produced bona fide in the lung tissue. Surprisingly, allergen-induced allergic airway inflammation failed to trigger local glucocorticoid synthesis despite the massive infiltration of neutrophils, eosinophils and T cells. In contrast to that in the intestinal epithelium local glucocorticoid synthesis in the lung was found to be dependent on the presence of adrenal glands as adrenalectomy abolished pulmonary steroidogenesis. In line with the notion that the lung metabolizes steroid precursors we found that ex vivo cultured lung tissue metabolized 3 H- During the last decade small RNA molecules has been identified as important regulators for gene expression. These microRNAs (miRNA) are single-stranded transcripts, which are expressed in many cell types, where they modulate RNA stability and translation and, therefore, controlling various cellular mechanism and tissue development. Against this background, in the present study the miRNA expression and potential target genes were studied in the human placenta as a tissue demonstrating various developmental changes in a limited period of time.

TaqMan®Array MicroRNA cards for profiling of 365 miRNAs in placentas of different gestation times revealed a significant expression of 277 miRNAs by comparing placentas of early gestation (<10.week), preterm (<30.week) and term (>37.week). When comparing the analyzed groups, 106 of these miRNAs expose a continuous up-(including miR-20a, miR-379) or downregulation (including miR-9, miR-200a). While comparing placentas of early gestation to term placentas, 30% (e.g. miR-9, miR-429) were up-and 70% (e.g. miR-126, miR-373) were downregulated. By focusing on the latter group with early preterm placentas the ratio is opposed. Here, 60% of miRNAs showed higher (e.g. miR-107) and 40% lower expression (e.g. miR-627, miR-191). With emphasize on these miRNAs, a computational prediction algorithm using the miRò database predicted a potential interaction between corticotropin-releasing hormone (CRH) and the miRNAs miR-9 and miR-429. Specific expression analyses validate an inverse expression between miRNAs and target with a reduced expression of miR-9 (93%) and miR-429 (94%) in term placentas, while CRH is upregulated 114fold. This interaction was verified on functional level by reporter gene assay. A significantly suppressed luciferase activity of the reporter plasmid containing the 3´-UTR sequence complementary to CRH was exhibit for the predicted miR-9 binding site. Here, the miRNA-mRNA-interaction reduces the luciferase activity by ~40%, whereas the decreased luciferase activity for the predicted miR-429 binding site is not significant.

The results demonstrate a gestation dependent expression of placental miRNAs, which may help to explain gestational changes in gene expression and highlight the potential of miRNAs as biomarker for pregnancy-related pathologies. Since Homo sapiens era wound treatment by early civilizations was based on the use of local flora. Scilla Indica Knuth Liliaceae (S.i) is a perennial herb with a pear shaped, tunicated bulb, bearing fibrous roots, white flowers and leaves on the stem resembling U. maritima. It is used as cardiac tonic, against inflammation, ulcers and sinus diseases. Traditionally the powdered bulb is used topically for warts treatment, while roasted and crushed bulbs are applied to corns of the feet soles. The plant contains steroid glycosides (bufadienolides 1-3%), glucoscillarene A, proscillaridine A, scillarene A, scillcyanoside ,scilliglaucoside ,mucilage and alkyresorcinol derivatives exerting skin healing properties similar to wheat bran products. The study is focused on the investigation of the restoration quality of a skin dorsal incision wound in Wistar rats In three groups, control (A1) and experimental (A2,A3 ) of rats weighing 350-450g local application of dichloromethane extract of S.i in vehicle olive oil preparations of 50 and 100mg were used. Animals were anaesthetized ( ether pro narcosi ), trauma 2 cm long and 2mm deep was performed by lancet on depilated dorsal area skin until the muscular aponeurosis and were treated for 4 days with 60λ of each preparation. Group A3 showed increased remodelling of trauma area (limited width). Granulomatous tissue was more pronounced in length, width and surface in group A2. The macroscopic ulcus dimensions were better in group A3 while the microscopic view were similar in A2 and A3 and better in comparison to control A1. A tendency to trauma remodelling was observed, without statistical significance (t =0.3) In recent years, it has been suggested that nanoparticles generated from combustion processes (e.g. diesel engine exhaust particles), may contribute to the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD). The aim of our current study was to investigate the effects of subchronic exposure to diesel engine exhaust (DEE) in the 5xFAD mouse model, which is characterised by progressive behavioural deficits as well as amyloid plaque formation and neuron loss. Ten weeks old female 5xFAD mice and their nontransgenic littermates were exposed by whole body inhalation to diluted DEE (~1 mg particles/m 3 ) or clean air (controls) for 3 or 13 weeks (5 days/week and 6 hour/day). Subsequently, all animals were subjected to a series of behavioural tests. At ten days post-exposure, mice were sacrificed to investigate lungs and brain tissues for pathological and biochemical and molecular-biological changes. In line with the expectations, the 5xFAD mice displayed typically age-dependent behavioural deficits and amyloid plaque formation in cortex and hippocampus. A significant DEE exposure-related effect was observed for the string suspension test, representing a measure of motor coordination/grip strength. DEE exposure was also associated with mRNA expression changes of markers of inflammation and oxidative stress in specific brain regions, including the olfactory bulb. Histopathology of plaqueload in cortex and hippocampus (from a limited number of animals investigated so far) did not reveal clear evidence for increased plaque formation due to the DEE exposures. Further research is needed to evaluate the effects of long term exposure to nanoparticles in the central nervous system. This work is supported by funds from the Research Committee of the Medical Faculty of the University of Düsseldorf (9772-365), the DFG Graduate School GRK1033 and the RIVM -Centre for Environmental Health, Bilthoven, Netherlands. Mono-glucosylation of (H/K/N)Ras by Clostridium sordellii lethal toxin (TcsL) blocks critical survival pathways, including the PI3K/Akt, the RalGEF/Ral, or the Raf/ERK, and results in apoptosis. In this study, Ras glucosylation is presented to result in expression of the cell death-regulating small GTPase RhoB based on transcriptional activitation. RhoB expression depends on K-Ras inhibition, as siRNA-mediated knock-down of specifically K-Ras (neither H-Ras nor N-Ras) provokes RhoB expression. RhoB expression further depends on inhibition of PI3K/Akt, as activation of PI3K/Akt using a PI3K activator prevents RhoB expression downstream of inactivated Ras. Newly synthesized RhoB is GTP-loaded and rapidly degraded in a proteasome-and a caspase-dependent manner, providing first evidence for caspase-dependent degradation of a Rho family protein. Although often characterised as a pro-apoptotic protein, RhoB suppresses caspase-3 activation in TcsL-treated fibroblasts. Conclusions: 1. Rapid and efficacious Ras inactivation by TcsL turns out to be particularly useful in the characterisation of Ras inactivation-induced RhoB expression as an immediate-early gene response. 2. The finding on the cytoprotective activity of RhoB in TcsL-treated cells re-enforces the concept that RhoB exhibits cytoprotective rather than pro-apoptotic activity on cellular background of inactive Ras.

The activity of the rhoB promoter is suppressed by RhoA (through a not yet identified pathway or Ras (through the PI3K/Akt pathway. Ras glucosylation by TcsL results in de-suppression of the rhoB promoter and RhoB expression. The calcium-binding protein Annexin A4 (ANXA4) is involved in diverse cellular processes including e.g. vesicular transport, ion channel regulation and transcriptional regulation. Upon Ca2+ binding ANXA4 undergoes conformational changes, which lead to oligomerization of ANXA4 to homotrimers and cause an increased affinity for membrane phospholipids, which in turn provokes the translocation from the cytosol and the nucleoplasm to plasma and nuclear membranes. In order to examine the effect of ANXA4 on CRE-and CREB-(cAMP response element-binding protein) dependent transcription, a series of transient transfections using a luciferase reporter gene driven by the CREB target promoter of the inducible cAMP early repressor (ICER) was carried out. When the luciferase reporter construct was co-transfected with ANXA4, there was significant reduction of basal (DMSO) luciferase activity ( The implantation of Drug Eluting Stents (DES) after coronary artery intervention was an important step treating coronary artery disease. Indeed, cytotoxic compounds like sirolimus used on DES are responsible for reduced in-stent restenosis in comparison with bare metal stents. Pimecrolimus, a potent anti-inflammatory drug has also been investigated for its efficacy on DES. Preclinical studies in pigs revealed promising antiproliferative effects of pimecrolimus on neointima formation. However, in humans, pimecrolimus coated stents exerted adverse effects. We hypothesize that compared to the highly active sirolimus, pimecrolimus may influence additional cellular processes leading to the worse outcome. In order to identify those processes we conducted in vitro studies in human coronary artery endothelial cells (HCAEC) and smooth muscle cells (HCASMC). BrdU in vitro assays of HCAEC treated with pimecrolimus examined an IC50 value of 6.787 µM [5.745 to 8 .017] which is much higher than IC50 values of sirolimus described in literature [0.1nM to 1nM]. GeneChip array analysis comparing gene expression in pimecrolimus and sirolimus treated HCAEC and HCASMC showed significant induction of several genes involved in interferon signaling. In detail, the expression of IFNβ activated genes like IRF9 and IFITM1 was up regulated in cells treated with pimecrolimus while no or oppositional effects were observed with sirolimus. Gene regulatory effects were validated by real time PCR. Incubation with IFNβ itself showed similar effects in up regulation of genes involved in interferon signaling. Furthermore, we were able to demonstrate a significant increase of IFNβ secretion in HCASMC and HCAEC treated with pimecrolimus. However, comparison of IFNβ and pimecrolimus on proliferation of HCAEC and HCASMC revealed different cellular responses. While IFNβ significantly decreased HCASMC and increased HCAEC proliferation, treatment with pimecrolimus lead to anti-proliferative effects on both cell types.

In conclusion, pimecrolimus activates pathways involved in interferon signaling but exerts different pharmacological effects, compared to the endogenous compound suggesting that INFβ secretion is not the major factor contributing to the difference in pimecrolimus function.

Identification of novel phospho acceptor sites of the mu opioid receptor regulating receptor internalization Illing S., Just S., Doll C., Schulz S. Uniklinikum der FSU Jena Pharmakologie und Toxikologie, Drackendorfer Straße 1, 07747 Jena, Germany

The opioid alkaloid morphine is among the most potent clinically used analgesics. However, the clinical utility of morphine to treat chronic pain is limited by its rapid development of tolerance and dependence. The stimulation of the mu opioid receptor (MOR) with DAMGO or morphine results in different patterns of receptor phosphorylation and trafficking. So far, the three major phosphorylation sites of the mu opioid receptor namely serine 363 (S363) threonine 370 (T370) and serine 375 (S375) have been identified using phosphosite-specific antibodies. However, mutations of these three residues to alanine (S363A/T370A/S375A) did not prevent agonist-dependent internalization. In the present study, we have constructed a series of phosphorylationdeficient mutants showing that mutation of at least six residues (S363A/T370A/S375A/T376A/T379A/T383A) was required to completely block agonistdriven endocytosis. Consequently, we generated phosphosite-specific antibodies to T376 and T379 which enabled us to provide direct evidence for agonist-dependent phosphorylation of these sites. Our analysis of time-and dose-dependent phosphorylation of MOR revealed that S375 was the primary phosphorylation site, whereas T370, T376 and T379 were secondary sites. Moreover, the partial agonist morphine induced only the phosphorylation of S375 but not of T370, T376 of T379. Our results show, that MOR phosphorylation occurs in a hierarchical and agonist-selective manner directly regulating receptor sequestration.

Assessment of MDA effects from toxicity studies with regard to endocrine modulation Jäger R. 1 Methylene dianiline (MDA; CAS No. 101-77-9) is on the USEPA List 2 for endocrine disruption screen testing. In a yeast androgen screening (YAS) assay (in vitro assay on receptor binding or blocking) MDA revealed a significant anti-androgenic activity at a concentration of 0.01 mM. To assess the biological relevance of this observation under in vivo conditions the existing data from animal toxicity studies with MDA were reviewed for indications of possible endocrine modulating (EM) effects (Weight-of-Evidence approach). In addition, literature was searched for relevant studies on MDA and structural analogues using the American Chemical Society SciFinder client-server software.

Structural analogues indicated several drivers for EM activity, notably the diphenolic ring structure. However, in vitro receptor binding assays showed MDA had no androgenic or anti-estrogenic activitiy. One report described a weak estrogenic binding at 0.2 mM MDA, while another described no effect at similar concentrations and a rat estrogen receptor binding study found no effect at 0.2 mM. Overall it is concluded that MDA does not bind to the estrogen receptor. Endocrine related effects of MDA were investigated in several species and dose routes in unvalidated research-type protocols. Guideline subchronic and chronic toxicity studies with rodents revealed neither adverse organ weight changes nor histopathological alterations of sex organs. The main systemic effects from the oral 13-week studies were body weight reduction and histopathological changes of the thyroid and bile duct (lowest LOAEL: 7.5 mg/kg bw). MDA was carcinogenic to rats and mice (thyroid and liver) after oral administration over 2 years. Non-neoplastic effects in the thyroid (rats and mice) were observed (lowest LOAEL for systemic toxicity: 9 mg/kg bw). MDA inhibits iodide oxidation which with concomitant decreased thyroid hormone formation is known to induce thyroid tumors.

In summary, in vitro screening tests revealed no consistent endocrine related effects of MDA. In vivo, there was an effect on the thyroid gland, possibly by enzyme inhibition. There were no histopathological changes of gonads and accessory sex organs and no evidence of sex hormone related EM. The evidence from the full dataset on MDA does not indicate androgenic or estrogenic effects. Overall, based on a weight of evidence assessment there are insufficient alerts for EM activity to suggest further testing should be done.

The GTPase ARFRP1 controls the assembly of ApoA-I to and the lipidation of chylomicrons in the Golgi of intestinal epithelium Jaschke A. 1 Background: The uptake and processing of dietary lipid by the small intestine is a multi-step process that involves luminal digestion, cellular uptake of fatty acids by the mucosa, and subsequent synthesis and export of chylomicrons. The GTPase ADPribosylation factor related protein 1 (ARFRP1) is a member of the ARF-family and controls the ARF-like 1 (ARL1)-mediated Golgi recruitment of GRIP domain proteins which in turn bind several Rab-GTPases. The aim of the study was to define the role of ARFRP1 in intestinal nutrient absorption. Methods: For the generation of intestine-specific null mutants Arfrp1 flox/flox mice were crossed with mice expressing the Cre recombinase under the control of the intestinespecific villin promoter (vil-Cre) and Arfrp1 expression was suppressed by siRNA in Caco2-cells. The phenotype in respect to lipid absorption and chylomicron production in the intestinal epithelium and in Caco2-cells, respectively, was analyzed. Results: Arfrp vil-/mice were viable but showed an early postnatal growth retardation (mean body weight of Arfrp1 vil-/was 43.3±5% lower than that of control mice at the age of 28 days) Arfrp1 vil-/mice displayed reduced triglycerides, free fatty acids and glucose plasma levels indicating that the growth retardation is the result of a malabsorption. Uptake of glucose and amino acids were unaffected by the deletion of Arfrp1. In contrast, lipid uptake as elucidated by oral fat tolerance tests was impaired in Arfrp1 vil-/mice. Arfrp1 vil-/enterocytes as well as Arfrp1 mRNA depleted Caco-2 cells absorbed fatty acids normally but secreted chylomicrons with a markedly reduced triglyceride content. In addition, while the release of apolipoprotein A-I (ApoA-I) was dramatically decreased ApoA-I accumulated in the Arfrp1 vil-/epithelium and was predominantly colocalized with Rab2.

Our results demonstrate that the growth retardation of Arfrp vil-/mice is a consequence of impaired intestinal fatty acid absorption. We suggest that ARFRP1 is required for the assembly of AopA-I to the chylomicrons and for the further lipidation of chylomicrons in the Golgi of intestinal epithelial cells. This finally leads to an secretion of chylomicrons with a markedly reduced triglyceride content.

RhoA influences adhesion and spreading of cardiac fibroblasts via complex regulation of cytoskeletal proteins Jatho A. 1 The monomeric GTPase RhoA is thought to be involved in the pathology of heart diseases, however, its role in cardiac cells is not well defined. Therefore we intended to analyze the effect of RhoA in cardiac fibroblasts by using a lentivirus-based knockdown approach. By doing so, we could show that the knockdown of this GTPase by about 50% resulted in a massive change in cell morphology, but displayed no effect on cell viability. The appearance of the cells in the 2D-culture changed from a fibroblast-typical stretched morphology with intracellular stress fibers to a more epithelial-like cell morphology. By morphometrical analyses we demonstrate that fibroblasts with reduced RhoA expression display an increase in cell surface by 2.2-fold and in perimeter by 1.5fold. Moreover, the depletion of RhoA significantly influences the adhesion velocity, as within the first hour after cell detachment about 20% of the RhoA knockdown cells reattach, but only 5% of the respective control cells. Based on these findings, we investigated the distribution and composition of different cytoskeletal proteins by immunofluorescence stainings and immunoblot analysis. We found, that the amount of b-actin is not reduced in RhoA knockdown cells, however, the distribution is markedly changed. In these cells internal star-shape bundles of actin could be found instead of the commonly appearing stress fibers in control cells. In contrast, the cortical actin fibers, mainly consisting of g-actin, were not affected. In addition, smooth muscle-actin, which is characteristic for myofibroblasts, was clearly reduced in RhoA knockdown cells compared to control cells by 33%. This reduction might be responsible for the more relaxed cell shape. In summary, the knockdown of RhoA influences cell adhesion and the morphological characteristics of cardiac fibroblasts, without obviously affecting cell proliferation and viability.

Using site-specific fluorescence labeling to study uptake of Pasteurella multocida toxin into eucaryotic cells Jehle D., Aktories K., Orth J. Institut für Experimentelle und Klinische Pharmakologie und Toxikologie Abteilung 1, Albertstraße 25, 79104 Freiburg, Germany Pasteurella multocida is an opportunistic pathogenic bacterium living in the nasal pharyngeal space of animals. P. multocida is of particular importance in the livestock management of pigs. Under special conditions infection of pigs with P. multocida leads to an atrophic rhinitis, which is characterized by the atrophy of nasal turbinate bones accompanied by a shortening and twisting of the snout. The causative agent of the atrophic rhinitis was found to be the bacterial protein toxin PMT (Pasteurella multocida toxin). After entering the cell the 146-kDa toxin activates various signal transduction pathways by stimulating heterotrimeric G proteins of the Gαq, Gα13 and Gαi family. The underlying mechanism of the activation of heterotrimeric G proteins is a deamidation of an essential glutamine residue leading to a constitutive activation of the G protein. The uptake of PMT into cells is not comprehensively understood. Therefore, we utilized a recently described technique, called "sortagging" (Popp MW et al. Nat Chem Biol. 2007; 3: 707) , to specifically couple fluorescence tags to the N-or C-terminus of PMT. The enzyme sortase A (SrtA) from Staphylococcus aureus attaches proteins to the bacterial cell wall. The substrates are recognized by an LPXTG motif. SrtA cleaves the peptide bond after the threonine and adds a glycine-containing nucleophile. We introduced these motifs into PMT to express SrtA-recognized toxin and coupled the toxin with fluorescence tags, respectively. Fluorescently labeled PMT was used to study the uptake of the toxin into eucaryotic cells by laser scanning microscopy. Munich Heart Alliance, München, Germany Cells within the myocardium communicate by secreted factors and this has been suggested to contribute to cardiac remodeling. To identify novel factors secreted by the myocardium, we have previously reported a genetic yeast screen which led to the identification of Protease Inhibitor 16 (PI16). Here we report the generation of a mouse line where PI16 can be deleted globally or conditionally using the Cre/LoxP system. After electroporation of the Pi16 floxneo targeting vector in embryonic stem cells and injection into murine blastocysts we gained a mouse line that carried the targeted modification of the Pi16 allele. Global PI16 deficiency (Pi16 lox/lox ) per se did not lead to a cardiac phenoytpe (HW/BW (mg/g): Pi16 +/+ = 5.4 ± 0.2 (n = 6), Pi16 lox/lox = 5.9 ± 0.7 (n = 4), P > 0.05; Fibrosis (%): Pi16 +/+ = 3.3 ± 0.2 (n = 7), Pi16 lox/lox = 3.9 ± 0.8 (n = 5), P > 0.05; Fractional Shortening (%): Pi16 +/+ = 29.8 ± 0.7 (n = 7), Pi16 lox/lox = 26.4 ± 2.5 (n=5), P > 0.05). In addition we carried out an immunohistochemical analysis of PI16 expression using PI16-deficient mice as negative controls. PI16 localized to cardiac fibroblasts, to the epididymis and the trachea. In the failing heart we detected accumulation of PI16 preferentially in fibrotic areas. We are currently applying cardiac stress models to gain a broader understanding of the function of PI16 and its potential as a therapeutic target molecule.

Enantioselective determination of R-and S-hyoscyamine in mammalian plasma and urine samples John Atropine (Atr) is the racemic mixture of the tropane alkaloids S-and R-hyoscyamine (hyo). S-hyo acts as a competitive acetylcholine antagonist at the muscarinic receptors (eutomer) inducing mydriasis, excitations, hallucinations, coma and ultimately death, whereas R-hyo does not (distomer). Atr is used for clinical intervention of poisoning with organophosphorus (OP) pesticides or nerve agents. Despite well known differences in pharmacological behavior, individual pharmacokinetics of both Atr enantiomers have rarely been addressed in the literature [1, 2, 3] . Therefore, we initially developed a nonchiral liquid-chromatography-electrospray tandem mass spectrometric method (LC-ESI MS/MS) that allows quantification of Atr and additional natural and synthetic tropane alkaloids from plasma after simple deproteinization [4] . To discriminate both Atr enantiomers the sample preparation step was expanded by an enzymatic pretreatment. Samples were incubated either with diluted human serum (not containing atropinesterase, AtrE, procedure A) or with diluted rabbit serum (procedure B). Rabbit serum contains AtrE (EC 3.1.1.10) which is suitable for stereospecific hydrolysis of Shyo into tropine and tropic acid while R-hyo remains unaffected. After sample precipitation, hyoscyamines were quantified by the LC-ESI MS/MS method. Following procedure A the concentration of total hyo and following procedure B remaining R-hyo were determined. S-hyo was calculated by the difference between these concentrations [3] . The impact of potential matrix ingredients that may appear in samples from OPpoisoned patients under Atr therapy were evaluated (oximes, OP agents, carbamates) [3] . The assay was applied to diverse toxicological and pharmacological samples. i) Measurement of natural S-hyo in an extract of atropa belladonna leaves as well as in plasma and urine of a female patient who was poisoned after ingestion of such leaves revealed that no biotransformation to R-hyo occurred. ii) Analysis of plasma obtained from an OP-poisoned female patient under Atr therapy revealed faster elimination of Shyo when compared to R-hyo [3] . iii) In contrast, no enantioselective differences were obvious in healthy anaesthetized swine after intravenous injection of Atr [5] . Data indicate that the enzymatic enantioselective procedure represents a useful tool to characterize in vivo behavior of R-and S-hyo allowing to reveal individual kinetics. Failing hearts are unable to adequately supply the body with blood and oxygen. Common therapeutic strategies interfere with cardiac remodelling, reduce cardiac preand afterload or aim at direct improvement of cardiac contractility. Cardiac contractility is mainly controlled by β1-adrenergic receptors. Resensitization of b-adrenergic receptors by inhibition of G-protein coupled receptor kinase 2 (GRK2) is discussed as a potential strategy to treat heart failure. We characterized Raf kinase inhibitor protein (RKIP) as a physiological inhibitor of GRK2 and found RKIP to increase contractility of neonatal cardiomyocytes. The present study evaluated the role of RKIP in heart failure. We assessed its effects on cardiac function in pressure overload induced heart failure and determined the expression patterns of RKIP in failing hearts of humans and mice. Transverse aortic contriction (TAC) was performed on 7-week-old C57/BL6 mice to induce heart failure by pressure overload of left ventricles. After three weeks of TAC, echocardiography showed distinct signs of decreased cardiac function in wild-type mice. Fractional shortening was reduced and left ventricular diameters were increased. Histological analyses revealed increased interstitial fibrosis, caspase-and TUNELassays indicated myocyte apoptosis. Western blot analysis showed significant upregulation of RKIP expression in failing hearts compared to non-banded control hearts. Interestingly, this upregulation of RKIP expression was also detected in failing human hearts and in samples of patients with aortic valve stenosis but not in healthy control samples.

To assess the effects of RKIP overexpression on heart failure, we analysed heart function and structure of RKIP transgenic mice and wild-type mice 3 weeks after TAC. While left ventricular hypertrophy was increased to similar extents in wild-type and RKIP trangenic mice, RKIP mice did neither develop dilatation of the left ventricle nor a decrease in fractional shortening. In contrast to wild-type mice, the expression of the heart failure markers BNP and ANP was not upregulated in banded RKIP transgenic mice after 3 weeks of TAC. Taken together, cardiac overexpression of RKIP prevented left ventricular dilatation and loss of contractile function in a mouse model of pressure overload induced heart failure. Therefore, increased RKIP expression may be an interesting target to prevent detrimental effects from increased left ventricular pressure. The main focus of this study was the chromatographic separation. The most frequently prescribed antibiotic drugs Clarithromycin, Erythromycin, Clindamycin, Cefuroxim, Doxycyclin, Amoxicillin, Levofloxacin, and Ciprofloxacin were selected for the screening method.

Method: A relatively short operation time and a sufficient separation were reached by column, eluent, and gradient optimization with POPLC (Phase Optimized Liquid Chromatography). In the first step columns with the five stationary phases C18 EPS 2, C18 SH 2, Phenyl 2, CN 2, and C30 were used to determine the retention times of the drugs in an isocratic mode. The stationary phases, the column length and the retention times were fed in the POPLC software and the optimal column was calculated. This column contained different stationary phases and was compared with customary columns.

Results: Using the optimal column a sufficient chromatographic separation of the eight antibiotic drugs was reached. That was not possible with the customary columns. With the optimal column the time of measurement was too long. Using a mobile phase gradient the measuring time could be reduced. Discussion: With LC-MS/MS a complete chromatographic separation of all analytes is not necessary. But when measuring many transitions in a biological matrix two problems should be excluded: ion suppression and a too small number of measurement points per peak. Especially when using positive and negative ionization in the MS a good separation is mostly necessary. To determine only the eight antibiotic drugs an optimized column is not necessary, but for a screening method with more than twenty drugs the POPLC system is very helpful. We have investigated the uptake mechanism of CDT and in particular the intracellular membrane translocation of CDTa. Our data indicate that CDT requires acidification of the endosomal lumen for translocation of CDTa across endosomal membranes into the cytosol. Bafilomycin A1, an inhibitor of endosomal acidification protects Vero (African Green Monkey kidney) cells from intoxication with CDT. Consistently, translocation of CDTa was observed when the acidic conditions of the endosomal lumen were mimicked at the cytoplasmic membrane of intact cells. Next, we tested whether host cell factors are involved in membrane translocation of the toxin. Radicicol, a specific pharmacological inhibitor of the chaperone heat shock protein Hsp90 as well as cyclosporine A, an inhibitor of cyclophilins delayed the intoxication of cells with CDT but not with toxins A and B [1] . This result was confirmed by analyzing the ADP-ribosylation status of actin from such cells in the presence or absence of the inhibitors. In addition, we excluded that the inhibitors of Hsp90 and cyclophilins have any effect on receptor binding, endocytosis or enzyme activity of CDT. The data strongly suggest that the participation of Hsp90 and cyclophilin is crucial for translocation of CDTa into the cytosol. Comparable results were obtained for the related binary iota toxin of C. perfringens. In vitro purified immobilized Hsp90 and cyclophilin A specifically bound to the enzyme components of CDT and iota toxins. In conclusion, the results imply a common Hsp90/cyclophilin A-dependent translocation mechanism for the family of binary actin-ADP-ribosylating toxins. Our current investigations focus on the participation of FK506-binding proteins (FKBPs), another group of peptidyl-prolyl cis/trans isomerases in the membrane translocation step of these toxins.

[1] Kaiser, E., Kroll, C., Ernst, K., Schwan, C., Popoff, M. R., Fischer, G., Buchner, J., Aktories, K. and Barth, H. (2011) . Complex multicellular in vitro coculture models represent a promising tool regarding e. g. cellular interactions with nanoparticles, since they more closely mimic the cellular composition of the body. Therefore, we used our developed coculture model of the alveolar-capillary barrier composed of lung epithelial cells (NCI H441) on top and microvascular endothelial cells (ISO-HAS-1) on the bottom side of a filter-membrane to study nanoparticle cytotoxicity and cellular uptake. With a coculture period of about 10 days the cells achieve a more differentiated and polarized state and develop a tight barrier, which can be measured via TER (transepithelial electrical resistance). Regarding cellular uptake of fluorescently labeled amorphous silica nanoparticles (aSNPs, 30 nm) the coculture took up much less aSNPs than conventional monocultures. Besides, we could not verify a specific uptake mechanism (e. g. clathrin-, caveolae-mediated endocytosis) via immunofluorescence staining of the cells. However, we detected aSNPs incorporated in flotillin-1 and -2 labelled vesicles. Former studies concerning cytotoxicity (lactate dehydrogenase assay) of amorphous silica nanoparticles (aSNPs, 30 nm) revealed that our coculture behaved much more robustly compared to conventional monocultures. However, regarding inflammatory responses (e. g. sICAM, IL-8 increase) the coculture responded more sensitively than conventional monocultures. In a further development we added a third cell type, the alveolar macrophage (AM), to our coculture. Since AMs embody the front-line of alveolar defense against inhaled pathogens and particles, they play a central role in regulating lung immunity. As model we applied the human acute monocytic leukemia cell line, THP-1 (prestimulated with 8 nM PMA for 4d) apically to the epithelial monolayer of the coculture. Our preliminary studies concerning inflammatory responses of the tripleculture (H441/ISO-HAS-1 with THP-1) revealed a higher sensitivity of the triple-culture compared to the double coculture. The triple-culture responded with an increased IL-8 release upon LPS or TNF-a stimulation. In conclusion, this triple-culture model offers a promising prospect to mimic more closely realistic cell interactions with nanoparticles in the distal lung. Ethanol is a component of many herbal fluid preparations [1] , since it is an excellent extraction solvent for the phytochemical components of herbal drugs and contributes to the stability of these medicines. Toxicological and pharmacokinetic evaluations [2] have shown that the small amounts of ethanol applied with therapeutic doses are safe even in children. Despite that these medicines have been used safely since many decades, they have occasionally been subject of discussion in the public, triggered by the increasing problem of recreational abuse of alcoholic beverages by children and young persons [3, 4] . Therefore, there is a growing need of a systematic evaluation of pharmacovigilance data on these medicines. For evaluating the experience gained from the therapeutic use of these medicines, 16 pro-and retrospective studies with 10 herbal medicinal products containing ethanol at doses of 40 to 240 mg per single dose, depending on the age group, have been analyzed, covering 49 816 patients of 0-12 years of age. In these studies, altogether 15 adverse drug effects have been described, none of which was attributable to the ethanol content of the medicines. In a survey of the worldwide use of these and other 6 herbal medicinal products it was shown that during the past few years, more than 764 Mio daily doses have been sold, corresponding to more than 33 Mio of patients (data obtained from manufacturers; figures available partly from 1993 onwards, partly from 2003/4 onwards). From the packages sold in Germany in the years between 2005 and 2009, 48.1 Mio were attributable to self-medication, 10.8 Mio to prescriptions reimbursed by health insurance (IMS, Frankfurt). As non prescription medicines are reimbursed in Germany only in children, at least the latter part of the prescriptions can be attributed mainly to children. All of these medicines are registered or licensed by regulatory authorities. Adverse effects are covered by the pharmacovigilance system, and no adverse effects attributable to the ethanol content have been reported. This set of data supports the conclusion drawn from the experience of a safe use over decades, i.e. that the ethanol content of herbal medicinal products does not give any causes for concern regarding their safety even in children. Dedication: This contribution is dedicated to Prof. Dr. Hilke Winterhoff, Institute for Pharmacology and Toxicology, University of Münster, who died on 9 May 2010. She has initiated this work. Prucalopride was introduced as a new selective 5-HT4 receptor agonist that is approved for treating obstipation. Whereas one could expect -due to the fact that 5-HT4 receptors are functionally expressed in the human heart -in clinical trials prucalopride did not show cardiac effects. This is quite surprising because other 5-HT4 receptor agonists have been withdrawn from the market just for that reason. In this study we used prucalopride for in vitro studies with atrial preparations from transgenic (TG) mice with cardiac myocyte-specific overexpression of the human 5-HT4a receptor. Isolated electrically driven (1 Hz) left and spontaneous beating right atria of TG mice were compared with those of wild type (WT) littermates. Moreover, we used isolated electrically driven (1 Hz) human right atrial preparations from patients undergoing cardiac surgery. Finally GR113808, a 5-HT4 receptor antagonist, was added. Prucalopride exhibited a dose dependent positive inotropic effect in left atria and a positive chronotropic effect in right atria of TG mice with a logEC50 of -6.8 and -7.1, respectively (p<0.05 vs. WT, n=3). In human atrial tissue prucalopride also acts as an agonist, leading to an inotropic effect. All effects could be antagonized by 10 µM GR113808. We could demonstrate that prucalopride acts as an agonist at the 5-HT4 receptor in our transgenic mice model and also in human right atrial preparations. These findings suggest that tachycardia and arrhythmias are possible side effects, which should be carefully looked for. The involvement of psychological factors, especially stress, are known to play an important role in functional gastrointestinal diseases (1, 2) probably by affecting the brain-gut axis. Based on the good correlation between stress & functional dyspepsia (FD), many animal models for FD have been developed where animals are subjected to various kinds of psychological stress either during the neonatal period or in adulthood. This stress was found to induce gastric motor dysfunction resembling symptoms of FD. Two models for stressinduced FD were performed in order to choose the more adequate one for studying sensitivity changes of the fundus to various mediators as well as changes in some relevant hormone levels in the blood for subsequently testing the efficacy of treatment with STW 5 (a 9 component herbal preparation of proven clinical efficacy in FD and in irritable bowel syndrome (3, 4) ). In one model, maternal separation (5) was performed on weanling rats starting from postnatal day 2 for 3 h each day for 3 weeks. Rats were then allowed to mature to an adult age. The other model was that of restraint stress (RS) (6, 7) . Adult animals were restrained for 90 min/ day for 1 week. The animals of both models were eventually sacrificed, the stomach fundus was isolated and its sensitivity in vitro to carbachol, potassium chloride, serotonin and adrenaline was tested. Blood samples were taken to assess levels of ghrelin, corticosterone releasing factor (CRF) and corticosterone. The sensitivity of fundus strips from restrained rats towards the agents tested, partly representing autonomic responsiveness, was more depressed than those from maternally separated ones. Levels of ghrelin, CRF and corticosterone were also more elevated in the RS model. That model was therefore chosen to test the efficacy of STW 5 in restoring the deranged parameters. A group of animals received STW 5 orally once daily starting treatment 1 week before exposing them to RS and continuing treatment for a further week during subjection to RS. STW 5 was effective in normalizing the depressed stomach fundus responses exhibited by animals subjected to RS and to normalize to a large extent the deranged blood levels of ghrelin, CRF and corticosterone. The findings lend further evidence to the role of the brain-gut axis in FD and gives supportive evidence for the first time for the clinical usefulness of STW 5 in this condition. There is good evidence that oxidative damage to DNA leads to down-regulation of transcription of affected genes and epigenetic gene silencing in a mechanism dependent on the 8-oxoguanine DNA glycosylase (OGG1), which generates harmful repair intermediates [1, 2] . We have recently shown that the magnitude of inhibition of transcription of an EGFP reporter gene by single 8-oxo-7,8-dihydro-deoxyguanosine (8-oxodG) varies between the opposing DNA strands of the gene [3] . We now have addressed the question, to which extent the transcription inhibitory potential of 8-oxodG depends on its position in the gene and on the DNA microsequence surrounding the modified nucleobase. To investigate the effect of position, we produced plasmid vectors containing single 8-oxodG or dG (underlined) in the second position of an AGC trinucleotide. Measurements of EGFP expression in transfected mammalian host cells showed that a single 8-oxodG caused a strong inhibition of gene transcription. The magnitude of this effect for 8-oxodG situated in the transcribed DNA strand of the gene was the same as in the non-transcribed DNA strand. Similarly, there was no quantitative difference between the effects of 8-oxodG present in the 5′-versus 3′-UTR of the gene. The results thus indicate that inhibition of transcription by this base modification does not depend on the position in the gene. Further comparisons were done between the effects of 8-oxodG nucleobases localised in the same DNA strand but within different sequence contexts. Gene expression analyses in the repair proficient host cells showed that the degree of inhibition of transcription caused by single 8-oxodG was dependent on the neighbouring nucleotides. Among three tested sequence motifs, the minimal effect on the gene transcription was found to correlate with a significantly less efficient base excision by the purified human OGG1 protein. The results thus support the initiatory role of OGG1 in the mechanism of transcriptional repression. In addition, the finding of the effect of DNA sequence on the base excision activity of OGG1 suggests that repair rates of single base modifications in genome could also be heterogeneous.

Allgayer J., Kitsera N., Epe B., Khobta A. Johannes Gutenberg University of Mainz Institute of Pharmacy and Biochemistry, Staudingerweg 5, 55128 Mainz, Germany

Interference of DNA base modifications with gene transcription is an important biological consequence of genotoxic damage [1] . An efficient method for incorporation of a single 8-oxo-7,8-dihydroguanine (8-oxoG) at a defined position in the EGFP gene in a plasmid vector was recently developed in our lab. The method relies on the availability of adjacent sites for a sequence-specific nicking endonuclease, which allow the insertion of a synthetic oligonucleotide containing 8-oxoG in a chosen position. We further showed that this single lesion inhibits transcription after excision by OGG1 [2] . In order to determine to which extend the observed effect depends on the position of the modified base in the gene, we constructed several new plasmid vectors which allow incorporation of the same DNA oligonucleotide containing 8-oxoG or G in different positions in different DNA-strands. DNA sequence cassettes were designed to contain a 5′-CATTGCTTCGCTAGCACG nucleotide sequence in different orientations, which was flanked by two unidirectional BsrDI recognition sites (5′-GCAATGNN). Adapters containing the restriction sites for directional cloning into the 5′-or the 3′-UTR of the plasmid-borne EGFP gene were added. The produced plasmid vectors thus allow the insertion of a single 8-oxoG in the same sequence context into opposing DNA strands in the 5'UTR and in the 3'UTR. Keratinocytes are the major cell type of epidermis and are responsible for the formation of an outer barrier, the statum corneum, to protect an organism against harmful environmental influences. For generation of this barrier, keratinocytes pass through a complex differentiation program that is accompanied by synthesis of lipids, like cholesterol and ceramides. Finally, the differentiation of keratinocytes leads to apoptosis. Another function of keratinocytes is to sense environmental factors, some of which are decoded by members of the transient receptor potential (TRP) ion channel family. TRPV3, for example, is predominantly expressed in keratinocytes, and decodes different chemical and physical stimuli like the terpenoid-derived ligands camphor and thymol or temperatures above 33°C. Less is known about the influence of cholesterol on TRPV3 signalling. We modified the cholesterol content of HEK293 cells stably transfected with TRPV3 and performed FLIPR-based calcium measurements. These experiments revealed that cholesterol enrichment robustly potentiates TRPV3 by sensitizing it to lower agonist concentrations. We verified these results with whole-cell patch-clamp measurements. In contrast, TRPV2, another heat-sensing channel, was not affected by cholesterol modification. Since former studies showed a defective formation of epidermal barrier in TRPV3 -/mice, our results imply that a cholesterol-regulated TRPV3 signalling may contribute to the progression of differentiation or initiation of apoptosis of keratinocytes. Ischemia-reperfusion injury causes severe problems in the early period after lung transplantation. Since transient receptor potential (TRP) channels are important regulators of vascular permeability and tone, we investigated the influence of a TRPC6 blocker on pulmonary function after simulated transplantation, using an ex vivo model of isolated perfused and ventilated rabbit lungs. To this end, heart-lung blocks were excised and mounted in an artificial thorax chamber. Negative pleural pressure ventilation was initiated, and lungs were perfused with albumin-containing Tyrode-Solution (100 ml min -1 ). After equilibration in a stable perfusion and ventilation mode for 30 min, lungs were flush-perfused with Perfadex ® solution, followed by an ischemic storage for 4 h on ice. Subsequently, ventilation and perfusion were re-initiated to simulate a transplantation situation. In the oxygenated group, pulmonary artery pO2 was adjusted to 120 mmHg, in the deoxygenated group, the perfusate inflow was gassed with nitrogen to achieve a pulmonary artery pO2 of 50 mmHg. Both transplantation conditions were conducted in the absence or in the presence of the TRPC6 blocker larixol acetate (5 µM). Hemodynamic and ventilatory parameters were continuously monitored. The weight of deoxygenated lungs steadily increased during 2 h of reperfusion from 22.1 ± 1.32 g to 35.4 ± 4.23 g. This weight gain was inhibited by supplementation of the TRPC6 blocker (from 21.4 ± 0.91 g to 27.4 ± 2.42 g 2 h after reperfusion). In contrast, oxygenated lungs showed no marked weight gain after reestablishment of perfusion (from 17.5 ± 1.51 g to 21.5 ± 2.29 g 2 h after reperfusion), and the TRPC6 blocker had no additional effect (initial 19.5 ± 1.28 g, 2 h reperfusion 21.3 ± 1.22 g). We conclude that a TRPC6-blocking compound to the lung perfusate during simulated transplantation counteracts the endothelial permeability increase and the resulting post transplant weight gain. The results indicate a role for TRPC6 in the regulation of pulmonary vessel permeability and support the concept that perfusion of donor lungs with TRPC6 blockers may prevent edema formation caused by ischemiareperfusion injury shortly after lung transplantation.

Regulation of human inducible nitric oxide synthase (iNOS) expression by noncoding RNAs (ncRNAs) Kleinert H., Schmitz K., Koch K., Hahn S., Pautz A. Universitätsmedizin der Johannes Gutenberg-Universität Mainz Institut für Pharmakologie, Obere Zahlbacher Str. 67, 55101 Mainz, Germany

The transcriptome analyses of human cells showed that additionally to the 30.000 protein coding sequences the human DNA codes for much more (450.000 ?) non-coding RNAs 1 . Beside the ribosomal, and transfer RNAs (rRNA and tRNA) involved in the mechanism of translation there are also short (snRNA, soRNA, miRNA) and long noncoding RNAs (ncRNAs) implicated in regulation of gene expression (splicing, translation, chromatin packaging etc.). Matsui et al. described regulation of IL-1β-induced iNOS expression by an antisense RNA (as-3-UTR-RNA) in rat hepatocytes 2 . A promoter located on the antisense strand 3' to the last exon (exon 27) of the rat iNOS gene drives the expression of an as-RNA complementary to the 3'-UTR of the rat iNOS mRNA. Using different sense primers with homology to the 3'-UTR sequences of the human iNOS gene for specific RT-PCR reactions (detecting only an as-RNA) we were not able to detect such an as-3-UTR-RNA in human cells. In addition, transient transfection analyses using constructs containing a 2.7 kb fragment of the 3'-flanking genomic sequences (as used by Matsui et al. in the rat system) of the human iNOS gene in front of a luciferase reportergene into DLD-1 cells revealed no promoter activity of these sequences. Korneev et al. described the expression of a 2 kb anti-iNOS-ncRNA in different brain tumors and showed reciprocal expression to the iNOS mRNA in human embryonic stem cells 3 . Analyzing the expression of this anti-iNOS-ncRNA in cytokine-treated DLD-1 cells also showed a basal expression of this as-RNA and an enhancement of the expression by cytokine-treatment. Downregulation of the anti-iNOS-ncRNA by siRNAs reduced whereas overexpression enhanced CM-induced iNOS expression in human DLD-1 cells. This indicates that this anti-iNOS-ncRNA regulates cytokine-induced iNOS expression in a positive manner. RNA 14, 2030 RNA 14, -2037 RNA 14, (2008 .

Using directed evolution to improve functional expression of class B G-protein coupled receptors Klenk C., Scott D. J., Plückthun A. Universität Zürich Institut für Biochemie, Winterthurerstrasse 190, 8057 Zürich, Switzerland

The class B of G-protein coupled receptors (GPCRs) comprises 15 peptide hormonebinding receptors which regulate important endocrine and neuroendocrine functions of the human body. Several of these receptors are implicated in the pathogenesis of severe diseases such as diabetes, osteoporosis, growth disorders and depression, which makes them attractive targets for drug therapy. To develop new compounds targeting these receptors a detailed understanding of the molecular structure is required which has not been succeeded to date. Structural studies of proteins by X-ray crystallography or NMR spectroscopy generally require large and homogenous quantities of protein. For GPCRs this prerequisite is difficult to achieve as the vast majority of GPCRs exhibits low endogenous expression and is very unstable in solution. Therefore, improved expression conditions are necessary for the efficient characterization of new GPCR structures. Here we present a method to optimize class B GPCRs for improved heterologous expression and increased thermostability by means of directed evolution. Libraries of class B GPCRs were obtained by random mutagenesis and were expressed in a heterologous expression system in which functional GPCR is targeted to the inner membrane of E. coli. Mutants that display increased receptor expression levels and ligand binding were selected by flow cytometry using fluorescently labeled ligands. Repetitive cycles of randomization and selection allow to gradually increasing the level of protein expression and stability. With this evolutionary approach key residues within the receptor sequence can be identified rapidly that are responsible for improved biophysical properties without affecting the pharmacological features of the receptor. Such GPCR mutants will become a valuable tool on the way to express high quantities of stable receptor protein for subsequent structural studies. Pasireotide (SOM230) is currently under clinical evaluation as a successor compound to octreotide for the treatment of acromegaly, Cushing's disease and carcinoid tumors. Whereas octreotide acts primarily via the sst2 somatostatin receptor, pasireotide was designed to exhibit octreotide-like sst2 activity combined with enhanced binding to other somatostatin receptor subtypes. Somatostatin and octreotide stimulate the complete phosphorylation of at least six carboxyl-terminal serine and threonine residues namely S341, S343, T353, T354, T356 and T359, which in turn leads to a robust endocytosis of the sst2 receptor. Surprisingly, pasireotide failed to phosphorylate the four threonine residues and induced only a detectable phosphorylation of S341 and S343. Somatoprim and KE108 are recently developed somatostatin analogs capable of binding to four of five somatostatin receptor subtypes. Here, we performed an in vitro study comparing the effects of pasireotide, somatoprim and KE108 on sst2 somatostatin receptor binding, phosphorylation, internalization and signaling. Further somatostatin, octreotide, pasireotide, somatoprim and KE108 were tested for functional selectivity at sst5 receptor mutants, which possess a carboxyl-terminal sst2tail. This approach allows detection of receptor activation by phospho-specific sst2 antibodies. Compared to octreotide, somatoprim activates sst2 but has a higher activity on sst5. KE108 and pasireotide are partial agonists at the sst2 receptor. Pasireotide, KE108 and somatoprim show comparable effects on sst5 receptor. However, none of these new pan-somatostatin analogs behaves like natural somatostatin on the sst5 receptor.

Cadmium modulates AhR-associated gene expression in the rat intestine after oral exposure Kluxen F. M. 1, 2 , Höfer N. Cadmium has been shown to mimic steroid estrogen effects in vivo and in vitro. We have recently identified cross-talk of estrogen receptor (ER) and aryl hydrocarbon receptor (AhR) in the rat uterus where 17beta-estradiol (E2) and CdCl2 modulate AhRassociated genes via ER after i.p. injection in a similar fashion (Kluxen et al., Arch Toxicol DOI 10 .1007/s00204-011-0787-x). However, the predominant route of exposure to cadmium in non-smokers is via diet. Moreover, uterus expresses mainly the receptor subtype ERalpha, whilst small intestine and colon express mainly ERbeta. Thus, we now investigated by real-time RT-PCR the effects of cadmium (2mg/kg b.wt CdCl2 (Cd2)) or steroid estrogen (0.1 mg/kg b.wt. 17alpha-ethinylestradiol (EE2)) on AhRassociated gene expression (i.e., Ahr, Cyp1a1, Gsta2, Nqo1) in the small intestine of rats after oral exposure (3 days gavage). The animals were also co-treated with Cd2 and pure anti-estrogen (2.5 mg/kg b.wt ZK191703 (ZK)) or EE2, to asseess whether ERmediated processes are involved. We also measured Cyp1a1 mRNA expression in two estrogen receptor negative colon cancer cell lines (HT-29 and CaCo2) treated for 5 days with CdCl2 (1µM) and E2 (0.01µM). The dose-dependency of cadmium induced AhR target gene modulation was studied in a second animal experiment, with administration of cadmium in drinking water for 28 days (0.4-9 mg/kg b.wt CdCl2 equivalent to 5, 50, and 150 ppm) and EE2 (0.08 mg/kg b.wt) as steroid reference.

In summary we present two major results: The metalloestrogen cadmium modulates dose-dependently the AhR-associated gene expression in the intestine after oral exposure. Yet, since the cadmium induced modulation of AhR target genes was not antagonized by anti-estrogen in the small intestine in vivo and was also found to occur in ER-negative colon cells in vitro, we propose that ER-independent mechanisms might play a role in this effect. MeG) is the most cytotoxic lesion. If not repaired by O 6 -methylguanine-DNA methyltransferase (MGMT), O 6 MeG/T mismatch is recognized by the mismatch repair system (MMR) that performs futile repair cycles. During this process secondary lesions (i.e. DNA single-strand brakes) are formed, which block DNA replication in the next replication cycle, leading to DNA double-strand breaks (DSBs). These DSBs eventually signal to apoptosis and other genotoxic endpoints. Here, we wished to address the question whether autophagy is part of the cellular response triggered by O 6 MeG. We also assessed whether autophagy influences apoptosis induced by TMZ in glioma cells. We show that TMZ induces autophagy in U-87 MG and LN-229 glioma cell lines. The maximum amount of autophagy was observed several days (96 h) after TMZ treatment and MGMT proficient cells did not display significant autophagy. Thus, the data show that MGMT protects against TMZ induced autophagy, pointing to O 6 MeG as the critical lesion responsible for induction of autophagy. Using colon cancer cell lines proficient and deficient in MMR, we show that MMR is required for TMZ-induced autophagy. Because DSBs, which emerge during the processing of O 6 MeG, are repaired preferably by homologous recombination (HR) LN-229 cells stably down-regulated for HR were tested for autophagy induction. The data indicate that DSBs are involved in TMZ-induced autophagy. Because autophagy following TMZ treatment occurs earlier than apoptosis we hypothesize that autophagy protects glioma cells against apoptosis. Using an early stage autophagy inhibitor 3-methyladenin we have shown, that autophagy inhibition sensitized glioma cells to TMZ-induced apoptosis. Taken together our data point out that TMZ induces autophagy in glioma cells and that autophagy protects glioma cells against TMZ-induced apoptosis. O 6 MeG is the lesion responsible for autophagy induction. Furthermore, the data also shows that MMR and DSBs are involved in the induction of autophagy after TMZ treatment. Work was supported by BMBF (02NUK016). Retinitis pigmentosa (RP) is a severe human retinal disease characterized by a progressive degeneration of rod photoreceptors and a secondary loss of cone function. In most cases, RP finally leads to legal blindness. Mutations in the regulatory subunit of the rod cyclic nucleotide-gated (CNG) channel (CNGB1a) have been found in patients suffering from RP. We used CNGB1-deficient (CNGB1 -/-) mice to establish a gene replacement therapy as a potential treatment for RP by means of recombinant adenoassociated viral (rAAV) vectors. The packaging limitations of rAAV vectors required a capacity-optimized vector of the large CNGB1a cDNA (approx. 4 kb). Therefore, we replaced regulatory elements within the expression cassette and used a short mouse rhodopsin promoter element for rod-specific expression. After injection of therapeutic rAAVs (serotype 8) into the subretinal space of 2-week-old CNGB1 -/mice, we assessed the restoration of vision by analyzing i) protein expression and localization, ii) retinal function and morphology and iii) vision guided behavior. We found that treated CNGB1 -/mice expressed full-length CNGB1a and CNGA1, which were previously downregulated. Both proteins co-localized in rod outer segments and formed regular CNG channel complexes in the treated area of the CNGB1 -/retina. Using electroretinography (ERG) we observed a distinct rescue of rod-driven responses. Moreover, CNGB1 replacement significantly preserved outer segment morphology and delayed retinal degeneration. Finally, treated CNGB1 -/mice performed significantly better than untreated mice in a modified water maze task designed to test for rod-mediated, vision-guided behavior. In summary, this work provides a proof-of-concept for the treatment of rod channelopathyassociated retinitis pigmentosa by rAAV-mediated gene replacement. Most endocrine disruptors interact with hormone receptors or steroid biosynthesis and metabolism, thereby modifying the physiological function of endogenous hormones.

Here, we present an alternative testing paradigm for detection of endocrine modes of actions that replace and reduce animal testing through refinement. Receptor mediated endocrine effects were assessed using the yeast based receptor mediated transcriptional activation YES/YAS assays and effects on steroid hormone biosynthesis were assessed using the human cell line H295R in the steroidogenesis assay. In our testing paradigm we propose to complement the in vitro assays with a single in vivo repeated dose study in which plasma samples are analyzed for their metabolome profile. The combination of these methods does not only contribute to refinement and reduction of animal testing, but also has significantly increased the efficient allocation of resources and allows for a sound assessment of the endocrine disruption potential of compounds. Thus, this proposal constitutes a potentially attractive alternative to EPA's Endocrine Disruptor Screening Program. Data on 14 reference substances for which the in vitro YES/YAS and steroidogenesis assays and the in vivo metabolome analysis were performed to assess their putative endocrine mode of action is presented here. The bovine corneal opacity and permeability (BCOP) test has been adopted by OECD for the identification of corrosive and severe ocular irritants (GHS category 1) for single component substances and multi-component formulations. Eye irritation tests (EIT) using human reconstructed tissue models (such as EpiOcular) have been described to predict ocular non-irritants (GHS no category). Thus the ultimate repaltement of the Draize rabbit eye irritation test (OECD TG 405) by a combined or tiered testing strategy could be possible. The purpose of this study was to evaluate whether the BCOP with additional corneal histology together with the EIT could be used to predict eye irritancy of agrochemical formulations according to different classification schemes including UN GHS and EPA systems. We have performed the BCOP (plus histology) and the EIT of 50 agrochemical formulations for which in vivo eye irritation data were already available (for registration purposes). Using the OECD TG guideline evaluation scheme for opacity and permeability in the BCOP was not predictive for the agrochemical formulations assessed here, while corneal histology grades and the EpiOcular tissue viabilities were useful predictors of eye irritancy potencies and could be applied for the different classification schemes.

The Nanomaterials offers extraordinary opportunities. The nano-structure can change the physical and chemical properties, and often also alters the biological effects. Hence the toxicity of a nanomaterial can differ from its larger-scale material; but as of today, no new quality of a general nano-specific toxic effect has been observed. Therefore the established testing methods are generally suitable. It is, however, difficult to apply the nanomaterials to in vitro test systems, since it is the nature of these materials to change their surface properties and agglomeration stateand the uptake and distribution in the body may differ from their larger-scale materials. While the methods for topical effects may be used for nanomaterials without further modification, the in vitro methods for genotoxicity testing require the dispersion in culture media. The use of reproducible and well-documented dispersion protocols andthe characterization of its particle size distribution is de rigueur . [1] . For many nanomaterials published genotoxicity studies did not give a consistent picture [2] and therefore there are rather effects of individual nanomaterials than nano-genotoxicity per se. Modern toxicology is based on the insight into toxic pathways. For nanomaterials a testing strategy will include testing for their primary effects (which might be only a handful: particle effects, catalyzing the formation of reactive molecules and ion release) and their uptake, distribution and clearance. The use of dermal penetration studies in vitro for the risk assessment of sunscreen nanomaterials has been demonstrated [3] . In vitro methods for specific effects (such as inflammation, pulmonary toxicity, sensitization) are currently awaiting validation (for both chemicals/molecules as well as nanomaterials). In the meantime alternative short-term in vivo studies with optimized biological readouts can deliver information on the toxic pathways as well as the biokinetics and dose-response relation of nanomaterials in the body. A short-term inhalation test for nanomaterials has already been used successfully [4] . Testing strategies based on those methods engage less animals and provides more significant data than classical testing. Moreover data from these methods will serve as a benchmark and a validation for the in vitro models under evaluation. Nanoparticles (NP) are increasingly used in various field of industry which necessitates evaluation of their safety. Also in the food industry, NPs have gained strong interest, for example as food additives or to improve food packing. However, the potential risks of ingested NP have been rarely investigated. Inhalation studies have revealed that inflammation and oxidative stress may represent unifying mechanism for the induction of adverse health effects of toxic NP. In the present study, a co-incubation model of human polymorphonuclear neutrohils (PMNs) and Caco-2 human intestinal epithelial cells was used as a model to address potential genotoxic effect of NP during intestinal inflammation. Oxidative DNA damage induction (measured by the Fpg-modified comet assay) was induced in the Caco-2 cells by activated PMN and this effect increased with increasing PMN to Caco-2 cell ratio. The crucial involvement of the phagocyte NADPH oxidase complex could be demonstrated using treatment of Caco-2 cells with bone marrow-derived PMN from NADPH oxidase deficient mice. DNA damage by PMN as well as H 2O2 was increased in buthionine sulphoximine (BSO) pre-treated Caco-2 cells, illustrating the importance of the cellular glutathione (GSH) status in these target cells. GSH depletion in Caco-2 cells could also be shown upon treatment with various types of NP. Our data suggests that ingested NP may increase the susceptibility of the colon mucosa to genetic damage during the occurrence of intestinal inflammation. The ingestion of seafood contaminated by acute toxic doses of the marine toxin okadaic acid (OA) is responsible for diarrhetic shellfish poisoning. It is recently known that both the rat and the human hepatic cytochrome P450 monooxygenases (CYP) are able to metabolize this toxin. Currently, there is a lack of data about the toxicity and mode of action of OA after xenobiotic metabolism. The aim of our study was the measurement of the toxicity and oxidative stress status in HepG2 cells incubated with OA in the absence and presence of S9 mix. Pure OA, as well as OA pre-activated with liver homogenisates (S9 mix) were used to treat human HepG2 cells that have nearly undetectable levels of functional CYP but express phase II enzymes. The experiments were performed with both human and rat S9 fraction plus cofactors of phase I enzymes. The cell viability was measured after 4 h using MTT-test and xCelligence real time cell monitoring system. Furthermore, levels of intracellular reactive oxygen species (ROS) were detected by 2´,7´-dichlorofluorescein diacetate and additionally by measuring the intracellular glutathione content.

In the presence of both human and rat S9 mix OA showed a higher toxicity than the parental substance. OA pre-incubated in rat S9 mix was toxic at 75 nM OA. Strong effects could be observed when OA was pre-activated with human S9-mix at a concentration of 50 nM OA. Pure OA was non-toxic in that concentration range. We could also detect an increase of oxidative stress in HepG2 cells treated with OA in the presence of all investigated S9-Mix. These results suggest that OA is activated after oxidative xenobiotic metabolism into metabolites which possess a higher cytotoxic activity and increase the amount of intracellular ROS in HepG2 cells.

Ballast Water Treatment -Emerging Health Risks Werschkun B., Banerji S., Krätke R. Bundesinstitut für Risikobewertung, Max-Dohrn-Strasse 10, 10589 Berlin, Germany

The introduction of invasive marine species into new environments by ships' ballast water, via ships' hulls and other vectors has severe impacts on the oceans. In 2004 the International Maritime Organisation (IMO) launched the International Convention for the Control and Management of Ships Ballast Water and Sediments which requires ballast water to be treated in order to eliminate alien aquatic species. Ballast water treatment may include mechanical, physical or chemical measures. Any ballast water management system using active substances needs IMO approval. Therefore identification of active substances, relevant chemicals and submission of specified datasets on their physical, chemical and toxicological properties is required in order to assess the safety for the aquatic environment and for human health. The BfR is the German federal agency responsible for health risk assessment and has been involved in more than twenty assessment and approval processes so far.

The majority of IMO approved systems are based on oxidative principles such as chlorination and ozonation. These methods can generate disinfection by-products (DBPs), which are a mixed group mostly of halogenated organic substances like trihalomethanes, haloacetic acids and haloacetonitriles. The formation of DBPs is well known from the disinfection of drinking water. Some DBPs are regulated under drinking water directives because of their long-term toxicity but many are unregulated and have unknown toxicological properties. The formation of DBPs may vary significantly depending on the treatment system as well as on environmental parameters like temperature, pH and composition of the organic matter within the aquatic environment.

In sea water, sources for DBP formation besides ballast water treatment are aquaculture and the cooling systems of coastal power plants.

In order to address possible health and environmental risks from DBPs formed during ballast water treatment a conference on emerging risks from ballast water treatment was held at BfR in October 2011. Here we summarise the main conference findings and identify areas for future research. Two presentations corroborated that significant amounts of DBPs can be formed in sea water and a presentation on the toxicological properties of DBPs pointed out that many have genotoxic properties. Accordingly, the determination of DBP species and generated concentrations under different ballast water treatment conditions was seen as a mayor task.

Different approaches for health and environmental risk assessments were also discussed. Appropriate human exposure scenarios and methods for exposure assessment, taking into account common approaches used in risk assessment were presented during the conference. A suitable approach based on derived PEC-values for exposure quantification was proposed in order to improve the procedure available for risk assessment of chemical agents used for ballast water treatment.

Agonist-Selective Signaling of µ-Opioid Receptors in T Lymphocytes Kraus J., Börner C., Lanciotti S., Koch T., Höllt V. Inst. für Pharmakologie und Toxikologie, Leipzigerstr. 44, 39120 Magdeburg, Germany

Opioids are the most potent analgesics and irreplaceable for the treatment of severe pain. In addition to their central effects, opioids modulate a great variety of immune effector cell functions, which may result in unwanted side effects during opioid treatment.

The effects of most of the commonly used opioids are mediated by µ-opioid receptors, which belong to the superfamily of G protein coupled receptors. Recent data support the concept that G protein coupled receptors function as dynamic entities, which may occupy multiple conformations and activate multiple signaling pathways in a ligand-dependent manner. Consequently, different ligands activating the same receptor may have different cellular effects, which has been termed "agonistselective signaling".

Little is known about agonist-selective signaling of µ-opioid receptors in immune effector cells. In a first attempt to understand if and why such different profiles among different opioids occur we investigated effects of different opioids in human Jurkat T cells. We report that the µ-opioid receptor ligands fentanyl, methadone, loperamide and betaendorphin induce internalization of a µ-opioid receptor-green fluorescent reporter construct, whereas morphine and buprenorphine did not induce internalization. The internalization was dependent on p38 MAPK and phospholipase D2. In line with this, we observed marked phosphorylation of p38 MAPK and activation of phospholipase D2 induced by the internalizing opioids, but no or little such activity by morphine and buprenorphine. As a physiological result, fentanyl, methadone, loperamide and betaendorphin treatment of primary human T cells and Jurkat T cells resulted in a strong, up to 100 fold induction of IL-4, which was dependent on p38. In contrast, morphine and buprenorphine only showed a weak, approximately one order of magnitude lower induction of IL-4. By inducing IL-4 opioids significantly modulate the T helper cell balance into the type 2 direction, which influences various immune responses, e. g. the antiviral, T helper cell type 1-mediated response. Considering the vital necessity of opioid use in humans, it is an intriguing goal to identify analgetically feasible opioids that have little or no immunosuppressive or -modulatory effects.

Modulation of cGMP signals by phosphodiesterases in smooth muscle cells Krawutschke C., Koesling D., Russwurm M. Ruhr-Universität Bochum Pharmakologie und Toxikologie, Universitätsstrasse 150, 44780 Bochum, Germany Within the cardiovascular system, cGMP mediates smooth muscle relaxation and inhibition of platelet aggregation. cGMP is formed by particulate guanylyl cyclases and nitric oxide-sensitive guanylyl cyclases, that are activated by natriuretic peptides or nitric oxide (NO), respectively. Besides the cGMP-forming enzymes, the cGMP-degrading phosphodiesterases strongly determine amplitude and shape of cGMP signals.

In vascular smooth muscle cells, three phosphodiesterases are considered to be responsible for cGMP degradation: PDE5, the cGMP-specific phosphodiesterase is activated directly by cGMP binding to its GAF domains; this activation if further stabilized by cGMP-dependent protein kinase-mediated phosphorylation. PDE1, the Ca2+/Calmodulin-stimulated PDE constitutes the majority of cGMP-hydrolyzing activity in smooth muscle cells at least in the presence of high intracellular Ca2+ concentrations. And lastly PDE3, the cGMP-inhibited PDE displays some cGMP-degrading activity, although cGMP binding to its catalytic domain is primarily thought to inhibit the cAMPdegrading activity of PDE3. cGMP signals measurable in radioimmunoassays require stimulation with cGMPincreasing vasodilator concentrations that are orders of magnitudes higher than those required for relaxation. Thus, we developed fluorescent sensors for real-time measurement of cGMP signals in single cells. By using these indicators, we analyzed the contribution of different cyclic nucleotide-degrading phosphodiesterases to the modulation of cGMP signals elicited by physiologically relevant vasodilator concentrations. Hyaluronan (HA) is a major component of extracellular matrices and is thought to control cellular phenotypes such as proliferation and migration. Therefore, HA synthesis may play an important role in the pathophysiology of atherosclerosis. There are three major HA-synthase isoenzymes (HAS1-3). The HAS3 gene is alternatively spliced. HAS3 transcript variant 2 (HAS3v2) encodes the smallest HAS isoenzyme which has a different C-terminus and contains only two transmembrane domains compared to HAS3 transcript variant 1. The aim of the present study was to investigate whether HAS3v2 is expressed by vascular cells, how it is regulated and where it is localized in cells and whether it indeed synthesizes HA. HAS3v2 mRNA expression was monitored by quantitative real-time RT-PCR. Protein expression was determined by Western blotting using a polyclonal antibody that was raised in rabbit. An N-terminal EYFP-HAS3v2 fusion protein and a DDK-tagged HAS3v2 construct were expressed for subcellular localization studies and co-immunoprecipitation (Co-IP). Endogenous HAS3v2 mRNA was expressed in both vascular smooth muscle cells (VSMC) and endothelial cells. Furthermore, Western blotting revealed HAS3v2 protein expression in VSMC and platelets. In VSMC HAS3v2 mRNA expression was strongly up-regulated in response to interleukin-1β (IL-1β, 10 ng/ml) whereas stimulation with interleukin-10 (10 ng/ml), platelet-derived growth factor-BB (10 ng/ml), transforming growth factor β (10 ng/ml), tumor necrosis factor α (10 ng/ml) and interferon-γ (10 ng/ml) had no effect. Transfection of HEK cells with EYFP-HAS3v2 fusion protein revealed localization to the endoplasmic reticulum but not to the plasma membrane. Furthermore, Co-IP experiments showed that tagged HAS3v2 proteins were precipitated together suggesting formation of multimeric HAS3v2 complexes. Transfection of HAS3v2 did not cause increased secretion of HA into the cell culture supernatant in HEK cells.

In conclusion, HAS3v2 is present in vascular cells and responds to inflammatory cytokines such as IL-1ß. Because of the intracellular localization and the lack of HA secretion in HAS3v2 transfected cells, HAS3v2 may serve intracellular functions apart from HA synthesis.

The tubulin antagonist pretubulysin shows strong vascular-disrupting properties in vitro Several epidemiological studies indicate a correlation of human exposure to ultrafine particulate air pollution caused by incomplete combustion processes and an increase in the incidence of pulmonary immune diseases like asthma. As a possible mechanism behind this pathological phenomenon, the adjuvant effect of lung inflammation induced by poorly soluble environmental particles has been hypothesised. The aim of our study was to investigate the causal link between carbon nanoparticle-induced lung inflammation and modulations of immune cell populations during processes leading to sensitization, and allergic immune responses of the airways. Therefore mice were treated with ovalbumin (OVA) alone or in combination with carbon nanoparticles (CNP) by pharyngeal aspiration. The induction of inflammation and the immune adjuvant activity were studied in the lungs and lung draining peribronchial lymph nodes (PBLN) at the level of sensitization, and at the level of the immune response. OVA-specific IgE antibodies were measured in blood serum, and the development of allergic airway inflammation was studied after OVA challenge. Results at the level of sensitization showed that CNP-induced immediate airway inflammation had immune adjuvant activity resulting in an increase of specific cell populations in PBLN and in a stimulation of asthma-specific Th2 cytokines. A specific reduction of the neutrophilic lung inflammation by application of the compatible solute ectoine significantly reduced the adjuvant effects of CNP. In OVA-sensitized mice, application of CNP 12 hours prior to allergen challenge, led to a significant increase in inflammatory cell infiltrate and respective cytokines in broncho-alveolar lavages. Coapplication of 1 mM ectoine together with CNP reduced the particle-induced effects. Our data show a link between neutrophilic lung inflammation and adjuvant effects of CNP. A specific reduction of neutrophils by the application of ectoine attenuated this NP induced adjuvant effect, indicating that particle-induced lung inflammation rather than the direct interaction of nanoparticles with immune cells is the critical step in environmentally modulated pulmonary immune diseases like asthma. Introduction: Drug-eluting stents (DES) are commonly used in the treatment of acute artery occlusion. However, even if released cytotoxic drugs reduced neointimal proliferation significantly there is still the risk of in-stent thrombosis. It is presumed that this is due to reduced reendothelialization. It has been suggested that coating the stent with biomolecules may provide a new approach to circumvent the lack of healing of the endothelial layer. One approach would be the use of biomolecular signals, such as (poly)peptides and growth factors. RGD and REDV are peptide motifs, known to enhance cell attachment and spreading. Aim: The aim of our study was to evaluate the efficacy of proteins, derived from elastin-like proteins (ELP) and artificial modified by incorporating with the amino acid motifs RGD,REDV and P15, in terms of endothelial healing on stents and other cardiovascular devices. Results: In this work, we generated vectors encoding for different biopolymers consisting of various bioactive signal molecule sequences. The peptides, e.g. based on the elastinlike matrix (VPGIG)2-VPGKG-(VPGIG)2, were synthesized using heterologous expression. After optimizing culture conditions and extraction procedures their biological activity was assessed using human umbilical vein endothelial cells (HUVEC). Elastin like proteins with differently incorporated bioactive signals (REDV, RGD and a small P15 peptide) were linked covalently via carbodiimide coupling to poly(L-lactide) (PLLA) films. HUVEC growth was determined on these modified surfaces using the BrdU assay (cell proliferation) and resazurin assay (cell viability). The chemically modified PLLA surfaces conferred higher cell viability after 1 h adhesion (60%) and an enhanced proliferation (63%, 1 h adhesion, 24 h cultivation) in comparison to the unmodified PLLA. These results indicate that the synthesized ELP incorporated with amino acid motifs promote an accelerated endothelialization of the biodegradable stent material PLLA. Discussion: In summary, we were able to generate elastin-like proteins modified by bioactive sequences. Those sequences enhanced endothelial cell proliferation and adhesion. Further studies are warranted to determine the activity on smooth muscle cells of these peptides. (1) The failing heart is characterized by excessive extracellular matrix production by myofibroblasts (MyoCFs) causing fibrosis and myocardial stiffening. MyoCFs represent phenotypically transformed cardiac fibroblasts (CFs) and are characterized by the expression of contractile proteins like a-smooth muscle actin (α-SMA) and enhanced secretion of growth factors (e. g. CTGF). Identification of intracellular enzymes that modulate this transformation process is desired to therapeutically modulate pro-fibrotic progression in heart failure. We show that PDE2A, a phosphodiesterase isoform, that is able to hydrolyse cGMP and cAMP, is markedly upregulated in failing hearts from patients with end-stage heart failure (2-3-fold, p<0.05, n=8). Notably, PDE2A protein abundance is 4-fold higher in MyoCFs compared to cardiomyocytes from neonatal rat hearts (p<0.05, n=7). To this end we tested whether PDE2A modulates the transformation of CFs isolated from neonatal rat hearts to MyoCFs. Indeed, as assessed by immunoblotting and fluorescent microscopy (α-SMA, phalloidin, DAPI), adenoviral PDE2A overexpression induced α-SMA expression (3.2-fold p<0.05, n≥8) and to a lower extent CTGF synthesis (1.5-fold, p<0.05, n≥5). Mechanistically, PDE2A showed preferential subsarcolemmal localisation with diminished total cGMP levels (-56%, p<0.05, n≥5). Consistently, parallel stimulation with atrial natriuretic peptide (ANP), a selective activator of membrane-bound guanylyl cyclase, normalized CTGF synthesis indicating that PDE2A controls cGMP in a discrete subdomain near the plasma membrane. Moreover PDE2A overexpression diminished the protein levels of vasodilator-stimulated phosphoprotein, a membrane cytoskeletal component (-60%, p<0.05, n≥5). These data implicate PDE2A-dependent subsarcolemmal cGMP regulation in myofibroblast formation and potentially cardiac fibrosis. Therefore, targeting PDE2A may lead to regression of the fibrotic remodeling associated with heart failure. Several anorganic nanoparticles (NP) causedhigher inhalation toxicity than the corresponding coarse particles (Oberdoerster et al. 2005) . We examined an organic pigment and a polymer dispersion each as nanomaterial and as the chemical identical coarse material in short-term inhalation studies in malerats. The polymer was an anionic acrylic ester copolymer containing free carboxylic groups. Three different particle sizes were synthesized by varying polymerization conditions: 12, 80 or 250 nm. Although polymeric acrylic ester was reported to be irritating to the respiratory tract at 3 mg/m3, all three tested polymers -including the NP (12 and 80 nm) -did not cause any changes in lavage fluid and in histopathology at 10 mg/m3. The organic pigment was a poorly soluble pyrrol with an intense orange color. The NP (10 to 50 nm width and 30 to 400 nm length) and the coarse pigments (70 to 200 nm width and 0.3 to 3 µm length) are both needle-like. They were tested at 1, 3, 10 and 30 mg/m3 for the NP and 3, 10 and 30 mg/m3 for the coarse materials. Mild and partly reversible morphological changes were observed in lung and lymph nodes at the highest concentrations, but the more pronounced effect were found in rats exposed to the coarse material. Likewise there was an increase of lavage parameters in rats exposed to thecoarse material but not to the NP. These data demonstrate that inhalation of finer NP is not necessarily associated with higher toxicity compared to the coarse material. The results were obtained with two organic particles of rather different size and composition but are in contrast to the more severe effects seen with several anorganic NP when compared to the corresponding coarse particles. Within the NanoCare Project a standard short-term inhalation test to examine the toxicity of inhaled aerosols from nanomaterials has been developed. The inhalation toxicity of nano-andpigmentary materials was studied: BaSO4, ZnO, CeO2, Al-doped CeO2, ZrO2, amorphous silica, surface-coated amorphous silica, Titania, carbon black and three multi-wall carbon nano tubes, all with complete phys-chem-characterization as planned for the OECD sponsorship program. Quartz dust TiO2 and ZnO were tested as pigmentary materials. Rats were exposed nose-only to three concentrations of one of these materials, 6 h a day for five consecutive days. Positive controls were exposed to quartz dust or pigmentary ZnO, negative controls to clean air. A wide range of endpoints for pulmonary toxicity were evaluated immediately after the last exposure and after 3 days and 3 weeks after the last exposure. Among these parameters, polymorphnuclear granulocyte count in bronchoalveolar lavage fluid is the most sensitive early parameter indicating inflammation process in lung, while histological examination reveals the type and localization of inflammation. Among these substances, we identified BaSO4 as having the lowest toxicity. All MWCNTs were most potent in producing progressive inflammation in the lung; granulomas in lung and lung associated lymph nodes were observed without indication for fibrosis. The NOAECs of the 16 substances ranged between < 0.1 and >50 mg/m 3 . Generally the material was only found in the lung (surface and macrophages) and in the draining lymph nodes. Surface modified amorphous silica was also found in the spleen. The data demonstrate that the method is able to differentiate the toxic potential of different nanomaterials and to indicate regression or progression of the effects effects. Moreover the lung burden and potential translocation to other tissues was detecable. Comparing the material properties and effects of the 16 materials, no general relationship between the toxicity and either particle size, specific surface area or aerosol particle number concentration was found. Hence we must not expect to find a gerneral "nanotoxicology" or a unifying dosimetry for all nanomaterials. We must rather be prepared to test individual nanomaterials for their effects. And to develop grouping concepts not only based on material properties but also on biopersistence, biokinetics and biological effects.

Part of this studes has been sponsored by BMBF (nanoCare).

Endpoint-centric search for toxicological information and data to support the information retrieval for regulatory programs Landsiedel R. 1 , Wächter T. The EU REACH regulation No 1907/2006 requires industry to ensure the safety of chemical use and manufacturing. All substances manufactured or imported in quantities above one tonne per year must be registered. Information requirements for the dossiers increase with increasing tonnage or once hazards are suspected. Searching for substance specific literature and the compilation of hazard data for safety assessments are highly challenging procedures. The novel web-based search engine Go3R, accessible free of charge at www.Go3R.org, has been created to allow quickly finding relevant hazard information and data. Go3R provides an endpoint-centered literature search to all scientists and regulatory authorities seeking for toxicological information. Furthermore, Go3R specifically highlights information on animal testing alternatives. Search results are presented automatically linked to an "intelligent table of contents" which enables the user to sort the literature listed in PubMed or the Toxicology Data Network (TOXNET) in a fast and comprehensive manner. Retrieved documents are automatically organized in categories relating to the IUCLID 5 chapters. Hereby, the user can browse directly through the entire 21 million documents without even having to start the search with an initial query. The semantically enriched platform supports the user during query formulation, allows for bibliographic analysis, and specifically highlights information related to the replacement, reduction, and refinement of animal experiments.

Search results in Go3R are shown in an dynamic table of contents (left) making them browsable for the contained information on animal testing alternatives and toxicologogical endpoints.

Towards a differentiation therapy of acute myelogenous leukemia with histamine H2-receptor agonists Laue S., Burhenne H., Seifert R. Hannover Medical School Institute of Pharmacology, Carl-Neuberg-Str. 1, 30625 Hannover, Germany Acute myelogenous leukemia (AML) is a devastating malignancy characterized by a differentiation block of myeloid progenitor cells. Recently, histamine dihydrochloride in combination with interleukin 2 has been approved as orphan drug for the consolidation treatment of AML (1) . It is assumed that histamine exerts its effects by activating the histamine H2-receptor (H2R) in human neutrophils, resulting in improved anti-tumor function of T killer cells by inhibiting NADPH oxidase-catalyzed superoxide formation. Previous studies had shown that histamine also induces myeloid differentiation (2) . Considering the fact that all-trans-retinoic acids constitutes a powerful differentiation therapy of acute promyelocytic leukaemia, a specific subtype of AML (3), we initiated a study to explore the possibility that H2R-mediated myeloid differentiation provides an alternative or complementary strategy to treat leukemias associated with differentiation blocks. As model system, we used HL-60 cells. In HL-60 cells, histamine and various H2-receptor agonists induced concentrationdependent increases in cAMP levels. Interestingly, ligands differentially increased cytosolic calcium concentration and extracellular receptor kinase (ERK) pathways, indicative for ligand-specific H2R conformations. H2R activation resulted in myeloid differentiation as assessed by enhanced formyl peptide receptor-mediated increases in cytosolic calcium concentration. H2R agonists showed no signs of cytotoxicity. Intriguingly, following H2R activation, the majority of the formed cAMP was exported into the extracellular space via multi-drug resistance protein (MRP) 4, indicating that export is a more important pathway for signal termination than cleavage of cAMP by phosphodiesterases. Despite effective cAMP export, even a short-term exposure (30 minutes) of cells was sufficient to induce expression of functionally active formyl peptide receptors. These data indicate that in contrast to previously held dogma, induction of myeloid differentiation does not require continuous presence of a cAMP signal. From a therapeutic point of view this is very important since "spike" therapy with cAMPincreasing substances may be sufficient to induce a therapeutic effect in AML, thereby also reducing toxic side effects. Currently, we are systematically exploring the effects of H2R agonists on signal transduction pathways and differentiation in various myeloid cell types to identify highly efficacious compounds. Introduction. Activation of Gαq/11 protein-coupled receptors of postsynaptic neurons can elicit the production of endogenous cannabinoids (endocannabinoids), which in turn inhibit transmitter release from axon terminals by activating presynaptic CB1 receptors. The aim of the present experiments was to study the mechanism of the endocannabinoid production. Specifically, we wanted to clarify the role of Ca 2+ release from intracellular stores in triggering endocannabinoid production. Methods. Patch-clamp-and Ca 2+ imaging experiments were performed on Purkinje cells in mouse cerebellar brain slices. Glutamatergic excitatory postsynaptic currents (eEPSCs) were elicited by electrical stimulation of parallel fibers. The Gαq/11 proteincoupled metabotropic glutamate receptor 1 (mGluR1) was activated by superfusion of (RS)-3,5-dihydroxyphenylglycine (DHPG) or -more physiologically -by burst stimulation of the parallel fibers. Results. Both DHPG superfusion and burst stimulation of parallel fibers elicited an increase in intracellular Ca 2+ concentration in the postsynaptic Purkinje cells. DHPG superfusion and burst stimulation suppressed eEPSCs, and this suppression was abolished in the presence of the mGluR1 antagonist CPCCOEt. The suppression of the eEPSCs was also sensitive to the CB1 receptor antagonist rimonabant, pointing to involvement of endocannabinoids and CB1 receptors. The suppression of the eEPSCs was attenuated after depletion of the endoplasmic reticulum Ca 2+ stores by thapsigargin, cyclopiazonic acid and IP3.

The results indicate that after activation of the Gαq/11 protein-coupled metabotropic glutamate receptor 1 (mGluR1) of the postsynaptic neuron Ca 2+ is released from the endoplasmic reticulum. This Ca 2+ release significantly contributes to the production of endocannabinoids. The endocannabinoids diffuse in the synaptic cleft retrogradely to the terminals of afferent axons and inhibit transmitter release there through presynaptic CB1 receptors.

The guanine nucleotide exchange factor Dock9 controls Reelin dependent Cdc42effects on radial migration Pichler M. 1 The regulation of blood glucose levels is under tight control of a complex system including hormone and neurotransmitter signalling. Many of these cellular signalling pathways are initiated by binding of the ligand to a G-protein coupled receptor (GPCR), e.g. noradrenaline inhibits insulin secretion upon binding to a Gi-coupled receptor. Upon GPCR activation the heterotrimeric G-protein is activated and both the α-subunit and βγdimers are released and interact with their specific target proteins. By the usage of Bordetella pertussis toxin (PTX) as a common Gαi inhibitor Gαi-dependent signalling pathways are interrupted which leads to increased insulin secretion, and significantly improves glucose tolerance. Since the Gαi-isoform specific roles in the regulation of glucose homeostasis are still debated we studied the glycemic control in Gαi2-deficient mice. Surprisingly and in contrast to the PTX data, glucose tolerance was unchanged in the Gαi2-deficient mice compared to wild type controls. However, the plasma insulin levels were significantly reduced upon glucose challenge. These findings point to disturbed islets function and improved peripheral insulin sensitivity. Analysing Gαi2deficient islets we show that islet size and number of nuclei are reduced. Nevertheless, in vitro insulin secretion is improved at low (3 mM) and high (16 mM) glucose concentrations and can be further stimulated upon PTX-treatment. These data indicate that Gαi2 proteins influence islet development and inhibit insulin secretion. In addition, these findings support our hypothesis that Gαi2-deletion influences peripheral insulin sensitivity. Therefore, we investigated glucose homeostasis and pAKT-levels after two hours feeding ad libitum in Gαi2-deficient mice. Under feeding conditions no differences in plasma insulin levels were visible although blood glucose levels were significantly reduced in Gαi2-targeted mice. pAKT-levels of liver and skeletal muscle were unaltered, whereas AKT phosphorylation in white adipose tissue was significantly increased, indicating improved glucose uptake of adipocytes.

In conclusion, Gαi2 is a negative regulator of both insulin secretion and peripheral insulin sensitivity and important for the maintenance of glucose homeostasis. 11689, 1992) in a radioligand binding test and to determine their functional effects with a membrane potential test using the dye R7260 (Molecular Probes, 0.125 mg/ml, excitation 505 nm, emission 530 nm). The affinity of compounds in radioligand binding was slightly higher in SUR2B than in SUR2A-type channels, but the enantiomeric ratio in SUR2A channels matched that one determined for the SUR2B-type indicating some conformity of the binding pockets of SUR2A and SUR2B-proteins. Surprisingly, however, the membrane potential tests revealed that the (3R,4S)-enantiomer acted as agonist (A) whereas the (3S,4R)-enantiomer acted as antagonist (B): (3R,4S)-BMS-191095 induced membrane hyperpolarisation whereas (3S,4R)-BMS-191095 repolarised cells prestimulated with submaximally effective concentrations of diazoxide. Concluding, BMS-191095 is not selective for SUR2A as compared to SUR2B-type K ATP channels. Its enantiomers activate and block SUR2-type KATP channels in a stereospecific manner.

Thieno-thiadiazine derivatives with full agonistic activity at SUR2B-type KATP channels act as partial agonists at cardiac SUR2A-subtypes Oldenhage C., Grittner D., Schmidt C., Lemoine H. Heinrich-Heine Universität, Inst. für Lasermedizin, Mol. Wirkstoff-Forschung, Universitätsstr. 1, 40225 Düsseldorf, Germany New potassium channel openers (KCO) of the thieno-thiadiazine(TTD)-type initially developed as agonists for the SUR1-type KATP channels (Nielsen et al., J Med Chem 45: 4171, 2002) were characterized in SUR2B-type KATP channels as agonists and antagonists, if R contains a quaternary (methyl-cycloalkyl) and a tertiary (R = cycloalkyl) carbon, respectively (Lemoine et al., this journal 375, R45, 2007) . To investigate the selectivity of TTD-derivatives for myocardial KATP channels the membrane potential actions of compounds were tested in HEK 293(Kir6.2/SUR2A)-cells and compared to HEK 293(Kir6.1/SUR2B)-cells as a model for smooth muscle-type KATP channels. Membrane potential was measured by fluorescence (excitation 505 nm, emission 530 nm) using 0.125 mg/ml of the dye R7260 (Molecular Probes). Standard-KCO induced hyperpolarisation with ~10-fold smaller potency (pEC50) in SUR2A. TTD-compounds with CH3-cycloalkyl residues not only lost potency but also intrinsic activity for channel activation (Emax) in SUR2A. Possibly, this loss of Emax would be much greater in native heart cells with a normal channel density. In contrast, TTD-compounds with cycloalkyl residues acted as antagonists of cells pre-hyperpolarized with diazoxide with similar affinity in SUR2A and SUR2B-type KATP channels. Concluding, selectivity of KCO for KATP channel-subtypes cannot only be achieved by a different affinity but also by a selective stimulation of the channel of interest. Small-conductance calcium activated potassium (KCNN/SK/KCa2) channels maintain neuronal calcium homeostasis, shape synaptic functions and prevent excitotoxic neuronal death. So far, little is known about the function of KCa2 channels in nonneuronal cells. The aim of this study was to investigate the expression of KCa2 channels in microglial cells and their potential function in microglial activation and maintenance. Expression of KCa2 channel subtypes in microglial cells was assessed by mRNA analysis, Western blots and immunocytochemistry. Lipopolysaccharide (LPS)-induced microglial proliferation was evaluated by the xCELLigence impedance-based system and MTT assays, and immunogenic activation of microglia was determined by measuring cytokines and nitric oxide (NO) release into the cell culture medium. The KCa2.2 and KCa2.3 channel activator CyPPA (25 µM) and specific inhibitory peptides (50 µM) were applied to distinguish effects mediated by the KCa2 channel subtypes. All KCa2 channel subtypes were detected on mRNA and protein levels in resting and in LPS-activated microglial cells. xCELLigence real-time measurements and MTT assays demonstrated that LPS (200 ng/ml) induced microglial proliferation. The KCa2.2/KCa2.3 channel activator CyPPA reduced LPS-induced microglial proliferation in a concentration-dependent manner. Specific peptide inhibitors of KCa2.3 channels, but not of KCa2.2 channels, reversed the CyPPA-effects on LPS-induced microglial proliferation. CyPPA alone did not alter the production of TNF-alpha or IL-6, but strongly reduced the LPS-dependent cytokine production. Interestingly, chelation of extracellular calcium by EDTA induced differential cytokine kinetics by decreasing LPS-dependent IL-6 production while TNF-a production was not affected. Moreover, using inhibitory SK3/KCa2.3 channel peptides, we demonstrated that SK3/KCa2.3 channels modulate LPS-induced cytokine IL-6 production in a calcium-dependent manner, while the TNF-a release was independent of extracellular calcium. In summary, the present study revealed that KCa2.3 channel stimulation reversed microglial activation. Thus, KCa2.3 channels may serve as a therapeutic target for reducing microglial activity and related inflammatory responses in CNS diseases.

(3R,4S)-(3S,4R)- Intracellular amyloid beta (Aß) oligomers and extracellular Aß plaques are key players in the progression of sporadic Alzheimer disease (AD). Still, the molecular signals triggering Aß production are largely unclear. We asked whether mitochondria-derived reactive oxygen species (ROS) are sufficient to increase Aß generation and thereby initiate a vicious cycle further impairing mitochondrial function.

Complex I and III dysfunction were induced in a cell model using the respiratory inhibitors rotenone and antimycin resulting in mitochondrial dysfunction and enhanced ROS levels. Both treatments lead to elevated levels of Aß. Presence of an antioxidant rescued mitochondrial function and reduced formation of Aß demonstrating that the observed effects depended on ROS. Conversely, cells overproducing Aß showed impairment of mitochondrial function such as comprised mitochondrial respiration, strongly altered morphology, and reduced intracellular mobility of mitochondria. Again, the capability of these cells to generate Aß was partly reduced by an antioxidant indicating that Aß formation was also ROS-dependent. Moreover, mice with a genetic defect in complex I, or AD mice treated with a complex I inhibitor, showed enhanced Aß levels in vivo.

Several lines of evidence show that mitochondria-derived ROS result in enhanced amyloidogenic amyloid precursor protein processing, and that Aß itself leads to mitochondrial dysfunction and increased ROS levels. We propose that starting from mitochondrial dysfunction a vicious cycle is triggered that contributes to the pathogenesis of sporadic AD.

Comparison of methods to derive health-based guidance or limit values for chemicals Licht O., Voss J. -U., Mangelsdorf I. Fraunhofer ITEM Chemikalienbewertung, Nikolai-Fuchs-Str. 1, 30625 Hannover, Germany Health-based guidance or limit values are derived for chemicals to compare measured or estimated exposure concentrations with these values. If the exposure is below the limit value, adverse effect for human health can be regarded as negligible, e.g. the exposure is expected to be tolerable. In Germany such values have been derived since years for chemicals that can be found in soil, water and air as well as human blood and urine (biomonitoring). In a research project sponsored by the German Umweltbundesamt several methods used by the agency are compared to the method laid in REACH guidance document R.8 to derive a Derived No Effect Level (DNEL).

The aim was to identify possibilities for standardization as well as to figure out specific elements in individual methods. In addition to extrapolation factors the public availability of guidance and specific derivations as well as procedures for consensus on the limit value were evaluated. The comparison of extrapolation factors revealed that, although they are named differently such as extrapolation, safety or assessment factors, they are used in a comparable manner. Factors for interspecies and intraspecies extrapolation are presented in more detail. The standard factor for such extrapolation is 10 in most cases.

In the REACH guidance this factor consists of a part for allometric scaling and remaining differences. Other factors are only defined in some methods, like a factor for extrapolation from LOAEL to NOAEL, data quality or data gaps. A factor for data quality is not laid down in the basic scheme for setting of indoor air guidance values, but is used in some of the recent derivations of limit values. Also the WHO guidelines for drinkingwater quality use comparable factors to account for adequacy of studies or database and nature and severity of effect. A transparent and documented derivation is necessary for acceptance of the value. The derivation methods as well as the evaluation document on a specific substance are available through publications or the internet in nearly all cases. For the DNEL only the numeric value is available at the ECHA website, but not any information on starting point and extrapolation factors. Although all guide or limit values are derived in a comparable way, differences, however, exist in some details. In most cases detailed explanation is lacking when deviating from standard or default assumptions. Often such deviation is based on expert judgement. Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world and has a poor prognosis with limited therapeutic options. Up to now, no curative systemic therapy exists emphasizing the high clinical importance of new therapies for HCC. Therefore, the identification and characterization of novel drugable targets is a relevant goal. Cyclin-dependent kinase 5 (Cdk5) is well characterized for its function in CNS development and disease. Recently, few reports indicate functions of Cdk5 in cancer. Cdk5 was shown to regulate tumor growth, and our group discovered that Cdk5 regulates angiogenesis. Since HCC is a highly vascularized tumor and anti-angiogenic treatment (Sorafenib) has shown some therapeutic benefit, we hypothesize that Cdk5 is an interesting target for HCC therapy. The aim of this study was to characterize the function of Cdk5 in HCC. Histology of tissue micro arrays indicates an increased expression of Cdk5 in human HCC tissue in comparison to healthy liver tissue of the same patient. To investigate the function of Cdk5 in HCC, we analyzed the impact of both pharmacological inhibition of Cdk5 and specific downregulation of Cdk5 with RNA interference on HCC cells. Pharmacological inhibition of Cdk5 with the small molecule roscovitine (R-Roscovitine, Seliciclib) decreased proliferation and clonogenic survival, induced G2/M cell cycle arrest and cell death, and reduced motility of HUH7 and HepG2 cells. Transient downregulation (siRNA) and stable knockdown (shRNA) of Cdk5 also reduced proliferation, clonogenic survival, migration and invasion of HUH7 cells. In a subcutaneous HCC xenograft model, treatment with roscovitine reduced tumor growth and angiogenesis, indicated by decreased tumor weight and volume, and reduced vessel density. Moreover, cotreatment of HCC cells with roscovitine and tumor necrosis factor related apoptosis inducing ligand (TRAIL) resulted in an over-additive additive effect on the induction of apoptosis. This coincided with reduced phosphorylation and activity of the anti-apoptotic transcription factor Stat3 at Ser727 that is directly phosphorylated by Cdk5, and Tyr705. In line with this, the expression of the antiapoptotic protein Mcl-1 is reduced by inhibition of Cdk5. Our results point to an important function of Cdk5 in HCC and suggest Cdk5 as an interesting pharmacologically druggable target for HCC therapy.

Delivery of mono-biotinylated RNaseA into macrophages with streptavidinconjugated Clostridium botulinum C3 toxin Lillich M. 1 , Chen X. Clostridium botulinum produces the ADP-ribosyltransferase C3, which modifies and thereby inactivates exclusively the small GTP binding proteins Rho-A,-B and -C. Recently, we discovered a specific endocytotic internalization of C3 toxin in macrophages and myeloid leukaemia cells, but not in epithelial cells [1] . Thus, C3 toxin provides a tool to target cells of the monocyte/macrophage lineage, which are involved in various diseases and are of great clinical interest. We used a biochemical crosslinking approach to design a delivery system based on an enzymatic inactive C3bot mutant (C3Mut) and streptavidin. The C3 portion mediates uptake of the transporter into monocytes/macrophages and streptavidin allows for binding of biotinylated cargo molecules to the transporter.

In vitro, the generated C3Mut-streptavidin bioconjugate showed specific and concentration dependent binding to biotinylated oligonucleotides as demonstrated by electrophoretic mobility shift assay. Cell fractionation experiments indicated an uptake of the bioconjugate into the cytosol of J774A.1 macrophages. In the next step, mono-biotinylated bovine pancreatic ribonuclease A (RNaseA) was used as a model cargo for the delivery of macromolecules by the bioconjugate. RNaseA is a highly stable, well studied protein which catalyzes the degradation of RNA. Mono-biotinylated RNaseA interacts in a specific and concentration dependent manner with the C3Mut-streptavidin bioconjugate in vitro as analysed with dot blot technique. The C3Mut-streptavidin bioconjugate efficiently mediates the internalization of biotinylated RNaseA into J774A.1 macrophages as analyzed with laser scanning microscopy in fixed cells. This finding was also confirmed by live cell imaging. Furthermore, cell fractionation showed a cytosolic delivery of biotinylated RNaseA in the presence of C3Mut-streptavidin. As expected we could not observe a cytotoxic effect of biotinylated wild-type RNaseA on J774A.1 macrophages, which is attributable to the presence of ribonuclease inhibitor protein in mammalian cells.

In summary, the C3Mut-streptavidin bioconjugate mediates the efficient internalization of biotinylated (macro)molecules into macrophage like cells, and therefore represents a useful tool for the transduction of exogenous molecules into macrophages. In addition, cytotoxic RNaseA mutants are available and will be used in further studies. Organometal compounds such as cisplatin or the second generation complexes carboplatin and oxaliplatin have become more and more important as antitumor agents. Nevertheless there is still an increasing demand for novel metal-based compounds. This is necessary due to severe side effects and the occurence of resistent tumour cells.

In this context we investigated the cytotoxic effects of imidazole-based phosphane gold(I) complexes as potential agents for cancer treatment. Initially we have used the MTT-Assay to examine the toxic potential of the gold complexes in H4IIE rat hepatoma cells. In this context CW60 (a diphosphane ligand with azoyl substituents R2P(CH2)2PR2, R= thiazol-2-yl) turned out to be the compound with the highest cytotoxic potential with an IC50 value of 6,5mM (24h incubation). Further investigations revealed that CW60 induced an apoptotic cell death in H4IIE demonstrated by the activation of caspase 3/7 (48h incubation with 10mM CW60). In addition the induction of apoptosis was confirmed by the DNA ladder formation (24h incubation with 5mM CW60). In connection with the molecular mechanisms of apoptosis induction we used the comet assay to analyse the generation of DNA strand breaks as well as the DCF-Assay to detect the formation of reactive oxygen species. However neither DNA strand breaks nor increased levels of reactive oxygen species were detected after 1h of incubation. Furthermore we analysed if the compound influences intracellular signalling pathways such as the JNK pathway and the PI3K/AKT but after 24h of incubation neither pAKT nor JNK were influenced. The imidazole based phosphane gold (I) complex CW60 shows strong toxic effects in H4IIE cells and turned out to be a promising compound as a potential agent for cancer treatment. 

The high and inappropriate intake of loop diuretics in hypertensive elderly reported in former studies has again been confirmed. Remembering that inappropriate intake of loop diuretics can lead to exsiccosis and electrolyte loss especially in elderly, better medical education has to follow these alarming results to improve the pattern of diuretic prescription. Furthermore, our results lead us to assume a high estimated number of unreported cases of torasemide use in uncomplicated arterial hypertension in elderly. This loop diuretic agent shows a longer duration of action compared with furosemide (elimination half-life: 3-4 hrs vs. 1 hr) and is effective in decreasing blood pressure in subdiuretic doses.

It must be pointed out that loop diuretics are still frequently inadequately prescribed because current guidelines recommend loop diuretics only in complicated arterial hypertension.

The role of cGMP/cGKI signaling and TrpC channels in regulation of vascular tone Loga F., Domes K., Hofmann F., Wegener J. Pharmakologie und Toxikologie For 923, Biedersteiner Str 27, 80802 München, Germany

Signaling by intracellular cGMP and cGMP-dependent protein kinase I (cGKI) is the major pathway in vascular smooth muscle, by which endothelial NO regulates vascular tone. Recent evidence suggests that TrpC channels are targets of cGKI in smooth muscle and mediate, at least partially, the relaxant effects of cGMP. We tested this concept by investigating the role of cGMP/cGKI signaling on vascular tone and peripheral resistance using cGKI-, TrpC6-, and TrpC3-, and TrpC3/C6-double knock-out mice. We found larger contractile responses to α-adrenergic stimulation in intact aorta from cGKI-, TrpC6-, and TrpC3/C6-double knock-out mice as compared to aorta from Ctr and TrpC3-knock-out mice indicating a functional link between cGKI and TrpC6 channels. No differences were found if the vasodilator tone, provided by the NO generation in the vascular endothelium, was inhibited by L-NAME. Likewise, no differences were observed in the increase in peripheral resistance by α-adrenergic stimulation using the hind limb perfusion system. Activation of cGKI by 8-Br-cGMP diminished aortic tone and peripheral resistance to a similar extent in control, TrpC6-, TrpC3-, and TrpC3/C6-double knock-out mice. No effect of 8-Br-cGMP was observed in preparations from cGKI -/mice. To test the co-localization of cGKI and TrpC channels, we performed immunocytochemistry on isolated smooth muscle and endothelial cells from aorta of Ctr, TrpC3-, and TrpC6-knockout mice. TrpC3 could be detected in both smooth muscle and endothelial cells whereas TrpC6 was only detected in endothelial cells. The results suggest that absence of cGKI or TrpC6 impairs the vasodilator tone induced by endothelial NO production but that cGKI and TrpC6 channels are not functionally coupled in vascular smooth muscle.

We thank Profs Birnbaumer (NIH) and Freichel (Homburg) for providing us with TrpC6 -/-, and TrpC3 -/mice and Prof. Flockerzi (Homburg) for the antibodies against the TrpC channels.

Whole genome microarray analysis of the effects of TCDD and PCB 153 in human hepatic cell models Lohr C. 1 , Neser S. After the treatment with TCDD, however, a total of 281 genes were more than 2-fold up regulated in HepG2 e.g. cytochrome P450 1A1 (CYP1A1) (32-fold) a sensitive marker for AhR activation. Additional up regulated genes in HepG2 were; arylhydrocarbon receptor repressor (AHRR) 15-fold, aldehyde dehydrogenase 3 A1 (ALDH3A1) 11-fold, and cytochrome P450 1B1 (CYP1B1) 10-fold. 44 genes were more than 2-fold down regulated in HepG2 cells e.g. -proprotein convertase subtilisin/kexin type 9. Markedly different findings were obtained in hHeps, i.e., 117 genes were up regulated, the highest up regulated gene was CYP1B1 with a 95-fold increase in gene expression, followed by CYP1A1 (41-fold) and ALDH3A1 (21-fold). Only a small group of genes were significantly down regulated (17 in total), e.g., solute carrier family 2 (facilitated glucose transporter). Comparing both human cell types, there was an unexpected small overlap of genes being up or down regulated. Interestingly, in both cell types, only 30 in common genes were up regulated, including CYP1A1, CYP1A2, CYP1B1 and ALDH1A3. Only platelet-derived growth factor receptor, beta polypeptide, was down-regulated in both HepG2 and hHeps. In conclusion, our data indicate pronounced differences in the patterns of TCDD-regulated genes between HepG2 cells and hHeps.

Detection of redox modified proteins in nociceptive processing Lorenz J. E. 1 , Kallenborn-Gerhardt W. Recent data indicate that redox modifications of proteins induced by reactive oxygen species (ROS) contribute to sensitization of pain pathways during persistent pain. However, little is known about the targets of ROS in pain processing, because the relatively unstable nature of many reversible protein oxidation states hampers the reliable detection and identification of modified proteins. Here, we used the quantitative thiol trapping technique termed OxICAT to identify proteins which are redox modified during nociceptive processing. We investigated spinal cords of untreated mice, after zymosan injection into a hindpaw (inflammatory pain model) and after spared nerve injury (neuropathic pain model). We identified several proteins with marked changes in their redox states after nociceptive stimulation. Our results show that the OxICAT method is an efficient method to detect redox modifications in proteins and that redox modifications seem to play a role in pain processing.

Supported by the Deutsche Forschungsgemeinschaft (SFB 815/A14).

Additive antinociceptive effects of a combination of vitamin C and vitamin E after peripheral nerve injury Lu R., Kallenborn-Gerhardt W., Geisslinger G., Schmidtko A. pharmazentrum frankfurt/ZAFES Institute of Clinical Pharmacology, Goethe University, Frankfurt am Main, Germany

Accumulating evidence indicates that increased generation of reactive oxygen species (ROS) contributes to the development of exaggerated pain hypersensitivity during persistent pain. In the present study, we investigated the antinociceptive efficacy of the antioxidants vitamin C and vitamin E in mouse models of inflammatory and neuropathic pain. We show that systemic administration of a combination of vitamins C and E inhibited the early behavioral responses to formalin injection and the neuropathic pain behavior after peripheral nerve injury, but not the inflammatory pain behavior induced by Complete Freund's Adjuvant. In contrast, vitamin C or vitamin E given alone failed to affect the nociceptive behavior in all tested models. The attenuated neuropathic pain behavior induced by the vitamin C and E combination was paralleled by a reduced p38 phosphorylation in the spinal cord and in dorsal root ganglia, and was also observed after intrathecal injection of the vitamins. Moreover, the vitamin C and E combination ameliorated the allodynia induced by an intrathecally delivered ROS donor. Our results suggest that administration of vitamins C and E in combination may exert synergistic antinociceptive effects, and further indicate that ROS essentially contribute to nociceptive processing in special pain states. -206, -213 and -214) replaced by leucine residues. Both amino acids are comparable in terms of hydrophobicity, volume and the preference for forming α-helices, but only methionine is oxidizable to a sulfoxide, in contrast to leucine.

In the present study we examined the protein-protein interaction (PPI) of recombinant AC 1, expressed in Sf9 insect cell membranes, with CaM, CaM-206, -213, -214 and -215 by measuring the catalytic activity of AC 1. CaM-mutants show a 3-4-fold lower potency than CaM, but they are more efficacious than CaM. Most prominently, CaM-215 was 133 % more efficacious than CaM. Such striking differences between CaM and CaM-mutants have not yet been observed for other mammalian effector proteins. As a result of the exchange of all methionine against leucine residues in CaM-215, it is more hydrophobic than CaM and this leads to a better PPI with AC 1.

In future studies we will examine the effects of CaM inhibitors, antidepressants and antipsychotics on CaM/AC 1 interaction. Furthermore we will analyze the effects of oxidized CaM and CaM-mutants on the catalytic activity of AC 1. Because oxidative stress is of great importance in aging, it is important to know more about the abovenamed interaction in view to the demographic change.

Taken together, our data point to a unique CaM/AC 1 interaction that may be selectively targeted by small molecules. In particular, enhancers of these interaction could be useful to improve memory and learning.

Gender differences in fat distribution and diabetes prevalence in NZO mouse Lubura M., Scherneck S., Zucker A., Schürmann A. Deutsches Institut für Ernährungsforschung Experimentelle Diabetologie, Arthur-Scheunert-Allee 114-116, 14558 Potsdam, Germany

Background: Excessive fat accumulation in visceral but not subcutaneous fat depots as well as ectopic fat storage in liver, skeletal muscle and pancreas are associated with an increased risk for the development of type 2 diabetes in humans. In this study we aimed to examine the influence of early fat distribution on onset of type 2 diabetes in mice. Methods: NZO mice are regarded as insulin resistant model in which only males become diabetic. We used male and female mice fed with high-fat and standard diet. We determined fat distribution by computed tomography for three times and conducted oral glucose tolerance tests on two different time points. Besides we assessed body weight and blood glucose levels on weekly basis. Results: Contrary to previous findings, we observed that not only male NZO mice on high-fat diet develop diabetes. Blood glucose levels at the 16 th week of age and total pancreatic insulin content indicated diabetes prevalence of 68% in males and 25% in females These results lead to the conclusion that high-fat diet counteracts protective action of estrogens against diabetes. Inversely to the findings in humans, female mice tend to store more fat in abdominal region than males. There was no relationship between early accumulation of fat in abdominal region and onset of type 2 diabetes. However, visceral fat was associated with liver fat in males as well as in females. Furthermore, at the age of ten weeks hepatic fat content correlated with blood glucose levels (r² = 0.69) indicating that the early hepatosteatosis is a predictor for hyperglycemia. However, there was no correlation between hepatic insulin sensitivity (indicated by quantitative insulin sensitivity index-QUICKI) and amounts of hepatic fat we conclude that early hepatosteatosis does not predict for glucose intolerance in NZO mice.

In the NZO mouse, the amount of liver fat but not the early fat distribution predicts for the later onset of type 2 diabetes. Further experiments are needed to examine the gender dependent differences in the diabetes prevalence of this mouse strain. With a prevalence of about 20-30% non-alcoholic fatty liver disease (NAFLD) represents the most common liver disorder in Europe. NAFLD manifestation ranges from steatosis through steatohepatitis (NASH) to fibrosis and cirrhosis, followed in some cases by liver failure and hepatocellular carcinoma. Fatty degeneration of liver cells, increased oxidative stress with concomitant lipid peroxidation and an induction of pro-inflammatory cytokines are proposed as possible causes for developing inflammation and fibrosis, but the exact pathogenesis of the progression of NAFLD into NASH is still unknown. Thus, besides life style modifications and weight reduction interventions, no established pharmacological therapy exists so far.

To gain further insights into the pathogenesis of NAFLD and NASH and to develop new therapeutic strategies, appropriate animal models are essential. Thus, in the present study three different dietary animal models for NAFLD were evaluated and compared to the biochemical and metabolic alterations seen with NAFLD and NASH in man.

Male adult Lewis rats were given standard food or one of three different diets: fatty liver diet [FLD] , methionine/choline deficient diet [MCD] or methionine/choline deficient plus high fat diet [MCD+HF] . After 1, 2, 4, 6 or 12 weeks of treatment, animals were sacrificed and body and liver weights, laboratory parameters (ASAT, ALAT) as well as histopathological changes in the livers and different parameters indicating oxidative stress or representing the biotransformation capacity of the livers were analyzed. With FLD and MCD+HF a normal body weight gain was observed, whereas with MCD body weight gain was strongly impaired. Liver weights were mainly increased after MCD+HF. Elevation of ASAT and ALAT values and hepatic steatosis were more pronounced after MCD and MCD+HF than after FLD. All three diets caused an increase in the oxidative stress in liver tissue, but especially with MCD a tremendous elevation in the hepatic levels of lipid peroxidation products was seen. With regard to liver biotransformation capacity, with all three diets mainly an induction of the cytochrome P450 2E1 and 4A1 isoforms expression and activity was observed, which was most pronounced after MCD and MCD+HF.

In summary, the changes induced by MCD or MCD+HF most closely resemble the alterations described in literature for NAFLD in man and thus should be preferred over FLD in future investigations on NAFLD and NASH. EP3 receptors for prostaglandin E2 convey stimulatory and inhibitory effects. E.g., their stimulatory effect leads to vasoconstriction in the human pulmonary artery and their inhibitory activity to reduction of neurotransmitter release from neuron endings. The aim of our study was (1) the pharmacological characterization of EP3 receptors in human pulmonary arteries and (2) the examination of the involvement of these receptors in the regulation of the neurogenic tachycardia in pithed rats. L-826266 served as the EP3 antagonist. Experiments were performed in human pulmonary arterial rings isolated from patients undergoing lobectomy during resection of lung carcinoma and in pithed and vagotomised rats. The EP1/EP3 agonist sulprostone (1 nM -100 mM) concentrationdependently contracted human pulmonary artery rings (pEC50 and Emax; 6.89±0.12 and 106.5±5.2%, relative to the contraction induced by KCl 60 mM). The concentrationresponse curve of sulprostone was not affected by the EP1 antagonist SC 19920 (10 µM) but shifted to the right by L-826266 (10 µM) (apparent pA2 6.22). Extending the exposure time to L-826266 from 0.5 to 3 h increased its antagonistic potency to 7.39 (Schild plot-based pA2; concentrations 0.1, 1 and 10 µM). In pithed rats electrical stimulation (0.66 Hz, 1 ms, 50 V for 5 s) of the preganglionic sympathetic nerve fibers or intravenous isoprenaline (0.15 nmol/kg) increased heart rate (HR) by 55 beats/min. Sulprostone (10 -1000 nmol/kg) did not affect the isoprenaline-induced increase in HR but inhibited the neurogenic tachycardia dose-dependently, maximally by 80%. L-826266 (3 µmol/kg) diminished the inhibitory effect of sulprostone 1000 nmol/kg on the neurogenic tachycardia by 20%.

In conclusion, EP3 receptors (1) located postsynaptically strongly contract human pulmonary arteries and (2) located presynaptically on sympathetic nerve fibres supplying the heart of rats strongly inhibit the neurogenic tachycardia.

-5-bromo-N- [3-(5- Voltage-gated Ca 2+ channels of the central nervous system control a multitude of Ca 2+ dependent processes such as neurotransmitter release, neuronal excitability, neurite outgrowth, synaptogenesis, plasticity and neuronal survival. The Cav2.1 Ca 2+ channelalso known as P/Q-type channel -belongs to the subfamily of high voltage activated Ca 2+ -channels. Ca 2+ influx via Cav2.1 Ca 2+ channels located at presynaptic nerve terminals triggers vesicle fusion and transmitter release at brain synapses and at the neuromuscular junction. Thus, Cav2.1 Ca 2+ channels play a crucial role in synaptic transmission. The global Cav2.1 knock-out phenotype is characterized by severe ataxia, dystonia and lethality during the first postnatal weeks and is therefore an unsuitable model to analyze the importance of Cav2.1 Ca 2+ channels for learning and memory. Therefore, we crossed a floxed Cav2.1 mouse line with Nex-Cre transgenic mice to establish a viable, forebrain specific knock-out mouse line (fbKO-mice). Results from Western blot analysis confirmed an efficient knock out of Cav2.1 in hippocampal and cortical preparations, whereas the expression level in the cerebellum was not altered. To investigate the specific role of Cav2.1 channels in hippocampus and neocortex dependent behavior, we performed tests for motor functions and sensory abilities and in particular learning and memory tasks. Mice with a forebrain specific Cav2.1 knock-out show significant deficits in spatial learning & reference memory and a significant reduced recognition memory as revealed by the Morris Water Maze and an object recognition task. The fbKO-mice exhibit no obvious locomotor deficits during behavioral tasks in the Open Field test and Elevated Plus Maze. Some fbKO-mice demonstrate episodes of seizures in the Morris Water Maze and during different Rotarod tasks. To assess motor-function of fbKO-mice in a stress reduced environment, we performed home cage based running-wheel motor-learning tasks. In summary, the diverse phenotypes of the forebrain specific knock-out mouse line emphasize the critical importance of Cav2.1 for learning and memory. Helicobacter hepaticus-infected Rag2 -/mice emulate many aspects of human inflammatory bowel disease (IBD), including the development of colitis and colon cancer [Erdman et al., 2009 , PNAS 106: 1027 -1032 . Toward the goal of elucidating mechanisms of inflammation-induced carcinogenesis and developing biomarkers of inflammation, we undertook a comprehensive analysis of macromolecular damage products during disease progression in H. hepaticus-infected Rag2 -/mice. Infected mice developed severe colitis and hepatitis, accompanied by infiltration of myeloperoxidase-positive neutrophils and F4/80-positive macrophages, by 10 wks postinfection (pi), progressing into colon carcinoma by 20 wks pi. qPCR array-based gene expression profiling revealed that pathophysiological changes were associated with characteristic alterations in the expression of genes related to inflammation, DNA repair, and oxidative stress response. To study inflammation-related macromolecular damage, colon and liver tissues were analyzed by isotope-dilution chromatography-coupled mass spectrometry to quantify a battery of 16 different DNA, RNA and protein damage products thought to represent the full spectrum of inflammation-related chemistries. Our data revealed a significant predominance of chlorinated DNA-, RNA-, and protein damage products by 20 weeks pi. In contrast, levels of damage products arising from oxidation, nitration and nitrosation changed only modestly or remained unchanged. Our analyses also revealed higher levels of damage products in RNA than in DNA and demonstrated organ-specific differences of oxidative damage products, such as 8-oxo-dG and its oxidation products spiroiminodihydantoin and guanidinohydantoin. Collectively, these results suggest that neutrophil and myeloperoxidase-induced chlorination chemistry may serve as a biomarker of IBD and may play important roles in the pathophysiology of IBD and colitis-associated cancer.

Characterization of a membrane protein expressed in mouse heart and brain Mannebach S. 1 Recently, a novel membrane protein in Drosophila was shown to be localized in presynaptic vesicles. It appears to mediate a Ca influx after vesicle fusion with the plasma membrane. Disruption of the corresponding gene leads to endocytic defects in drosophila [1] . Apparently, this protein plays a role in exo-and endocytosis and could serve as a Ca channel supplying Ca required for endocytosis. We have identified a protein in mouse, c90rf7, which shares 26,3% amino acid sequence identity with the Drosophila protein. It covers 171 amino acid residues. Using RT-PCR the full length transcripts could be identified in brain, kidney, pancreas, heart, spleen, thymus and mast cells. Coexpression of c90rf7 and the Ca V2.2 channel in Xenopus oocytes reduced the amount of the α1B and CaVβ3 subunits of the Ca 2+ channel in the plasma membrane but did not affect the gating properties of the CaV2.2 channel. Expression of c90rf7 alone did not yield any channel activity. We therefore started to produce recombinant protein using the His-SUMO-prokaryotic expression vector. The protein was efficiently expressed as His-SUMO-c9orf7-fusion in E.coli (yield 2mg at 1mg/ml). We are currently preparing the c9orf7 part of the His-SUMO-c9orf7fusion protein by Ulp1-protease digestion followed by various chromatographic steps. The purified recombinant protein will be used to immunize rabbits to get antibodies. In parallel we generated antisera by immunizing rabbits with peptide fragments derived from the c9orf7 sequence. We could not identify any homologues of C9orf7 in the mouse genome and to analyze its function we are currently generating C9orf7 deficient mouse lines by gene targeting. We have chosen a strategy for conditionally inactivation of the gene with the option to study the cellular localization of C9orf7 by expression of the bgalactosidase gene under the control of the endogenous C9orf7 promoter. By southern blot analysis we´ve already identified 210 homologous recombinant embryonic stem cell clones out of 300 analyzed ones and we will proceed with blastocyst injection to get chimeric mice and finally mice carrying the introduced mutations in the c90rf gene. PARPs are involved in various biological processes such as regulation of DNA repair, cell cycle progression, and cell death. Consequently, several PARP inhibitors are currently in clinical development as chemo-and radiosensitizers as well as monotherapeutic agents following the concept of synthetic lethality. Pharmacological and toxicological studies call for an accurate analysis of PARP activity in terms of a detailed knowledge of the structure of PAR and a reliable method for its quantification. We have developed a sensitive, precise, and accurate bioanalytical method based on liquid chromatography coupled to electrospray tandem mass spectrometry (LC/MS-MS) to characterize and quantify PAR with femtmol sensitivity: PAR is extracted from cells and hydrolysed to specific monomeric units, i.e., ribosyladenosine, which is characteristic for linear PAR, diribosyladenosine, which is characteristic for branching points, and adenosine, which represents the terminal part of the polymer. Using this method, we are currently analyzing PAR levels in different cell lines and in primary human peripheral blood mononuclear cells (PBMCs) both under physiological conditions as well as upon genotoxic stress and in the presence of potent PARP inhibitors. We expect that after completing method validation this assay will be useful for a wide range of applications in pharmacology and toxicology.

Gene mutagenic potential and metabolite profile of 17β-estradiol in cultured V79 cells expressing human cytochrome P450 1A1 Martínez Jaramillo D., Lehmann L. University of Wuerzburg, Institute of Pharmacy and Food Chemistry Section of Food Chemistry, Am Hubland, 97074 Wuerzburg, Germany

Oxidative metabolism of the female sex hormone 17β-estradiol (E2) is considered to play a major role in the initiation of hormone-induced carcinogenesis. In extrahepatic tissues, E2 undergoes metabolic activation by cytochrome P450-dependent monooxygenase (CYP) isozyme 1A1 to 2-hydroxy-(2-HO) and to a lesser extent to 4-HO-E2. If not conjugated, these catecholestrogens (CE) can further oxidize to electrophilic quinones (Q), which may react with DNA and induce thereby mutations. Conjugation of these CE in extrahepatic tissues is mainly catalyzed by catechol-Omethyltransferase.

In order to identify possible mutagenic metabolites (i) the induction of gene mutations by E2 was determined in male Chinese hamster lung fibroblasts (V79 cells) expressing human (h) CYP1A1 and (ii) the metabolite profile of E2 in these cells was analyzed via gas chromatography/mass spectrometry after solid phase extraction of the cell suspension in the culture medium. (i) Gene mutations were assessed using the hypoxanthine-guanine phosphoribosyltransferase assay. The promutagen benzo[a]pyrene (BaP) served as positive control requiring metabolic activation by hCYP1A1 and dimethylsulfoxide as solvent control. V79 hCYP1A1 were treated with 100 nM E2 for 3 weeks and the resulting 6-thioguanine (6-TG) resistant mutants selected at weeks (w) 2 and 3. The frequency of spontaneous 6-TG resistant mutants per 10 6 colony-forming cells ranged from 5 ± 1 (w2) to 9 ± 4 (w3). As expected, 0.25 µM BaP induced a significant increase in mutant frequency (MF, 266 ± 13, w2 and 132 ± 46, w3) . Treatment with 100 nM E2 resulted in a 3-fold (17 ± 2, w2) and a 2-fold (23 ± 4, w3) increase in MF, suggesting slight mutagenic activity.

In culture medium of V79 hCYP1A1 treated with 100 nM E2, 2-HO-E2, 2-methoxy-(MeO)-E2, 3-O-methyl-2HO-E2 and 4-MeO-E2 (suggesting intracellular formation of 4-HO-E2) were detected. While 4-MeO-E2 concentration remained constant over the exposure period, the concentration of the other metabolites increased in a timedependent manner. The maximum concentration increase was reached at w3 for methylcatechols and at w2 for 2-HO-E2, correlating with the maximum increase in MF, observed after 2 weeks as well.

In conclusion, E2 possessed a slight mutagenic potential after hCYP1A1-mediated activation to 2-, 4-HO-E2 and their corresponding methylcatechols.

Cumulative effects of three triazole fungicides in a broad dose range in vitro Rieke S., Kneuer C., Bumke Scheer M., Lampen A., Hirsch-Ernst K., Marx-Stoelting P. Bundesinstitut für Risikobewertung Chemikaliensicherheit, Max-Dohrn-Str., 10589 Berlin, Germany Consumers are exposed to multiple residues of different pesticides via the diet. This raises questions concerning potential cumulative effects, especially for substances causing toxicity by a common mode of action. The aim of this work was to investigate potential combination effects of the three triazole fungicides epoxiconazol, tebuconazol and flusilazol for selected parameters in a broad dose range in vitro. Parameters investigated were cytotoxicity, hormone synthesis (17-β estradiol, progesterone and β-hCG), expression of a panel of androgen-or estrogen-responsive genes in the human placental choriocarcinoma cell-line Jeg-3 and transactivation via estrogen receptors α and β in stably-transfected HEK 293 cells.

The ability to inhibit steroidogenesis was analysed by measuring the concentrations of 17β-estradiol and progesterone in cell culture supernatants of Jeg-3 cells. Additionally, the placental peptide hormone β-hCG was measured. While no change in β-hCG and 17β-estradiol concentrations were observed, all triazoles induced a dose-dependent decrease in progesterone concentration and a cumulative effect was observed implying dose additivity at individual doses of >1.7 µg triazole/ml. Significant activation of ERβ by the three triazoles, especially by flusilazol, was observed at 5 µg triazole/ml and combined exposure showed additive effects, while no significant activation of ERα was observed. Based on the data, our findings suggest dose-additivity of triazole pesticides with the same mode of action for selected parameters in vitro. No significant effects were observed at lower doses [1ng -1µg triazole/ml] neither for substances applied individually nor in combination.

Transient receptor potential channels as mediators of catecholamine release Mathar I. 1 TRP proteins form cation channels that are regulated through strikingly diverse mechanisms. Recently, genetic association studies identified many Trp genes including Trpm4 as risk factors for disease states such as arrhythmias, hypertension and cardiomyopathy. The melastatin TRP channels TRPM4 and TRPM5 have distinct properties within the TRP channel family; they form non-selective cation channels activated by intracellular calcium ions and are expressed in heart, aortic endothelial cells, kidney and adrenal gland. Disruption of the TRPM4 gene in mice leads to increased basal blood pressure without evidence for impairment of endothelium-or smooth muscle-dependent regulation of contractility of peripheral resistance vessels, the renin angiotensin aldosterone system, basal cardiac output or body fluid homeostasis. Instead, TRPM4-deficient chromaffin cells exhibit increased acetylcholine-induced exocytosis of catecholamines which is associated with elevated level of epinephrine in the plasma and its metabolites in the urine. This indicates that TRPM4 serves as an inhibitory regulator of exocytotic catecholamine release, at least in chromaffin cells. Whether catecholamine release is also regulated by TRPM4 in other cells of the sympathetic nervous system such as perivascular neurons still needs to be clarified as well as the molecular mechanism underlying how TRPM4 regulates catecholamine release. Besides TRPM4 we recently identified transcripts encoding additional TRP channels including TRPC5 and TRPC6 in chromaffin cells isolated by laser capture microdissection but their functional role in these cells is still unknown. Measurements of the time course of the intracellular calcium concentration before and during acetylcholine stimulation (10µM) of catecholamine release as well as the analysis of the number of released vesicles in chromaffin cells relvealed no changes in TRPC1/C5/C6 triple knock out mice compared to wildtype controls. Although it seems that these TRPC proteins are not directly involved in catecholamine release from chromaffin cells induced by acetylcholine application in our hitherto existing experiments, their contribution to the modulation of catecholamine release by agonists of Gq-coupled receptors still needs to be analysed. Aims: Sulfonylureas (SUs) are among the most widely used oral hypoglycaemic drugs that stimulate insulin secretion. In addition, SUs have pleiotropic effects on other tissues. Regarding the effects of SUs on adipocytes conflicting findings were reported. We have now investigated the actions of glimepiride and glibenclamide (=glyburide) in primary human adipocytes. Methods: Primary cultured human white pre-adipocytes were differentiated in vitro according to a standard protocol. Lipid accumulation was assessed by Oil Red O staining and determination of triglyceride content; gene expression was measured by Real-Time PCR and Western blotting. Results: We initially characterized the genes regulated during human preadipocyte differentiation by a global microarray analysis. Treatment with glimepiride and glibenclamide caused a strong accumulation of lipid droplets and an increase in triglyceride content. Genes involved in lipid metabolism were induced, chemokine expression was decreased. Interestingly, the effects of SUs were over all qualitatively and quantitatively similar to pioglitazone. In direct comparison glibenclamide was more potent than glimepiride in respect to the induction of FABP4 (EC 50 0.32 vs. 2.8 µM), an important adipocyte marker gene. SU-induced differentiation was virtually completely blocked by the PPARγ-antagonist T0070907 but not affected by diazoxide, indicating PPARγ activation by SUs. Repaglinide, causing insulin liberation like SUs but being structurally different, had no effect on adipocytes. Conclusions: In primary human pre-adipocytes, glibenclamide and glimepiride strongly induced differentiation, apparently by activating PPARγ . Thus, SUs but not repaglinide may be used to influence insulin resistance beyond their effect on insulin liberation.

The role of AT1A and AT1B receptors as mechanosensors in myogenic vasoconstriction Blodow S. 1 , Schneider H. Arterial myogenic tone denotes the intrinsic property of vascular smooth muscle cells to constrict in response to an elevated intraluminal blood pressure. This physiological reaction is more distinct in small resistance arteries than in large conduit arteries. Understanding the underlying mechanisms should provide useful information for the treatment of diseases like anaphylactic shock and systemic hypertension in which this reaction is altered. Whereas the underlying signaling cascade has been extensively studied, the molecular identity of the mechanosensory elements still remains elusive. Recent studies at the cellular level suggest a sensory function for a subgroup of Gprotein coupled receptors (GPCRs) coupling to Gq/11-proteins. By determining mRNAexpression levels of selected GPCRs in consecutive pairs of resistance and conduit vessels, we could identify a subset of Gq/11-coupled receptors such as angiotensin II AT1B, vasopressin V1A, endothelin ETA and ETB and α1A adrenoceptor significantly enriched in resistance vessels. By pharmacological blocking of those highly expressed GPCRs by different antagonists and inverse agonists, we evaluated their influence on the formation or the intensity of myogenic tone, as measured in isolated murine mesenteric arteries ex vivo. While blocking of V1A receptor and α2A and α2AB adrenoceptors showed no differences of myogenic tone, blocking of AT1A and AT1B receptors by losartan and candesartan, ETA receptor by BQ123 and α1A adrenoceptor by prazosin caused significant reductions of the vascular response. Analyzing the myogenic response of AT1A -/mice with and without additional blocking of AT1B receptors by candesartan suggested that especially AT1B receptors play a dominant role for mechanosensitivity in mice. This was further supported by investigating the myogenic response of AT1B -/mice. These findings suggest that mechanosensitive Gq/11-protein coupled receptors, especially AT1B receptors, play a dominant role for the development of myogenic vasoconstriction. TRPM3 ion channels are activated by steroidal compounds and noxious heat and are considered to be involved in insulin secretion and pain perception. The expression of the TRPM3 gene generates a variety of different transcripts which arise by alternative splicing and the use of different promoters [1] . They encode a substantial variety of isoformes and so far we have identified more than 20 distinct TRPM3 proteins in mouse and rat each varying in exons 1, 2, 8, 13, 15, 17 and 24 . These variants differ enormously in their biophysical properties. For example splicing within exon 24 affects the channel pore and causes significant changes of the ionic selectivity of TRPM3 channels [2] , whereas splicing of 54 nucleotides encoded by exon 13 leads to dormant TRPM3 proteins. However, the frequency of these different isoformes in TRPM3 expressing tissues is completely unknown. To get insight into the significance of the different TRPM3 isoformes we investigated the abundance of alternative TRPM3 transcripts in different tissues and cell types by Reverse Transcription quantitative PCR (RT-qPCR). We found that the frequency of splicing within exon 13 ranges from 5 up to 20 % in different cell types and tissues. Furthermore we analyzed the TRPM3 transcriptome in the choroid plexus of the brain and the pituitary gland, tissues in which TRPM3 transcripts are most abundant. For that purpose we sequenced more than 120 clones, each. Corresponding to our RT-qPCR result, we found a significant number of transcripts lacking exon 13. In cells of the choroid plexus nearly all (124 /126 clones) carried the short Ca 2+ permeable pore. Furthermore, we identified seven variants spliced in exon 20 encoding truncated TRPM3 proteins. However, the composition of the TRPM3 transcriptome in the choroid plexus and pituitary gland differed enormously, indicating the importance of alternative splicing for TRPM3 function in different tissues. The concept of "thresholds of toxicological concern" (TTC) defines tolerable dietary intakes for chemicals without toxicity data and is widely applied to chemicals present in food in low concentrations such as flavorings. Based on a statistical evaluation of the results of many toxicity studies and considerations of chemical structures, the TTC concept derives a maximum daily oral intake without concern of 1800, 540 or 90 µg/person/day for non-genotoxic chemicals depending on the allocation to so-called Cramer classes I, II or III. For substances with a structural alert for genotoxicity a TTC value of 0.15 µg/person/day might be used. Recently, it has been investigated, whether the TTC values, which were derived based on mostly chronic oral dietary rodent studies would cover all relevant toxicities (neurotoxic, repeated dose, reproductive and developmental, immune effects and endocrine-related effects). Several authors using different specific databases have confirmed that the TTC values derived using Cramer classes are also covering immunotoxic, neurotoxic, reproductive and developmental effects. A respective decision tree is going to be presented, also considering substances or substance classes which shall be excluded from the TTC approach. There are several areas in which the TTC concept is already used, or a TTC approach is considered useful, to assess low-level human exposures, or help in prioritizing toxicological testing; as for example the assessment of plant metabolites and degradates of pesticide active substances, feed and food additives, chemicals with a low exposure profile under REACH, residues, metabolites and impurities in plants, chemicals, plant protection products or pharmaceuticals. If no structural alert for genotoxicity is given or standard genotoxicity tests are negative the Cramer class III value of 90 µg/person/day, which corresponds to a dose of 1.5 µg/kg bw is considered to represent a chronic tolerable daily intake of the test substance. Examples for current and future uses of the TTC concept in regulatory toxicology are presented. Objective: Hyaluronan (HA), synthesized by three HA-synthases (HAS1, -2, -3), is a prominent matrix component of atherosclerotic lesions. The aim of the present study was to identify the HAS isoenzyme that is associated with HA-matrix remodeling in inflammatory regions of atherosclerotic plaques. Furthermore the underlying regulatory pathways were determined and functional aspects of this regulation in vascular smooth muscle cell (VSMC) were addressed. Methods and Results: During atherosclerosis in ApoE deficient mice the peak of macrophage invasion at 14 weeks coincided with HA deposition and induction of HAS3 in aortic root plaques. In human symptomatic carotid artery plaques HAS3 was by far the most prominent HAS isoenzyme as determined by quantitative real time RTPCR. In vitro, in human vascular smooth muscle cell (VSMC) HAS3 was specifically induced via activation of NFkB by interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNFa) as shown by ChIP assay and utilization of NFkB inhibitor Bay 11-7082. HAS3 was also upregulated in a co-culture system by activated macrophages via paracrine release of TNFa and IL-1β as verified by neutralizing antibodies. In human atherosclerotic lesions NFkB positive VSMC were frequently detected in close proximity with HA and F4/80 positive macrophages as shown by immunohistochemistry. To study the effects of HAS3 mediated HA synthesis in human coronary VSMC, lentiviral overexpression and knockdown of human HAS3 were employed. Overexpression of HAS3 resulted in increased migration and proliferation whereas knock down had the opposite effect. The effects of HAS3 were mediated by both PI3K signaling and MAPK signaling via hyaluronan receptors CD44 and RHAMM. Conclusion: The present results suggest that HAS3-dependent HA synthesis is induced in human VSMC by inflammatory cytokines released from activated macrophages. Moreover, HAS3-mediated HA production induced phenotypic activation of VSMC. Pulmonary inflammation and airway remodeling are major features of chronic obstructive lung disease (COPD). In addition, pulmonary hypertension is a common comorbidity, which is associated with a poor prognosis of the disease. Recent studies in a guinea pig model of allergic asthma have shown that increased arginase activity, which converts Larginine into L-ornithine and urea and competes with nitric oxide synthases for the common substrate, contributes to allergen-induced airway inflammation, hyperresponsiveness and remodeling. There is evidence that cigarette smoke and lipopolysaccharide (LPS), both involved in the pathogenesis of COPD, increase the expression of arginase, however, its role in the pathogenesis of COPD is currently unknown. This study aimed to investigate the role of arginase in pulmonary inflammation and remodeling, using a guinea pig model of LPS-induced COPD. To this aim, guinea pigs were instilled intranasally with LPS or saline twice weekly for 12 weeks and were pretreated by inhalation of the arginase inhibitor (2)S-amino-boronohexanoic acid (ABH) or PBS. Repeated LPS exposure increased lung arginase activity, resulting in increased Lornithine/L-arginine and L-ornithine/L-citrulline ratio's. Both ratio's were reversed by ABH treatment. Repeated LPS exposure also induced increased IL-8 levels, neutrophils, goblet cells, hydroxyproline and airway collagen content in the lung, which were all abrogated by ABH. Moreover, repeated LPS exposure increased right ventricular mass, indicative of pulmonary hypertension, which was similarly prevented by ABH. In conclusion, increased arginase activity contributes to pulmonary inflammation, airway remodeling and right ventricular hypertrophy in a guinea pig model of COPD, indicating that arginase inhibitors may have therapeutic potential in the treatment of this disease. (Supported by MSD).

Behavioral abnormalities in HCN3-deficient mice Michalakis S., Schöll-Weidinger M., Mader R., Cao-Ehlker X., Fenske S., Wahl-Schott C., Biel M. Center for Integrated Protein Science Munich (CIPSM) Department of Pharmacy -Center for Drug Research, Ludwig-Maximilians-Universität München, Butenandtstr. 5-13, 81377 München, Germany HCN3 encodes a hyperpolarization-activated and cyclic nucleotide-gated channel, which is expressed in various brain regions including thalamic, hypothalamic and habenular nuclei as well as brain stem and olfactory bulb. In this study we performed a comparative analysis of HCN3 -/and HCN3 +/+ mice using a battery of behavioral tests and telemetric biopotential measurements to evaluate a potential role of HCN3 in central nervous system function. In general, the knockout mice showed normal motor function as assessed by the rotarod and open field tests. Telemetric home cage activity and core body temperature measurements confirmed a normal circadian behavior, but revealed a lower basal activity that concurred with decreased body temperature during the light phase and the light-dark transition phase. Hippocampus-dependent spatial learning was normal. By contrast, HCN3 knockout mice showed more immobility than control mice on day two of the Porsolt forced swimming test, which could reflect increased depressionlike behavior. However, center exploration in the open field test as well as performance in the light-dark transition and the elevated-plus maze tests was normal in HCN3 -/mice. This suggests that general anxiety was not changed in the knockout mice. In addition, HCN3 knockout mice were less active on the second day of the open field test, which supports a habituation phenotype. Finally, HCN3 -/mice had higher burying scores in the marble-burying test, which is a test for certain aspects of obsessive compulsive disorder in rodents. Taken together, genetic deletion of HCN3 in mice results in distinct behavioral abnormalities related to behavioral despair and expression of repetitive behaviors in response to mild stressors. Mielke H. 1 , Gundert-Remy U. Alcohol consumption when breast feeding is discussed controversially. Some groups recommend breast pumping before alcohol consumption and feeding the stored milk instead of breast feeding after drinking alcohol. This study was performed to simulate the blood concentration in the breastfed baby and to assess the health impact. Method: We established a physiologically based kinetic model. Its parameters were calculated (partition coefficients tissue/blood ; Schmitt, 2008) Silva et al.,1993) . We simulated 1. the alcohol concentration in a breastfed neonate and a 3-month-old suckling infant after the nursing mother had consumed alcohol,2. the alcohol concentration in utero/fetal compartment during pregnancy assuming the identical alcohol consumption of the pregnant woman 3. the alcohol concentration during infant´s treatment of bloating by an approved herbal drug containing alcohol. Results: Peak maternal alcohol concentration was 0.59 ‰ after consuming 0.25 L of wine, peak concentration was 0.0033 ‰ in the newborn, 0.0038 ‰ in the 3-month-old infant and 0.38 ‰ in the utero/fetal compartment. The peak concentration after herbal drug treatment was 0.015‰ in the neonate and 0.015‰ in the 3-month-old infant, respectively. We discuss the results of the simulations and compare it with doses and published concentrations measured in experimental animals or in vitro studies. Conclusions: We conclude that the recommendation "1 to 2 glasses of wine on occasion" (Agence Nationale d'Accréditation et d'Évaluation en Santé, ASSANTE 2002) is in accordance with the simulation results presented here whereas stricter rules are not scientifically sound. (2002) http://www.has-sante.fr/portail/upload/docs/application/pdf/ breastfeeding_guidelines.pdf da Silva et al. (1993) Adenylyl cyclases (AC) mediate physiological responses in virtually all cells, where their regulation through receptors and G proteins results in the modulation of cAMP. In the present study we focused on the kinetics of interactions between the alpha-subunit from inhibitory G protein type 1 (Gαi1) and adenylyl cyclase type V (AC5). These proteins were labeled with CFP and YFP, respectively. The dynamics of their interactions was monitored by means of high temporal resolution FRET imaging in HEK cells expressing unlabeled a2A-receptor and Gβg subunits. To activate the signaling pathway, we applied agonist using a rapid superfusion device. Application of norepinephrine resulted in the development of a FRET signal, indicating interaction between Gai1-CFP and YFP-AC5. After withdrawal of agonist the FRET signal recovered with a remarkably slow time course compared to the deactivation kinetics of Gi proteins reported previously (Bünemann et al. 2003) . To further analyze the properties of the dissociation between Gai1 and AC5 we measured in parallel the offset kinetics of the interaction between Gai1-YFP and Gβg-CFP (Gi1-FRET) after agonist withdrawal under comparable conditions. In addition we tested to what degree the coexpression of RGS4 accelerated the deactivation of Gi proteins and the dissociation of Gai1-CFP from YFP-AC5. These experiments revealed that in the absence of RGS4 the dissociation of Gai1 from AC5 after agonist withdrawal takes about 3 times longer than the deactivation of Gi proteins. In the presence of RGS4 this difference is even larger due to the pronounced acceleration of G protein deactivation. The dissociation of Gai1 from AC5 was only marginally accelerated by RGS4. These observations lead us to hypothesize, that AC5 might trap activated G protein-subunits and thereby affect the G protein cycle by shifting the equilibrium towards activated G proteins. If this hypothesis is true, it should result in a left-shifted dose response curve compared to G protein activation dose response. In support of this hypothesis we found that the concentration response curve for Gai1-AC5 interaction was several-fold leftward-shifted compared to the concentration-response curve of Gi-protein activation under very similar conditions. Influencing the dynamics of the G protein cycle by effectors may represent a novel and powerful mechanism for finetuning the sensitivity of receptor evoked responses in an effector-specific manner. Obesity, the excessive accumulation of white adipose tissue (WAT), has reached pandemic dimensions. The factors that determine fat mass are not fully understood, but adipocyte hypertrophy and adipokine secretion are thought to be important. In present study, we investigated the role of the cyclic GMP (cGMP) signaling pathway focusing on cGMP-dependent protein kinase I (PKGI) in white adipocytes. PKGI is expressed in WAT, preadipocytes and differentiated adipocytes as demonstrated by real-time PCR, western blot and immunochemistry. Differentiation of PKGIfl/fl preadipocytes, using an optimized protocol, resulted in an enhanced lipid accumulation as evidenced by Oil Red O staining. Deletion of PKGI in PKGIfl/fl adipocytes infected with a Cre lentivirus (LV-Cre, PKGI0/0) exhibited reduced differentiation. Analysis of the triglyceride (TG) content revealed a significant decrease of TG levels by 65% ± 1% in PKGI0/0 as compared to PKGIfl/fl adipocytes. Western blot analysis of white adipocytes showed a significant decrease of C/EBPalpha (19% ± 4.8%), PPARgamma (66% ± 2.9%) and aP2 (37% ± 10.6%) expression in PKGI0/0 cells as compared to PKGIfl/fl. Treatment of 3T3-L1 cells with cGMP resulted in increased lipid accumulation and enhanced expression of fat marker genes. Lentiviral overexpression of PKGI further increased differentiation. Importantly, PKGI significantly induced mitochondrial biogenesis in 3T3-L1 cells. Concomitant activation of PKGI in 3T3-L1 preadipocytes and treatment with the demethylating agent 5-aza-deoxycytidine significantly increased expression of uncoupling protein-1 (UCP-1) -a unique protein of brown fat cells. We found RhoA as major target of PKGI signaling with increased phosphorylation of RhoA at Ser-188 in PKGI overexpressing cells. Moreover, PKGI-dependent phosphorylation counteracts the effects of RhoA on insulin signaling as well as adipokine expression. Taken together, PKGI is a key player in white adipocyte differentiation that regulates cell size and has an anti-inflammatory effect. PKGI decreases the secretion of proinflammatory adipokines via inhibition of RhoA signaling. In addition, activation of PKGI can establish a brown fat cell like phenotype during white adipocyte differentiation if the UCP-1 promoter is accessible. The Rag GTPases, RagA, RagB, RagC, and RagD form a subfamily GTPases of the Ras-related superfamiliy. Rag proteins are characterized by a modified Ras-like GTPbinding domain and a unique C-terminal region lacking a lipid modification motif. Interestingly, Rag proteins have been proposed to function as heterodimeric complexes consisting of RagA or RagB associated with RagC or RagD. Rag GTPases have been implicated in the control of mammalian target of rapamycin (mTOR) function, in particular in regulation of the nutrient-stimulated and/or hormone-regulated mTOR activity. The protein kinase mTOR is found as the catalytic subunit of two larger protein complexes referred to as mTOR complex 1 and 2, mTORC1 and 2. Under amino-acidrich conditions, activated mTORC1 promotes protein synthesis and inhibits autophagy, while under starvation autophagy inhibition is released. Increasing evidence suggests that activation of Rag GTPases contributes to mTORC1 function. Thus, Rag proteins were found to be associated with a protein complex termed Ragulator, a major regulatory protein of mTORC1 function and guanine nucleotide exchange of Rag GTPases within the Rag-Ragulator-complex were described to promote mTORC1 translocation to its functional lysosomal compartment. However, the guanine nucleotide exchange properties of Rag proteins are poorly characterized, and it is currently unknown, how amino acids promote Rag proteins to facilitate the formation of the active, raptor-binding state of the Rag heterodimers. To characterize the guanine nucleotide exchange properties of the Rag GTPases as momomers or heterodimers in more detail, recombinant Rag proteins were expressed in bacteria and purified from this source to near homogeneity. First, the parameters of GDP/GTP exchange of each of these proteins were compared using the non-hydrolysable GTP analogon GTPgS. The results showed that the Rag isoforms are distinct in their guanine nucleotide exchange activities. In particular, nucleotide exchange on RagA and RagC, but not on RagB and RagD, was only observed at low concentrations of GDP and MgCl 2 in extraction and assay buffers, i.e. conditions favoring the GDP/GTP exchange. These findings may indicate that guanine nucleotide exchange on RagA and RagC is controlled by guanine nucleotide exchange factors and suggest specific functions of the individual Rag GTPases within individual Rag heterodimers. In in-vitro studies on rat and canine mast cells and human mast cell leukemia cells HMC1.2 BZ at micromolar concentrations inhibited mediator release which appeared to be related to an inhibition of the intracellular cAMP pathway. In order to identify potential targets on/in mast cells at which BZ may cause an inhibitory effect on mast cell activation, the 1,4-BZ flunitrazepam (flu), clonazepam (clo) and 4chlorodiazepam (4-cd) were selected because of their different affinity and selectivity to/for the GABA-A-receptor and the translocator protein (TSPO): flu and clo bind with nanomolar affinity to GABA-A receptors, whereas 4-cd is a selective ligand at TSPO with nanomolar affinity to TSPO but only micromolar affinity to GABA-A receptors. Flu also possesses nanomolar affinity to TSPO, whereas clo has no or only micromolar affinity to TSPO. After incubation of HMC1.2 cells with 4-cd, flu and clo for 1, 6 and 24 hours up to 712 genes were significantly differently expressed in a substance-specific and timedependent manner. Comparison of the genes differently expressed at 6 hours revealed that the expression of 217 genes was regulated by both flu and clo but only 8 genes were regulated by both 4-cd and flu suggesting that flu and clo induce gene expression by acting at a target site different from that of 4-cd. The difference between the gene regulation by flu and clo on the one hand and that of 4-cd on the other hand is also reflected in pathway analysis. Since it was conceivable that the beneficial effects of the 1,4-BZ could be mediated by the recognition sites targeted by the 2,3-BZ, i.e. the GRIA2-encoded ionotropic glutamate receptor AMPA2, we investigated by quantitative PCR whether HMC1.2 cells express GRIA2, TSPO, the genes encoding the subunits of the GABA-A receptor and the GABA-forming enzyme glutamic acid decarboxylase. TSPO, GABRA3, GABRB3, GABRE and GABRD were moderately expressed. In addition, there was a week or very week expression of GABRA2, GABRA4, GABRB2, GABRG3 and GABRR2. Expression of GRIA2 was not detectable. Taken together, it cannot be decided yet from our data whether the inhibitory effect of benzodiazepines on mast cell activation is due to an action at TSPO or at GABA-A receptors of a novel subunit composition.

Monien B. H., Glatt H. German Institute of Human Nutrition (DIfE) Department of Nutritional Toxicology, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany 5-Hydroxymethylfurfural (HMF) and furfuryl alcohol (FFA) are common constituents of foodstuffs in which they are formed by heat-and acid catalyzed reactions from carbohydrates. HMF and FFA have been reported to induce the formation of hepatocellular adenomas in female mice and renal tubule neoplasms in male mice, respectively. We studied whether the carcinogenic effect of these hydroxymethylsubstituted furans may originate from sulfotransferase (SULT)-catalyzed formation of electrophilic esters. HMF was inactive in in vitro mutagenicity tests using standard activating systems. In contrast, it was mutagenic in V79 cells genetically engineered for expression of human SULT1A1 suggesting that HMF is converted into the reactive 5sulfooxymethylfurfural (SMF). Following incubation of mutagenic SMF with porcine liver DNA in vitro, specific methylfurfural adducts were detected using liquid chromatography tandem mass spectrometry (LC-MS/MS), i.e., N 6 -((5-formylfuran-2-yl)methyl)-2'deoxyadenosine (N 6 -FFMdA) and N 2 -((5-formylfuran-2-yl)methyl)-2'-deoxyguanosie (N 2 -FFMdG). These adducts were also detected in DNA from V79-SULT1A1 cells incubated with HMF. In order to determine sulfo conjugation of HMF in mice in vivo, we conducted pharmacokinetic measurements showing that about 500 ppm of the HMF dose was converted to SMF and reached the circulation. Like HMF, FFA was negative in the standard Ames test and various other in vitro genotoxicity tests. We showed that FFA is mutagenic in Salmonella typhimurium TA100 engineered for expression of human SULT1A1. The putative mutagen 2-sulfooxymethylfuran was synthesized and incubated with porcine liver DNA, in which various nucleoside adducts were found. The main adducts, -MFdA were detected in DNA of FFA-exposed Salmonella strain TA100-SULT1A1 and in DNA of liver, lung and kidney of FVB/N mice that had received about 390 mg FFA/kg body weight per day via the drinking water for 28 days. In summary, both furan derivatives form mutagenic sulfate esters in vitro and in vivo. In the future, we will use genetically engineered mice to characterize the role of single murine and human SULT forms in the bioactivation of the furan derivatives and the contribution to tumor induction. Background: MicroRNAs are small non-coding RNAs that can negatively regulate gene expression on a post-transcriptional level and have been shown to interact with epigenetic mechanisms like DNA methylation. MeCP2 (methyl CpG binding protein 2) is a protein that binds methylated DNA CpGs in the promoter region of genes and can thus regulate their expression. Otherwise, MeCP2 is known to be a target gene for several microRNAs including the cluster miR-132/212 in the brain. Recently, our group could show that MeCP2 expression is downregulated in human heart failure suggesting that MeCP2 might be involved in cardiac pathogenesis. The aim of this project is to study the upstream regulation of MeCP2 by the cluster miR-132/212 in the heart during cardiac hypertrophy in-vitro and in-vivo. Methods and Results: To test whether hypertrophic stimuli can induce miR-132/212 expression, we treated cultured NRCMs with 40 µM norepinephrine for 72 hours. This induced cardiomyocyte hypertrophy and expression of the hypertrophy marker Nppa, but also of miR-132 (1.79 ± 0.14-fold of untreated cells, p<0.01) and of miR-212-3p (1.73 ± 0.26-fold of untreated cells, p<0.05) and downregulated Mecp2 mRNA and protein levels (0.80 ± 0.04-fold of untreated cells, p<0.05). To check whether MeCP2 downregulation also occurs by direct miR-132/212 activation we increased levels of miR-132 and miR-212 in cardiac myocytes by transfecting precursor miR-132 and miR-212-3p molecules. Again, we observed NRCM hypertrophy, Nppa mRNA upregulation and Mecp2 mRNA and protein downregulation (0.75 ± 0.05-fold of control, p<0.05) after miR-132 overexpression. Similar results were obtained by overexpression of miR-212-3p. To test the effects of adrenoceptor activation on the miR-132/212-MeCP2 axis in-vivo, wild-type mice received isoprenaline and phenylephrine via osmotic minipumps (30 mg/kg/day each). After 7 days, cardiac ventricles were analyzed. Nppa gene expression (2.81 ± 0.32 -fold of control animals), miR-132 and miR-212-3p levels (3.67 ± 0.5 and 2.62 ± 0.4 -fold of control animals, p<0.01 and p<0.05, respectively) were increased while MeCP2 protein levels decreased to 77% (p<0.05) Conclusion: These results suggest that in-vitro and in-vivo adrenoceptor stimulation leads to the activation of miR-132/212 expression and to downregulation of MeCP2 in cardiac myocytes in-vitro and in-vivo. Leopold-Franzens-Universität, Innsbruck, Austria AT-1 receptor antagonists block the angiotensin II-enhancing effect on noradrenaline release from sympathetic neurons. In a cell-free assay the binding affinity of the AT-1 receptor antagonists telmisartan and valsartan to the gamma peroxisome proliferatoractivated receptor (PPARγ) is close to that of the PPARγ selective agonists thiazolidinediones (TZDs). We tested whether the TZDs rosiglitazone and pioglitazone would also modify the prejunctional facilitatory effect of angiotensin II. Left ventricular slices of rats were incubated with tritiated noradrenaline, perifused and electrically stimulated. The negative logarithm of the drug concentration that caused a 30% increase of control (pEC30%) was calculated. Angiotensin II caused a concentration-dependent increase of tritium overflow induced by electrical stimulation [pEC30%=8.6±0.2 (mean±SEM, n=18); maximum increase=110±8%]. Neither rosiglitazone nor pioglitazone (0.3-3 µM) had a direct effect. The concentrationresponse to angiotensin II in the presence of fixed concentrations of rosiglitazone was shifted to the left with increase of the maximum (pEC30%=8.8±0. 2, 9. 2±0.2 and 9.3±0.3; maximum increase=118±14%, 146±13% and 148±16%, in the presence of 0.3, 1 and 3 µM of rosiglitazone, respectively, n=4-6, each). In contrast, pioglitazone in concentrations up to 3 µM had no effect on the release-enhancing effects of angiotensin II. Results show that rosiglitazone but not pioglitazone potentiates the noradrenalinerelease enhancing effect of angiotensin II. This action might contribute to the risk for myocardial infarction from rosiglitazone use but not from pioglitazone use. Deleted in Liver Cancer 1 (DLC1) is a tumor suppressor whose allele is lost in 50% of liver, breast, lung and 70% of colon cancers. Despite its significance, the molecular mechanisms that drive cancerous transformation upon DLC1 loss remain unclear. We found that the transcriptional coactivators Megakaryoblastic Leukemia 1 and 2 (MKL1/2) are constitutively localized to the nucleus in hepatocellular and mammary carcinoma cells that lack DLC1. Moreover, DLC1 loss and MKL1 nuclear localization correlated in primary human hepatocellular carcinoma. Nuclear accumulation of MKL1 in DLC1-deficient cancer cells was accomplished by activation of the RhoA/actin signaling pathway and concomitant impairment of ERK-mediated MKL1 phosphorylation. DLC1 loss led to constitutive activation of the MKL-dependent, tumor-relevant target genes CTGF, Cyr61, Myl9 and Myh9. Furthermore, we identified a novel target gene, Integrin a5, with a key role in cell migration and metastasis, that exhibited a DLC1-and MKLdependent regulation. Depletion of MKL1/2 suppressed not only cell migration, but also cell proliferation and anchorage-independent cell growth induced by DLC1 loss. Our data provide insight into the mechanism by which DLC1 loss initiates tumorigenesis. As MKL1 and 2 have a key role in this process, this pathway may provide promising pharmacological targets for cancer therapy.

Universität Bonn, Pharma-Zentrum Bonn Pharmazeutisches Institut, Pharm. Chemie I, An der Immenburg, 53121 Bonn, Germany

Membrane receptors activated by purine and/or pyrimidine nucleotides ("P2 receptors") are widely distributed in the body and constitute novel (potential) drug targets. They are subdivided into G protein-coupled P2Y receptors (P2Y1, 2, 4, 6, 11, 12, 13, 14) , and homo-or heterotrimeric ligand-gated ion channel or P2X receptors (subunits: P2X1-7). We have been interested in the identification and development of potent and subtype-selective ligands -as tool compounds and potential drugs -for the various P2Y and P2X receptor subtypes. Our strategy involves (i) establishment of a proprietary compound library consisting of synthetic small molecules and natural products; (ii) development of screening assays suitable for medium throughput screening; (iii) careful analysis of structure-activity relationships at each target and systematic optimization of the lead structures; (iv) pharmacological evaluation of selected compounds. This approach has led to new biological tools for several targets, including P2Y and P2X receptors [1] [2] [3] [4] [5] [6] . Fine particles in particulate matter (PM) are effective vehicles to transport toxicants into the lung; depending on their size, smaller particles may reach the bronchiolar or alveolar space. In recent years the PM fraction PM2.5 has especially been correlated with both pulmonary and cardiovascular diseases. In order to better characterize PM emission and distribution of environmental tobacco smoke (ETS) from cigarettes (reference cigarette (RC), brand cigarette (BC)) we have developed an ETS emitter to simulate human smoking emission and measured PM2.5 concentration in a telephone booth (1,75 m3 volume) as an example for small indoor spaces like cars. Fine particulate matter was measured using an aerosol spectrometer with 6 sec time resolution; laser scatter allowed a size resolution from 0,25 µm to 32 µm. For the PM2.5 concentration the following values were calculated: cumulative PM2.5 concentration as AUC-PM2.5 (µg/m3/sec), peak PM2.5 concentration as cmax-PM2.5 (µg/m3) and average PM2.5 concentration Cmean-PM (µg/m3). In closed door condition both cigarettes produced particulate AUC-PM2.5 values of 59 000 ± 15 000 µg/m3/sec (RC) to 85 000 ± 31 000 µg/m3/sec (BC After myocardial infarction (MI) inflammatory cells and cardiac fibroblasts (CF) determine the remodeling response. Interleukin-6 (IL-6) is induced in the ischemic myocardium and is known to stimulate the differentiation of fibroblasts to myofibroblasts. Hyaluronan (HA) is an extracellular matrix component synthesized by HA-synthase isoenzymes (HAS 1-3) and is also known to control fibroblast phenotypes. However, it is presently unknown whether IL-6 participates in the remodeling of the HA-matrix or whether the HA-matrix modulates the responses to IL-6. Therefore, the aim of the present study was to elucidate whether IL-6 regulates the expression and function of HA-matrix in CFs. Cells were isolated from C57Bl/6J mice and used during passage 2-3 for experiments. CFs were stimulated with IL-6 or Hyper-IL-6 which is a fusion protein of IL-6 and soluble IL-6 receptor (sIL-6R). After 10 and 30 min signal transducer and activator of transcription 3 (STAT3) was phosphorylated in response to Hyper-IL-6 but not in response to IL-6. RT-PCR revealed rapid upregulation of HAS 1 (5.94 ± 2.49 fold of unstimulated control, 1h) in response to Hyper-IL-6. HAS2 was induced to a lesser degree (2.04 ± 0.55 fold of unstimulated control, 1h) whereas HAS3 was not responsive (1.49 ± 0.42 fold of unstimulated control, 1h). In contrast, IL-6 had no effect on transcript levels of HAS isoenzymes. In turn, expression of HAS1 and HAS2 in response to Hyper-IL-6 was inhibited by AG490, which indicates the involvement of STAT signaling. Interestingly, despite induction of HAS1 and HAS2 the amount of secreted HA as determined by an ELISA-like assay was not affected by Hyper-IL-6. This may indicate that IL-6 regulates the cell surface associated HA-matrix of CFs.

In conclusion, the present data demonstrate that cardiac fibroblasts respond to IL-6 trans-signaling (Hyper-IL-6) via the soluble IL-6R and subsequent STAT3 signaling with increased HA-synthesis. The fact that IL-6 had no significant effect suggests that the expression of the non-signaling membrane-bound IL-6 α-receptor (IL-6R) in cultured murine cardiac fibroblasts is not sufficient to induce HAS 1 and -2 gene expression. Therefore, IL-6 trans-signaling mediated by IL-6 and the circulating sIL-6R might be necessary to mediate the IL-6-induced HAS expression in vivo.

MRGPRD receptor endogenously expressed in dorsal root ganglia: evidence for an activation by 3-aminoisobutyric acid Müller S., Hoffmann K., von Kügelgen I. Universität Bonn Institut für Pharmakologie und Toxikologie, Sigmund-Freud-Straße 25, 53127 Bonn, Germany

The GPCR MRGPRD (mrgD) is highly expressed in small diameter dorsal root ganglion (DRG) neurons and has been implicated to play a role in nociception. The receptor was previously shown to respond to β-alanine. In the present study we searched for agonistic activity of structural analogues of β-alanine.

For further characterization of the receptor we used Fura-2 fluorimetry, a NFAT luciferase reportergene assay and the determination of the inhibition of forskolin-induced cAMP production ([ 3 H]-cAMP affinity assay). First, we confirmed the activation of the receptor by β-alanine and GABA. In reportergene experiments we then identified 3-DLaminoisobutyric acid as an agonist, with similar potency but weaker affinity when compared to β-alanine (Ec50 165 µM). Fura-2 fluorimetry showed an increase in intracellular Ca 2+ levels by 3-DL-aminoisobutyric acid (300 µM). Moreover, 3-DLaminoisobutyric acid reduced the forskolin-induced cAMP production by up to 65 % (Ec50 195 µM). In addition to 3-DL-aminoisobutyric acid, we identified 3-DL-aminobutyric acid as a weak agonist acting at the MRGPRD. Other closely related substances failed to show significant responses. Next to the agonists we further characterized antagonists inhibiting the response to βalanine mediated by MRGPRD. Chlorpromazine shifted the concentration-response curve of β-alanine to the right with an apparent pKB of 4.9 (NFAT assay), thioridazine with an apparent pKB of 5.4 (NFAT assay) and 5.3 (cAMP assay) and rimcazole with an apparent pKB of 5.2 (NFAT assay) and 5.2 (cAMP assay). In conclusion we show for the first time that 3-DL-aminoisobutyric acid is an agonist at the MRGPRD and that the structure-activity relationship of agonists at MRGPRD is very close. The SDF-1-chemokine receptor CXCR4 plays a key role during embryogenesis and regulates functions of immune and stem cells in adult life. Furthermore, CXCR4 is involved in disease states including inflammation and cancer. It is well established that SDF-1-stimulated CXCR4 receptors activate Gi protein-dependent signal transduction pathways and undergo C-terminal phosphorylation and internalization. Because the CXCR4 C-terminal domain contains 15 serine and 3 threonine residues, it is incompletely understood which of the potential phosphorylation sites contribute to homologous and heterologous regulation of CXCR4. Here, we analyzed the phosphorylation pattern of CXCR4 at 3 C-terminal sites after stimulation of the receptor with SDF-1 and after PMA-induced activation of the PKC pathway as a model for heterologous receptor phosphorylation. Using phospho-specific antibodies against S324/325, S338/339 and S346/347 in immunoblot analyses, we showed that the 3 sites were phosphorylated after stimulation with SDF-1 or PMA. Stimulation with EGF or forskolin did not induce phosphorylation at these sites. SDF-1-induced phosphorylation at S324/325, S338/339 and S346/347 was reversible after wash out of the ligand. Time course analyses revealed that phosphorylation occurred first at S346/347 and then at S324/325 and S338/339. Taken together, these results indicate that the C-terminus of CXCR4 is phosphorylated at multiple sites by homologous and heterologous pathways and that phosphorylation at the different sites may be hierarchically organized. Human milk represents the best form of nutrition for infants early in life. However, it can also contain toxic contaminants that may adversely affect infant's development. The nephrotoxin ochratoxin A (OTA) is present in human milk (Tab. 1 in [1] ), but information on transfer from maternal blood to milk is scarce: Published data [2] indicate that levels of OTA in milk are roughly one tenth (0.1) of those in blood. But, the efficiency of the OTA-transfer at various stages of breastfeeding may vary since studies in animals revealed that transfer of OTA is apparently time-and dose-dependent. Thus, the aim of this study was to assess the OTA transfer from blood to milk at different stages of breastfeeding in humans. In a small Chilean cohort, 18 lactating women were asked to provide blood and milk on the same day. These samples were collected on four different occasions within the first months after delivery and analyzed using HPLC with fluorescence and/or tandem mass spectrometric detection [1] . The transfer of OTA from blood to milk was quantitatively assessed by measuring the milk to plasma ratio (M/P). The average OTA level in blood plasma was 235 ± 129 ng/L, and no major variations were observed over time (p = 0.19). On the other hand, OTA levels found in colostrum (85 ± 60 ng/L) were higher than in mature milk (p < 0.05). In line with these data, higher M/P ratios (Table) were obtained with samples collected in the first six days after delivery. This study showed that the transfer of OTA from blood to milk was more efficient with colostrum (M/P 0.43 ± 0.26) than with mature milk. Thus, a higher exposure to OTA can be expected for neonates than for infants at later stages of breastfeeding. Moreover, the lactating women have lower average OTA levels in plasma than non lactating women from Chile [3] , indicative of milk as additional excretion route.

Acknowledgement: This work has been supported by a stipend from CONICYT/DAAD to KM. Exposure of infants to ochratoxin A (OTA) deserves particular attention since OTA is nephrotoxic, and one of the most potent rodent renal carcinogen studied to date [1] . Moreover, infants may be more vulnerable to the toxic effects of contaminants than adults. OTA-levels in plasma of infants are indicative of an early exposure in life [2] . But blood sampling is an invasive method not readily applicable for breastfed infants. Thus, the aim of this study was to implement a non invasive biomonitoring method to assess OTA-exposure in this group.

To assess the exposure to OTA, breast milk and infants' urine specimens were collected, from two different cohorts: Chile (n= 28) and Turkey (n=10, only urine). Analysis of the samples was performed using enzymatic hydrolysis prior to extraction and HPLC-MS/MS [3] . The magnitude of infants' exposure was assessed by calculating the OTA-daily intake with human milk and relating it also to urinary OTA levels.

Calculations of the daily intake with human milk [4] showed that infants may be exposed to OTA at high levels, exceeding the tolerable daily intake (TDI) of 5 ng/Kg-bw/day set for adults [1] . In both cohorts, most of the urine samples tested positive for OTA (Chile 72%, Turkey 80%). OTA levels observed in urine samples from the Turkish infants (range: 116 -1,361 ng/L) were 10 fold higher than levels found in Chilean samples (range positive samples: 17 -320 ng/L). Further analysis of phase II metabolites in urine confirm the excretion of OTA as conjugate (glucuronide) in highly exposed infants.

In conclusion, OTA exposure of infants early in life was documented. Given that OTAintake by several infants exceeded the TDI for adults, further biomonitoring in this vulnerable group is advised including also suitable biomarkers of effect. A mixture of (E)-and (Z)-Clomiphene citrate is the first line therapy of female infertility. However, up to 30% of patients do not respond. (E)-Clomiphene is structurally closely related to another selective estrogen receptor modulator, tamoxifen which is frequently used for the treatment of hormone receptor-positive breast cancer. Like tamoxifen, clomiphene is extensively metabolised by the cytochrome P450 system. Using the estrogen receptor response assay (E)-4-hydroxyclomiphene and (E)-4-hydroxy-N-desethylclomiphene (EC50: 2.5 and 1.4 nM, respectively) turned out to be 100 times more active at the ER compared to the parent drug isomers and de-ethylated metabolites. Using recombinant expressed human CYP isoforms and inhibitory antibodies, CYP2D6 revealed to be the major isoenzyme involved in the formation of 4-hydroxlated metabolites. N-deethylation was catalysed by CYPs 3A4/5, 2D6, 2C19 and 2C8. Rates of 4-hydroxylation in microsomes from 30 human liver donors correlated with the number of functional CYP2D6 genes. These in vitro results were confirmed in a pharmacokinetic study with female healthy volunteers receiving a single dose of clomiphene. In carriers of two non-functional CYP2D6 genes (poor metabolizers) Cmax of (E)-4-hydroxyclomiphene and 4-hydroxy-Ndesethylclomiphene was 8 and 12 times lower, respectively, when compared with subjects with at least one fully functional CYP2D6 allele. In contrast, half-life of (E)-clomiphene and (E)-N-desethylclomiphene was 10 and 50-fold higher, respectively, in poor metabolizers. Our data provide first evidence of a pharmacogenetic rational for the variability in the response to clomiphene treatment. Among the tested compounds, compound 1 proved to be the most active derivative, showing a significant toxicity at a concentration of 2,5 µM. Compounds 2 and 3 showed significant toxic effects at a concentration of 5 µM. The compound 4 showed no toxicity up to a concentration of 100 µM. All derivatives 1, 2 and 3 have a EC50 between 10 and 15 µM. We further proved the induction of apoptosis by Apo-ONE assay (caspase 3/7 activity) and life/dead-assay (fluorescence microscopy). In conclusion, these gold complexes exhibit an example of interesting potential candidates for future anticancer pharmaceuticals due to relatively high cytotoxicity.

Gene regulating effects in mouse liver subsequent to treatment with selected dioxin-like compounds and PCB 153 using whole genome microarray analysis Neser S. 1 , Lohr C. 1 , van Ede K. I. 2 , Andresen K. 3 interaction with the aryl hydrocarbon receptor (AhR), with 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD) being the most potent congener amongst the AhR agonists. Recent risk assessments have employed the toxic equivalency factor (TEF) concept. The current EU-project SYSTEQ aims at developing, validating, and implementing human systemic TEFs as indicators of toxicity for DL-compounds. At present, the best known parameter of AhR mediated effects is the induction of cytochrome P450 isoenzymes (CYPs), i.e., CYP1A1, 1A2, and 1B1. One of the major objectives of the SYSTEQ project is the identification of novel quantifiable biomarkers. In a three day study, female C57BL/6 mice were treated with single doses of six DL-congeners (TCDD, PCB 118, PCB 126, and PCB 156) , and the 'non-dioxin-like' (NDL) PCB 153. Quality tested (Agilent ® Bioanalyzer) mRNA isolated from livers was analyzed using the Agilent ® mouse whole genome array (4x44K) system. The quantity of genes affected (≥ 2fold) was highly heterogeneous amongst the DL-compounds. Whereas TCDD-treatment upregulated 89 genes, and down-regulated 66, 4-PnCDF-treatment had impact on 3208 (up), and 2396 (down). Treatment with PCB 118 led to marginal numbers of 16 up and 6 down-regulated genes. With 64 (up), and 38 (down) genes shared, the most extensive overlap occurred between TCDD-and 1-PnCDD-treatment. No overlap was found due to treatments with the NDL PCB 153 (21 up, 1 down) and TCDD. When comparing the effects of all DL-congeners, minor numbers of genes of 19 up, and 5 down-regulated remain, most of them being related to drug metabolism. While PCB 118 regulated only genes involved in drug metabolism, omission of PCB 118-regulated genes resulted in consistently 51 (up), and 24 (down) regulated among DL-compounds. In conclusion, our findings suggest that the pattern of gene regulation in mouse liver elicited by PCB 153 was strictly different from TCDD, while a very limited coincidence of genes was found even among DL-compounds. Comparison of these 'core' genes with data from human models is required with respect to determination of novel biomarkers. Introduction: Proper use of antibiotics is essential with regard to effective treatment of bacterial infections. Providing adequate information for patients can contribute to achieve this aim. Materials and methods: Data was collected from the relational database of the drug information service at Dresden University of Technology. The patients, who used the service, were interviewed concerning socio-demographic characteristics, reason for enquiry, number and kind of drugs taken, and diseases. Possible contact paths were phone, e-mail or letter. In the present evaluation, all enquiries from the years 2009 and 2010 were analyzed descriptively focussing primarily on systemic antibiotics as reason for the enquiry. Results: In the evaluated period, 4454 enquiries were registered in total. In 4.7% of those enquiries systemic antibiotics were named with a total number of 283 drugs. 50.9% of those antibiotics were found to be the direct reason for the enquiry. Most common information requested by patients corresponded to adverse drug reactions (50.7%), diagnosis/treatment (35.4%), drug application (29.2%) and drug-druginteractions (19.4%). The majority of the requesting patients (61.5%) was born before 1970. A correlation between incidence of enquiries especially concerning antibiotics and quarterly statistics could not be detected. Conclusion: Mainly patients aged 40 years or more seem to need or search for further information about antibiotic medication. Advice is required especially regarding adverse drug reactions and diagnosis or treatment. In order to this, the advisory service can help patients to lose their insecurity and to gain more confidence in handling antibiotic drugs. Colon cancer is one of the most frequent cancers in the industrialized nations. Epidemiological studies show a correlation between highly processed meat and the development of colorectal tumours. It is assumed that the risk of developing colorectal cancer, among various different factors, is related to the uptake of toxic substances contained in food such as heterocyclic aromatic amines that arise during the processing of fish and meat. PhIP is the most abundantly formed heterocyclic compound, and therefore has the biggest impact. In a previous study, we measured the absorption of PhIP in different intestinal segments of the rat. In the present study we focussed on the potential mechanisms by which PhIP is reabsorbed. The unidirectional PhIP transport from the mucosal to the serosal compartment (J ms) and in the opposite direction (Jsm) was examined using the Ussing chamber technique and 14 C-PhIP as a radiotracer. The proximal jejunum and distal colon of male Fischer 344 rats in short-circuit current chambers was clamped, so that mucosal and serosal compartments were built. The PhIP flux rates were determined at defined intervals over 120 min. The experimental conditions were selected in such a way that negative net flux rates (Jnet = Jms-Jsm) were indicative of an active secretion. Both intestinal segments showed large differences. While in the jejunum Jms and Jsm of PhIP were not significantly different, there was an active secretion in the colon. In a next step the transport proteins involved in this process should be examined. Introduction: Human organic anion transporter 2, OAT2 (SLC22A7), is abundantly expressed in kidney and liver and mediates the sodium-independent uptake of clinically relevant drugs like 5-fluorouracil, paclitaxel, bumetanide, tetracycline, and zidovudine. While immunohistochemical studies have localized human OAT2 to the basolateral membrane of kidney proximal tubules, its hepatic localization is currently unknown. We, therefore, firstly determined OAT2 localization in human liver. Because interindividual variability of OAT2 expression may affect hepatobiliary drug uptake and elimination, we next systematically investigated the influence of genetic and non-genetic factors on hepatic OAT2 expression.

Methods: An expression profile of OAT2 for 20 human tissues was determined by realtime quantitative polymerase chain reaction (TaqMan). OAT2 mRNA expression was analyzed in well-characterized human liver samples from 150 Caucasians that were accompanied by detailed demographic and clinical data. OAT2 was localized in human liver cryosections using a commercial rabbit polyclonal antibody and hepatic OAT2 protein levels were determined.

Resequencing, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and genome-wide single nucleotide polymorphism microarray technology served to genotype variants in the SLC22A7 gene region.

Results: OAT2 mRNA was expressed in several human tissues, including liver. Moreover, a new alternatively spliced variant of OAT2 was identified in human liver. Hepatic expression of full-length OAT2 mRNA and OAT2 protein varied 37-fold and 41fold, respectively. OAT2 mRNA and protein levels did not correlate with each other. OAT2 was localized to the sinusoidal membrane of human hepatocytes. No novel variants in the 9 exons, the 5'-flanking region, or the 3'-untranslated region of the SLC22A7 gene were identified. Univariate analysis showed that OAT2 mRNA is reduced in patients diagnosed for cholestasis (p=0.0012) and is affected by genetic variants.

Whereas the influence of genetic variants on hepatic OAT2 expression appears to be limited, cholestasis significantly contributes to the variable interindividual OAT2 expression. This indicates consequences for hepatic drug elimination of and response to OAT2 drug substrates such as paclitaxel or tetracycline.

The life threatening toxicity of organophosphorus (OP) nerve agents is caused by the inhibition of the acetylcholine esterase (AChE). Oximes were shown to be potent reactivators of inhibited AChE, but in poisoning by some compounds, e.g. soman, they have only a small therapeutic effect. For such cases, an alternative new strategy may be the intervention at nicotinic acetylcholine receptors (nAChR). Previous studies with the bispyridinium non-oxime MB327 demonstrated therapeutic effects against soman in vitro and in vivo which was partly attributed to its direct interaction with nAChRs [1] . We investigated the interaction of MB327 and several structure analogous at the orthosteric binding site of human α7 nAChR (hα7 nAChRs), a subtype which appears to be widespread in the human body, and compared the results with data obtained from Torpedo-nAChRs, which show a high degree of homology with human muscle-type nAChRs.

Interaction of compounds with the orthosteric binding site of hα7 nAChRs were investigated with radioligand binding experiments performed as high-throughput method [2] . Membrane preparations of GH 4C1 cells stably expressed hα7 nAChRs were incubated with the nAChR agonist [³H] epibatidine and appropriate concentrations of the unlabelled competitors e.g. bispyridinium compounds. After incubation, bound and free [³H] epibatidine were separated by rapid vacuum filtration. Ki values of the competing compounds were calculated with nonlinear regression.

Three bispyridinium compounds, MB442, MB456 and MB770 exhibited Ki values at micromolar concentrations while three other compounds, MB327, MB583 and the pharmacological inactive MB424 (negative control) did not show any interaction with the orthosteric binding site of hα7 nAChRs. With Torpedo-nAChRs, Ki values were in similar orders of magnitude -except MB442 which indicated significant subtype selectivity. Interestingly, the affinity of monomeric pyridinium derivates did not correlate with their bispyridinium structure analogues.

Obviously, no correlation between the affinity to the orthosteric binding site and the functional improvement of neuromuscular transmission exists, although species-related differences cannot be excluded. In this study, we analysed the cytotoxic and clastogenic effects of the anticancer drug nimustine (ACNU) in cells deficient in repair proteins involved either in homologous recombination (HR) or non-homologous end-joining (NHEJ). We show that HR mutants are extremely sensitive to ACNU as measured by the induction of apoptosis and colony formation as well as the induction of chromosomal aberrations. The NHEJ mutants were slightly sensitive to ACNU and differed in their sensitivity, with the Ku80 mutants being moderately sensitive and the DNA-PKcs mutant resistant, comparable to the wild-type (wt). Cell death was mostly executed via the caspase-dependent apoptotic pathway with involvement of caspase-3 and -7, and necrosis was also induced. Further, we investigated the kinetics of DNA double-strand break (DSB) formation that resulted from the repair of ACNU-induced interstrand cross-links by means of γH2AX and 53BP1 foci analysis in wt and mutant cells. Cells mutant in HR did not repair DSB and went into the apoptotic or necrotic pathway, whereas wt cells were able to repair most of the DSB. Cells deficient in Ku80 formed at early times after ACNU treatment less γH2AX and 53BP1 foci compared to the corresponding wt, which might be due to a reduced capacity of recognising DSB. At later times after treatment, Ku80 mutant cells show foci levels similar to the wt indicating restitution of H2AX phosphorylation. We also analysed whether DSB formation after ACNU treatment was replication-dependent using synchronised cells. We determined the formation of γH2AX and other DSB marker in wt cells that passed through the first cell cycle after demecolcine synchronization. The level of γH2AX foci increased significantly in the S-phase and remained at a high level during G2 where a fraction of cells remained arrested. Rad51, ATM, MDC-1 and RPA-2 foci were also formed and shown to co-localize with γH2AX. These foci were ameliorated significantly in S-and G2-phase, which was similar to the time course of γH2AX foci formation. In western blots, we confirmed a higher phosphorylation level of ATM and ChK2 and less phosphorylation of CHK1 in HR mutants. The data indicate that ACNUinduced DNA cross-links give rise to cyto-and genotoxicity via the formation of DSBs that activate the cellular DNA damage response. The endocannabinoid system has been established as a mediator of numerous central and peripheral biological functions. Cannabinoids have emerged as attractive alternatives or supplements to therapy with opioids for chronic pain states. However, in human the activation of cannabinoid receptors is associated with side effects. For clinical exploitation of the analgesics properties of cannabinoids, a major challenge is to devise strategies that reduce or abolish their adverse effects on cognitive, affective and motor functions without attenuating their analgesics effect. In animal studies, the anti-nociceptive efficacy of cannabinoids has been unequivocally demonstrated in several models of inflammatory and neuropathic pain. However, there are marked inconsistencies between different reports with respect to the locus of these pain-protective effects. We are working towards establishing the contribution of CB1 receptors expressed on the peripheral terminals of nociceptors to cannabinoid-induced analgesia. Using CB1 globally knock-out animal as background, we induce the expression of CB1 specifically in nociceptive neurons localized in the peripheral nervous system and test the analgesic effects of cannabinoid systemical delivery in these mice. Our results support the development of peripherally acting CB1 analgesic agonist with reduced central side effects. Furthermore, we are utilizing proteomics approach to identify protein complexes that interact with CB1 receptor which hold promise in understanding cannabinoid signaling in health and disease. Most chemoattractants, including chemokines, complement C5a, fMLP, and leukotriene B4 are signaling through heterotrimeric G proteins of the Pertussis toxin (PTx)-sensitive Gi family. The functional inactivation of all Gαi proteins with PTx leads to a fulminant decompensation of the immune system, whereas the constitutive inactivation of a single Gαi coding gene results in mild phenotypes in mice. We are mostly interested in the nonneuronally found Gαi2 and Gαi3 isoforms and their redundant and specific roles in immune function and infection. For this purpose cellular in vivo and ex vivo models and in vivo infection model with Listeria monocytogenes are being used. Macrophages were isolated from the peritoneal cavity of wild type (wt) and Gαi-deficient mice 4 days after i.p. injection of 4% thioglycolate that induces peritonitis in vivo. We confirmed previous observations that in Gαi2-deficient mice the migration of macrophages into the peritoneal cavity was reduced after induction of peritonitis. Regarding the expression levels of Gαi and Gβ isoforms in the lavage samples, the predominant Gαi isoform Gαi2 was upregulated in Gαi3-deficient macrophages. Vice versa Gαi3 was upregulated in Gαi2-deficient macrophages. Concerning Gβ isoforms, both Gβ1 and Gβ2 were strongly reduced in the Gαi2-deficient macrophages which resulted in a reduced total amount of Gβ. Surprisingly, the Gαi3-deficient macrophages showed reduced Gβ1 protein levels only which caused a change in the Gβ2/ Gβ1 quotient in favour of Gβ2.

We are currently establishing an in vivo infection model with L. monocytogenes in Gαi2and Gαi3-deficient mice. Our previous in vitro infection studies in mice embryonic fibroblasts provided us with information about possible distinct roles of these two isoforms as far as the uptake of L. monocytogenes in the cells is concerned. Challenging the immune system of Gαi-deficient mice with this pathogenic organism will give us new insights into the systemic immune response in these mice upon bacterial infection.

Our data indicate that we may surmount the redundancy between these two isoforms and focus on their distinct and specific roles in pathogen defense.

FRET-based β-Arrestin2 biosensors reveal conformational changes upon binding to the β2-adrenergic receptor in real time and living cells Nuber S., Zabel U., Ziegler N., Hoffmann C., Lohse M. J. Institut für Pharmakologie und Toxikologie Pharmakologie, Versbacherstr.9, 97078 Würzburg, Germany β-Arrestins are multifunctional adapter proteins that regulate seven transmembranespanning receptor (7TMR) signaling and initiate also alternative signaling pathways. Studies have shown that β-Arrestins undergo conformational changes upon receptor stimulation, which are thought to be necessary for its downstream actions. To investigate these conformational changes in living cells we constructed FRET based biosensors of β-Arrestin2, in which CFP was fused to the C-terminus and the FlAsHbinding motif (CCPGCC) was inserted to different positions within the N-or C-domain of β-Arrestin2. Upon β2-adrenergic receptor (β2AR) stimulation we observed a decrease of the intramolecular FRET signal between CFP and FlAsH at the N-domain (β-Arrestin2FlAsH2), indicating a conformational change moving the C-terminus and the Ndomain of β-Arrestin2 relative to each other. Kinetic analysis revealed that this conformational change immediately follows β-Arrestin2/β2AR interaction on a timescale of seconds. A β2AR mutant that was previously shown not to interact with β-Arrestin2 was utilized as control and did not induce a conformational change in the β-Arrestin2 molecule. Our data provide evidence that β-Arrestin2 changes it`s conformation upon binding to the activated β2AR in living cells. The β-Arrestin2FlAsH2 sensor could serve as universal biosensor for GPCR activation.

Studies on the physiological role of annexin A4 in the heart Nunes F. 1 The calcium binding protein annexin A4 has been examined in the context of heart failure in the past. Annexin A4 expression level was found to be elevated in ventricles of human failing heart in comparison to expression levels in non-failing ventricles. Furthermore the intracellular localization pattern in atrial cardiomyocytes was found to be altered in the failing human heart (Moravec and Matteo, Cardiovasc Res 2000). In order to gain insight into the possible physiological significance of these findings we utilized an annexin A4 gene trap model (GT) in which the annexin A4 protein content was not detectable in ventricles and atria. Measurements of sarcomere shortening and calcium transient kinetics in isolated ventricular cardiomyocytes revealed a prolonged calcium transient decay at stimulation frequencies of 0.5 Hz, 1Hz and 2 Hz as well as an increased sarcomere shortening at 1 Hz and 2 Hz in anxa4 gene trap animals in comparison to wild type (WT) ( The effects of the β-adrenoreceptor agonist isoprenaline (Iso) on the shortening of ventricular cardiomyocytes was increased in GT as compared to WT (0,2±0.013 vs. 0.17±0.012, *=p<0.05 vs. WT; N=14-18/5). Western blot analyses indicated that the expression of the sarcoplasmic reticulum (SR) Ca 2+ -ATPase (SERCA2a) and the phosphorylation status of its regulator protein phospholamban (PLB) did not differ between groups (n=7). However, co-immunoprecipitation experiments suggest, that anxa4 is able to interact with HAX1, which acts as a repressor of SERCA2a (n=4). We performed force measurements in isolated and electrically stimulated left atria in response to rising isoprenaline concentrations (10 -9 M-10 -5 M). The positiv inotropic effect of isoprenaline was significantly increased in GT atria (rel. force at 10 -4 M Iso [%]: WT: 433±76; GT: 699±63 *= p<0.05 vs WT; n=8-10). In conclusion, annexin A4 contributes to the regulation of cardiomyocyte contractility. The anxa4 up-regulation might therefore contribute to diminished cardiac performance in heart failure.

Matteo RG, Moravec CS. Immunolocalization of annexins IV, V and VI in thefailing and non-failing human heart. Cardiovasc Res. 2000 Mar;45 (4) Background: Pregnane X receptor (PXR) is considered the most important sensor of natural and anthropogenic xenobiotics in vertebrates. In contrast, the amphibian ortholog is involved in neural development and irresponsive to xenobiotics. Instead, the Xenopus laevis constitutive androstane receptor (CAR) was recently found to possess PXR-like properties, featuring low basal activity and a pronounced ligand spectrum. Thus a structural and functional characterisation of X. laevis CAR may provide further insights into human CAR basal and ligand-induced activity. Methods: The time-point of origin of CAR genes was determined by macrosynteny analyses of CAR, PXR, and VDR (vitamin D receptor) gene loci, which form the NR1I subfamily of nuclear receptors. Based on a 3-dimensional protein model of Xenopus laevis CAR, docking studies with structurally diverse agonists were conducted. Proteinligand-interactions as well as sequence comparisons were performed in order to select amino acids to be mutated towards human CAR. The organ response to CAR activators was determined in Xenopus laevis using RNA microarrays. Results: CAR emerged together with PXR and VDR from an ancestral NR1I gene in early vertebrates via two whole-genome duplications. This was followed by losses of CAR from the fish lineage and of PXR from Sauropsida (reptiles and birds). Amino acids important for ligand binding were identified. Structural features responsible for the pronounced basal activity in human constitutive androstane receptor are not present in X. laevis CAR. In human PXR the inter-helical loop in front of helix 3 is part of the ligandbinding pocket and supposed to be responsible for the wide substrate spectrum. In amphibian CAR this inter-helical loop plays no role in ligand binding. CAR agonists resulted in a pronounced induction of antimicrobial peptides in the ovary. Conclusions: CAR emerged already in early vertebrates and it is conserved in land vertebrates, whereas xenosensing PXR is found only in the fishes and mammals. We provide a comprehensive modeling and mutational analysis of this first reported amphibian xenosensor. The induction of antimicrobial peptides by CAR activators suggests a link between xenosensing and innate immunity. The latter one may play a previously unrecognized role in the amphibian reproduction. Background: Retigabine belongs to a novel class of potent anticonvulsant drugs and is currently being investigated in clinical routine. The therapeutic range of retigabine serum concentration is unknown. A therapeutic drug monitoring (TDM) is used for most other anticonvulsant drugs. The aim of this study was to develop a method for the determination of retigabine in serum of patients and to compare the effect and the side effects of retigabine with the blood levels of the drug. Method: A HPLC method with tandem mass spectrometric detection for the sensitive determination of retigabine was developed. Solid-phase extraction (SPE) of 250 µl serum with Oasis HLB cartridges allowed a reliable quantification down to 5 ng/ml. In order to develop an assay with high sample throughput and to obtain maximum response for the analytes we required the shortest possible retention time. To implement the determination of retigabine in a second step in the routine TDM of anticonvulsant drugs the corresponding HPLC method was selected: a Purospher RP18 column (55 mm x 2 mm; 4 µm, MERCK) and a mobile phase with a steep acetonitrile gradient. Results: The great advantage of having analytes with different molecular masses and similar retention times in combination with MS/MS detection enabled us to aim at a minimum separation that might remove some salts or matrix components that can suppress or interfere with the analyses from the target components, while maintaining good sample throughput. The method was validated. The assay is precise, accurate, fast, sensitive, and selective. Discussion: The developed method is suitable for therapeutic drug monitoring of retigabine. The correlation of the serum concentration and the effect of the drug and thus the necessity of TDM have to be tested.

Targeting inflammatory T lymphocytes with conditional chemokine receptor antagonist expression for a tissue-specific therapy of chronic inflammatory disorders Ogrissek N., Giegold O., Pfeilschifter J., Radeke H. H. Uniklinikum der Goethe-Universität pharmazentrum / ZAFES, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany Chemokines and their receptors are known to be involved in the pathogenesis of chronic inflammation and autoimmune diseases. Several approaches tried to use chemokine receptor antagonists as therapeutics to reduce exagerrated immune response, however, due to compensation and systemic side effects clinical trials often failed.

In previous experiments our group identified three promising antagonists. CXCL11(4-79) has antagonistic function for CXCR3, CXCL12(P2G2) is able to inhibit CXCR4 and the herpesvirus 8 encoded protein vMIP-II interferes with CCR1, -2 and -5 as well as with CXCR3, -4 and CX3CR1. Their expression and secretion was confirmed in Pichia pastoris and antagonistic function has been proven by a reduction of T cell migration. The aim of this project is to develop a cell-based therapy for chronic inflammation with a treatment that is based on the collective effect of CXCL11(4-79), CXCL12(P2G2) and vMIP-II. With targeting of stable transduced memory T cells these antagonists should be conditional expressed and secreted directly in the centre of inflammation, resulting in inhibition of further inflammatory T cell accumulation. To realize this project we first cloned constitutive lentiviral constructs containing these antagonists and optimized transduction of T cells, such as the OVA-specific memory Th-1 cell clone IF12 with the potential to initiate antigen specific nephritis in SCID mice. Next we investigated expression and secretion of CXCL11(4-79), CXCL12(P2G2) and vMIP-II with PCR, Western Blot and ELISA. At the moment we want to measure the inhibition efficiency of T cell migration in vitro with chemotaxis and flow chamber assays. Construction of an inducible lentiviral vector plasmid to ensure expression of the antagonists only upon T cell activation, is also part of our current work. Finally we would like to test the chemokine receptor antagonists in vivo in two relevant mouse models of type-1-diabetes and contact dermatitis. Small Heterodimer Partner 1 (SHP-1) is a member of the superfamily of nuclear receptors (NRs). In contrast to other NRs this orphan receptor lacks the DNA binding domain. However, SHP-1 is known to inhibit activity of several NRs by direct proteinprotein interaction. Importantly several of the interacting NRs have been shown to directly regulate SHP-1 expression, suggesting that SHP-1 is involved in negative feedback loops of various metabolic pathways, such as cholesterol-, bile acid-and drug metabolism and glucose homeostasis. Recently binding sites for NRs were identified in the promoter region of SHP-1, including HNF4α, LRH1, LXR, FXR, SREBP1c and PPARγ. The aim of our study was to identify single nucleotide polymorphisms (SNPs) in the promoter region of SHP-1 and to determine their impact on the transactivation of SHP-1. 120 DNA samples from subjects of the population based cohort Study of Health in Pomerania were analyzed by Sanger sequencing, thereby we identified four SNPs namely -594T>C (rs71636795), -413G>C, -423 C>T (rs78182695) and del-195CTGA (rs145613139). Subsequently those polymorphisms were tested for their functional consequence performing cell based reporter gene assays testing all above mentioned modulators (LRH1, LXR, FXR, SREBP1c and PPARγ) of SHP-1 expression. Only the transactivation by HNF4α was decreased in the presence of the -423 C>T polymorphism to 69% and the -413G>C polymorphism to 75%. In conclusion we described SNPs with impact on transactivation. It will be aim of future studies to determine the potential impact on physiological processes or disease development. Autosomal recessive polycystic kidney disease (ARPKD) is a rare genetic disease, afflicting about 1 in 20.000 individuals. ARPKD is characterized by cystic fusiform dilatations of the renal collecting ducts leading to massive enlargement of the kidneys and ultimately loss of renal function. In addition, the patients suffer from congenital hepatic fibrosis (CHF), possibly leading to portal hypertension and liver enlargement. So far, there is no cure for ARPKD. Therapy is focussing on controlling the disease symptoms [1] . Mutations in the PKHD1 gene cause ARPKD. More than 300 different mutations in this gene have been reported, all leading to the same phenotype, though there are differences regarding the severity of the disease [2] . In animal models of autosomal dominant polycystic kidney disease (ADPKD) as well as ARPKD elevated levels of cAMP were shown [1] [2] [3] . In isolated kidney cells cAMP stimulates Cl-secretion and activates the B-raf /MEK/ERK pathway. These both are important factors for cyst development and disease progression [2, 3] . Intracellular cAMP regulation is based on conversion of ATP to cAMP by adenylyl cyclases (ACs) and degradation by phosphodiesterases . Referring to this, we asked the question if there are differences in the activation and expression pattern of ACs in PCK rats, an animal model of ARPKD [4] and in Sprague Dawley rats. Therefore, we examined membranes in a radioactive AC activity assay using various stimulatory compounds, e.g. forskolin, a direct AC activator, or hormones like glucagon and vasopressin to characterize ACs. Furthermore, we examined AC isoform expression on the mRNA level via RT-PCR. We observed that in PCK rats AC activity was decreased in general in comparison to Sprague Dawley rats. In future experiments we are aiming to obtain further knowledge about the influence various hormones exhibit on PCK rat ACs and to biochemically characterize ACs. The major pathogenicity factors TcdA and TcdB from Clostridium difficile monoglucosylate and thereby inactivate small GTP-binding proteins of the Rho subfamily after entering host cells via receptor-mediated endocytosis. Although the intracellular mode of action of the toxins is well understood, far less is known about binding structure and internalization pathway of TcdA and TcdB. Since antibodies directed against the C-terminal located clostridial repetitive oligopeptides (CROPs) are able to neutralize toxin cytotoxicity the CROP domain is acknowledged to mediate receptor binding. However, we recently demonstrated that CROP deletion mutants of TcdA (TcdA 1-1874 ) and TcdB (TcdB 1-1852 ) enter host cells and exhibit full cytopathic potency though lacking the proposed receptor binding domain. We therefore refute the accepted opinion of a solely CROP-mediated toxin uptake and re-evaluate the role of the CROPs in toxin endocytosis. TcdA 1-1874 and TcdB 1-1852 induced time and concentration dependent cell rounding and Rac1-glucosylation. However, depending on the cell line, truncated toxins exhibit up to 10-fold reduced potency towards host cells compared to the respective full length toxin. The observed difference in toxin potency might reflect the recognition of different receptor structures or the use of various endocytotic routes. Interestingly, pre-incubation of cells with the isolated CROP domain enhances binding as well as cytotoxicity of subsequent applied truncated TcdA indicating that the CROPs primarily determine toxin uptake. In fact, competition experiments revealed that TcdA and TcdA 1-1874 predominantly use different receptor structures corroborating the notion of alternative internalization processes utilized by TcdA. Different routes for cellular uptake might enable the toxins to enter a broader repertoire of cell types leading to the observed multifarious pathogenesis of C. difficile. Thus, characterization of alternative endocytotic pathways used by the C. difficile toxins might therefore be the basis to investigate the opportunity of toxin uptake inhibition as therapeutic option. In neurodegenerative diseases, such as Alzheimer´s disease and Parkinson´s disease, mitochondrial pathways of apoptosis are considered as major features of the underlying neuronal cell death. Such mitochondrial mechanisms of apoptosis are mediated by the BH3-only protein Bid, a member of the Bcl-2 family that triggers mitochondrial permeabilization and the subsequent release of death-promoting proteins into the cytosol. The pivotal role of Bid in apoptotic cascades of neuronal cells has been shown in our previous studies showing a neuroprotective effect of Bid siRNA and small molecule Bid inhibitors such as BI6c9 in vitro. In vivo, however, the available Bidinhibitors failed to protect brain tissue likely because the compounds were not bioavailable or did not cross the blood brain barrier. Therefore, chemical modifications of BI-6c9 were generated resulting in new structures and molecules with different pharmacophors. The aim of the present study is to identify novel potent Bid inhibitors available for applications in model systems of brain damage in vivo. For the first screening of 80 compounds we used a model of glutamate toxicity in immortalized mouse hippocampal neurons (HT-22 cells). In this model system, glutamate induces a decrease of intracellular glutathione levels resulting in lipoxygenase activity and enhanced formation of toxic reactive oxygen species (ROS). To investigate the compounds' ability to prevent glutamate induced cell death, we first analyzed the cell viability by the MTT assay. In addition, we examined the cell survival by using real time monitoring of cell impedance (xCELLigence System) to determine the neuroprotective potency of the new structures. Using these assays, we identified 10 novel molecules that significantly prevented glutamate-induced toxicity in HT-22 cells. Further we were able to express and to purify recombinant Bid in a high amount. In the ongoing study the purified Bid protein will be used for co-crystallization with the identified neuroprotective structures for further optimization of novel Bid inhibitors for therapeutic applications in experimental models of neurodegenerative diseases in vivo.

Polymorphic enzymes, urinary bladder cancer risk and structural change in the local industry Ovsiannikov D. 1 , Selinski S. In the 1990s, an uncommonly high percentage of glutathione S-transferase M1 (GSTM1) negative bladder cancer cases (70 %) was reported in the greater Dortmund area (Golka et al., 1997) . The question arose whether this uncommonly high percentage of GSTM1 negative bladder cancer cases was due to environmental and occupational exposure decades ago. Thus, 15 years later, another study on bladder cancer was performed in the same area after the coal, iron and steel industries had finally closed in the 1990s. In total 196 bladder cancer patients from the St.-Josefs-Hospital Dortmund-Hörde and 235 controls with benign urological diseases were investigated by a questionnaire and genotyped for GSTM1, GSTT1 and the N-acetyltransferase 2 (NAT2) tag SNP rs1495741. The frequency of the GSTM1 negative genotype was 52 % in bladder cancer cases and thus much lower, compared to a previous study performed from 1992-95 in the same area (70%). NAT2 genotypes were distributed equally among cases and controls (63% slow acetylators). Less GSTT1 negative genotypes were present in cases (17%; controls 20%). Apoptosis inducing factor (AIF) has been identified as a key factor in intrinsic pathways of caspase-independent neuronal death in model systems of acute brain injury and neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease. AIF is a mitochondrial intermembrane flavoprotein with the capacity to translocate to the nucleus where it induces chromatin condensation and large-scale DNA fragmentation. Previous studies revealed that AIF deficiency leads to protective effects in different models of neuronal death in vitro and in vivo. However, AIF also plays an important physiological role for the integrity and function of the mitochondrial respiratory chain.

Thus, AIF deficiency may significantly alter mitochondrial functions and metabolic homeostasis thereby preconditioning the cells to tolerate subsequent stress stimuli. The present study addresses this hypothesis and investigates whether neuroprotection by AIF depletion was attributed to a preconditioning effect, i.e. protecting mitochondrial function and integrity. As model system we use glutamate induced oxytosis in immortalized mouse hippocampal HT-22 neurons. Silencing of AIF expression by siRNA (20nM) protected mitochondrial morphology and integrity against glutamate induced damage. Microscopy analysis of the mitochondrial morphology revealed that AIF siRNA prevented mitochondrial fission. Furthermore, FACS analysis confirmed that mitochondrial membrane potential was stable in cells with AIF silencing. This protection of mitochondrial morphology and integrity by AIF depletion was associated with preserved ATP levels and inhibition of cell death as detected by an MTT assay. Pronounced formation of lipidperoxides as another indicator of mitochondrial damage was also attenuated in cells preconditioned by AIF siRNA. These protective effects of AIF siRNA were highly similar to effects obtained with low doses of rotenone (20nM), which was applied as an inhibitor of complex I and mediated comparable preconditioning effects in the HT-22 cells. Overall, these findings support the conclusion that AIF depletion mediates a preconditioning effect protecting neuronal cells from a subsequent glutamate toxicity. In order to link these preconditioning effects to complex I functions, protein expression and functional analysis of complex I are being analysed to identify the molecular mechanisms of AIF dependent control of neuronal life and death. -dependent inactivation and display very slow voltage-dependent inactivation. Both properties are of crucial importance in ribbon synapses of retinal photoreceptors and bipolar cells, where sustained Ca 2+ influx through Cav1.4 channels is required to couple slowly graded changes of the membrane potential with tonic glutamate release. Mutations in the gene coding for Cav1.4 cause severe impairment of retinal circuitry function and have been linked to congenital stationary night blindness type 2A (CSNB2), Aland Island eye disease (AIED) and cone-rod dystrophy type 3 (CORDX3). The clinical phenotypes of these eye diseases vary substantially regarding the ratio of rod to cone functional impairment. The reasons for this variability are not known. To gain more insights into the pathophysiology caused by loss of Cav1.4 function we analyzed the visual phenotype of Cav1.4-deficient mice. To this end, we combined immunohistochemistry, electroretinography (ERG) and vision-dependent behavioral testing. Immunohistochemical analysis using synaptic and postsynaptic markers revealed severe synaptic defects in Cav1.4-deficient mice. Heterozygous Cav1.4 mice showed mosaic synaptic defects most probably caused by random X-chromosomal inactivation of the healthy allele. Electroretinography revealed a loss of scotopic and photopic photoreceptor function. This loss of retinal network function resulted in impaired performance of Cav1.4 knockout mice in a water maze-based behavioral test of rod and cone function.

In conclusion, loss of Cav1.4 channels strongly impairs rod and cone retinal function and vision in mice.

LSR is the host cell receptor for clostridial iota-like toxins Papatheodorou P., Aktories K. Albert-Ludwigs-Universität Freiburg Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Albertstr. 25, 79104 Freiburg, Germany

The human enteric pathogen Clostridium difficile is the most serious cause of antibioticassociated diarrhea and pseudomembranous colitis. Hypervirulent strains of the pathogen, associated with more severe disease and increased death rates, produce the binary actin-ADP-ribosylating toxin CDT (C. difficile transferase) in addition to the Rho glucosylating toxins A and B. CDT is member of the family of clostridial iota-like toxins, including C. spiroforme toxin (CST) and the eponym C. perfringens iota toxin. The toxins induce depolymerization of the actin cytoskeleton and the formation of microtubulebased cell protrusions that increase adherence and colonization of Clostridia. Using a haploid genetic screen, we identify the lipolysis-stimulated lipoprotein receptor (LSR) as the target molecule for entry of CDT into host cells. In addition, we present evidence that LSR is shared as a cell entry point by all members of the iota-like toxin family. Identification of the toxin receptor provides a most valuable basis for antitoxin strategies. Bisphenol A (BPA) is a chemical of high interest due to its endocrine activity. Controversy exists concerning the blood concentration due to normal exposures. Some authors claimed to have measured concentrations in the ng/ml range which is in contrast to kinetic properties of BPA. BPA is excreted in the urine as glucuronide and sulfate metabolites. Recently, data on the in vitro metabolism of BPA by recombinant UDPglucuronyltransferase 2B15 enzymes (UGT2B15) revealed that UGT2B15.2 and UGT2B15.5 had markedly lower intrinsic clearance as compared to UGT2B15.1 (Hanioka et al., 2011) . Using the in vitro metabolism data, we scaled the kMand Vmaxvalues in an established human physiologically based toxicokinetic (PBTK) model (Mielke and Gundert-Remy, 2009, Mielke et al., 2011) to the values of the variants. For oral doses at relevant exposure levels, the maximum blood concentration (Cmax) for the UGT2B15.2 variant (V2) was 5 fold and those of the UGT2B15.5 variant (V5) was 7 fold higher than that of the UGT15.1 variant. With dermal exposure at a relevant exposure level, the Cmax values were 1.4 (V2) and 1.6 fold (V5) of UGT15.1 variant. A combined exposure of oral and dermal exposure, an exposure scenario, which occurs in daily life, resulted in 2.4 fold (V2) and 3.2 fold (V5) higher Cmax values as compared to UGT15.1 variant. The values for the area under the blood concentration time curve (AUC) were for a relevant oral dose 5.7 fold (V2) and 8.6 fold (V5), for relevant dermal exposure 1.4 fold (V2) and 1.6 fold (V5), and for combined exposure 1.9 fold (V2) and 2.5 fold (V5) of UGT15.1 variant. From the results we conclude: (1) Polymorphism of UDPglucuronyltransferase (2B15.2 and 2B15.5) has an impact on the blood concentrations which, however, is less than 10 fold for Cmax and for AUC. The effect is more pronounced for oral as compared to dermal or combined exposure. (2) Polymorphism of metabolism does not explain the blood/plasma concentrations in the ng/ml range measured by some authors.

Hanioka N, Oka H, Nagaoka K, Ikushiro S, Narimatsu S. Effect of UDPglucuronosyltransferase 2B15 polymorphism on bisphenol A glucuronidation. Arch Toxicol. 2011, 85(11) :1373-81. Mielke H, Gundert-Remy U. Bisphenol A levels in blood depend on age and exposure. Toxicol Lett. 2009 8; 190(1) :32-40. Mielke H, Partosch F, Gundert- The heart responds to maladaptive pro-hypertrophic stimuli by stimulating intrinsic signals that contrast and dampen the onset and development of hypertrophy. Cyclic guanosine monophosphate (cGMP) and its downstream effector cGMP kinase I (cGKI) have been suggested to be an important anti-hypertrophic signaling pathway (1) . Intracellular levels of cGMP can be raised by the action of nitric oxide (NO) and natriuretic peptides (ANF, BNF), or by inhibiting cGMP-degrading phosphodiesterases (PDE). A growing body of evidence suggests that the PDE5 specific inhibitor Sildenafil (Sil) prevents and reverses hypertrophy and chamber remodelling in the heart of mice subjected to thoracic aorta constriction (TAC) by elevating cGMP levels and cGKI activation (1) . In contrast, using a mouse model that lacks cGKI expression in every cell type except smooth muscle cells (βRes mice; see ref.

2), we recently showed that the absence of this kinase does not alter the onset of hypertrophy induced by TAC or isoproterenol infusion (2) . Sil is believed to increase cardiac cGMP levels, although it is unclear, if its target (PDE5) is expressed in CM (2). Sil may act on other PDEs, such as PDE1C which is abundant in CMs. It is also unclear if Sil effects are mediated by other cardiac cell types, in particular by cardiofibroblast.

To answer these questions, we are currently investigating whether Sil is able to prevent hormone induced cardiac hypertrophy in the absence of cGKI in CM. Preliminary results on βRes mice show that even in the case of chronic AngII infusion, lack of cGKI In CM does not alter the Induction of hypertrophic response, at least in the initial phase (7days of AngII infusion at 2mg/kg/day). Interestingly, βRes mice showed impaired cardiac function, as indicated by decreased Fractional Shortening. Sil was able to partially block the onset of cardiac hypertrophy in WT animals, but not in βRes mice, indicating a requirement of cGKI in this process. In particular, Sil was able to block the transcription of pro-fibrotic genes such as TGFβ, CTGF, CollagenI and Fibronectin. The overexpression of the somatostatin receptors sst2 and sst5 in neuroendocrine tumors provides the molecular basis for therapeutic application of the stable somatostatin analogs octreotide and pasireotide. Whereas the phosphorylation of the carboxyl-terminal region of the sst2 receptor has been studied in detail, little is known about the agonist-induced regulation of the human sst5 receptor. Here, we have generated phosphosite-specific antibodies for the carboxyl-terminal threonines 333 (T333) and 347 (T347), which enabled us to selectively detect either the T333-or the T347-phosphorylated form of sst5. We show that agonist-mediated phosphorylation occurs at T333, whereas T347 is constitutively phosphorylated in the absence of agonist. We further demonstrate that the pan-somatostatin analog pasireotide and the sst5-selective ligand L-817,818 but not octreotide or KE108 were able to promote a clearly detectable T333 phosphorylation. However, none of these compounds was able to stimulate T333 phosphorylation and sst5 internalization to the same extent as the natural somatostatin. Agonist-induced T333 phosphorylation was dose-dependent and selectively mediated by G protein-coupled receptor kinase 2 (GRK2). Like that observed for the sst2 receptor, phosphorylation of sst5 occurred within seconds. However, unlike that seen for the sst2 receptor, dephosphorylation and recycling of sst5 were complete within minutes. We also identify protein phosphatase 1g (PP1g) as sst5 receptor phosphatase. Together, we provide direct evidence for agonist-selective phosphorylation of carboxyl-terminal T333. In addition, we identify GRK2-mediated phosphorylation and PP1g-mediated dephosphorylation of T333 as key regulators of rapid internalization and recycling of the human sst5 receptor. Termination of signaling of activated G protein-coupled receptors (GPCRs) is essential for maintenance of cellular homeostasis. Although the regulation of agonist-induced phosphorylation has been studied in detail for many GPCRs, the molecular mechanisms and functional consequences of receptor dephosphorylation are far from understood. Recent studies have shown that phosphatase inhibitors, such as okadaic acid and calyculin A, can block the dephosphorylation of a number of GPCRs including the ß2 adrenergic receptor, D1 dopamine receptor, parathyroid hormone receptor 1, thromboxane A receptor and the vasopressin receptor 1. However, a specific phosphatase has not been identified so far. In present studies, we have examined the mechanism and function of receptor dephosphorylation using the sst2A somatostatin receptor and the µ-opioid receptor (MOR) as models. Within those analyses, we have identified protein phosphatase 1beta (PP1ß) as the phosphatase for the cluster of phosphorylated threonines (353TTETQRT359) within the sst2A somatostatin receptor carboxylterminus using siRNA knock down screeening. Those phosphorylation sites mediate ß-Arrestin binding. We have also identified protein phosphatase 1gamma (PP1γ) as MOR phosphatase that catalyzed T370 and S375 dephosphorylation at or near the plasma membrane within minutes after agonist removal. Here, we show the different activated phosphatases with functional selective mutants. We examined tailswap mutants which specify the different phosphatase activities. Therefore we produced a MOR-rsst2A chimera with the rsst2A C-terminal tail and a rsst2A-MOR chimera with a MOR C-terminal tail.

Detoxification by conjugation of glutathione? Formations of DNA adducts of patulin activated by glutathione Pfenning C., Lehmann L. University Wuerzburg, Institute of Pharmacy and Food Chemistry Section of Food Chemistry, Am Hubland, 97074 Wuerzburg, Germany As a frequent contaminant in apple juice, the mycotoxin patulin (PAT) has shown mutagenic potential in cultured mammalian cells at concentrations which are equivalent to those found in marketable foods. This fact is in contrast with the assumption that conjugation to the major intracellular nucleophile glutathione (GSH) leads to detoxification of the electrophile PAT. Although PAT reacts readily with GSH, previous studies showed that co-incubation of PAT with model thiols and amine compounds increased the reactivity of PAT towards amines forming mixed-type adducts. Thus, we hypothesise that the potential to react with DNA bases after being activated by GSH might contribute to the mutagenicity of PAT. Adduct formation of DNA bases (adenine, guanine or cytosine) with PAT in the presence and absence of GSH was studied under neutral conditions. Liquid chromatography coupled with electrospray ionization tandem mass spectrometry was applied for identification and structure elucidation of putative adducts. Besides published as well as hitherto unknown PAT-GSH adducts, several PAT-DNA base adducts were formed both in presence and absence of GSH. In addition, with each of the three DNA bases one product exhibiting a mass to charge ratio and fragmentation pattern suggesting a mixed thiol-amine adduct was detected. Based on the fragment ions of adducts formed with GSH and chemically modified derivatives, we postulate a cyclic structure of the PAT-GSH-DNA base adducts, resulting from the reaction of the α-amino group of the glutamic acid residue with the C7-carbonyl function of PAT. The exocyclic amino group of the DNA base is linked to C1 of the PAT backbone by an amid bond. Thus, the present study demonstrates the reactivity of PAT towards DNA bases and the participation of GSH in adduct formation. The postulated structures of DNA adducts could be used as biomarkers for the determination of the internal exposure to PAT in humans. Prescribing information detailed in the Summary of Product Characteristics (SPC) forms the officially approved basis for safe prescribing of drugs. In a project funded by the German Federal Ministry of Education and Research (BMBF) we aimed to derive an internationally valid data set for safe prescribing of psychiatric drugs and therefore analyzed and compared the content of internationally available prescribing information.

A team of pharmacists and clinical pharmacologists performed an in-depth comparison of the German, Swiss, British and US-American SPCs of 10 top prescribed psychiatric drugs. For 7 drugs (of identical pharmaceutical form) the SPCs from the same manufacturer were available for all countries, whereas for three drugs SPCs were only available from different companies. In these cases the most recent prescribing information from each country was included in the comparison.

In 40 SPCs 2220 individual data points (55.5±17.4 per individual SPC) were compared. Between countries the timeliness of prescribing information for an individual drug varied by a median of 18.5 (range: 6-134) months. The respective SPCs covered on average 71.4±30.3% (range: 12.5-100%) of all mentioned indications and 70.1±24.4% (range 15.4-100%) of all mentioned contraindications. The warnings and precautions section of an individual SPC covered on average 59.5±17.1% (range: 12.5-93.3%) of all mentioned warnings and precautions for that drug. The variation observed was only marginally improved when restricting the analysis to the 28 SPCs of the 7 drugs available in all four countries from the same manufacturer.

Across countries, the Summary of Product Characteristics of individual psychiatric drugs show substantial variation in crucial prescribing information. As different manufacturers are unlikely to explain much of the observed variation, these data argue for a better international cooperation and standardization of the content of Summary of Product Characteristics. This project is supported by the German Federal Ministry of Education and Research (BMBF), project grant No. 01 EX1015B. Protein kinase CK2 (former name 'casein kinase 2') is a highly conserved serine/threonine kinase, which acts as a component of regulatory networks implicated in many cellular processes but is also linked to various types of human cancer [1, 2] . Elevated CK2 activity has been associated with aggressive tumor behavior and results in growth advantage, enhanced survival and dynamic adaption to stress of cancer cells [3] . CK2 is a heterotetramer consisting of two catalytic subunits (CK2α) attached to a dimer of regulatory subunits (CK2β) [4] . CK2β stabilizes CK2α against denaturation and modulates the substrate specificity of the catalytic subunit [5] . Due to the relatively small and hydrophobic CK2α-CK2β interface (832 Ų) [4] , low molecular weight inhibitors are able to interfere with the CK2 subunit interaction and thus affect the kinase activity [6] . Such inhibitors might exhibit an increased specificity in comparison to those compounds interacting with the conserved ATP binding site [3] . We have developed an ELISA-based CK2α-CK2β binding assay using recombinant human CK2 subunits. Different blocking reagents were analyzed to minimize nonspecific binding. The optimized binding assay was then applied to screen for inhibitors of the CK2 subunit interaction. Primary hits were further characterized by determination of the parameters IC50 and Ki as well as by comparing the results from the binding assay with literature-known or recently obtained crystal structures.

Numerous epidemiological studies have shown associations between exposure to ultra fine particles and an increase in cardiovascular diseases such as atherosclerosis, coronary heart disease and myocardial infarction. Ultra-fine particles have an aerodynamic diameter of < 0.1 µm and are highly diverse with impurities of transition metals and organic compounds (e.g. polycyclic aromatic hydrocarbons). Posttranslational modification (PTM) of proteins, particularly phosphorylation, is a key element in the regulation of cell function and any disturbance can lead to multiple diseases. The present study focused on the proteomic-based identification of phosphorylated proteins to understand the mechanism behind ultrafine particle exposure and cardiovascular disease development. As one of the major sources for UFP emissions are diesel exhaust, therefore to mimic the diesel particles, carbon black (CB) and benzo[a]pyrene loaded carbon black (CB+) were used in the present study.

Cells of the endothelial cell line EA.hy926 were exposed for 14 days to 100 ng/mL CB and CB+. Phosphoprotein extraction of whole cell lysates was carried out by the method developed in our lab 1 . The obtained proteins were then separated by two-dimensional gel-electrophoresis followed by MALDI-ToF-MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) analysis of differently expressed proteins. To further validate the results invasive potential of cells were monitored by plating exposed cells for 24 hrs on top of Matrigel-coated inserts.

Differential expressions of 20 phosphoproteins were found in CB-treated cells while an altered expression of 33 phosphoproteins was observed in CB+-treated cells. The MALDI-ToF analysis revealed proteins involved in the regulation of the endothelial permeability and the cellular plasticity such as vimentin, actin and transitional endoplasmic reticulum ATPase. Further, the invasion assay supported these results as the CB-exposed cells showed a high invasive potential as compared to control. [1] Pink M. et. al. , Precipitation by lanthanum ions: A straightforward approach to isolate phosphoproteins, J.Protomics, 75, 2011, [375] [376] [377] [378] [379] [380] [381] [382] [383] 302 Application of the TTC approach in cosmetics Platzek T. Bundesinstitut für Risikobewertung, Max-Dohrn-Straße 8-10, 10589 Berlin, Germany Regulatory toxicologists in Europe have been discussing the TTC approach since more than a decade, e.g. the previous Scientific Committee on Food in 1996. Since then, the concept was further developed and is now applied in the EU for the assessment of flavouring substances by EFSA. Two committees are discussing possible applications: The EFSA Scientific Committee prepared an opinion exploring options for the application in food and feed, e.g. for impurities of food additives, thermal reaction products, food contact materials, contaminants etc. In addition, an EU non-food expert Committee consisting of members of SCCS, SCHER and SCHENIR discussed the TTC concept in general as well as additional possible fields of application with the focus on cosmetics and an opinion was already published for public consultation. The proposal to apply the TTC approach also for cosmetic ingredients was introduced by a paper published in 2007 by a COLIPA (The European Cosmetics Association) supported working group. Major aspects to be considered are the following: 1.

Applicability domain. The chemical space of the TTC dataset (> 600 compounds) has to be compared with that of cosmetic ingredients (> 10 000 compounds).

Route to route extrapolation. Since no adequate dermal toxicity database is available both data on oral intake used in the TTC approach and on dermal exposure to cosmetic ingredients have to be transfomed to internal exposure figures. Gastrointestinal and dermal bioavailability as well as route specific differences in metabolism have to be integrated.

Exposure. The reliability of exposure estimation is the second pillar of the TTC approach. Compared to food, data on exposure to substances in cosmetics and consumer products is scarce. A pragmatic step forward is the comparison of TTCs and NOAEL-derived safe exposure levels for cosmetic ingredients. This work was already done with substances in food contact materials and chemicals from the ELINCS list. For cosmetic ingredients a similar European project is ongoing. Further refinement of the TTC approach is needed taking into account the up-to-date toxicological knowledge. With cosmetics specific problems may arise in praxi: according to the new EU cosmetic legislation the safety of cosmetic products available on the market has to be assessed by the manufacturer or importer and also assessors with limited toxicological experience may apply the TTC approach, e.g. by running the TOXTREE software.

Plöttner S., Marczynski B., Käfferlein H. U., Welge P., Groth H., Engelhardt B., Schmitz K., Erkes A., Brüning T. Institut für Prävention und Arbeitsmedizin der Deutschen Gesetzlichen Unfallversicherung -Institut der Ruhr-Universität Bochum (IPA) Toxikologie, Bürkle-dela-Camp-Platz 1, 44789 Bochum, Germany Polycyclic aromatic hydrocarbons (PAHs) comprise several hundred compounds with different carcinogenic potentials, typically occurring in complex mixtures. Due to a lack of data risk estimations for PAH mixtures are usually based on those for benzo[a]pyrene (B[a]P). The aim of our present study was to explore the suitability of a permanent human lung cell line as tool for future studies on genotoxicity of PAH mixtures. In this pilot study we investigated the time-and concentration-dependent generation of specific anti-benzo[a]pyrene -7,8-diol-9 ,10-epoxide (anti-BPDE)-DNA adducts as well as cytochrome P450 (CYP) 1A1/1B1 enzyme activities after B[a]P-incubation in vitro. We used metabolically competent A549 lung carcinoma cells which display several characteristics of alveolar epithelial type II cells. After 24 h and 48 h incubations with different B[a]P-concentrations cytotoxic effects were assessed with the neutral red assay and CYP1A1/1B1 activities using luminescent tests. The formation of specific anti-BPDE-DNA adducts was determined by HPLC with fluorescence detection. A time-and concentration-dependent formation of anti-BPDE-DNA adducts was observed with maximum rates of 340.5 ± 39.1 and 599.6 ± 132.4 anti-BPDE/10 8 nucleotides after incubation with 1 µM (24 h) and 3 µM B[a]P (48 h), respectively. However, the mean adduct rates decreased at higher B[a]P-concentrations. The reduction was more pronounced after 24 h than after 48 h. Increased CYP1A1/1B1 activities were observed at > 0.1 -1 µM (24 h) and > 0.1 -3 µM (48 h). A clear decrease of enzyme activities was observed at higher concentrations for both incubation times. In the neutral red assay no more than 10% cytotoxicity in relation to the negative control were found after 24 h incubation with ≥ 10 µM B[a]P and after 48 h with ≥ 1 µM B[a]P. Overall, incubation of A549 cells with B[a]P resulted in a time-and concentration-dependent increase of CYP-activities and anti-BPDE-DNA adducts. This clearly shows that A549 cells are able to generate mutagenic DNA-adducts. Thus, the in vitro model used in the present work appears suitable for genotoxicity studies of individual PAHs and PAH mixtures, and therefore may be a useful tool for research on syncarcinogenesis. The β subunit of the Cav1.2 channel complex has been shown to play a key role in Cav1.2 channel trafficking and channel characteristics like opening probability. Furthermore, the last exon of the CACNB2 gene coding for Cavβ2, exon 14, contains several potential phosphorylation sites, e.g. for protein kinase A or Ca 2+ /calmodulin dependent CaMKII. PKA-dependent phosphorylation mediates β-adrenergic stimulation of Cav1.2. Potential phosphorylation sites are Ser478 and Ser479 (in the β2 subunit in rat, Perez-Reyes et al. 1992 a ) and Ser1928 in the C-terminus of the poreforming α1C subunit (Lemke et al. 2008 b ) . In cardiomyocytes CaMKII regulates Ca 2+ release and reuptake from and into the SR and is involved in the facilitation of the calcium channel. Potential interaction sites between the Cav1.2 channel complex and CaMKII are Thr498 in the β2 subunit (in rat, Grueter et al. 2006 c ) and Ser1512 and Ser1570 in the Cterminus of the α1C subunit (Blaich et al. 2010 d ) . However, the exact pathways remained widely unclear up to now. To clarify these mechanisms and to identify the relevant phosphorylation sites for PKA and CaMKII we established a mouse line carrying a stop codon in exon 14 after aa Pro501. This mutation prevented translation of the Cavβ2 C-terminus containing the corresponding potential phosphorylation sites mentioned above (Cavβ2stop mouse). These mice were viable, showed unaltered expression of the truncated of Cavβ protein and unchanged ECG and echocardiography. Electophysiological analysis of isolated cardiomyocytes showed no differences in current density, the effect of isoproterenol, the time course of inactivation and the facilitation property when compared to cells isolated from littermate controls. For further investigations we bred the Cavβ2stop mice with S1928A mice (Lemke et al. 2008 b ) lacking the PKA phosphorylation site S1928 in the α1C subunit (S1928Aβ2stop mouse) or with SF mice (Blaich et al. 2010 d ) lacking the CaMKII phosphorylation sites S1512 and S1570 in the α1C C-terminus (SFβ2stop). Both mouse lines were viable and showed unchanged echocardiography recordings compared to their control littermates and unaltered ECG for S1928Aβ2stop mice. Electrophysiological investigations on cardiomyocytes of S1928Aβ2stop mice showed unchanged β-adrenergic stimulation with isoproterenol compared to littermate controls. These results suggest that β adrenergic regulation of the cardiac Cav1.2 channel is not mediated by these phosphorylation sites. Introduction. Chronic atrial fibrillation (cAF) is marked by increased fibrosis which contributes to the perpetuation of the disease. In addition to the role of fibrosis in structural remodeling of cardiac tissue, fibroblasts can couple with cardiomyocytes via gap junction thereby altering the electrophysiological properties of the later and potentially participating in atrial electrical dysfunction. In order to understand the importance of fibroblasts in the pathophysiology of cAF, we compared the electrical properties of atrial fibroblasts isolated from patients in sinus rhythm (SR) and cAF. Methods. Fibroblasts were isolated by outgrowth culture from right atrial biopsies and cultivated in medium containing 10% fetal calf serum. We used whole-cell patch clamp techniques to investigate ion currents and membrane potential. Results. SR and cAF fibroblasts showed similar capacitance (SR: 43.6 ± 4.6 pF, n=33; cAF: 54.7 ± 5.1 pF, n = 17) and membrane potential (SR: -21.0 ± 4.3 mV, n = 14; cAF: -27. 4 ± 4.8 mV, n = 16) . In both groups, we observed fast activating outward currents with a mean threshold at -20 mV. Interestingly, current amplitude was significantly larger in SR than cAF cells (SR: 23.8 ± 4.2 pA/pF, n = 15; cAF: 6.1 ± 1.0, n = 6; p < 0.05). When maintained in culture for 3-5 weeks, cells from both groups developed Na + currents. Surprisingly, the fraction of cAF cells displaying such currents was larger than the SR counterpart (cAF: 38%; SR: 15%). Furthermore, Na + current amplitude was significantly larger in cAF fibroblasts (SR: 6.1 ± 2.0 pA/pF, n = 5; cAF: 17.4 ± 4.4 pA/pF, n = 6; p < 0.05). Na + currents were not altered by 100 nM Tetrodotoxin (TTX), but 10 µM TTX reduced current amplitude to 42% of control, suggesting that the channel involved is the cardiac TTX-resistant isoform Nav1.5. Conclusion. In the context of cAF, fibroblasts undergo electrophysiological changes which need to be thoroughly described. Understanding whether those changes contribute to the AF substrate might provide new therapeutic targets for the treatment of cAF.

The initial recruitment of Gi to the Alpha2A-adrenergic receptor is affected by Gprotein-coupled receptor kinases and arrestins Prokopets O. S., Krasel C., 35043 Marburg, Germany Most G-protein-coupled receptors undergo homologous desensitization after agonist stimulation. In this process, agonist-activated receptors are phosphorylated by Gprotein-coupled receptor kinases (GRKs), followed by binding of arrestins to the still agonist-occupied, phosphorylated receptors. It is assumed that arrestin competes with heterotrimeric G-proteins for the receptor molecule and thereby causes desensitization of G-protein-mediated responses. We tested this idea by investigating the effect of GRKs and arrestins on the recruitment of G-proteins by the alpha2A-adrenergic receptor. HEK293T cells were transfected with YFP-tagged alpha2A-adrenergic receptor and the three subunits of Gi1. The G(beta) subunit was CFP-tagged. Upon stimulation with noradrenaline, a very rapid, robust increase in FRET between the receptor and the G(beta) subunit was observed, confirming previous observations that the alpha2Aadrenergic receptor recruits Gi with a half-life of around 150 milliseconds. When arrestin3 and GRK2 were also co-transfected, the half-life of Gi recruitment was substantially delayed, increasing to around 2 seconds. There seemed to be no effect of GRK2+arrestin3 cotransfection on a second stimulation; neither the kinetics nor the extent were altered compared to the first stimulation. Interestingly, GRK2 alone seemed to cause a similar but slightly less pronounced delay of G(beta) recruitment to the alpha2A-adrenergic receptor. In corresponding experiments, we measured the recruitment of arrestin3-CFP to YFP-tagged alpha2A-adrenergic receptors in the presence of GRK2. Upon stimulation with noradrenaline, we observed a robust increase in FRET between the receptor and arrestin3, confirming previous observations that the alpha2A-adrenergic receptor recruits arrestin3. Co-transfection of the three subunits of Gi1 had no effect on the kinetics of arrestin3 binding to the alpha2A-adrenergic receptors. Based on previous results with other receptors, an attenuation of Gi recruiting to the alpha2A-adrenergic receptor is quite unexpected. Our data suggest that the relation between receptors, G-proteins, GRKs and arrestins may be more complex than previously postulated. Introduction: Human urinary bladder expresses mRNA of the three known badrenoceptor (b-AR) subtypes (b1, b2, b3) in detrusor and urothelium. We have shown previously that only b3-AR is involved in human detrusor relaxation. To investigate the urothelium-induced modulation of b-AR-mediated relaxation, we have examined systematically whether other b-AR subtypes are involved.

Human detrusor tissue samples were obtained from patients undergoing radical cystectomy for the treatment of bladder cancer. Detrusor strips were studied with and without an intact mucosa layer. Muscle strips were precontracted with 1 µM carbachol and relaxation was studied in response to the b-AR agonist NE. a-AR mediated processes were blocked with the a-AR antagonists phentolamine and prazosin. Selective b-ARs antagonists were used to investigate b-AR mediated relaxation. At the end of each experiment 10 µM forskolin was used to determine maximum cAMP-mediated relaxation. 

The presence of intact urothelium reduces potency but not effectivity of NE indicating involvement of urothelium-mediated processes not only during detrusor contraction but also during relaxation. NE-mediated detrusor relaxation is mediated through b3-AR in the absence of urothelium. But in intact detrusor strips b2-ARs seem to have an additional inhibitory effect. Affinity of the selective b3-AR antagonist L748,337 is unchanged, therefore the intact urothelium does not interact with the function of b3-ARs.

Aldosterone causes oxidative stress and DNA damage independent of blood pressure in vivo Queisser N., Schupp N. Universität Würzburg Institut für Toxikologie, Versbacherstr. 9, 97078 Würzburg, Germany

Background: An inappropriate increase of the mineralocorticoid aldosterone (Ald) can be induced by a stimulated renin-angiotensin-aldosterone system. Epidemiological studies exploring the connection between hypertension and cancer found higher cancer mortality and an increased risk to develop kidney cancer in hypertensive individuals. We recently showed that Ald produces oxidative stress, activates transcription factor NF-kB and is genotoxic in kidney tubule cells.

Objectives: This study investigated the capacity of Ald to induce oxidative/nitrosative stress, DNA damage, DNA repair, apoptosis, cell proliferation and the activation of NF-κB in rat kidneys. Methods: Mineralocorticoid-dependent hypertension was induced by Ald/salt in Sprague Dawley rats. DNA damage and oxidative/nitrosative stress markers were detected immunohistochemically.

Results: Ald/salt treatment caused increased blood pressure compared to untreated rats. Tempol, an antioxidant, and hydralazine, a vasodilator acting independent of the renin-angiotensin-aldosterone system, could lower the blood pressure, while the mineralocorticoid receptor (MR) antagonist spironolactone was administered in a subtherapeutical dose not lowering the blood pressure. Ald/salt treatment caused oxidative and nitrosative stress, structural DNA damage, double strand breaks, DNA repair and NF-κB activation. Spironolactone decreased these markers significantly. Tempol was also able to reduce these markers, while hydralazine had no effect. Ald/salttreated kidneys showed a tendency to lower apoptosis and to increased cell proliferation compared to control rat kidneys. Discussion: This study provides a first hint of blood pressure-independent effects of Ald. The MR and the production of ROS seem to be crucial for the damaging effects of Ald. An aberrant or long-term activation of NF-κB by persistently high Ald levels could support resistance to apoptosis and the survival of cells with damaged DNA, and increase cell proliferation. These actions could contribute to the increased cancer incidence in hypertension by initiating carcinogenesis. Grant support by the DFG is gratefully acknowledged. CREB regulating transcriptional coactivator 1 (CRTC1) is a transcriptional coactivator of the transcription factor CREB. We have recently shown its expression in cardiomyocytes and its activation by beta-adrenergic signaling. Beta-adrenergic signaling contributes to the pathogenesis of cardiac hypertrophy, leading to heart failure, as evidenced by the therapeutic success of the beta-adrenoceptor antagonists. In order to investigate if CRTC1 is involved in this process, we investigated the expression of CRTC1 in hypertrophied myocardium from mice and humans. Methods: Protein lysates from mouse and human samples were investigated for CRTC1 protein expression. We distinguished between an acquired and an inherited form of cardiac hypertrophy. Acquired cardiac hypertrophy is an adaptation of the heart to an increased cardiac workload and can be found in patients with an aortic valve stenosis. In mice this kind of hypertrophy can be evoked by Transverse aortic constriction (TAC). The inherited form of cardiac hypertrophy is caused by mutations in genes coding for proteins of the sarcomeric apparatus and is referred to hypertrophic cardiomyopathy (HCM). As a model for HCM, transgenic mice with a mutation in the Mybpc3 gene, coding for the sarcomeric protein cardiac myosin-binding protein C (cMyBP-C), were used. These transgenic mice were characterized by a hypertrophied heart. In case of human samples we distinguished between patients with an acquired form of hypertrophy due to an aortic valve stenosis, and patients with a hypertrophic obstructive cardiomyopathy (HOCM), a special form of HCM. Results: In the TAC mice and in the MYPPC3 mutant mice the expression of CRTC1 was significantly higher than in the controls (2.1-and 1.9-fold, respectively; n=3). In both forms of human hypertrophy, we found a significantly 5-fold upregulation of CRTC1 protein level in comparison to human samples from non-failing myocardium or samples from patients with a dilatative or ischemic cardiomyopathy (n=7-10 for each group).

Conclusions: CRTC1 is upregulated in cardiac hypertrophy with acquired or geneticbased origin. The evaluation of the role of CRTC1 in the heart may help to elucidate the role of beta-adrenergic signaling in the development of cardiac hypertrophy.

Microarray gene expression profiling reveals up-regulation of the cardiac lipid metabolic process at the onset of heart failure Fu X., Abd Alla J., Quitterer U. ETH Zürich Molekulare Pharmakologie, Winterthurerstrasse 190, 8057 Zürich, Switzerland Atherosclerosis and chronic pressure overload are major cardiovascular risk factors for the development of heart failure in patients. To mimic those risk factors in experimental models we used atherosclerosis-prone apolipoprotein E (ApoE)-deficient mice, and chronic pressure overload was imposed by abdominal aortic constriction (AAC). Cardiac function was monitored by echocardiography. Severe atherosclerosis in aged ApoEdeficient mice or chronic pressure overload induced signs of heart failure as evidenced by a significantly reduced cardiac ejection fraction (<30%). To investigate pathomechanisms underlying the development of heart failure, microarray gene expression analysis and QT-RT-PCR were performed of failing heart tissue relative to age-matched controls. Gene ontology analysis of the microarray data revealed that the onset of heart failure, in two different experimental models, was characterized by a strong up-regulation (≥2-fold) of the cardiac lipid metabolic process and lipid overload. Lipid metabolism genes were involved in lipid synthesis, storage and oxidation. The major palmitate-synthesizing enzyme, fatty acid synthase, was causally related to the development of cardiac dysfunction by enhancing cardiomyocyte apoptosis. Taken together the data support that the onset of experimental heart failure is characterized by a dysfunction of the cardiac lipid metabolism promoting cardiomyocyte death. AT1-receptor blockers (ARBs) are established for the treatment of high blood pressure and new onset of diabetes is reduced by ARBs. In the past years evidence increased that body weight may also be lessened particularly in rats and mice. However, less data are available whether ARBs still reduce weight, when treatment was initiated not until animals became obese by diet. Prior to drug treatment, spontaneously hypertensive rats were fed for 6 months with chow but also with a cafeteria diet (CD) to develop obesity. Controls received only chow (CONchow). CD-fed SHR were treated for 3 months with telmisartan (TEL 8mg/kg/d) or amlodipine (AML, 12 mg/kg/d), whereas controls received vehicle (CONCD). Systolic blood pressure (SBP), feeding behaviour, body weight, abdominal fat mass (by MRT) and energy expenditure (by indirect calorimetry) was monitored. Leptin sensitivity was assessed by measuring energy intake and expenditure after repetitive injections (s.c.) of leptin. Insulin sensitivity was functionally determined by glucose and insulin tolerance tests. Due to CD feeding body weight was increased after 6 months by more than 60 g. TEL normalized SBP whereas it remained >200 mmHg in CONCD and CONchow. TEL additionally reduced CD-induced increase of body weight and abdominal fat mass. Food intake was diminished during the first 4 weeks, but raised beyond control levels during the last 4 weeks of treatment. The shift of the respiratory index to lower levels indicated improved energy expenditure. In response to exogenous leptin, the food intake of CONCD was higher compared to CONchow, indicating a leptin resistance. This assumption is further supported by high triglyceride concentrations of CONCD. After TEL, leptininduced food intake was reduced and energy expenditure was increased compared to CONCD, indicating that leptin sensitivity was at least partially restored. Accordingly, triglycerides were reduced. Compared to CONCD, the insulin sensitivity was improved by TEL since maximal increases in plasma concentrations of glucose and insulin in response to glucose challenge were reduced, but glucose response to insulin challenge was diminished. Even though reduction in blood pressure was almost similar between TEL and AML, metabolic and antiobese efficacies of AML were markedly attenuated. We conclude that telmisartan reveals wide efficacies in improving all symptoms of the Metabolic Syndrome. The pleiotropic effects are not related to the hypotensive action of TEL.

A β2-adrenoceptor -cAMP mediated, immediate stimulation of β2-adrenoceptor gene expression in human lung fibroblasts is opposed by a delayed up-regulation of inhibitory factors Racké K., Lamyel F., Kämpfer N., Schütz I., Warnken M. Univ. Bonn Dept. Pharmacol. &Toxicol., 53105 Bonn, Germany

Based on their bronchodilatory effects, β2-adrenoceptor agonists constitute essential elements in the treatment of bronchial asthma and COPD. However, treatment with long-acting β2-adrenoceptor agonists has been associated with possible worsening of airway hyper-reactivity, possibly because of loss of β-adrenoceptor function. Therefore, the molecular regulation of β2-adrenoceptor expression was addressed here. MRC-5 human lung fibroblasts were cultured for up to 48 h in absence or presence of test substances, followed by β2-adrenoceptor mRNA determination by qPCR. β2-Adrenoceptor mRNA decreased with a half-life of 25 min after inhibition of mRNA synthesis with actinomycin D (30 µM), but increased by 333±85%, 502±52% and 640±165% (means±SEM) within 1.5, 4 and 6.5 h, resp. after inhibition of protein synthesis by cycloheximide (30 µM). The β2-adrenoceptor agonists formoterol and olodaterol (1-100 nM) induced a rapid increase in β2-adrenoceptor mRNA (maximally within 1 h by 100±19% and 110±19% at 10 nM, resp.). However, after 4 h exposure to 10 nM formoterol or olodaterol a reduction in β2-adrenoceptor mRNA by 59±8% and 58±6%, resp., was observed. Both, the stimulatory and inhibitory effects of β2adrenoceptor agonists were mimicked by forskolin (10 µM, increase by 88±14% and inhibition by 49±4%) and cholera toxin (5 ng/ml, increase by 76±12% and inhibition by 77±7%). The formoterol-induced up-regulation of β2-adrenoceptor mRNA was blocked by actinomycin D, but not by cycloheximide. Moreover, in presence of cycloheximide, β2adrenoceptor agonist induced inhibition was converted into a marked stimulation.

In conclusion, expression of β2-adrenoceptors in human lung fibroblasts is highly regulated at transcriptional level. The observations with cycloheximide indicate that the β2-adrenoceptor gene is under strong inhibitory control of short-living, not yet identified suppressors. Although both, the time-dependent up-and down-regulation of the β2adrenoceptor gene expression by β2-adrenoceptor activation appears to be mediated via adenylyl cyclase -cAMP signalling, only the stimulatory effect appears to be a direct action on the β2-adrenoceptor gene. ET-1 appears to be involved in the pathogenesis not only of pulmonary hypertension, but also in fibrotic remodeling associated with chronic obstructive airway diseases. Since human lung fibroblasts (hLF) are a source of ET-1 and have been shown to be controlled by muscarinic receptors and β-adrenoceptors, a possible muscarinic and βadrenergic modulation of ET-1 expression in hLF was explored. MRC-5 hLF were cultured for up to 24 h in absence or presence of test substances, followed by prepro-ET-1 (ppET-1) mRNA determination by qPCR. The muscarinic agonist oxotremorine (10 µM) induced an increase in ppET-1 mRNA by 180%, an effect prevented by 10 nM tiotropium. The β2-adrenoceptor agonist olodaterol (up to 100 nM) caused a reduction of ppET-1 mRNA expression by 45%. The effect of 10 nM olodaterol was prevented by ICI 118,551 (1 µM), but not affect by CGP 20712 (3 µM) . The PKA agonist 6-Bnz-cAMP (500 µM) caused a reduction in ppET-1 mRNA expression by 65%, whereas the Epac agonist 8-CPT-2'-O-Me-cAMP (100 µM) caused only a marginal inhibition by 22%. Olodaterol (10 nM) strongly opposed the stimulatory effect of 10 µM oxotremorine. An increase in ppET-1 mRNA expression by 185% caused by 0.3 ng/ml TGF-β was effectively opposed by 10 and 100 nM olodaterol, resulting in an inhibition comparable to that in absence of TGF-β. However, the increase in ppET-1 mRNA caused by a maximally effective concentration of TGF-β (1 ng/ml, increase by 620%) was not significantly affected by 10 or 100 nM olodaterol. Likewise, the PKAagonist 6-Bnz-cAMP (500 µM) opposed the increase in ppET-1 mRNA expression caused by 0.3 ng/ml TGF-β, but not that caused by 1 ng/ml TGF-β. TGF-β caused, with an IC 50 of 0.3 ng/ml, a marked down-regulation of β2-adrenoceptor mRNA expression, maximally by 90% within 6 h. ET-1 expression in hLF is stimulated by muscarinic receptors and inhibited by β2adrenoceptors. The effect of β2-adrenoceptors may be mediated via PKA. ET-1 expression in hLF is markedly up-regulated by TGF-β, but only effects of sub-maximally effective concentrations of TGF-β are opposed by the β2-adrenoceptor -PKA pathway, in part because of TGF-β-induced down-regulation of β2-adrenoceptors. Since ET-1 can promote pro-fibrotic features in hLF, inhibition of ET-1 expression could contribute to long-term beneficial effects of long-acting β2-adrenoceptor agonists such as olodaterol and long-acting muscarinic antagonists such as tiotropium.

Cardiac hypertrophy leads to up-regulation of dipeptidyl aminopeptidase-like proteins in human and rat Radicke S., Hutschenreuther A., Schaefer M. Universität Leipzig Rudolf-Boehm-Institut für Pharmakologie und Toxikologie, Härtelstr.

Cardiac hypertrophy is a major risk factor for heart failure and associated morbidity and mortality. Functional down-regulation of K + currents is a prominent feature of cells isolated from failing ventricles. A marked decrease in the transient outward potassium current Ito has been shown in various models. Changes in the K + channel expression differ depending on the species, and the mechanism of induction of heart failure. To study the regulation of Ito channel subunits we compared the hypertrophic responses in human ventricular tissues from failing hearts with doxorubicin-induced hypertrophy in rats and in H9c2 embryonic rat cardiac cells. Specifically, we quantified mRNA expression of the pore-forming subunits Kv4.3 and Kv4.2, the cytosolic β-subunit KChIP2 and the transmembrane subunits DPP6 and DPP10 using RT-PCR. Treatment with doxorubicin (2 µM) induced hypertrophy and increased the mRNA expression of the hypertrophy marker genes ANF, BNP and beta-MHC in H9c2 cardiac myoblasts. While Kv4.3 was detected in H9c2 cells and hearts from human and rat, Kv4.2 mRNA was only expressed in adult rats. During hypertrophy Kv4.3 was downregulated in human tissue as well as in doxorubicin-treated H9c2 cells compared to the controls. In rat hearts Kv4.2 expression was increased after doxorubicin treatment. Interestingly, Kv4.2 was also found to be up-regulated in rat heart tissues and H9c2 cells after treatment with doxorubicin. As previously shown, KChIP2 mRNA expression was significantly reduced in tissues of failing hearts. In contrast, KChIP2 mRNA was upregulated in hypertrophic rat hearts and H9c2 cells. The expression of DPP6 and DPP10 was observed only in human hearts. But mRNA levels of both were significantly increased in failing tissues. DPP6 and DPP10 were not expressed in the adult rat heart or H9c2 cells, whereas in rats with doxorubicin-induced cardiac hypertrophy and in doxorubicin-treated H9c2 cells, the mRNA of DPP6 and DPP10 was up-regulated.

H9c2 cells showed almost identical hypertrophic responses to those observed in human ventricles and rat hearts. This finding validates H9c2 cells as a model for in vitro studies of cardiac hypertrophy. In further studies we will investigate the consequences of a knock-down of DPP6 and DPP10 in doxorubicin-induced hypertrophic H9c2 cells. In preliminary experiments specific short-hairpin RNA, targeting DPP6 and DPP10, has been designed and tested in heterologous expression systems.

The nonsynonymous c.521T>C germline genetic variation in the liver-specific organic anion transporter SLCO1B1 is associated with methotrexate pharmacokinetics in pediatric acute lymphoblastic leukemia Radtke S. 1 Background: Methotrexate (MTX) plasma concentration is related to its clinical effect. Transport proteins, such as ABCC2, SLCO19A1, and SLCO1B1, have been implicated in the disposition of MTX. Here we investigated whether common reduced-function variants in ABCC2, SLCO19A1, and SLCO1B1 contribute to the interindividual variability in methotrexate pharmacokinetics in children with acute lymphoblastic leukemia (ALL).

We analyzed MTX pharmacokinetics (MTX plasma concentration at the end of infusion C24h, MTX AUC24-48h, and MTX clearance Cl) in an unselected population of 419 children with ALL from the ALL-BFM 2000 trial (ClinicalTrials.gov: NCT00430118) who received 1676 courses of MTX at 5 g/m 2 as 24 h infusions. The contribution of genes (genetic component, rGC) to the interindividual variability in MTX pharmacokinetics was estimated according to the method of Kalow et al. (1998) . ABCC2 c.-24C>T (rs717620), SLCO19A1 c.80G>A (p.His27Arg, rs1051266), SLCO1B1 c.521T>C (p.Val174Ala, rs4149056) and SLCO1B1 388A>G (p.Asn130Asp, rs2306283) genotypes were analyzed by TaqMan polymerase chain reaction.

There was substantial interpatient variability in average (± SD) MTX C24h (50.95 ± 24.15 µmol/l), AUC24-48h (57.44 ± 37.52 h*mg/l), and Cl (390.72 ± 223.95 ml/min/m²). The rGC values of C24h, AUC24-48h, and Cl ranged from 0.61-0.71 suggesting that variation in MTX pharmacokinetics has a substantial genetic component. After adjustment for age and sex by multiple regression, the SLCO1B1 c.521T>C SNP was significantly associated with C24h (P<0.001), AUC24-48h (P<0.001), and Cl (P=0.011) of MTX. Compared with the wildtype genotype, in patients with the TC genotype C24h and AUC24-48h increased by 18% (P=0.009) and 28% (P=0.003), respectively, whereas Cl significantly decreased by 15% (P=0.012). Pharmacokinetic variables significantly changed with increasing number of variant c.521T>C alleles (P<0.02, Jonckheere-Terpstra), suggesting a per allele effect consistent with a co-dominant model of association. In contrast, the ABCC2 c.-24C>T, SLCO19A1 c.80G>A, and SLCO1B1 388A>G polymorphisms did not show an association with MTX pharmacokinetics.

Conclusions: The nonsynonymous c.521T>C polymorphism in SLCO1B1 contributes to the variability of MTX pharmacokinetics in this study of high-dose MTX in pediatric ALL. This project is supported by the Johannes und Frieda Marohn-Stiftung. The antitumorigenic mechanism of the selective cyclooxygenase-2 (COX-2) inhibitor celecoxib is still a matter of debate. Using human lung cancer cell lines (A549, H358, H460), the present study investigates the contribution of COX-2 and peroxisome proliferator activated receptor γ (PPARγ) to apoptosis elicited by celecoxib. Celecoxib was found to cause apoptotic cell death in a concentration-dependent manner (10 -50 µM), whereas structurally-related COX-2 inhibitors (etoricoxib, rofecoxib, valdecoxib) were inactive in this respect. Apoptotic cell death by celecoxib was suppressed by preincubation of tumor cells with the selective COX-2 inhibitor NS-398, the PPARγ antagonist GW-9662 and by siRNA targeting COX-2 or PPARγ. Celecoxib-induced apoptosis was paralleled by a time-and concentration-dependent upregulation of COX-2 and PPARγ at both mRNA and protein level. Using an established COX-2 activity assay monitoring immediate conversion of exogenously added arachidonic acid to the respective prostaglandins (PGs), NS-398 was shown to suppress celecoxib-induced COX-2 activity when added prior to arachidonic acid. Among the COX-2-dependent PGs analyzed, PGD 2 and its dehydration product 15-deoxy-∆ 12,14 -PGJ2 were found to induce cytosol-to-nucleus translocation of PPARγ as well as PPARγ-dependent apoptosis. Celecoxib-elicited translocation of PPARγ was inhibited by preincubation of cells with NS-398 which itself did not alter celecoxib-induced total PPARγ protein expression. Finally, a COX-2-and PPARγ-dependent proapoptotic mechanism of celecoxib was confirmed in primary tumor cells obtained from brain metastases of two lung cancer patients. Together, our data demonstrate a proapoptotic mechanism of celecoxib involving initial upregulation of COX-2 and PPARγ and a subsequent nuclear translocation of PPARγ by COX-2-dependent PGs. Uncoagulated PPP contained 40-60 nM thrombin throughout. FXa was initially measured in clots at 10 nM. This level declined over time while clot-conditioned PBS accumulated FXa. Exposure of human aortic SMC to clots or native unclotted PPP for 24h only marginally influenced SMC apoptosis but increased mitogenesis over 15-fold. This was reduced by all 4 inhibitors. Clot-stimulated induction of TNFα and interleukin-6 mRNA was also attenuated by the inhibitors. Denatured PPP (no protease activity) increased SMC mitogenesis to a level seen in SMC exposed to clot and combined hirudin + DX9065a, reflecting the well-known mitogenic actions of serum alone. Conclusion: Coagulation of human plasma generates nanomolar amounts of thrombin and FXa, sufficient to stimulate the proliferative and inflammatory properties of adjacent SMC. Our observations validate the use of purified thrombin and FXa at nanomolar concentrations for in vitro studies, and support the individual and coagulationindependent roles of these proteases in cell proliferation and inflammation. Antithrombotic therapy with argatroban or rivaroxaban may limit the cellular effects of clot-derived thrombin and FXa, while normal anti-platelet therapy would not. This aspect should be considered in the clinical use of these agents, specifically in healing processes after vessel injury.

RhoGEF17 mediates cGMP/cGK induced adherence and relaxation of vascular smooth muscle cells Rauch J. 1 , Stephan-Schnatz K. The guanine nucleotide exchange factor RhoGEF17 is the only GEF known so far to be directly activated by cGMP-dependent kinase. It is expressed in various types of smooth muscle cells and has been shown to play a role in the regulation of cell integrity. In a previous investigation we showed that the knockdown of RhoGEF17 by a shRNA approach caused a loss of actin stress fibers and a subsequent change of smooth muscle cell morphology that finally resulted in cell rounding. We now provide evidence that the expression level of RhoGEF17 influences the re-attachment of cultured rat aortic smooth muscle cells (RASMC) to a surface after detachment. Although RhoGEF17 depleted RASMC were still able to adhere and spread, their cell surface area remained considerably smaller than that of control cells in the first 24 hours after seeding. Cell counting revealed that 6 to 12 hours after seeding the percentage of adherent cells was significantly lower in the RhoGEF17 knockdown group compared to the control group.

These data indicate a delay in attachment. Interestingly, the knockdown of RhoGEF17 was paralleled by a loss in RhoA and cadherine expression. As RhoGEF17 mediated a cGMP-induced activation of the small GTPase RhoA in RASMC, we studied the effect of a stable cGMP analogon (8-pCPT-cGMP) on the adhesion process. In accordance with previously published data, cGMP treatment accelerated the attachment of RASMC to the surface within the first 12 hours. In contrast, the adhesion of RASMC after RhoGEF17 knockdown was no longer stimulated by cGMP. As these data indicate that RhoGEF17 mediates cGMP-dependent signalling in a physiological process we wondered whether this protein might also play a role in the regulation of cGMPdependent relaxation of vascular smooth muscle cells. Thus, we performed a collagenbased contraction assay with RhoGEF17 depleted RASMC. While the contraction in response to serum was not affected by the depletion of RhoGEF17, we observed a slight decrease in basal contractility. Interestingly, cGMP was not able to counteract the serum-driven contraction of RhoGEF17 knockdown cells. There was no cGMP-induced relaxation in these cells. We conclude that RhoGEF17 is involved in the cell adhesion of vascular smooth muscle cells and likely promotes the expression of specific proteins. Its activation is required to mediate cGMP-induced signalling in terms of vascular smooth muscle cell adherence and relaxation. Human eosinophil and neutrophil granulocytes are cells of the innate immune system. They both express formyl peptide receptors (FPR) und histamine H2 receptors (H2R). Activation of FPR leads to a release of reactive oxygen species (ROS). H2R activation results in an increase of intracellular 3'-5'-cyclic adenosine monophosphate (cAMP) concentration and inhibition of FPR-mediated ROS release via adenylyl cyclase activation. In this study we compared the effects of various H2R ligands on cAMP accumulation and formyl peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)induced ROS release in isolated eosinophils and neutrophils. cAMP concentration was determined by HPLC/tandem mass spectrometry, and ROS release was assessed by monitoring superoxide dismutase-inhibitable reduction of ferricytochrome C. In eosinophils, histamine, amthamine and 5-methylhistamine exhibited similar potencies and efficacies with regard to cAMP accumulation and inhibtion of ROS release. In marked contrast, in human neutrophils, we observed dissociations in potencies and efficacies of ligands at increasing cAMP accumulation and inhibition of ROS production. Our data suggest that in human eosinophils, but not neutrophils, cAMP mediates inhibtion of ROS production. In a broader context our data provide a compelling example of the context-dependency of the pharmacological properties of G-proteincoupled-receptors. Specifically, one has to be cautious when extrapolating experimental observations from one cell type to another, even when they are very closely related to each other. Comonomers and monomers are used as dental restorative materials (e.g. in dental composites). Unconverted compounds can be released from dental composites and can enter the body in humans. Comonomers can induce various side effects in humans. This study was evaluated to qualify and to quantify eluted compounds from various dental composites. Following composites were tested (producer in parentheses): Els extra low shrinkage (Saremco), Synergy Duo Shade (Coltène), Grandio (VOCO), Tetric Evo Ceram (Vivadent), Venus (Kulzer), Gradia (G.C.), and Premise (Kerr).Polymerized composites (100 mg) were incubated in GC vials with 1 ml dest. water or 1 ml methanol, each at 37 °C for 72 hours. Aliquots were taken, and eluted compounds were analyzed with the method of gas chromatography/mass spectrometry (GC-MS) and liquid chromatography/mass spectrometry (LC-MS).From all composites 18 different compounds were found. Following comonomers were quantified (µg/ml; mean ± s.d.; n=4)( fig. 1 ).Following range of the eluted and detected comonomers from dental composites was found (dest. water; decreasing elution): Venus > Gradia > Synergy Duo Shade > Tetric Evo Ceram Premise > Grandio > Els Extra low shrinkage. * n.d. = not detectable (below limit of detection). Triphenylstibane was detected in Tetric Evo Ceram (5 ± 2 µg/ml).

Reimann C., Lupp A., Schulz S. Institut für Pharmakologie und Toxikologie Universitätsklinikum Jena, Drackendorfer Str.

Objective: CXCR4 is a plasma membrane chemokine receptor which is involved e.g. in organogenesis, hematopoesis and inflammation. In the adult organism CXCR4 is physiologically expressed on various cell types, in particular on lymphocytes. With respect to neoplastic tissues, in the current literature an over-expression of CXCR4 is described in different types of tumors, especially in breast and prostate cancer. Additionally, an involvement of CXCR4 in tumor metastasis is discussed. Subsequently, detection of CXCR4 expression in a given human tumor sample would provide a valuable predictive information on disease prognosis and possible therapeutic intervention. However, previous attempts to localize CXCR4 using poorly characterized mouse monoclonal or rabbit polyclonal antibodies have yielded predominant nuclear and occasional cytoplasmic staining, but did not result in the identification of cell surface receptors. Thus, the aim of the present study was to reassess the CXCR4 expression in a panel of formalin-fixed and paraffin-embedded human normal and neoplastic tissue samples by means of immunohistochemistry using the well characterized novel rabbit monoclonal anti-CXCR4 antibody UMB-2. Methods: In comparison to negative and positive control samples (CXCR4-knockout and wild-type mouse embryos) the extent of staining in the different normal and neoplastic tissue specimens was scored from zero (no expression) to three (high expression). Results: CXCR4 was found to be expressed in all neoplastic tissue entities analyzed. In many cases, the receptor was predominantly localized at the plasma membrane of the tumor cells. However, in all CXCR4-expressing tumor entities a huge interindividual variability both in the percentage of positive cells and in the intensity of staining was noted which strongly differed also between the various types of cancer. The most intense (score three) staining was found in the samples of (small cell) lung cancer, ovarian cancer and of pheochromocytoma. Additionally, lymphatic organs such as lymph nodes, spleens and tonsils were CXCR4 positive, with mainly B cells displaying a distinct staining of the plasma membrane.

The rabbit monoclonal antibody UMB-2 may prove to be of great value in the assessment of CXCR4 expression in different human tumor entities and of the mechanisms underlying the formation of metastases, thus helping to find new targets and strategies in cancer therapy.

Link between β2-adrenoceptor-mediated inhibition of formyl-peptide-induced O2 β2-Adrenoceptor (ADRB2)-agonists are in daily use for asthma therapy. Although most cases of asthma are controlled by standard medication, a subpopulation of asthmatics remains difficult to treat. The ADRB2 gene contains a total of 49 polymorphisms. This variability could cause part of the ~70 % genetically-determined differences in therapy response. Genetic and corresponding functional data on ADRB2 can help to understand the complex disease and, in cases of severe asthma, optimize therapy with ADRB2agonists for each individual. (Ortega et al. 2007; Chung et al. 2011) Our present study connects sequence data with pharmacological data of prototypical ADRB2-ligands, namely, (R)-isoproterenol, (R,R)-and (S,S)-fenoterol, (R)-and (S)salbutamol and (R,R)-formoterol. As a pathophysiologically relevant cell type, we analysed human neutrophils from peripheral blood of healthy volunteers. Formyl-peptide-induced O2 .production and its inhibition by the agonists are examined in a 96-well cytochrome-c assay.

Characteristic pharmacological values (pIC50, Emax) are obtained for each individual. The data-set for each individual is supplemented by a differential blood cell analysis and an asthma-related questionnaire. Most importantly, each volunteer's ADRB2-sequence variant is determined by Sanger-sequencing. Complete determination of a 1,490 bp sequence, including the entire ADRB2 exon and part of the flanking 5'-and 3'-untranslated regions, allows mapping of the most common, but also of new or rare polymorphisms. First data demonstrate the inter-day and intra-individual robustness of the functional data. In the next step, we will link sequence variants and functional differences within a population of sixty volunteers with sufficient statistical power. Collectively, this study represents a straightforward approach to link functional and genetic data of a clinically relevant receptor. Cyclic nucleotide phosphodiesterases (PDEs) are classified into eleven families and are essential for second messenger metabolism in human cells. 1 Recently, we have shown that several human PDEs possess a much broader substrate-specificity than previously assumed, being capable of hydrolyzing not only the purine nucleotides cyclic adenosine 3',5'-monophosphate (cAMP) and cyclic guanosine 3',5'-monophosphate (cGMP), but also pyrimidine nucleotides such as cyclic uridine 3',5'-monophosphate (cUMP). 2 These data were obtained using a highly sensitive HPLC mass spectrometric assay which is quite expensive and whose technical requirements are available only in few laboratories. In our present study we developed a fluorimetric PDE activity assay using 2'-O-(N'methylanthraniloyl) (MANT)-substituted cyclic purine and pyrimidine nucleotides that can be used more broadly in the scientific community. Human PDE3A is important for the regulation of platelet aggregation, oozyte maturation, vascular smooth muscle relaxation and contractility of cardiac myocytes. 1 Moreover, this PDE shows a broad substrate-specificity, hydrolyzing cAMP, cGMP, cUMP and cyclic inosine 3',5'-monophosphate (cIMP). 2 Using this enzyme, here, we demonstrate that various MANT-substituted cyclic nucleotides are substrates of PDE3A and undergo a significant change in fluorescence whilst being hydrolyzed, thus allowing a quantitative analysis of catalysis via fluorescence detection. In fact, not only native cUMP but also MANT-cUMP is a substrate of PDE3A. This finding is consistent with data published by Hardman and Sutherland 3 who described a cUMP-degrading PDE activity in homogenates from beef and dog heart, leading to the assumption that the PDE activity described there could be attributed to PDE3A. As cUMP has furthermore been proven to be present in mammalian cells 4 , to differentially activate cAMP-and cGMP-dependent protein kinases 5 and to be synthesized from UTP by mammalian soluble guanylyl cyclase 6 , our present study supports the hypothesis that this cyclic nucleotide could play an important role in cell metabolism. The newly established fluorescence assay with MANT-cUMP facilitates future studies on PDE3A and the assumed second messenger function of cUMP. RhoA is reportedly involved in STAT-dependent transcription. However, the pathway connecting the GTPase and STAT signaling has not been characterized. We made use of bacterial toxins, which directly activate Rho GTPases to analyse this pathway. Cytotoxic necrotizing factors (CNFs) are produced by pathogenic Escherichia coli strains and by Yersinia pseudotuberculosis. They activate small GTPases of the Rho family by deamidation of a glutamine, which is crucial for GTP hydrolysis. We show that RhoA activation leads to phosphorylation and activation of STAT3 and identify signal proteins involved in this pathway. RhoA-dependent STAT3 stimulation requires ROCK and JunKinase activation as well as AP1-induced protein synthesis. The secretion of one or more factor/s activate/s the JAK-STAT pathway in an auto/paracrine manner. We identify CCL1/I-309 as an essential cytokine, which is produced and secreted upon RhoA activation and which is able to activate STAT3-dependent signaling pathways. The knowledge about the connection between RhoA and STAT signaling is crucial for understanding several deseases, especially cancer.

Acid sphingomyelinase-deficient mice are protected from the lethal cardiovascular effects in TNF-induced septic shock Reiss L. K. 1 Christian-Albrechts Universität Institut für Immunologie, Michealisstrasse 5, 24105 Kiel, Germany Introduction: The cytokine tumor necrosis factor (TNF) is a mediator of septic shock. Sepsis is a major cause of acute respiratory distress syndrome (ARDS), a heterogeneous lung disease with a mortality of about 50%. The present study was designed to investigate the effects of high systemic TNF-levels on the lung and on the systemic circulation in wildtype and acid sphingomyelinase-deficient (ASM -/-) mice. The enzyme acid sphingomyelinase generates the signaling molecule ceramide that plays a critical role in edema formation and vasodilatation. Material and Methods: ASM -/and wildtype mice were ventilated mechanically at VT=8mL/kg and f=180min -1 with FiO2=0.3 and PEEP=2cmH2O while lung mechanics were followed. Half of the mice received 50µg of murine TNF intravenously. Blood pressure was stabilized by intra-arterial fluid support and body temperature was kept at 37°C to prevent lethal shock and to allow investigation of blood gases, lung histopathology, pro-inflammatory mediators and microvascular permeability 6 hours after TNF application. Results: TNF induced septic shock in wildtype mice, as indicated by metabolic acidosis, high serum levels of the sepsis marker procalcitonin, decreasing blood pressure and reflex tachycardia. Interestingly, ASM -/mice were protected from the TNF-induced cardiovascular effects and mortality. In the present study, circulating TNF failed to cause lung injury. Lung mechanics stayed stable during ventilation in all groups and also pulmonary histopathology, cytokine levels and microvascular permeability were unaffected. Conclusion: Circulating TNF alone is not sufficient to cause acute lung injury. We conclude that the cardiovascular effects in TNF-induced septic shock are partly mediated by acid sphingomyelinase.

Cyclophilin A siRNA provides mitoprotection and prevents AIF-dependent neuronal cell death Reuther C. 1 Cyclophilin A (CypA) is a peptidyl-prolyl-cis-trans isomerase which is localized in the cytosol. Recent data suggested that neuronal cell death involved cytosolic CypA translocation to the nucleus, where it formed a pro-apoptotic complex with apoptosis inducing factor (AIF). This CypA-AIF complex induced caspase-independent chromatin condensation, DNA degradation and cell death in various paradigms of apoptosis. On the basis of these data, the selective inhibition of the AIF-CypA complex was proposed as a potential strategy to prevent AIF-dependent cell death in neurons. Therefore, the aim of this study was to determine effects of CypA silencing in a model of glutamate toxicity in immortalized hippocampal HT22 neurons. First, we addressed the interaction of AIF and CypA by immunoprecipitation and their translocation to the nucleus by immunohistochemistry and confocal fluorescence microscopy. After exposure of HT-22 cells to glutamate the translocation of AIF and Cyp A occurred prior to cell death. CypA siRNA attenuated glutamate-induced cell death as detected by the MTT-assay, impedance measurements (xCELLigence system), and by FACS analysis after Annexin V/ Propidium Iodide staining. Most intriguingly, CypA siRNA also preserved the mitochondrial membrane potential as shown by FACS analysis after TMRE staining. Further, confocal microscopy showed that CypA silencing prevented mitomorphology alterations and blocked the release of mitochondrial AIF to the nucleus. The inhibition of the AIF translocation to the nucleus was also shown by Western blot analysis. In summary this study demonstrates that silencing of CypA prevents mitochondrial disruption and attenuates glutamate toxicity in vitro. Thus, CypA is a promising target for mitoprotection as a basis for novel strategies of neuroprotection. Up to now the question is unresolved how the ingested dose influences the absorption and metabolism of chlorogenic acids (CGA) from food. So far no studies have been performed on the impact of the dose on CGA absorption, circulation and excretion. Recently we performed a dose-response study in a randomized, double-blinded, crossover design with five ileostomy subjects. In three trials the volunteers consumed after a two day polyphenol free diet coffee with varying CGA content (HIGH 4525 µmol; MEDIUM 2219 µmol; LOW 1053 µmol). The CGA concentrations in plasma, ileal effluent and urine were subsequently identified and quantified by HPLC-ESI-MS, HPLC-ESI-MS/MS and HPLC-DAD. The results showed that the consumption of higher CGA concentrations lead to a faster ileal excretion measured in the ileal effluents. This corresponded to the renal excretion of 8.0 ± 4.9% (HIGH), 12.1 ± 6.7% (MEDIUM) and 14.6 ± 6.8% (LOW) of total CGA and metabolites. We found that CGAs with a caffeic acid moiety are predominantly sulphated and those with a ferulic acid moiety are predominantly conjugated via glucuronidation prior renal excretion. Furthermore, in the ileal effluents, sulphation of both structural units dominated. In plasma samples (after enzymatic deconjugation) the AUC values were determined by the major CGA classes in coffee, the caffeoylquinic acids: 551.5 ± 93.8 nM*h -1 (HIGH); 299.3 ± 79.6 nM*h -1 (MEDIUM) and 222.8 ± 91.3 nM*h -1 (LOW). No major differences in the metabolic pattern were observed. Additionally, we were able to identify new metabolites of CGA in urine and ileal fluids.

We conclude that the consumption of high concentrations of CGA via coffee might influence the gastrointestinal transit time and consequently affect CGA bioavailability.

This study was supported by the Nestlé Research Centre (Lausanne, Switzerland).

Interaction of antagonists with the ATP binding pocket at the human P2X3 ion channel Helms N., Riedel T., Illes P. Rudolf-Boehm-Institut für Pharmakologie und Toxikologie, Härtelstraße 16-18, 04107 Leipzig, Germany

The homomeric P2X3 receptor (P2X3R) is a rapidly activating and desensitizing cation channel, gated by extracellular ATP. It consists of three homomeric subunits. This representative of the P2X receptor family is highly expressed on sensory afferent neurons and plays a significant role in chronic pain, bladder reflexes and taste sensation. Therefore, the development of selective antagonists for P2X3 receptors and knowledge about the binding of these antagonists are of great significance for future pain therapy and therapy of urge incontinence. To simulate the shape of the rapidly desensitizing agonist-induced current responses via P2X3 receptors, we created a specific Markov model to describe the binding of agonists and competitive antagonists. This model can be used to prove the competitive character of inhibition and to calculate the association and dissociation constants of the antagonists. Furthermore we use this model to fit current responses at P2X3 wild type receptors and their mutants to α,βmethylene ATP in the presence of different antagonists. Whole-cell patch-clamp recordings were performed on HEK 293 cells, heterologously expressing the human P2X3 receptor, to determine the concentration-response relationship of different antagonists. By applying increasing concentrations, differences of antagonist potency could be observed at the wild type receptor. Afterwards, we chose amino acid residues for replacement by alanine, which seem to be important for agonist binding and should be so for competitive antagonist binding as well, based on our homology model, developed from the zebrafish P2X4R crystal structure and previous mutagenesis studies. We intend to identify those amino acids which are important for competitive antagonist binding by monitoring the altered antagonist potency on the mutated receptor when compared with the wild-type receptor.

Analysis of the P2X3 agonist binding site by double mutant cycles Riedel T., Wiese S., Illes P. Rudolf-Boehm-Institut für Pharmakologie und Toxikologie, Härtelstraße 16-18, 04107 Leipzig, Germany Purinergic P2X receptors belong to the family of ligand-gated ion channels. They are non-selective cation channels, activated by extracellular ATP. One of the seven members of the P2X receptor family, the P2X3 receptor, is localized at the plasma membrane of sensory neurons and is involved in pain perception. Therefore, this receptor is a possible target for new drugs in pain treatment. The development of such drugs can be supported by an exact knowledge of the receptor structure and function. There are many hints to the ATP binding site, but the interaction of the P2X receptor with its agonists and antagonists remains still unknown. In this study, we investigated the effects of single alanine substitutions of amino acid residues in the supposed ATP binding site of the homomeric human P2X3 receptor on the effect of nucleotide analogues. The mutant receptors were expressed in HEK293 cells and the nucleotide effects were measured by means of the whole-cell patch-clamp method. Modifications in the receptor binding site changed the concentration-response dependency as well as the current kinetics during fast pulsed agonist applications. Based on this fact, we were able to distinguish binding from gating, conductance, and desensitisation, using a Markov model that describes the complete channel behaviour by a matrix of rate constants. The results were also checked for consistency with a structural hP2X3 model that we developed from the known zebra fish P2X4 crystal structure in the closed state.

Voltage-dependent modulation of alpha2A adrenergic receptor signaling Rinne A., Birk A., Bünemann M. Philipps-Universität Marburg Institut für Pharmakologie und Klinische Pharmazie, Karlvon-Frisch Str. 1, 35034 Marburg, Germany G protein-coupled receptors (GPCRs) are proteins that regulate numerous signaling pathways by activation of intracellular G proteins. GPCRs are activated by extracellular stimuli, such as light, hormones and neurotransmitters. Recent evidence suggests that some GPCRs exhibit voltage-sensitivity leading to a modulation of their activity by the membrane potential (VM). We used a FRET-based biosensor of the α2A adrenergic receptor to analyze receptor activation at defined membrane potentials in HEK 293 cells by means of voltage-clamp recording. The biosensor was stimulated either with the partial agonist clonidine or with the full agonist norepinephrine (NE) and receptor activation was measured as decrease in the ratio of acceptor-/donor-fluorescence. Receptor stimulation by NE was inhibited at depolarizing membrane potentials but enhanced by hyperpolarization. Inhibition of NE activated receptors was strong at low concentrations (500 nM: 60 % inhibition) but almost absent at saturating agonist concentrations (100 µM: 9 % inhibition). Both agonist-induced and hyperpolarizationinduced receptor activation exhibited a similar monoexponential time course and speed of activation was primarily dependent on agonist concentration for both activation modes. The latter indicates that depolarization lowers the apparent affinity of the NE receptor interaction and thus causes receptor deactivation by means of NE release. Application of clonidine (1 µM, VM=-90 mV) resulted in a FRET response that was inhibited by 40 % at +60 mV. In contrast to NE, strong receptor inhibition at +60 mV was present even at super-saturating concentrations of clonidine (100 µM), suggesting that voltage alters the equilibrium between active and inactive conformations of the receptor. Voltage-dependence of the a2A adrenergic receptor also modulated downstream receptor signaling: G protein activation or the recruitment of arrestins, which we determined in FRET assays that directly detect Gαi protein activation or receptor-arrestin interactions, were both substantially inhibited at VM = +60 mV. Therefore we conclude that negative membrane potentials promote active conformations of the a2A adrenergic receptor, increase affinity of full agonists and enhance receptor signaling. In conclusion the present data show that SCC cells extrude more HA, possibly related to increased levels of HAS3, in comparison to keratinocytes. Increased amounts of HA appear to be essential for the UVB induced tumourgenesis of SCCs in mice. This effect might be related to the pro-proliferative property of high molecular weight HA. Furthermore biological active HA fragments derived from HA degradation by hyaluronidases (Hyal1,2) are thought to be pro-angiogenetic, anti-apoptotic and proinflammatory, thus possibly also promoting tumour growth and malignancy.

Estradiol induced paracrine release of EGF from keratinocytes protects the dermal hyaluronan/versican matrix during photoaging Röck K. 1 , Meusch M. Hyaluronan (HA) and versican are key components of the dermis and are responsive to UVB induced remodeling. The aim of the present study was to investigate the molecular mechanisms of estrogen (E2) mediated effects on HA-rich ECM during actinic aging. 10 weeks of UVB irradiation (3 x 1 MED (80 mJ/cm 2 ), weekly) of hairless Skh-1 mice caused a marked decline of dermal HA, which was aggravated by ovariectomy (OVX). Subcutaneous substitution of estrogen (E2) by means of controlled release pellets abolished these effects confirming the stimulatory role of E2. The increase of dermal HA correlated with induction of HA synthase HAS3 by E2. In addition the HA-binding proteoglycan versican was induced by UVB and further increased by E2. However in cultured skin fibroblasts E2 reduced the expression of versican and had no effect on HAS3. Therefore, direct upregulation of HAS3 and versican in fiborblasts by E2 was excluded. However, E2 increased the expression of EGF in UVB irradiated skin in vivo and in keratinocytes in vitro. EGF in turn upregulated the expression of HAS3 and versican in dermal fibroblasts. Furthermore the supernatants of Estradiol treated keratinocytes led to the same effects in dermal fibroblasts, which could be abolished by previous treatment of the supernatant with neutralizing EGF antibody or treatment of the fibroblasts with EGF receptor blocker erlotinib. Functionally, dermal HA and versican induction by E2 correlated positively with proliferation and negatively with accumulation of inflammatory macrophages in the dermis. Collectively these data suggest that E2 treatment increases the amount of dermal HA and versican via paracrine release of EGF which may be implicated in the pro-proliferative and anti-inflammatory effects of E2 during photoaging.

Differential pro-and eukaryotic toxicity of silver released from nanocomposite surfaces increases the therapeutic window of silver in antibacterial treatments Röhl C. 1 , Hrkac T. Silver has been used since ancient times as antimicrobial agent. Recently, silver gained new attention due to its higher effectiveness in its nanoform. This led to new developments of silver nanomaterials, e.g., for medical devices and consumer products. Though, it is generally assumed that silver is less toxic for eukaryotes than for prokaryotes, concern is raised, if nanosilver at the same time might also increase mammalian cytotoxicity. In our study we examined the toxicity of silver released from nanocomposite surfaces with that of silver from AgNO3 solutions for adherent bacteria and mammalian cells. Therefore, we established an in vitro reference system which enabled us to compare the therapeutic window between prokaryotic toxicity and eukaryotic integrity in both exposure settings. We focussed especially on the comparability of the bacterial and mammalian cell systems and the development of characterized Ag/TiO2 nanocomposite coatings with well-defined silver filling factors and silver surface release, which could be varied over a wide concentration range. As reference cells the E. coli SAR 18 strain and human dermal fibroblasts, which are of special relevance in the context of medical devices like implants or wound dressings, were chosen. Bactericidal effects were determined by direct growth visualization of the GFP-producing E. coli strain by epifluorescence microscopy. Mammalian cell growth and toxicity was determined by the MTT assay, protein measurements and phase contrast microscopy. The Ag/TiO2 samples were prepared by sputter co-deposition from two separate magnetron sources. The silver surface concentration release was determined by XPS and the silver release by ICP-MS. In solution a concentration-dependent constant silver concentration could be determined between 2 and at least 72 hours at the surface. While lowest bactericidal and cytotoxic concentrations of Ag + from AgNO3 solutions with 0.64 and 0.95 mg/cm 2 , respectively, differed only slightly, the therapeutic window increased significantly if Ag + was released from the nanocomposite surface. While the toxicity on the fibroblasts was unchanged the bactericidal potency increased at least one order of magnitude. Taken together, it can be concluded that local exposure factors i) can be modulated by silver nanocomposites and ii) play an important role for the differential toxicity of surface silver on bacteria and mammalian cells. Charite -Universitätsmedizin Institut für Integrative Neuroanatomie, Funktionelle Zellbiologie, Philippstr. 2, 10115 Berlin, Germany C3 exoenzyme (C3bot) a clostridial ADP-ribosyltransferase does not possess a cellbinding/-translocation domain. Nevertheless, C3 is able to efficiently enter intact cells, including neuronal cells but the mechanism of uptake is not yet understood. In the present work, binding of C3bot to the hippocampus-derived HT22 cell line was characterized by means of binding and blot overlay assays as well as mass spectrometry analysis to identify binding partners of C3bot. The binding assays established that C3bot bound in a concentration-dependent manner to HT22 cells. In the overlay assay we detected one clear band of 55 kDa. To elucidate whether glycosylation is important for the C3bot-protein interaction, HT22 cells were incubated with glycosidase F resulting in a decreased binding of C3 to the 55 kDa band. To explore the involvement of phosphorylation in the binding of C3 to the putative binding protein, blot was pre-treated with CIP (Calf Intestinal Phosphatase) before overlay with C3bot. Pretreatment greatly reduced the C3bot-protein interaction. Moreover, inhibition of dephosphorylation by vanadate before in intact cells showed an increased level of C3botprotein interaction in the following overlay. Thus, interactions between C3bot and HT22 cell proteins may require phosphorylation. To further characterize the 55 kDa band as binding target of C3bot, the 55 kDa band was digested with trypsin and then subjected to LC-Orbitrap mass spectrometry analysis. From this 55 kDa single gel band 141 proteins were identified. Further analysis of the identified proteins will provide a possible interaction partner of C3bot. In sum, protein overlay assays revealed that phosphorylation and glycosylation are critical for efficient C3bot-protein interaction. S76 335 Solvent effects on enzyme kinetics in vitro Rokitta D., Pfeiffer K., Gerwin H., Streich C., Fuhr U. Uniklinik Köln Institut für Pharmakologie, Gleueler Str. 24, 50931 Köln, Germany Kinetic parameters provide essential quantitative information for characterisation of drug metabolising enzymes. Such enzymes are located in an a partially queous environment, but to solve potential lipophilic substrates for in vitro measurements organic solvents are regularly needed. To preserve the enzymes from denaturation and other solvent related effects, the concentration of these solvents must be kept low. Data on nature and extent of such solvent effects is sparse. In this study, we investigated the effects of methanol, ethanol, acetonitrile and dimethylsulfoxide (1% to 4%) on the assessment of k m, Vmax and Clint with regard to the 1-hydroxylation of midazolam via CYP3A4 and the CYP1A2 catalyzed metabolism of caffeine to paraxanthine in vitro. The presence of acetonitrile showed the highest Vmax value for paraxanthine formation but the lowest values for 1-hydroxymidazolam formation. The km value for midazolam showed no systematic effects of organic solvents, while for caffeine km was up to eightfold lower for solvent free samples compared to solvent containing samples. The present example suggests that the presence of organic solvents may considerably influence enzyme kinetic parameters beyond a mere change in apparent activity. These effects are differing between enzyme-substrate systems and solvents. It remains to be determined to which extent such effects compromise in vitro -in vivo extrapolations, and which solvents are most appropriate.

Atrial remodeling and arrhythmia induced by the transcription factor ER81 Rommel C. 1 , Rösner S. Introduction: The transcription factor ER81 (ETS related 81) belongs to the large family of ETS-transcription factors that are involved in developmental processes and in the pathogenesis of cancer. ER81 is activated by Gq-and Gs-coupled receptors leading to a phosphorylation of the transcription factor by MAP-kinases and protein kinase A, respectively. Cardiac ER81 mRNA expression is increased in failing human hearts. However mechanical unloading by a left ventricular assist device leads to normalization of ER81 expression. Thus, the aim of the present study was to investigate the cardiac function of ER81 in genetically modified mouse models.

We previously generated transgenic mice overexpressing ER81 under control of the cardiomyocyte-specific α-myosin heavy chain gene (αMHC) promoter by pronuclear injection and established independent transgenic lines. Electrocardiography (ECG) was assessed in mice at day 5 after birth (P5) and in adult mice (3 months) during isoflurane anesthesia and by ECG telemetry in awake mice, respectively. ECG analysis revealed no differences between the genotypes at day 5 after birth. However, we found a decreased heart rate, a replacement of regular P-waves by an undulating baseline and frequent supraventricular extrasystoles in adult ER81 αMHC transgenic mice. Next, isometric contractile force measurements on isolated left atria were carried out in organ baths. While WT left atria responded to increasing concentrations of isoprenaline, NKH477 and calcium with an increase in contractility, the maximal positive inotropic responses to these substances were severely blunted in ER81 αMHC atria. We performed Western blots to identify potential aberrations of calcium handling and regulatory proteins. Phosphorylation of serine 16 of phospholamban (PLN) was reduced in ER81 αMHC mice. In addition, protein phosphatase 1 (PP1) expression was significantly increased in ER81 αMHC mice, which is consistent with the increased dephosphorylation of phospholamban. Furthermore, we found a decreased expression of calsequestrin and Serca2a protein in ER81 αMHC atria. Electron microscopy revealed the significant structural remodeling of ER81 αMHC atria at 3 months of age. Conclusion: Increased cardiac expression of the ETS-transcription factor ER81 leads to structural and electrical remodeling of the atria. Thus, ER81 may play an important role in the pathogenesis of cardiac arrhythmias in chronic heart failure.

Signal transduction pathway of ATP and UTP in neonatal rat cardiac myocytes Rothkirch D., Gergs U., Neumann J. Institute for Pharmacology and Toxicology Medical Faculty, Magdeburger Str. 4, 06097 Halle (Saale), Germany Extracellular ATP and UTP can be released from the heart during pathological conditions such as ischemia or hypoxia. In humans, ATP and UTP levels are increased during myocardial infarction. ATP and UTP can act via P2-purinoceptors which are further divided in P2X1-7 and P2Y1-14-receptors. As previously shown ATP and UTP can induce inotropic effects in cardiac preparations of mice and man. For rat articular chondrocytes and human intestinal cells it has been demonstrated that the MAPK cascade can be activated by ATP and UTP. Therefore, the cardiac effects of ATP and UTP on force of contraction probably occur via the MAPK pathway. To investigate the signal transduction pathway involved, we studied the effects of ATP and UTP on MAPK phosphorylation in isolated neonatal rat cardiac myocytes using phosphorylation-specific antibodies. 100 µM ATP as well as UTP transiently increased phosphorylation of ERK 1/2 and p38 MAPK with a maximum effect at 5 to 10 minutes after application of ATP and UTP in neonatal cardiac myocytes (n=3 preparations each). The maximum phosphorylation of p38 increased with ATP to 284% ± 68% (p<0.05) at 10 minutes and with UTP up to 204% ± 21% (p<0.05) at 5 minutes. The phosphorylation with ERK 1/2 MAPK increased with ATP to 234% ± 46.5% (p<0.05) at 5 minutes and to 337% ± 27% (p<0.05) with UTP at 10 minutes of basal values, respectively. After 20 minutes, predrug values of MAPK phosphorylation were reached again. In summary, we noted an ATP-and UTP-induced phosphorylation of ERK 1/2 and p38 MAPK in isolated neonatal rat cardiac myocytes. The involved receptor subtype(s) and the link between MAPK phosphorylation and inotropic effect of ATP and UTP need to be elucidated.

HaMeeL: use of eLearning in teaching pharmacology and toxicology -the Halle experience Rulf K. 1 , Gergs U. Introduction: During the past three years, our faculty has started to integrate items of eLearning into the standard curriculum of a classical medical school: the "Hallesches Medizinisches eLearning -HaMeeL". Our hypothesis was that these new eLearning tools would improve the willingness of students to spend more time into learning and this would lead to an improved outcome (in multiple choice tests).

Methods: Hence, we offered medical students (5 th or 10 th semester) additional learning environments. The courses for students (experimental pharmacology and toxicology or clinical pharmacology) were existed of a weekly lecture and in addition tutorials (problem-based-learning style, paper cases) or classical seminars. Furthermore, we offered the possibility to use an online multiple choice quiz (involving 8 -12 previously used tests) and/or an online module on heart failure each week. We used the learning management system ILIAS software in combination with the content management system Stud.IP. All students were subjected to an introductory test (to assess knowledge prior to our teaching section and allowing us to exclude a conceivable bias due to previous knowledge, involving basic items from prior teaching opportunities), a mid-term test and a final test to assess gain of knowledge. A maximum of 60 points could be obtained as a sum of both tests. Results: In the means 40% of students used the new eLearning tools (quizzes, heart failure module). However, there was no association between the use of self-assessment quizzes and examination results. The usage of the online quizzes increased in the periods before the exams. However, usage of the heart failure module was accompanied by significantly increased scores in exams. Moreover, in a formalized evaluation system, students positively commented on our eLearning efforts. Conclusions: While usage of our quizzes did not improve test marks, another more sophisticated clinically oriented eLearning module seemed to be improving test outcomes marginally.

Targeting of ERK Thr188 phosphorylation attenuates cardiac hypertrophy but preserves the anti-apoptotic effects of ERK1/2 Ruppert C., Vidal M., Lohse M. J., Lorenz K. Institut für Pharmakologie und Toxikologie Pharmakologie, Versbacher Str. 9, 97078 Würzburg, Germany Background and aims: The extracellular regulated kinases 1 and 2 (ERK1/2) play an important role in cardiac hypertrophy and cell survival. ERK1/2 are phosphorylated at the so-called TEY motif, which in turn activates ERK1/2. Hypertrophic stimuli lead to an additional autophosphorylation threonine188 (Thr188). This autophosphorylation of ERK1/2 stimulates activation of nuclear ERK targets, which are known to induce hypertrophy. The aim of this study is to investigate whether specific targeting of ERK Thr188 phosphorylation affects both ERK functions -ERK mediated hypertrophy and cardioprotective cell survival. Methods and results: For the analysis of cardiomyocyte hypertrophy in vitro, we stimulated cardiomyocytes with phenylephrine and measured the incorporation of tritiated isoleucine. Cardiac hypertrophy was assessed by echocardiography before and after transverse aortic constriction (TAC). For analysis of cell survival, caspase activity and DNA fragmentation was determined upon hydrogen peroxide stimulation in vitro and in response to TAC in vivo. To differentiate between inhibition of ERK1/2 activity and prevention of ERK Thr188 phosphorylation, we either inhibited ERK activity with PD98059 or overexpressed a mutant of ERK2, which cannot be phosphorylated at Thr188 phosphorylation. While inhibition of overall ERK activity with PD98059 attenuated cell survival and hypertrophy in vitro, specific targeting of ERK Thr188 phosphorylation by overexpression of the phosphorylation deficient mutant (ERK2 T188A ) attentuated phenylephrine induced hypertrophy, but preserved the anti-apoptotic effects of ERK. Cardiac overexpression of ERK2 T188A significantly reduced TAC-induced hypertrophy compared to wild-type ERK2 overexpressing mice. In line with the in vitro experiments, ERK Thr188 inhibition only prevented hypertrophy in the TAC model without promoting apoptosis. Conclusions: These results show that blockade of ERK Thr188 phosphorylation attenuates cardiomyocyte hypertrophy but preserves anti-apoptotic effects of ERK1/2. Therefore, specific targeting of ERK Thr188 phosphorylation might be a promising strategy for the treatment of pathological hypertrophy. Intracellular cAMP levels are determined by interplay of cAMP formation by adenylyl cyclases and cAMP degradation by phosphodiesterases (PDE). Eleven families of PDEs are known. One of the most recently identified PDEs is PDE10, a PDE in principle capable of hydrolysing cAMP as well as cGMP. PDE10 contains a tandem of so called GAF domains in its N-terminal regulatory domain that mediate activation by cAMP. Because current knowledge about the tissue distribution of PDE10 was mostly based on the analysis of mRNA distribution, we generated antisera against PDE10 to analyze tissue distribution of the protein level. Using these antibodies, we found a prominent occurrence of the enzyme in testis and in brain, where it was confined to the striatum. Thus, PDE10 displays a comparably restricted tissue distribution which is in contrast to that of many other PDEs. Low cAMP levels in so called medium spiny neurons of the striatum have been implicated in schizophrenia. Furthermore, studies using the nonspecific PDE10 inhibitor papaverine as well as specific PDE10 inhibitors suggest PDE10 as a target for the treatment of schizophrenia. Here we set out to analyze the contribution of PDE10 to cAMP degradation in striatum, to identify the physiological pathways PDE10 is involved in and to clarify the functional impact of the proposed phosphorylation of the enzyme.

Identification of cGMP-dependent kinase I substrate complexes Salb K., Schlossmann J. Universität Regensburg Lehrstuhl Pharmakologie, Universitätsstrasse 31, 93053 Regensburg, Germany

The cGMP-dependent kinases (cGKs) are components of the NO/cGMP/cGK-signalling pathway and have a great physiological importance in a multitude of tissues and organs such as smooth muscles and platelets. Two isoforms of the cGKI and the cGKII are known. cGKIα and cGKIβ differ only in their first ~ 100 amino acids which constitute the leucine zipper and the autoinhibitory domains. The N-terminal leucine zipper domains mediate homodimerization of the kinase and the interaction with diverse substrate proteins. Since cGKIα and cGKIβ express different N-termini they interact with different substrates. The cGKIβ isoform is assembled in a macrocomplex at the endoplasmic reticulum (ER) with the intracellular calcium release channel Inositoltrisphosphate receptor I (InsP 3R-I) and the Inositol-trisphosphate receptor associated cGMP kinase substrate (IRAG). We investigated, whether IRAG also interacts with the InsP3R-II and the InsP3R-III in murine platelets and tissues. Additionally, we analyzed the interaction between the 52 amino acid peptide Phospholamban (PLB), which is also located at the ER and regulates the ER calcium reuptake by the sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA), and the two cGKI isoforms. We performed cGMP-agarose experiments with murine WT and IRAG-KO platelets to examine the IRAG-InsP3R interactions. The InsP3R-II isoform was neither bound to cGMP-agarose nor detected in the anti-IRAG immunoprecipitate. On the other hand, InsP3R-III from WT but not from IRAG-KO platelets was bound to cGMP-Agarose. Hence, InsP3R-III interacts directly with IRAG but not with cGKIβ in murine platelets. However, in colon smooth muscle lysate, InsP3R-III not only interacted with the IRAG protein but was also detected in the anti-cGKIα-immunoprecipitate. Phospholamban from WT and IRAG-KO platelets was also bound to cGMP-agarose. Subsequent immunoprecipitation experiments with the respective antibodies against the two cGKI isoforms revealed that PLB interacted both with cGKIβ and cGKIα. These results were supported by analysis of colon smooth muscle tissue from WT and IRAG-KO mice. In conclusion, IRAG interacts with InsP3R-I and InsP3R-III but not with InsP3R-II in murine platelets and colon smooth muscle tissue. Moreover, Phospholamban is an interacting partner of both the cGKIα and the cGKIβ isoform.

The human immunodeficiency virus type 1 enhancer binding protein 1 (HIVEP1) is regulated by proinflammatory stimuli and statins Salomon A. 1, 2 , Schmitz B. Objective: HIVEP1 binds NF-ĸB and other proinflammatory consensus sequences, and is suggested to be involved in inflammatory processes. We recently identified two tagging SNPs, one positioned 90 kb upstream (rs169713) and another in exon 4 (rs2228220) of the HIVEP1 gene, to be replicatively associated with venous thrombosis in GWAs and follow-up studies (AJHG, 2010; PLoS ONE, 2011) . Methods: Total RNA isolation was performed after treatment of vascular endothelial cells (EA.hy926) with proinflammatory cytokines or statins (24h). Serial HIVEP1 promoter deletion constructs were cloned into the pGL3-Basic vector, a potential enhancer fragment, harbouring rs169713C/T, into the pGL3-Promoter vector. In EA.hy926 cells and THP1 monocytes, reporter gene assays were performed by transient transfection and overexpression of transcription factors. ChIP and bandshift assays were performed to identify candidate transcription factors. Results: In EA.hy926 cells, endogenous HIVEP1 expression was increased by proinflammatory cytokines TNFα and IL-1β. Simvastatin (1.2 and 2.4 µM) and atorvastatin (9 µM) -but not pravastatin or aspirin -both dose-dependently decreased basal and TNFα-stimulated HIVEP1 expression. The construct harbouring rs169713T exerted significantly higher transcriptional activity (TA) compared to rs169713C (P<0.001). For an intronic modulator, reporter gene assays demonstrated a regulatory effect on HIVEP1 expression in EA.hy926 and THP1 cells. Cotransfection of SP1 and EGR1 led to an increase in TA, while WT1 exclusively upregulated TA of constructs comprising the intronic modulator. ChIP and bandshift assays combined with specific antibody detection revealed binding of SP1 to the 5'-flanking region and the intronic modulator of HIVEP1. Conclusion: Increased HIVEP1 expression during inflammatory conditions can be repressed by simvastatin and atorvastatin, and not by pravastatin or aspirin. Basal HIVEP1 expression is regulated by SP1 combined in a transcription factor module with EGR1 and WT1 under basal and/or inflammatory conditions. The rs169713 site harbours potential activational capacity for HIVEP1 gene transcription and may communicate with the SP1/EGR1/WT1 module. To date, the treatment of various movement disorders of the central nervous system is still insufficient. In most cases this is due to the sparse knowledge of the pathophysiology. L-DOPA-induced dyskinesias (LID) represent a severe complication of long-time pharmacotherapy in Parkinson's disease that deserves novel therapeutics. An increased activity of striatal projection neurons, which express KV7.2/3 channels, seems to be involved in the pathophysiology of these spontaneous involuntary dystonic and choreatic movements. Previous studies demonstrated an antidyskinetic effect of the KV7.2-7.5 channel opener retigabine after acute and chronic treatment in a rat model of LID. In order to clarify if this effect was based on the modulation of KV7.2/3 channels, we examined the acute effects of the preferred KV7.2/3 channel opener ICA 27243 on LID in this animal model. Four weeks post 6-OHDA lesioning of the left forebrain bundle, dyskinesia was induced by chronic treatment with 10 mg/kg L-DOPA and 15 mg/kg benserazide for 20 days. Three subtypes of dyskinesia (limb, axial and orolingual) were rated according to a score system from 0 to 4 over 180 min. For drug testing, ICA 27243 (5, 10 and 15 mg/kg) was administered intraperitoneal additionally to L-DOPA (or vehicle). Effects of drug action in comparison to vehicle controls were detected by adding up the severity scores of each observation time. Additionally, effects on parkinsonian symptoms were examined 20 min after drug administration using the block and the stepping test. ICA 24273 reduced the severity of dyskinesia significantly at all doses while no negative impact on the antiparkinsonian effect of L-DOPA was observed. Whereas the antidyskinetic effect was restricted to the first 20 min after the application of 5 mg, it lasted up to 110 min in rats treated with 10 mg ICA 27243. A higher dose of 15 mg did not further enhance the antidyskinetic effect. The results of our study suggest that the antidyskinetic effect of the K V7 channel opener retigabine was based on its action on striatal KV7.2/3 channels. In line with the results of previous studies with retigabine, this action does not seem to interfere with the antiparkinsonian effect of L-DOPA.

This study was supported by the Micheal J. Fox Foundation. Background: Skeletal muscle toxicity is the major side effect of HMG-CoA-reductase inhibitors (statins) and can be simulated in engineered skeletal muscle. Statins are known to exert "pleiotropic" effects, e.g. reducing endothelial dysfunction by inducing NO synthases and NO production. The role of NO synthases in skeletal muscle under statin treatment is largely unknown. Interestingly, some skeletal muscle pathologies (e.g. Duchenne muscular dystrophy) may be exacerbated by increased iNOS activity. Here we tested whether or not statin-induced skeletal muscle toxicity would be associated with enhanced NO synthesis.

We generated engineered skeletal muscle (ESM) from rat skeletal muscle cells, matrigel and collagen. ESMs displayed typical skeletal muscle properties (differentiated muscle fibres, tetanic contractions). Under baseline conditions ESM expressed eNOS most abundantly, followed by iNOS and nNOS (n=4-5). Myotoxic Cerivastatin (0.01, 0.1, 1 µM for 5 days) caused a concentration-dependent decrease of contractile force (p<0,05, n=17-20) paralleled by an increase in iNOS transcript (mean±SEM: 0.01 µM 4±0.9-fold, n=3 p<0.05; 0.1 µM 9.3±2.8-fold, n=3 p<0.05) and protein (0.01 µM 5.1±2-fold, n=4 p<0.05; 0.1 µM 7.8±0.8-fold, n=3 p<0.05). Mevalonic acid fully prevented the iNOS increase suggesting that the induction is HMG-CoA reductase-dependent. To test whether iNOS may contribute to the decrease in contractile force we co-treated ESM with 1400W, a specific iNOS inhibitor. We applied 5 µM of 1400W, a concentration found to potently reduce lipopolysaccharide (LPS)induced NO-production in cultured myotubes. However, we did not observe a rescue effect (n=9-15). Also, L-NAME (10 mM), an unspecific NOS inhibitor, did not improve contractile function, instead we observed increased myotoxictiy (n=6-13, p<0.05). To further investigate the role of NO for muscle function we treated the ESMs with increasing concentrations of the NO-donor SNP. Only high concentrations of SNP (10 µM) caused a reduction of contractile force. Combined treatment with cerivastatin and 0.1 µM SNP showed a tendency towards improved force development in ESM. Conclusions: Statins increase iNOS activity in our skeletal muscle model (ESM). However, this does not seem to functionally contribute to myopathy in ESM. Increased production of NO may in fact be a protective measure. ESM may help to dissect clinically relevant functional changes in statin myotoxicity.

Characterization of primary skin fibroblasts of patients with 3M syndrome and mutations in the CUL7 gene Meyer K., Hieber M., Engelhardt S., Sarikas A. Technische Universität München Institut für Pharmakologie und Toxikologie, Biedersteiner Str. 29, 80802 München, Germany

3M syndrome is an autosomal-recessive disorder characterized by pre-and postnatal growth retardation (< -4 SD), facial dysmorphism and skeletal anomalies. The majority of patients harbor missense mutations of the CUL7 (76%) or OBSL1 (16%) gene, respectively. CUL7 constitutes an E3 ubiquitin ligase that is involved in the regulation of the insulin-like growth factor 1 (IGF-1) signaling pathway via ubiquitin mediated degradation of insulin receptor substrate 1 (IRS-1).

To investigate the role of CUL7 mediated IRS-1 degradation in the pathogenesis of 3M syndrome.

Primary skin fibroblasts of seven 3M syndrome patients (six with CUL7 mutations, one with a OBSL1 mutation) and control fibroblasts were analyzed for proliferation rate (cell counter), cell cycle profile (FACS), cell morphology and cellular senescence (histochemistry), IRS-1 protein concentrations and activation of the IGF-1 signaling pathway (Western Blot). The proliferation rate of 3M patient fibroblasts was significantly increased when compared to control cells. In contrast, IRS-1 protein levels and activation of the PI3K/Akt and ERK MAPK pathway were only increased in a subset of 3M cells that carried CUL7 mutations, but not in cells from a patient with the OBSL1 mutation. No significant differences in cell cycle profile, cell morphology or cellular senescence were observed in 3M patient fibroblasts when compared to control cells. To determine the pathogenetic contribution of increased IRS-1 levels to the observed phenotype, human IMR90 fibroblasts were stably transfected with retroviral vectors encoding IRS-1. Despite 20-fold overexpression of IRS-1 compared to empty vector controls, no significant effect of IGF-1 stimulation on proliferation rate or PI3K/Akt and Erk MAPK signaling was observed.

Skin fibroblasts of 3M patients with CUL7 mutations displayed an increased proliferation rate and enhanced activation of the IGF-1 signaling pathways. Despite accumulation of IRS-1 in fibroblasts from a subset of 3M patients with CUL7 mutations, no pathomechanistic role for IRS-1 could be demonstrated. Collectively, our data indicate that a dysregulated IGF-1 signaling may contribute to the pathogenesis of 3M syndrome, yet in an IRS-1 independent manner.

Pharmacases.de -a student-centered eLearning project of clinical pharmacology Zollner B., Berg C., Gros N., Muß N., Oestreicher D., Engelhardt S., Sarikas A. Technische Universität München Institut für Pharmakologie und Toxikologie, Biedersteiner Str. 29, 80802 München, Germany

Pharmacases.de is a novel e-learning website of clinical pharmacology that presents clinically relevant aspects of pharmacology and toxicology in an interactive and multimedial manner.

The aim of the project Pharmacases.de was to develop an innovative concept for creating high quality eLearning content that i) integrates and promotes the theoretical and cooperative skills of final year medical students and ii) is easily adoptable by cooperating institutes and hospitals.

A peer-teaching concept was developed in which final year medical students with the elective pharmacology (PJ Wahlfach Pharmakologie) independently researched and wrote eLearning lessions ("pharmacases"). Subject-specific expertise was acquired by consulting elective students of other disciplines. At present (11/2011) , this "peer network" consists of elective students of nine cooperating institutions (pathology, microbiology, radiology, cardiology, psychiatry, dermatology, neurology, ophthalmology, pediatrics) at the Technische Universität München. The average time for the generation of one eLearning lession by the peer network was 10 days. To date, the website consists of 49 pharmacases that are available to all students online (http://www.pharmacases.de). The website also contains a discussion forum and evaluation form for direct feedback. On average, Pharmacases.de has 1000 visitors per month with the following evaluation results: "excellent": 76%, "good": 15% and "satisfactory": 9% (n=33).

The didactic concept of Pharmacases.de enabled the efficient generation of high quality eLearning content in a student-centered and interdisciplinary manner. The peer-teaching approach supports the collaborative skills of final year medical students and facilitates the transfer of theoretical pharmacological knowledge into clinical practice.

Improved glucose tolerance, less chronic adipose tissue inflammation and reduced adipose tissue mass in mice with adipocyte-specific loss of TAK1 Sassmann A., Offermanns S., Wettschureck N. Max-Planck-Institut für Herz-und Lungenforschung Pharmakologie, Ludwigstr. 43, 61231 Bad Nauheim, Germany TGF-β activated kinase 1 (TAK1) is known to be involved in numerous inflammatory processes by linking receptors for inflammatory stimuli like LPS, interleukin-1 or TNFa to IKK, p38 and JNK activation. Chronic inflammation of white adipose tissue is one of the major causes for the development of insulin resistance and impaired glucose tolerance in states of obesity. To investigate the role of TAK1 in white adipose tissue, we crossed the tamoxifen-inducible white adipocyte-specific Cre mouse line AdipoqCreER T2 with animals carrying floxed alleles of the TAK1 gene. AdipoqCreER

; TAK1 fl/fl animals and Cre negative control littermates are viable and fertile and do not show any developmental defects. After tamoxifen induction and high fat diet feeding adipocytespecific TAK1 knockout mice show improved glucose tolerance and lower fasting insulin levels compared to control animals. In line with this, serum levels of the adipose tissuespecific hormone resistin are reduced in adipocyte-specific TAK1 knockout mice. These findings are accompanied by a lower state of chronic inflammation of adipose tissue as indicated by a dramatic reduction of adipose tissue macrophage number and lower serum levels of TNFα and interleukin-6. Stimuli like TNFα, interleukins and TGF-β released from macrophages and adipocytes are known to promote obesity-related adipose tissue inflammation. When stimulated with these substances TAK1 deficient adipocytes show reduced activation of JNK and p38 which both play an important role in the development of insulin resistance. Interestingly, we observe a lean phenotype in adipocyte-specific TAK1 knockout mice when fed a high fat diet which reflects a reduction of white adipose tissue mass. Currently we are investigating the molecular mechanisms underlying the reduced adiposity and lower state of chronic inflammation in adipose tissue. Growth of small cell lung cancer (SCLC) cells is regulated via the autocrine stimulation of G protein coupled receptors (GPCRs), i. e., neuropeptide and muscarinic acetyl choline (ACh) receptors. The activation of Gq/11 and calcium-dependent GPCR pathways results in the stimulation of ERK signaling which is necessary for the mitogenic effects of neuropeptides or ACh on SCLC cells. In contrast, the role of calcium-independent GPCR signaling and its interplay with Gq/11-regulated pathways in SCLC cells are less well defined. The aim of our studies was to characterize the molecular make-up and the interaction of these pathways, and to delineate the phenotypic effects of calciumdependent and -independent signaling cascades in SCLC cells. Using a panel of SCLC cell lines, we found that the stimulation of neuropeptide receptors led to an increase of calcium which was independent of extracellular calcium and could be prevented by depleting internal calcium stores. This calcium increase was sufficient to activate the tyrosine kinase Pyk2 and subsequently the ERK1/2 cascade. The role of Pyk2 for the growth of SCLC cells was further supported by the fact that inhibition of Pyk2 using a siRNA approach or a novel specific inhibitor, PF431396, exerted pronounced cytotoxic effects on SCLC cells, whereas non-SCLC cells were less sensitive. Interestingly, the inhibition of G 12/13 signaling by siRNA-mediated G(alpha)12 or G(alpha)13 knockdown also markedly reduced the growth of SCLC in vitro or in subcutaneous tumor xenografts, and increased the sensitivity of SCLC cells towards certain cytostatics. To further define the role of calcium-dependent signaling via Pyk2 versus the role of calcium-independent signaling via G12/13, we tested the effect of Pyk2 inhibition in cells with impaired G12/13 signaling. Notably, Pyk2 and G12/13 double inhibition led to an even increased proliferation. Thus, we propose that dysbalanced G protein signaling favoring either Pyk2 activation or G12/13-dependent cascades inhibits the growth of SCLC cells, whereas the parallel inhibition of both pathways restores again the balance and the growth capacity in this tumor entity. Dendritic cells (DCs) are essential for the initial immune response and for the defence against inhalated pulmonary toxins and carcinogens in lung. To differentiate DCs, the cell line THP-1 were used for 7 days and stimulated with various cytokines (IL-4, GM-CSF, TNF-a, Ionomycin). The DCs were characterized by flow cytometry with different typical dendritic cell markers (for example CD11c, CD209, CD83) and by immunfluorescence compared to monocytes. The bronchial tract contains up to 800 DCs per mm² and therefore we established a triple culture model to mimic the situation in vivo. The triple culture consists out of primary human epithelial cells from small bronchi (HBEC) and lung fibroblasts which are cultured under air-liquid conditions on filter membranes for 4 weeks and DCs which were added after the differentiation phase of the bronchial cells. During the cultivation time the HBEC formed an epithelial layer expressing both tight and adherens junctions. They also produced mucus, formed functional cilia with a beat frequency of between 16 to 20 Hz and the transepithelial resistance values were stable between 600 to 800 Ω·cm². Pathomechanisms of pulmonary toxicity in vivo are difficult to investigate, so the tripleculture model is the basis for investigations of the toxic effects at cellular level. Lungtoxic substances such as organophosphates are usually absorbed through inhalation.

Organophosphates are dangerous nerve agents for the human organism. At high concentrations organophosphates damage in the coculture without DCs the cell-cell contacts of the epithelial layer. In the triple culture DCs firstly respond to inhaled organophosphates and seem to compensate effects on the other cells.

In summary, it is very important to understand the pathogenic mechanisms of lung injury in relation to the role of dendritic cells in lung. They could play an essential role in therapy against damage of organophosphates in the lung.

Co-purification of ARF GTPase-activating protein GIT1 and Cavb3 Schalkowsky P., Wissenbach U., Fecher-Trost C., Flockerzi V. Universität des Saarlandes Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Kirrbergerstraße, 66421 Homburg, Germany

High-voltage activated Ca channels are assembled from pore-forming α1 subunits and two distinct types of auxiliary subunits, Cavβ1-β4 and, maybe, α2δ1-δ4. By a Cavβ3-specific antibody based affinity chromatography the Cavβ3 protein was highly enriched from rat brain microsomal membranes. Proteins associated with Cavβ3 were identified by mass spectrometry (LC-ESI-MS/MS) and include α1-subunits, α2δ-subunits and β-subunits. In addition to these expected interacting proteins additional proteins were co-purified with the Cavβ3 protein, including the G protein-coupled receptor kinase-interactor 1 (GIT1). The 770aa GIT1 is a ubiquitously expressed multidomain protein which may serve as a scaffold to bring together molecules to form signaling modules controlling, for example, vesicle trafficking, cytoskeletal organization and cell migration. In rat brain lysates the GIT1 and Cavβ3 proteins were co-immunoprecipitated by the antibodies for Cavβ3 and GIT, respectively. We cloned the GIT1 cDNA from mouse brain and co-expressed it with the Cavβ3 subunit in HEK cells. Like in brain lysates the GIT protein was retained by Cavβ3 precipitated by the antibody for Cavβ3 and Cavβ3 was retained by the GIT1protein precipitated by the antibody for GIT1. Both proteins, Cavβ3 and GIT1 are endogenously co-expressed in mouse embryonic fibroblasts (MEF). We could not observe potassium-induced voltage-activated Ca influx in these acutely prepared cells. Accordingly, MEFs can be used as a model system to study the impact of Cavβ3-GIT1 interaction in the absence of functional Cav channels. In addition, using MEFs from Cavβ3-deficient mice enables us to control the impact of Cavβ3 on GIT1 function. Vice versa down-regulation of GIT1 by specific siRNAs might allow to control the impact of GIT1 on Cavβ3 function. As read-outs we use cell migration assays and monitor receptor-dependent and receptor-independent calcium signaling in these cells.

Effects of sphingosine-1-phosphate and FTY720 on epidermal hyperproliferation and inflammation in an imiquimod induced mouse model of psoriasis The sphingolipid sphingosine 1-phosphate (S1P) is a mediator that modulates various physiological functions of skin cells. S1P has distinct direct effects on keratinocytes as it diminishes proliferation and induces differentiation which is a classical goal of psoriasis therapy. Furthermore, S1P modulates the function of various immune cells, mainly to an anti-inflammatory direction. Thus, the strategy of targeting immune cells with locally acting S1P was explored in an experimental animal model of psoriasis vulgaris, the recently established imiquimod induced psoriasis mouse model and in the mouse tail test. Topical administration of imiquimod onto back and ear skin led to a distinct inflammatory response characterized by epidermal hyperproliferation, scaling and redness which was scored with a modified PASI (psoriasis area and severity index). The positive control diflorasone diacetate and S1P, but not FTY720 reduced the epidermal hyperproliferation by topical administration onto ear skin, indicating a mode of action for S1P via the S1P2 receptor, which is not activated by FTY720. There was also a moderate reduction of inflammatory cell influx and edema formation in ear skin by S1P treatment, which was even more pronounced by treatment with diflorasone diacetate. The PASI determined on back skin was, however, only significantly reduced by diflorasone diacetate. The discrepancy between outcome on ear and back skin remains elusive.

In the mouse tail assay, the influence of S1P in stratum granulosum formation (orthokeratosis) was tested compared to the positive control calcipotriol. Whereas topical administration of calcipotriol led to the expected significant increase of stratum granulosum in mouse tail epidermis, S1P lacked such an effect, indicating a different mode of action in epidermal differentiation. Taken together, these results imply that topical administration of S1P might be a new option for the treatment of mild to moderate psoriasis lesions. Inhalation of toxicants such as sulphur mustard (SM), an alkylating chemical warfare agent, cause pulmonary complications like respiratory failure, pulmonary edema and secondary pneumonia. In order to investigate pathomechanisms of pulmonary toxicity, an in vitro alveolar-capillary co-culture model has been established recently by our group. In this model the human lung adenocarcinoma epithelial cell line (H441) is mimicking the epithelial site of the alveoli while the human hemangiosarcoma cell line (Iso-Has) represents the endothelial site. Acute respiratory injuries are accompanied by disruption of the alveolar-capillary barrier that can be detected by the use of biochemical markers (e.g. LDH) and electrochemical indicators (e.g. transepithelial resistance). SM-mediated pulmonary injury is characterized by the increased secretion of proinflammatory mediators (e.g. IL-6). A shortcoming of this model is the missing inflammatory component in the lung. Aim of the present project is the addition of macrophages to the established co-culture model to improve the model and to investigate the relevance of inflammatory processes in toxic lung injury. The effect of SM on this triple-culture model is characterized with special regard to the interaction of epithelial cells and macrophages. The human acute monocytic leukemia cell line (THP-1) was stimulated to allow differentiation into macrophages. Validation of the cellular differentiation was checked by specific clusters of differentiation (e.g. CD206) using flow cytometric analysis. After successful differentiation into macrophages, these inflammatory cells were added to the co-culture model before and after exposure with SM, respectively. The cytotoxicity of SM on the triple-culture model was evaluated by XTT assays and TER measurements. Furthermore, immunohistochemical staining of tight junction proteins (e.g. ZO-1) and of adherens junction proteins (e.g. E-cadherin) was conducted to enhance the knowledge of the function of the intercellular junction in injured and rejuvenated regions as well as the interaction of epithelial cells and macrophages. For the contact allergen para-phenylenediamine (PPD) we showed that concentrations above 50 µM are accompanied with inhibition of NAT1 activity in human keratinocytes [1] . In the following we investigated the impact of PPD on NAT1 activity in antigenpresenting cells using dendritic cell-like cells, namely the monocytic THP-1 cells. Measured NAT1 activity of THP-1 was comparable to those found in primary keratinocytes. A 24h treatment of THP-1 cells with physiologically relevant concentrations of PPD (10-200 µM) led to a 47% reduction of NAT1 activity. Comparable results were found for mono-acetylated PPD (MAPPD) whereas di-acetylated PPD demonstrated no inhibition. Time-dependent studies found a significant decrease in enzyme activity already 8h after application of PPD or MAPPD while NAT1 mRNA levels were not modified. These results are indicative for a substrate-dependent inhibition. Further investigations concentrated on the restoration of NAT1 activity after treatment with PPD or MAPPD. Here we found that N-acetylation capacities were restored after 24h cultivation of the treated cells in fresh medium. Independent of the enzymatic activity, certain compounds are known to oxidise the catalytic cysteine or form adducts with the NAT1 protein. Therefore we studied whether PPD and/or oxidised PPD including the trimer Bandrowski´s Base interact additionally with recombinant NAT1 protein itself in the absence of acetyl-coenzyme A. We found that all compounds but MAPPD bind to NAT1 protein after 2h. The greatest inhibition was found for oxidised PPD (up to 50%). Due to the greater inhibition by oxidized PPD we propose that oxidation products interact with the protein whereas PPD itself modulates NAT1 enzyme activity in a substrate-dependent mode of action. Overall we demonstrated that PPD can inhibit NAT1 in two different ways. The work was partially financed by Federal Office of Public Health (FOPH), Switzerland and Stiftung zur Förderung begabter Studierender und des wissenschaftlichen Nachwuchses Objective: Fabry's disease is a rare progressive multisystem disorder resulting from deficiency of the lysosomal enzyme alpha-galactosidase A (GLA, EC 3.2.1.22). We hypothesize that genetic GLA variants, especially those in its promoter region are of pathophysiological relevance for the development and progression of Fabry's disease phenotypes. This study focuses on the characterization of the GLA promoter, identification of functional genetic variants and impact of transcription factor EB (TFEB), a regulator of lysosomal genes.

We screened 4011 bp of the 5'-flanking region of GLA in 60 patients with Fabry's disease and 60 controls for genetic variants. Serial promoter deletion constructs for reporter gene assays were designed and identified genetic variants were introduced by site-directed mutagenesis. Constructs were transiently transfected into immortalized human kidney epithelial (IHKE) cells and human vascular endothelial cells (EA.hy926) to determine transcriptional promoter activity (TA).

Sequencing of patients' DNA revealed five genetic variants in the 5'flanking region of GLA, significantly more frequent in Fabry's patients compared to control group (rs2071225; rs3027580; rs3027579; rs59647857; rs3027575; all minor alleles p<0.0027). We identified two regions, a proximal one between -110 and -425 and a distal region between 1106 and -1421 with significant TA, in both cell lines. Cotransfection with TFEB activated TA of both regions significantly up to 5.3-fold (p<0.001).

In IHKE cells, insertion of the minor T allele (rs2071225) significantly enhanced basal TA of the proximal promoter region (p=0.0006), while insertion decreased basal TA (p<0.0001) of the distal promoter portion. The combined insertion of the minor C alleles (rs3027580; rs3027579), which were in complete linkage disequilibrium, significantly increased basal TA of the distal promoter region (p=0.0037).

Our results indicate that three genetic variants, overrepresented in Fabry's patients, are located within transcriptionally active regions, possibly altering TF binding sites and therefore, affecting GLA expression. Future analysis will assess the impact of GLA promoter variants and GLA regulation by TFEB with respect to Fabry's phenotypes. Multiple sclerosis (MS) and its animal counterpart experimental autoimmune encephalomyelitis (EAE) have a major inflammatory component that drives and orchestrates both diseases. Ceramides (Cer) are known as mediators of inflammatory processes, but until now their role in MS was not elucidated. We measured the ceramide levels in the cerebrospinal fluid of MS patients and control patients using LC-MS/MS. Interestingly, the C16:0-Cer levels were 1.9 fold increased in MS patients. This translates into the finding that C16:0-Cer levels were also significantly elevated in the lumbar spinal cord of EAE mice. The raised C16:0-Cer levels in the lumbar spinal cord were caused by a transiently increased expression of ceramide synthase (CerS) 6 in macrophages. Nitric oxide (NO) and tumor necrosis factor alpha (TNF-α) secreted by interferon gamma (INFγ ) induced macrophages play an essential role in the development of MS. Astonishingly, RNAi experiments reveal that CerS6 and its product C16:0-Cer are mediators of INF-γ induced NO/TNF-α release in RAW macrophages. Moreover, treatment of EAE mice with L-cycloserine prevented the increase of C16:0-Cer and of iNOS/TNF-α expression and caused a remission of the disease. In summary, CerS6 plays a critical role in the initial phase of MS, most likely by regulating the NO and TNF-α synthesis. This let us speculate, that a substance designed to inhibit CerS6 and therefore to limit the inflammatory effects of C16:0-Cer may represent a new drug in MS therapy.

Role of cGMP-dependent protein kinase I for kidney fibrosis Schinner E. 1 cGMP is synthesized via nitric oxide-or natriuretic peptide-stimulated guanylyl cyclases and exhibits pleiotropic regulatory functions also in the kidney. Hence, the integration of cGMP signaling via cGMP-dependent protein kinases (cGK) might play a critical role for renal physiology. Both isozymes were detected in arterioles, mesangium and within the cortical interstitium. In contrast to cGKIα, the β isoform was not detected in the juxtaglomerular apparatus and medullary fibroblasts.

Here, we focused on the function of cGKI in the renal interstitium emphasizing a functional differentiation of both isoforms. The interstitium exists mainly of fibroblasts playing a prominent role in the interstitial fibrosis. Accordingly, cGKI could also be involved in this pathophysiological process. Therefore, we studied whether cGKI influences renal fibrosis which was induced by unilateral ureter obstruction (UUO). At first we analysed the role of the NO/cGMP signaling by application of cGMP increasing YC1 or ISDN. Thereby we detected antifibrotic effects of these substances. Subsequently we tested whether these effects are mediated by cGKI by using mutant mice. On the one hand we examined αSM-rescue mice (expressing cGKIα only in smooth muscle under the control of the SM22 promotor with a cGKI-KO background) and cGKI-KO mice (expressing no cGKI). On the other hand we used tgtg mice expressing more cGKIα in smooth muscle than wt mice (transgenic cGKIα under the control of the SM22 promotor). Due to the steeply increased use of nanomaterials for commercial and industrial applications, toxicological assessment of their potential harmful effects is urgently needed. Moreover, the continuous development of novel materials requires the implementation of hazard-predicting models to prevent potential health effects resulting from human exposure. In the present study, we studied the toxic potential of a set of nanoparticles (NP) with varying physicochemical properties in human A549 lung epithelial cells, HepG2 liver epithelial cells and HK-2 proximal tubule epithelial cells. The used nanomaterials incorporated five TiO 2 samples, two ZnO samples (i.e. uncoated and coated), two multi-walled carbon nanotube (MW-CNT) samples and a nanoparticulate Ag sample. Cells were treated with NP at doses ranging from 0.3 to 80 µg/cm 2 for cytotoxicity and from 06 to 40 µg/cm 2 for genotoxicity. DNA damage was evaluated using the alkaline comet assay while concurrent cytotoxicity was determined by the WST-1 assay. Marked contrasts in cytotoxic and DNA damaging properties were observed among the different materials. The overall strongest responses were observed with the uncoated ZnO-NP sample and with Ag-NP, although effects were found to depend on the cell type. Notably, the DNA damaging effect of Ag-NP could at least partly be attributed to its dispersant. Present results form part of a growing data set which are generated in the framework of the EU FP7 project ENPRA (FP7-NMP) to establish dose-response relationships and a mathematical model to predict the hazard of nanoparticles.

Increased spontaneous HPRT mutant frequency in V79 cells expressing human cytochrome P450 1B1 Schlechtweg A., Esch H. , Martínez Jaramillo D., Lehmann L. University of Wuerzburg/Institute of Pharmacy and Food Chemistry Section of Food Chemistry, Am Hubland, 97074 Wuerzburg, Germany

The hypoxanthine-guanine phosphoribosyltransferase (HPRT) assay in Chinese hamster V79 lung fibroblasts (V79 cells) represents a widely-used mammalian test system to detect gene mutations. Since V79 cells do not express any cytochrome-P450dependent monooxygenase (CYP) isozymes, usually an activating system has to be added. Therefore, V79 cells expressing human (h) CYP isozymes have been commercialized. To test these V79 cells for their use in the HPRT test, V79 h1A1 and h1B1 cells were characterized regarding (i) spontaneous frequency of 6-thioguanineresistant clones per 10 6 clonable cells (SMF), (ii) the stability of which over 4 weeks (w), and (iii) the mutational spectrum (MS) of cDNA from mutant clones. MS of cDNA was determined by isolation of total RNA, reverse transcription/amplification of the coding region by polymerase chain reaction and Sanger sequencing of the amplification product. Activity of CYP isozymes was verified by ethoxyresorufin-O-deethylase (EROD) assay. (i)/(ii) Whereas the SMF of V79 cells (w2:13±4; w4:2±1) and V79 h1A1 (w2:17±4; w4:3±0) only varied within the range of historical controls, SMF of V79 h1B1 increased continuously over time (w2: 36±6; w4: 68±13). (iii) Although the SMF of V79 and V79 h1A1 were similar, the mutational spectrum of V79 cells was characterized by as many transversions as transitions and deletions of exon 4 or exon 4+5, whereas the mutational spectrum of V79 h1A1 was characterized exclusively by transversions and deletion of exon 7+8. Surprisingly, with 57 out of 59 cDNAs derived from V79 h1B1 mutant clones, no amplification product was detected. First results indicate that there is at least one gene mutation in the untranslated region before and behind the coding region precluding amplification with the original primers. (ii)To reduce the SMF of V79 h1B1, cells with wildtype HPRT activity were cloned and one clone with an EROD activity which did not differ significantly from the original cell population was further characterized. Initially, SMF of the clone varied between 0.7±0.6 and 1.2±0.0. Yet its SMF was unstable reaching up to 104±6. In conclusion, the mutational spectrum differed between the V79 cell lines. Furthermore, h1B1 expression seemed to enhance SMF in V79 cells. Even though a temporary reduction of the SMF by cloning was possible, SMF of V79 h1B1 cells was unstable. We wanted to investigate the possible antithrombotic effects and elucidate the chemical identity of the active principles involved in inhibitory effects against ADP-induced aggregation of human platelets by wild garlic, Allium ursinum L. Method: Bioassay-guided isolation procedure was used followed by spectrometric identification of pure active compounds. For the bioassay, blood was taken from healthy human volunteers and platelet rich plasma (PRP) was prepared for turbidimetric platelet aggregation tests. PRP, stimulated with 20µM ADP, was treated with extracts of different polarities, fractions and isolated single compounds from Allium ursinum. The extracts were investigated by thin layer chromatography, HPLC, mass spectroscopy, ESI-MS and 1d/2d 1H/13C-NMR spectroscopic techniques. For references the ADT-Antagonist MeS-AMP was used. Result: Fresh Allium ursinum leaves were extracted with ethanol, which was the potent form that effectively inhibited ADP-induced aggregation of human platelets. This ethanolic extract was subjected to liquid-liquid partition. Whilst the aqueous phase containing the moiety of cysteine sulphoxide and thiosulphinate derivatives showed only weak activity on platelet aggregation, the ethyl acetate and especially the chloroform partitions showed highest aggregation inhibiting potency. Thus, in our bioassay effects of alliins/allicins could be neglected. The chloroform phase, possessing the strongest activity, was separated into 28 fractions by gradient elution open CC on silica gel. The most active fractions 11-17 were separated again yielding 10 subfractions. This afforded 1,2-di-O-α-linolenoyl-3-O-β-D-galactopyranosyl-sn-glycerol and β-sitosterol-3-O-β-Dglucopyranoside, the structures of which were determined by ESI-MS and 1d/2d 1H/13C-NMR spectroscopic techniques. Furthermore, the diminutive amounts of volatile oil of A.ursinum leaves obtained by steam distillation according to Ph.Eur. could be evaluated as a third aggregation inhibiting principle. Conclusions: At the first time two active, non-sulphur-containing constituents of wild garlic, namely a galactolipid and a phytosterol, could be identified exhibiting inhibitory action on ADP-induced aggregation in human blood platelets. As a major constituent, the galactolipid 1,2-di-O-α-linolenoyl-3-O-β-D-galactopyranosyl-sn-glycerol, not yet found in Allium spec., appears as a new, highly useful marker substance for A.ursinum drugs, or their pharmaceutical preparations.

In recent years, public attention focused more and more on risk factors which may impair sperm quality and thereby human reproduction. In this context, for example pesticides, alcohol, cigarettes, and even mobile phones are discussed. A variety of parameters exists including sperm counts as well as sperm motility, which are considered to be two of the most important parameters to evaluate sperm quality in animal models with the final aim to assess human risk. In recent years Computer Assisted Sperm Analysis (CASA) devices mostly replaced the formerly used manual counting and manual motility assessment. However, although CASA offers multiple opportunities and can allow for an objective and more detailed evaluation, several pitfalls exist which can alter the results profoundly and consequently compromise the quality of the data and ultimately the validity of a study.

The aim of the present study was to establish and validate the CASA device TOX IVOS Sperm Analyzer from Hamilton Thorne and thereby to gain detailed knowledge about the practical advantages but also intricacies which may alter the obtained results. In this regard healthy adult male rats (10-12 weeks old) were used. Ultrasonic sound resistant sperm heads were isolated from the testis and in addition, sperms were isolated from the Cauda epididymis. Testicular sperm head counts and sperm motility were assessed using different isolation procedures and/or instrument settings. Results Different instrument settings modulate both -sperm motility and testicular sperm counts. In this regard, a wide range of results including slight changes as well as false positive/negative results were obtained. In addition, the modification of the isolation procedure can lead to variable results especially for sperm motility. Conclusion Isolation procedures as well as instrument settings can alter the results. Consequently, in an experimental setting, potential adverse effects can be confounded with methodologically mediated apparent findings exerted via inappropriate use of the device -depending on the respective conditions in the test laboratory. This study demonstrates the relevance of standardization of testing conditions adopted for computer assisted sperm analysis and the need for a robust validation prior to use in experimental settings. Orai and STIM proteins have been identified as central components of the highly Ca 2+ selective, store-operated current in immune cells (ICRAC). The molecular basis of selective Orai-mediated activation of the calcineurin/NFAT pathway and the crosstalk with other channel and scaffold molecules of the TRPC family are still incompletely understood. Using patch clamp recordings complemented by fluorescence and TIRF microscopy we investigated interactions between Orai1 and TRPC3 in plasma membrane microdomains of RBL-2H3 mast cells. Orai1-mediated CRAC currents, activated by passive store depletion, were found significantly reduced by over-expression of TRPC3. This negative impact of TRPC3 on ICRAC was independent of channel function as the TRPC3 pore dead mutant (E630K) inhibited ICRAC to a similar extent as wild type TRPC3. Importantly, despite a reduction in ICRAC, NFAT translocation in TRPC3 overexpressing RBL cells remained unchanged, or was even slightly promoted. Store depletion-induced NFAT translocation in RBL cells was as well unaffected by TRPC3E630K but substantially reduced by TRPC3 mutants with either i) eliminated FKBP12/calcineurin binding (P704Q) or ii) deficiency in PKC phosphorylation (S712A). Moreover, inhibition of PKC phosphorylation by (GFX109203X; 3 µM) strongly suppressed NFAT signaling. We suggest TRPC3 as a scaffold that links Orai-mediated Ca 2+ -entry to NFAT/calcineurin signaling within plasma membrane microdomains.

Neurally-induced bronchoconstriction in human and guinea pig precision-cut lung slices Schlepütz M. 1 , Rieg A. D. Introduction: Precision-cut lung slices (PCLS) are well suited to study peripheral airway responses in different species. Airway tone is under close control of the autonomic nervous system and dysregulation may contribute to airway hyperresponsiveness as observed in human lung diseases such as asthma. Hence, the aim of the present study was to characterize neurally induced bronchoconstriction (BC) in guinea pigs (GP) and to compare the results with those in human PCLS. Methods: PCLS were prepared from GP or human lung tissue. Nerve endings in PCLS were activated by electric field stimulation (EFS) or capsaicin addition. Cholinergic nerve responses were proven by atropine. Capsaicin was used to show excitatory nonadrenergic non-cholinergic (eNANC) responses. Ruthenium red or SKF96365 were used to confirm transient receptor potential (TRP) channel contributions upon eNANC activation. Results: GP and human PCLS were both sensitive to EFS and airways contracted to 39±26% of the initial airway area (%-IAA) and 63±21%-IAA, respectively. In frequency response curves half maximal response was found at 7.0±1.2 Hz for guinea pig PCLS and 8.1±0.8 Hz for human PCLS. EFS-induced BC was inhibited by atropine in both species. Capsaicin contracted GP to 18±15%-IAA. 60% of human PCLS were responsive to capsaicin and airways contracted to 72±10%-IAA, respectively. In GP Ruthenium red and SKF96365 blocked capsaicin-as well as EFS-induced BC. Conclusion: GP and human PCLS contain atropine sensitive cholinergic and capsaicin sensitive eNANC nerve endings. Since GP PCLS were sensitive to TRP channel inhibitors, the involvement of those channels can be characterized with respect to lung diseases. In conclusion, GP PCLS resemble the human distal lung innervation and represent a useful model to study neural airway pharmacology.

The Erk1/2-pathway is involved in PKC-induced Nox4 up-regulation Schlufter F., Xia N., Förstermann U., Li H. Universitätsmedizin Mainz Institut für Pharmakologie, Obere Zahlbacher Straße 67, 55131 Mainz, Germany NADPH oxidases (Nox) are major producers of reactive oxygen species in the vascular wall and Nox4 is the most abundant Nox isoform in human endothelial cells. We have previously shown that treatment of human EA.hy 926 endothelial cells with phorbol 12myristate 13-acetate (PMA) for 48 h leads to an up-regulation of Nox4 expression. This effect of PMA is mediated by protein kinase Cα, because it is preventable by the PKC inhibitor Gö 6983 and by PKCα-siRNA. The present study is aimed to investigate the signal transduction cascade downstream of PKCα. PMA-induced Nox4 up-regulation can be attenuated by PD 98,059 (an Erk1/2 inhibitor), but not by SP 600125 (a JNK inhibitor), indicating in the involvement of Erk1/2. Consistently, PMA treatment leads to a sustained activation of Erk1/2, and siRNAmediated knockdown of Erk1/2 markedly reduces the PMA-induced Nox4 up-regulation.

H89, an inhibitor of the mitogen-and stress-activated protein kinases (MSKs) has no effect on the PMA-stimulated Nox4 expression, indicating that MSKs are not the target molecules of Erk1/2 in this scenario. On the contrary, knockdown of the transcription factor Elk-1 by siRNA significantly reduces the PMA-induced Nox4 up-regulation. In conclusion, Erk1/2 and Elk-1 are involved in the PKCα-induced Nox4 up-regulation.

Determination of spontaneous mutation frequencies in normal human mammary gland tissue using the Random Mutation Capture technique Schmalbach K., Lehmann L. University of Wuerzburg Section of Food Chemistry, Am Hubland, 97074 Wuerzburg, Germany Annually, over 57,000 women develop breast cancer in Germany. The accumulation of genetic mutations in mammary gland tissue during lifetime may be reasonable for developing breast cancer. In particular mutations in tumor suppressor genes, e.g. p53, seem to play an important role in developing cancer. Up to now, lack of a method sensitive enough to determine the expected very low spontaneous mutation frequency (SMF) in normal mammary gland tissue precluded the investigation of the role of spontaneous mutations acquired in the p53 gene in epidemiological studies. The only test with the potential to determine low SMFs was the Random Mutation Capture (RMC) assay, a genotype selective method which detects mutants that render the mutational sequence non-cleavable by the TaqI restriction enzyme after accumulation of the target sequence. Therefore, the suitability of the RMC assay to determine SMF in p53 gene in normal human mammary gland tissue was evaluated. Thus, the RMC assay was optimized concerning (i) DNA isolation, (ii) PCR conditions, and (iii) amount of mammary gland tissue. (i) Genomic DNA from normal human mammary gland tissue, obtained from healthy women who underwent mamma reduction surgery for cosmetic reasons, was isolated using an extended proteinase k digestion prior to chloroform extraction. (ii) The target sequence in intron 6 of p53 gene was captured by hybridization with a complementary uracil-containing DNA-probe synthesized via polymerase chain reaction (PCR), followed by magnetic separation from the remaining genomic DNA. The copy number of the target sequence was quantified by competitive PCR. The number of mutants was detected after cleavage of the target DNA with TaqI by means of PCR with a primer set flanking the restriction site. (iii) With 2 g of normal mammary gland tissue a SMF of 2.2±1.4x10 -7 per base pair was determined indicating the RMC assay suitable for SMF determination. In conclusion, the SMF in the p53 gene in normal human mammary gland tissue was determined for the first time, enabling the future investigation of factors influencing the SMF during breast cancer development. Cigarette smoke-induced release of pro-inflammatory cytokines including interleukin-8 (IL-8) from inflammatory as well as structural cells in the airways, including airway smooth muscle (ASM) cells, may contribute to the development of chronic obstructive pulmonary disease (COPD). Despite the wide use of pharmacological treatment aimed at increasing intracellular levels of the endogenous suppressor cyclic AMP (cAMP), little is known on its exact mechanism of action. We report here that next to the β2-agonist fenoterol, direct and specific activation of either exchange protein directly activated by cAMP (Epac) or protein kinase A (PKA) reduced cigarette smoke extract (CSE)-induced IL-8 mRNA expression and protein release by human ASM cells. CSE-induced IκBαdegradation and p65 nuclear translocation, processes that were primarily reversed by Epac activation. Further, CSE increased extracellular signal-regulated kinase (ERK) phosphorylation, which was selectively reduced by PKA activation. CSE decreased Epac1 expression, but did not affect Epac2 and PKA expression. Importantly, Epac1 expression was also reduced in lung tissue from COPD patients. In conclusion, Epac and PKA decrease CSE-induced IL-8 release by human ASM cells via inhibition of NF-κB and ERK, respectively, pointing at these cAMP effectors as potential targets for antiinflammatory therapy in COPD. However, cigarette smoke exposure may reduce antiinflammatory effects of cAMP elevating agents via down-regulation of Epac1.

Polycyclic aromatic hydrocarbons (key marker substance benzo[a]pyrene (BaP)) have been assumed to play a role in the development of bladder cancer. The objective of the present study was to unravel cellular and in particular cytoskeletal response to BaP. To follow the sequential steps of chemical carcinogenesis the differential proteomic profile was analyzed at early and late time points. The study was carried out in a superficial human bladder cancer cell line (RT4) exposed to 0.5 µM BaP, a subacute concentration based on results of proliferation (BrdU) and DNA damage (TUNEL) tests.

Cells of a human bladder cancer cell line (RT4) were exposed to 0.5 µM BaP for 24 h (n=5), 4 wk (n=7) and 8 wk (n=3). Proteins of whole cell lysate were separated by twodimensional electrophoresis (Fig. 1) . Differentially expressed proteins were identified by matrix-assisted laser desorption/ionization-time of flight analysis. Cortactin, actin and tubulin were immunohistochemical stained. Changes in migration and colony forming ability were assessed by scratch wound-healing assay and soft-agar colony formation.

Results: By using several databases (UniProt, Reactome, Panther) the identified proteins were categorized into different functional classes such as mRNA processing, translation, protein metabolic process, or several areas associated with the organization of the cytoskeleton. 45 % of the differentially expressed proteins after 24 h of treatment are involved in the processing of pre-mRNA (20 %) and protein metabolism (25 %). This pattern changed after 8 wk of treatment. Then, 48 % of the proteins affected the cytoskeleton whereas still 26 % were categorized to protein metabolism and only 7 % to pre-mRNA processing. In the immunhistochemical staining, the treated cells appeared after 8 wk of exposure larger and rounder predominantly due to the modifications of the actin cytoskeleton. Merged images of actin and cortactin revealed that both proteins colocalized in invadopodiae. After 8 wk, a higher number of treated cells moved toward the centre of the wound and they formed more soft-agar colonies compared to vehicle conditions, suggesting a transformation of the cells. In conclusion, BaP exposure causes in an early phase an activation of the spliceosome which can led to an epithelial-tomesenchymal transition. Two coordinators of a cell-type-specific splicing program, Epithelial Splicing Regulatory Proteins 1 and 2, are currently being validate by PCR. fused master gel : Representative 2-DE gel of RT4 cells exposed to 0.5 µM BaP for 8 wk. Protein spots which were differentially expressed compared to control and identified were marked.

Cannabinoids stimulate mesenchymal stem cell migration via a mitogen-activated protein kinase pathway Schmuhl E. 1 , Ramer R. Mesenchymal stem cells (MSCs) are known to be involved in various regenerative processes such as cardiac, ocular, skin and bone tissue healing. However, little is known about the pharmacotherapeutical options aiming at tissue healing steps such as the mobilization and homing of MSCs. Here, we show that cannabidiol (CBD), a nonpsychoactive cannabinoid, stimulates the migration of human adipose-derived MSCs in both Boyden chamber and in vitro scratch wound assays. In Boyden chambers CBD (0.01 -3 µM) was shown to promote cell migration in a time-and concentration dependent manner. This promigratory action was inhibited by AM-630 (CB2 receptor antagonist) and by O-1602 (G protein-coupled receptor [GPR] 55 agonist). Moreover, CBD activated the mitogen-activated protein kinase (MAPK) pathway as evidenced by increased phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Blockade of ERK activation by PD98059 prevented CBD-stimulated MSC migration, whereas inhibition of p38 MAPK by SB203580 was inactive in this respect. Furthermore, AM-630 and O-1602 were found to attenuate CBD-induced ERK activation. An ERK-dependent promigratory action was likewise demonstrated for the phytocannabinoid ∆ 9 tetrahydrocannabinol and for the hydrolysis-stable anandamide analogue R(+)methanandamide. We conclude that CBD promotes MSC migration via receptordependent ERK activation, possibly contributing to tissue healing. The Duffy Antigen Receptor for Chemokines (DARC) binds promiscuously many inflammatory chemokines without showing intracellular signal transduction. It is mainly expressed on endothelial cells of postcapillary venules and on red blood cells, where it acts as a transendothelial transporter of chemokines and as a chemokine sink, respectively. Surprisingly, as shown for human and mouse brain, DARC is also expressed at high density in the cerebellum. However, nothing is known about the function of DARC in this location. We addressed this question by subjecting C57Bl/6 wildtype and DARC-deficient mice to a series of behavior experiments including Morris water maze-, elevated plus maze-, rotarod-and actometer tests. While the results from the water maze experiments are ambiguous, elevated plus maze trials show a strong aversion of DARC -/mice to walk to the end of the open arm, which is consistent with anxiety-like behavior. Moreover, DARC -/mice show greatly reduced locomotor activity, which is at least partly caused by episodes of reduced mobility occurring more frequently than in the corresponding wildtype controls (elevated plus maze, actometer). Finally, DARC -/mice spend a significantly reduced time on the rotating rod compared to C57Bl/6 wildtype controls, which may indicate an impaired cerebellar function. We conclude that DARC in fact modulates brain function. Surprisingly, this appears to be happening under homeostatic conditions, although DARC binds for the most part to inflammatory chemokines. It remains to be elucidated, how this effect can be caused by a non-signaling chemokine receptor. It may be an indirect consequence of altered brain chemokine concentrations or of as yet unknown signaling pathways activated by DARC.

Transporter gene expression in human head-neck squamous cell carcinoma and epigenetic regulation mechanisms Schnepf R. 1 Hals-Nasen-Ohren-Klinik, Kopf-und Halschirurgie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Waldstraße 1, 91054 Erlangen, Germany Background: Membrane transporters may affect the disposition and thereby treatment efficiency of anticancer drugs in human head-neck squamous cell carcinoma (HNSCC). The gene expression profile of transporters in HNSCC, however, is unknown and was evaluated in this study. Moreover, we evaluated mechanisms by which transporters are regulated in HNSCC. We focused on the role of the nuclear pregnane X receptors (PXR, NR1I2) and epigenetic mechanisms.

Methods and results: Real-time RT-PCR revealed a significantly increased mRNA expression of SLCO1A2 and SLCO1B3 and a significantly decreased expression of transporters such as SLCO2B1, SLCO2A1 and ABCC3 in human HNSCC tissue samples compared to adjacent normal mucosa. Moreover, an association between SLCO2B1 mRNA levels in tumor tissues and five-year survival of HNSCC patients was observed (χ 2 =6.59; P=0.010; n=34). Bisulfite sequencing revealed that promoter CpG islands of ABCC3 and SLCO2A1 were not methylated and thus these genes were not epigenetically silenced in HNSCC tissues. In the HNSCC-derived UMSCC-1 and SCC-15 cell lines, transcript expression of transporters (e.g., ABCC3, SLCO2A1; P<0.001) and PXR (NR1I2; P<0.001) was markedly induced by the DNA methyltransferase inhibitor decitabine. Cotreatment with the prototypical PXR activator rifampicin significantly reversed decitabine-induced ABCC3 and SLCO2A1 expression.

Conclusions: Transporter expression profiles significantly differed between HNSCC and normal mucosa and expression levels of SLCO2B1 may serve as a marker for prognosis. Modulation of the epigenome with the anticancer drug decitabine substantially affects transporter expression in UMSCC-1 and SCC-15 cells, suggesting epigenetic regulation mechanisms. Moreover, interactions between epigenetic and nuclear receptor-mediated mechanisms in transporter regulation occur. This work was in part supported by the Johannes und Frieda Marohn Foundation.

The role of HCN2 in neuropathic and inflammatory pain Schnorr S. 1 , Eberhardt M. The pacemaker current Ih is carried by hyperpolarization-activated cyclic nucleotidegated cation channels (HCN1-4) and contributes to cellular excitability in the heart and the nervous system. HCN1 and HCN2 are the two most abundant HCN subunits in peripheral sensory neurons with HCN2 being the prevalent isoform in nociceptive small sized C-fibre dorsal root ganglion (DRG) neurons. We examined the role of HCN2 for peripheral sensitization and spontaneous neuronal activity in neuropathic and inflammatory pain. We generated a conditional deletion of HCN2 by using a nociceptor specific Cre-transgene driven by the Nav1.8 promoter. The nociceptor-specific knockout of HCN2 in DRG neurons (nospHCN2KO) was confirmed by quantitative RT-PCR and western blot. Immunohistochemical staining revealed that the deletion of HCN2 was mainly restricted to small sized DRG neurons. The conditional loss of HCN2 resulted in a significant reduction of Ih positive small diameter DRG neurons pointing to a central role of this isoform to the HCN current in nociceptive neurons. Behavioral studies showed that the lack of HCN2 did not influence basal pain responses but led to a significant reduction in mechanosensation in both neuropathic and inflammatory pain models. However, thermosensation of the mutants was only decreased in neuropathic pain conditions. In wild-type animals, intraperitoneal, intraplantar and even intrathecal injection of the HCN channel blocker ZD7288 nearly eliminated tactile allodynia caused by inflammation in contrast to thermal hyperalgesia which remained unaffected. In contrast, pain thresholds in nospHCN2KO mice did not significantly increase after pharmacological block of Ih. Additionally, experiments revealed that the inflammatory condition induced an upregulation of HCN2 protein in the spinal dorsal horn compared to saline injected mice. Our results suggest that HCN2 might be a new target in the treatment of neuropathic and inflammatory pain. The proper functioning of the central, as well as the peripheral nervous systems is of outstanding importance to the survival and well-being of humans. Yet, the integrity of neuronal systems is constantly challenged by a plethora of environmental and occupational toxins. Some of these toxins preferentially target neural cells. These neurotoxins can exert their devastating effects by very different modes of action. Neurotoxins may induce apoptosis or necrosis of neurons, or interfere with axon growth and elongation. These processes can be identified by specialized in vitro tests. Furthermore, neurotoxins have been described to alter glial function which may compromise the viability of surrounding neurons. As another important mode of action, several neurotoxins act on neurotransmitter receptors, thereby altering signal propagation within neuronal networks. Countless natural and synthetic substances have been characterized for their effects on neurotransmitter receptors and today can be used for detailed studies of receptor function. However, environmental toxins of anthropogenic origin and occupational toxins that both represent constant sources for human exposure are still poorly studied with respect to their effects on neurotransmitter receptors. Thus, the need for a better understanding of the susceptibility of neurotransmitter systems for toxic effects exerted by these substances is of outstanding importance for the protection of human health.

Here, we introduce an imaging-based approach for the screening of the effects of potential and known neurotoxins on neurotransmitter receptors of intact cells in vitro. Different neuronal cells were tested for their sensitivities for classical neurotransmitters using life-cell imaging experiments. In more detail, we examined the proportion of responding cells and determined the EC50 values for the most prominent neurotransmitters in cell lines widely used for in vitro neurotoxicity studies on the one hand, namely SH-SY5Y and LUMES cells, and primary mouse neurons on the other hand. With these data at hand, we are now able to identify and characterize the effects of neurotoxins on receptor function in chronic, as well as acute exposition paradigms. The use of an in vitro imaging-based physiological test system is at the interface between non-functional in vitro approaches and in vivo toxicity tests, thus, giving mechanistic insight into neurotoxic processes without requiring animal experiments.

Apomorphine acts on TRPA1 channels Scholze A., Schaefer M., Hill K. Universität Leipzig -Universitätsmedizin Rudolf Boehm-Institut für Pharmakologie und Toxikologie, Härtelstr. 16-18, 04107 Leipzig, Germany

Apomorphine is a non-narcotic derivative of morphine which acts as a dopamine agonist and is clinically used to treat "off-states" in patients suffering from Parkinson´s disease. Adverse effects of apomorphine treatment include dopaminergic effects such as nausea, but also ulceration and pain at the injection site. We wanted to test whether an activation of TRP (transient receptor potential) channels in sensory neurones contributes to the perception of pain after apomorphine injection. While the warm/heat receptors TRPV1, TRPV2, TRPV3, and TRPV4 and the cold receptor TRPM8 were insensitive towards apomorphine treatment, TRPA1 could concentration-dependently be modulated by apomorphine. Low micromolar apomorphine concentrations potently activated heterologously expressed TRPA1 channels in a stably transfected cell line (HEK293-TRPA1), as well as natively expressed TRPA1 in cultured dorsal root ganglion neurones. On the other hand, when using higher concentrations of apomorphine, we observed inhibition of TRPA1 activity. Previous studies have shown that subcutaneously administered apomorphine produces a biphasic dose response relationship in rats, inducing hyperalgesia at low doses whereas high doses of the substance cause antinociception. From our studies we conclude that such in vivo effects of apomorphine are presumably mediated by activation/inhibition of TRPA1 expressed in sensory neurones Agonist binding to a G Protein-coupled receptor (GPCR) induces a conformational change of the receptor protein, which results in the activation of receptor-associated heterotrimeric G proteins [1] . In radioligand binding studies, conducted to investigate ligand binding to specific GPCRs, receptors are usually probed with radioantagonists. As in other GPCRs [2] , agonists of the muscarinic M2 receptor exhibit biphasic kinetics and biphasic competition curves with radioantagonists, indicating a more complex situation probably caused by G protein interactions.

Here, we present a detailed study of the binding of agonists to muscarinic M2 receptors including the novel super-high affinity agonist iperoxo and a differential chemical knockout of G proteins. In addition to membrane homogenates living cells were employed. We demonstrate that the high affinity fraction in biphasic curves does not differ between selected full agonist and is sensitive to pertussis toxin, thus indicating that this receptor population is associated with Gi proteins. However, despite promiscuous signalling properties of M2 receptors, the low affinity fraction is not associated with any other G protein, since low affinity binding is insensitive to high concentrations of guanylnucleotides and cholera toxin. Moreover, high affinity agonist binding appears solely in membrane homogenates but not in experiments conducted with living cells, probably due to their high intracellular concentration of guanylnucleotides. Taken together the chemical knock-out of G proteins revealed that the high affinity binding of agonists in membrane homogenates is associated with the interaction of the muscarinic M2 receptor with Gi proteins. The low affinity binding cannot be related to another G protein, although the muscarinic M2 receptor exhibits promiscuous G protein signalling properties. Interestingly data obtained with living cells do not reveal any high affinity binding of agonists. Prolonged stress leads to a dysregulation of the hypothalamus-pituitary-adrenal (HPA)axis and may affect the sensitivity of pain perception. However, it is not yet known whether the alterations of HPA-axis and increased pain sensitivity are related. To create a long lasting stressful situation, male Wistar rats were exposed to a restraint-stress for 1 h daily over a period of two weeks. The effect of stress on the HPA-axis was determined by adrenal morphology and stress hormone levels, the influence on mechanical pain sensitivity was evaluated by the Randall-Selitto paw pressure test. On day 15 the animals exhibited a significant mechanical hyperalgesia. They also showed increased ACTH and corticosterone plasma levels and an enlarged zona fasciculata of the adrenal gland, indicating a dysregulation of the HPA-axis.

For testing the correlation of HPA-axis dysregulation and hyperalgesia a persistent increase in plasma corticosterone in Wistar rats was generated by the administration of corticosterone via the drinking water for two weeks. These animals also showed an increased mechanical nociceptive sensitivity with an accompanied decrease of the adrenal glands and reduced ACTH levels.

The results show that chronic stress leads to a dysfunction of the HPA-axis with an accompanied mechanical hyperalgesia which can be mimicked by oral administration of corticosterone. Thus, this in-vivo test system may provide a new animal-friendly pharmacological model for stress-related pain disorders.

The Alternaria mycotoxins AOH and AME induce CYP1A1 and apoptosis in murine hepatoma cells dependent on the aryl hydrocarbon receptor Mycotoxins are secondary metabolites of fungi including the genus Alternaria (black mold). Alternaria fungi are known to infest different types of foodstuffs and produce diverse toxins amongst them the mycotoxins alternariol (AOH) and alternariol methyl ether (AME) which are potential carcinogens. As planar compounds, AOH and AME are preferentially metabolized by cytochrome P450 (CYP) 1A1 and 1A2. The most prominent regulator of CYP1A1 is the dimeric transcription factor complex AhR/ARNT, which is activated by planar ligands. Therefore we studied the activation of AhR/ARNT by AOH and AME and monitored CYP1A1 induction in murine hepatoma cells (Hepa-1c1c7). Indeed, AOH and AME enhanced the levels of CYP1A1 in Hepa-1c1c7 cells but not in cells with inactivated AhR (Hepa-1c1c12) or ARNT (Hepa-1c1c4). Furthermore, we studied the cytotoxicity of AOH and AME. By using a fluorescence-based microscopic readout we measured effects on cell counts, apoptosis, senescence and micronuclei formation. Both AOH and AME reduce the cell number and the cell nuclei show drastic morphological changes e.g. enlargement after AOH treatment or micronuclei formation. The observed effects where, except for the induction of apoptosis, independent of AhR/ARNT. In summary, AOH and AME activate the AhR/ARNT pathway to induce CYP1A1 expression and apoptosis. However, the predominant cytotoxic effect of AOH and AME in hepatoma cells is a profound reduction in cell numbers, which is independent of the AhR/ARNT pathway. Special purpose databases are the first place for researchers in the life sciences to obtain expert curated data. Naturally, such resources are limited in terms of timeliness and comprehensiveness. The literature database PubMed alone lists more than 20,000,000 scientific abstracts, and 700,000 are newly added every year. The protein sequence database UniProtKB stores over 10,500,000 sequences, a hundred times more than ten years ago. Turning these data into meaningful information and making it accessible to both humans and computers have become an essential part of biological discovery and biomedical research. Text mining techniques have proven useful to extract the missing links in areas such as drug-target and drug-disease prediction, the extraction of mutation-phenotype associations, or the prediction of protein-protein and protein-ligand interactions. By systematically extracting information from available literature, reports or patents, text mining techniques can help to refine existing categorical knowledge stored in databases, and hence will support drug repositioning, the discovery of novel cancer biomarkers, or help to understand causes of hereditary diseases. In the area of regulatory toxicology we developed Go3R, the first knowledge-based search engine for alternative methods to animal experiments. The system not only helps retrieving information on the availability of alternative methods that allows for replacing, reducing or refining animal experiments, but also provides an endpoint-centered literature search to all scientists and regulatory authorities seeking for toxicological information and data. The up-to-date taxonomicstructured "table of contents" provided by Go3R allows for search in the literature listed in PubMed or the Toxicology Data Network (TOXNET) in a fast and comprehensive manner. The semantically enriched platform supports the user during the query formulation, allows for bibliographic analysis, and reveals existing relations to replacement, reduction, and refinement of animal experiments.

Impaired cardiac excitation-contraction-coupling in mice with complete inactivation of the CREM gene Schulte J. S., Tekook M., Schmitz W., Müller F. U. Westfälische Wilhelms-Universität Institut für Pharmakologie und Toxikologie, Domagkstraße 12, 48149 Münster, Germany

The structurally related transcription factors cAMP response element binding protein (CREB) and cAMP response element modulator (CREM) mediate a regulation of gene transcription in response to cAMP and are expressed in the heart. Mice with complete inactivation of CREM display impaired cardiac contraction and relaxation, decreased expression of SERCA and down-regulation of β1-adrenoceptors. To elucidate the underlying functional mechanisms on the cellular level we here investigated cellular electrophysiology and Ca 2+ -cycling in ventricular cardiomyocytes from CREM KO mice (KO).

Adult ventricular cardiomyocytes were isolated from KO and wildtype (WT) mice (age 16-20 weeks) and subsequently used for experiments within 6 hours after isolation. Action potentials (APs) were recorded from ventricular cardiomyocytes (perforated patch, whole cell current clamp 

Inactivation of CREM seems to have no consequences for AP duration and possibly associated ion channels but leads to impairment of Ca 2+ cycling and sarcomere shortening under basal conditions well explaining the previous findings in vivo. Our results show that CREM is essential for a regular excitation-contraction coupling in the mouse heart. (supported by the IZKF Münster)

New mechanistic insights in NO/cGMP actions in the vasculature Schulte K., Koesling D., Universitätsstr. 150, 44781 Bochum, Germany Hypertension, a major risk factor for cardiovascular diseases, is associated with vascular changes resulting in increased vascular contractility und vascular peripheral resistance. A prominent factor in the development and maintenance of hypertension is the reninangiotensin-aldostrerone system. Angiotensin II (Ang II) is the main mediator of this system and a powerful vasoconstrictor. Ang II increases the intracellular Ca 2+ concentration thereby activating myosin light chain (MLC) kinase, which enhances MLC phosphorylation and subsequent vascular contraction. Opposite to angII-induced vascular contraction, NO/cGMP pathway promotes vascular relaxation by decreasing Ca 2+ concentration and lowering MLC phosphorylation. Responsible for MLC dephosphorylation is the MLC phosphatase (MLCP), whose activity is regulated by different phosphorylations. Phosphorylation of MLCP via RhoA-activated Rho-kinase enhances phosphatase activity while phosphorylation via the cGMP-dependent protein kinase has been proposed to decrease enzymatic activity. To investigate the interplay of AngII with the NO/cGMP pathway, we treated wild-type and KO mice lacking the cGMP forming NO receptor, NO-GC1, with AngiotensinII (1.44 mg / kg BW / d) for two weeks. In addition to various cardiovascular parameters, physiological changes in vascular reactivity of aortic rings of AngII-treated WT and NO-GC1 KO mice were assessed in organ bath experiments and correlated with biochemical parameters as the phosphorylation state of MLC, MLCP and Rho-kinase activities examined by immunoblot analysis. Analysis of cGMP levels revealed that AngII treatment decreased cGMP in WT mice to levels comparable to those of the KO mice which were unaltered by the treatment. Our study will provide further mechanistic insights in the molecular interactions between constrictor and dilator stimuli in the vasculature. Nanomaterials are already used today and offer even greater use and benefits in the future. The progress of nanotechnology must be accompanied by investigations of their potential harmful effects. For airborne nanomaterials, lung toxicity is a major concern and obviously the particle size is discussed as a critical property directing adverse effects. While standard toxicological test methods are generally capable of detecting the toxic effects, the choice of relevant methods for nanomaterials is still discussed. We have investigated two genotoxic endpoints -alkaline Comet assay in lung tissue and micronucleation in polychromatic erythrocytes of the bone marrow -in a combined study 72 hours after a single instillation of 18 µg Gold nanoparticles (NP) into the trachea of male adult Wistar rats. The administration of three test materials differing only in their primary particle size (2-, 20-and 200-nm) did not lead to relevant DNA damage in the mentioned tests. The measurement of clinical pathology parameters in bronchoalveolar lavage fluid (BALF) and blood indicated neither relevant local reactions in the animals' lungs nor adverse systemic effects. Minor histopathology findings occurred in the lung of the animals exposed to 20-nm and 200-nm sized nanomaterials. In conclusion, under the conditions of this study the different sized Gold NP tested were non-genotoxic and showed no systemic and local adverse effects at the given dose.

Platelet dense granule secretion mediates platelet-dependent enhancement of tumor cell transmigration and formation of metastases Schumacher D., Strilic B., Wettschureck N., Offermanns S. MPI für Herz-und Lungenforschung Offermanns, Ludwigstr. 43, 61231 Bad Nauheim, Germany Tumor cell metastasis to distant organs is the primary cause of mortality in cancer patients. Tumor cells leave the primary tumor, intravasate, survive in the circulation and extravasate through the endothelial cell layer to grow in the target organ. It has long been known that blood platelets play an important role in tumor cell survival and dissemination, but the mechanism by which platelets promote metastasis remained unclear. Given that platelets are found closely associated with tumor cells shortly after vascular arrest, we explored whether platelets can facilitate the transmigration of tumor cells through the endothelium and thereby promote extravasation of tumor cells into the organ parenchyma. The ability of various mouse and human tumor cells like Lewis-Lung carcinoma cells (LLC1), B16F10 melanoma cells or human neuroblastoma cells (SH-SY5Y) to transmigrate through an endothelial cell layer was strongly enhanced by seeding tumor cells together with mouse or human platelets onto the endothelial cell layer. This indicates that platelets facilitate tumor cell transmigration in vitro. We found that platelet granule secretion is involved in this process as supernatant from platelets incubated with tumor cells but not from resting platelets was sufficient to enhance tumor cell transmigration. Additionally, no platelet-mediated increase of tumor cell transmigration was observed in dense granule secretion-defective platelets of Munc13-4 deficient mice. Thus, dense granule secretion is required for platelet-dependent tumor cell extravasation in vitro. While the growth and weight of primary tumors after subcutaneous injection of LLC1 and B16 cells was indistinguishable between wild-type mice and animals lacking Munc13-4, the number of metastases in the lung was strongly reduced in Munc13-4-deficient animals. The strong decrease in formation of metastases in Munc13-4 deficient mice was also observed after i.v. injection of LLC1 and B16F10 cells. Thus, platelet dense granule secretion plays a critical role in tumor cell metastasis by enhancing tumor cell transmigration through the endothelial cell layer.

Formation of DNA adducts in mouse tissues by the Brassica ingredient 1methoxy-3-indolylmethyl glucosinolate and its break-down product 1-methoxy-3indolylmethyl alcohol Schumacher F. 1 , Engst W. Glucosinolates are secondary metabolites present at substantial levels in cruciferous vegetables, such as broccoli and cabbage. After injury of plant tissue they are activated by the enzyme myrosinase to form various electrophilic degradation products like isothiocyanates. We previously showed that 1-methoxy-3-indolylmethyl (1-MIM) glucosinolate (or neoglucobrassicin) is a potent genotoxicant in bacterial and mammalian cells after activation by myrosinase. The induction of mutations could be correlated with the formation of DNA adducts [1] . We have identified and synthesized the major DNA adducts N 2 -(1-MIM)-dG and N 6 -(1-MIM)-dA. Moreover, we developed a highly sensitive UPLC-ESI-MS/MS method for selective MRM quantification of these adducts using stable-isotopic labeled adducts as internal standards. While the plant enzyme myrosinase is probably almost completely inactivated after cooking the vegetables, the glucosinolates reach the gut mostly intact due to their good heat and pH stability. Enzymes of individual intestinal bacteria are able to cleave the glycosidic bond of the glucosinolates, which leads to the formation of reactive metabolites within the gut lumen. We were able to detect significant levels of N 2 -(1-MIM)-dG and N 6 -(1-MIM)-dA in a dose-dependent manner in the large intestine of mice treated orally with isolated 1-MIM glucosinolate. The peak levels of N 2 -(1-MIM)-dG and N 6 -(1-MIM)-dA in the murine large intestine were reached 8 h after a single administration of 600 µmol 1-MIM glucosinolate/ kg body weight. The oral application of the relatively stable metabolite 1-MIM alcohol to mice led to the formation of identical DNA adducts. This benzylic alcohol can be activated by sulfotransferases to an electrophilic sulfo conjugate. In contrast to the intact glucosinolate the orally administered 1-MIM alcohol generated significant levels of N 2 -(1-MIM)-dG and N 6 -(1-MIM)-dA not only in the large intestine but also in other tissues, such as the liver, of mice.

[1] H. Glatt, C. Baasanjav-Gerber, F. Schumacher, B. H. Monien, M. Schreiner, H. Frank, A. Seidel, W. Engst, Chem.-Biol. Interact., 192 (2011) 

Human Pregnane X Receptor genotype of the donor but not of the recipient is a risk factor for delayed graft function after renal transplantation Schwab M. 1, 2 , Schaeffeler E. Delayed graft function (DGF) is an important immediate complication in renal transplantation significantly contributing to decreased long-term allograft survival. In addition to donor-and recipient-related risk factors altered renal excretion of xenobiotics by membrane transporters may influence DGF as well. Using recipients' and donors' DNA, we assessed the impact of genetic variants on DGF for the transporter proteins, Pglycoprotein (ABCB1) and multidrug resistance protein 2 (ABCC2), and the nuclear pregnane X receptor (PXR/NR1I2), regulating the transcription of drug metabolizing enzymes and membrane transporters. In our local cohort of transplant patients (n=178) DGF occurred in 27.5 %. Logistic regression analysis using four different genetic models (i.e. co-dominant, dominant, recessive and log additive) indicates that only the donor's PXR rs2276707 8055TT genotype was significantly associated with DGF (recessive model: OR, 9.0; 95%CI, p=0.004 unadjusted) , even after correction for multiple testing (p=0.035 Holm-adjusted). When we performed multivariate analysis including genetic and 16 clinical co-variates (i.e. age, gender, HLA mismatches, panelreactive antibodies, immunosuppression using CNI, T cell-depleting agents, anti-IL-2 receptor antibody and steroids, cold or warm ischemia time, living vs deceased donors or graft loss) again DGF was significantly associated only with the PXR rs2276707 TT genotype of the donor (recessive model: OR, 16.08; 95% CI, 2.0-129.46; p=0.0046 unadjusted) which held true after correction for multiple testing (p=0.04). For ABCC2 variants only the donor rs17222723 3563T>A genotype correlated with DGF by univariate (OR, 4.65; 95%CI, p=0.005 unadjusted) as well as by multivariate analysis (OR, 5.5; 95%CI, p=0 .01; Table 4 ) but not after correction for multiple testing (p=0.12). For variants in the ABCB1 gene no significant associations with DGF were detected for both the donor's and the recipient's genotype. In summary, our findings suggest for the first time that PXR may be a risk gene for the development of DGF independently from previously identified risk factors.

Supported by the Robert-Bosch Foundation, Stuttgart, Germany, the BMBF grant 03IS2061C (Berlin, Germany) and by the Ferdinand Eisenberger grant of the German Society of Urology (ID KrS1/FE-10).

Formation, morphology and structural requirements for formation of microtubule protrusions by Clostridium difficile toxin CDT Schwan C., Kruppke A. S., Nölke T., Aktories K. Institut für Experimentelle und Klinische Pharmakologie und Toxikologie I, Albertstr. 25, 79104 Freiburg, Germany Clostridium difficile is an anaerobe, gram-positive pathogen. It causes antibioticassociated diarrhoea and pseudomembranous colitis by production of the Rho GTPaseglucosylating toxins A and B. Recently emerging hypervirulent Clostridium difficile strains additionally produce the binary ADP-ribosyltransferase toxin CDT (Clostridium difficile transferase). CDT is taken up via receptor-mediated endocytosis after binding to the Lipolysis Stimulated Lipoprotein Receptor (Papatheodorou et al., PNAS 2011) . In the cytosol, CDT ADP-ribosylates actin at Arg 177, thereby actin polymerization is blocked, resulting in disruption of the F-actin network. CDT and other binary actin-ADP-ribosylating toxins induce redistribution of microtubules in the cell interior and formation of long (>150 µm) microtubule-based protrusions at the surface of intestinal epithelial cells which increase bacterial adherence (Schwan et al., PLoS Pathog 2009 ). The clostridial actin-ADP-ribosyltransferases influence the dynamicity of microtubules and their capture at the cell cortex by indirectly affecting different microtubule regulating proteins like CLASP2 and ACF7. Besides the influence of CDT on microtubule regulatory proteins, the formation of protrusions depends on plasma membrane composition. Depletion of cholesterol, the breakdown of sphingomyelin or inhibition of sphingolipid-synthesis reduce the formation of microtubule-based protrusions. Surprisingly, most of the CDT-induced processes contain membrane-tubules derived from the endoplasmatic reticulum (ER). The remodeling of the ER is microtubule dependent and is mainly mediated by Stim1 that usually functions as a calcium sensor in the ER and activates the store operated Orai1 calcium ion channels in the plasma membrane. The data suggest that toxin-induced changes of the microtubule system including alterations of the ER, may affect trafficking and ER-dependent signalling. Bilobalide is a neuroprotective constituent of Ginkgo biloba with an unknown mechanism of action. In the present study, we first used microdialysis in mice to evaluate changes in the extracellular fluid of the brain during and after stroke. Microdialysis probes were implanted into the striatum of CD-1 mice, and dialysates were obtained while a monofilament was inserted for 60 min via the common carotid artery (CCA) to block perfusion through the middle cerebral artery (MCA). While glucose levels dropped immediately upon middle cerebral artery occlusion (MCAO), glutamate concentrations in the microdialysates -as measured with a CMA 600 analyzer -rose extensively during ischemia to more than 2000% of baseline level. Both glucose and glutamate levels recovered rapidly when MCAO was terminated after 60 min. When bilobalide (10 µM) was perfused into the striatum through the microdialysis probe during MCAO, glucose levels dropped but the neurotoxic rise of glutamate was significantly attenuated and reached only 500% of baseline level (p<0.01). In the following experiments, we investigated the activity of mitochondria in ischemic brain. Ischemia was induced by MCAO, and ischemic as well as "healthy" tissue from the opposite hemisphere was obtained. Mitochondria were isolated and mitochondrial respiration was monitored using the Oroboros ® oxygraph. Significant deficits of respiration were observed after ischemia. In the healthy hemisphere, the respiratory states (leak I+II, complex I+II+IV, oxidative phosphorylation (OXPHOS) and electron transport system (ETS) capacity) showed a decrease of oxygen consumption to 60-70% of sham-operated mice. In the ischemic hemisphere, several values were lower at 40% of sham-operated mice (leak I+II, complex II+IV,OXPHOS and ETS) whereas complex I showed a remarkably low respiratory capacity of 16% of baseline. Direct addition of bilobalide (10 µM) to post-ischemic mitochondria caused a 2-fold increase of complex I activity in vitro. Pretreatment of mice with bilobalide (10 mg/kg i.p.) one hour before MCAO caused a significant, 3-fold improvement of complex I respiration when measured ex vivo. These data clearly indicate that bilobalide targets mitochondrial processes within the respiratory chain, preserving complex I function during ischemia. This action likely explains its neuroprotextive activity in vivo. Unreacted resin monomers such as 2-hydroxyethyl methacrylate (HEMA) are environmental stressors released from dental composites after incomplete polymerization. The production of reactive oxygen species (ROS) is a major response of cells to monomer exposure. Moreover, adverse effects of monomers including delayed cell differentiation or mineralization processes, DNA damage or apoptosis are associated with increased ROS production. The intracellular redox homeostasis is controlled by the major non-enzymatic antioxidant glutathione (GSH), and antioxidant enzymes. Here, we hypothesized that cells exposed to HEMA responded by a differential expression of antioxidant enzymes such as superoxide dismutase (SOD-1), catalase (CAT) or glutathione peroxidase (GPx1/2). RAW246.7 mouse macrophages were exposed to HEMA (0-8mM) for 24h, and protein expression was analyzed by Western blotting. To study the influence of intracellular GSH on enzyme expression, GSH synthesis was reduced by the inhibitor buthionine sulfoximine (50µM BSO), or enhanced by 2-oxothiazolidine-4-carboxylate (5mM OTC) and N-acetyl cysteine (10mM NAC). Expression of SOD-1 found in untreated cultures was decreased in the presence of HEMA and even further reduced by BSO. In contrast, NAC counteracted HEMAinduced inhibition of SOD-1 expression. CAT expression was not detected in untreated cells, however, the enhanced expression of CAT in cells exposed to HEMA indicated the decomposition of abundant levels of hydrogen peroxide. The minor influence of BSO or OTC showed that expression of CAT was independent of GSH levels while a decrease of HEMA-induced CAT expression in the presence of NAC indicated reduced oxidative stress. GPx1/2 was expressed in untreated cultures, and its down-regulation by BSO indicated that this enzyme was primarily responsible for H2O2 decomposition. The inhibitory effect of HEMA on GPx1/2 expression was enhanced by BSO but counteracted by OTC or NAC. These findings indicate that H2O2 is the predominant reactive oxygen species generated in the presence of dental resin monomers like HEMA. Abundant H2O2 production leads to the activation of CAT expression and a feed-back inhibition of SOD-1 expression. The HEMA-induced reduction of GPx1/2 expression is most likely a consequence of reduced GSH levels because of the formation of glutathione disulfide (GSSG) or by GSH-HEMA adducts. The life-threatening effects of certain organophosphorus compounds such as soman or fenamiphos cannot be antagonized adequately by the treatment with atropine and oximes. Alternative approaches are necessary. Since the adequate restoration of disturbed muscle function is considered to be life-saving, a model is needed for screening of potentially therapeutic substances. An established model for the development of such new therapies is the diaphragm preparation. However, this model requires a large number of animals and experimental available time frame is limited to some hours. Here, the organotypic nerve-muscle co-culture may be an appropriate alternative, because a large number of specimens with low numbers of animals and a long period of investigation over several days is possible. In the present study, the restoration of VX paralysed muscle function with obidoxime was investigated by using both models.

Slices of spinal cord and muscle tissue were dissected from mice embryos (E 14-15) , fixed to coverslips and incubated in roller tubes for about 2-4 weeks. Spontaneous muscle activity was recorded by video microscopic techniques and was quantified offline. Muscle force production in mice diaphragm preparations was elicited by indirect field stimulation technique in a 12 chamber organ bath and quantified as time-force diagrams (AUC) that were expressed as relative changes of the muscle force compared to the control data. Application of the nerve agent VX (0.75 µM) resulted in a strong reduction of muscle activity in the co-culture and of muscle force production in the diaphragm muscle. After obidoxime (10 µM) was added spontaneous muscle activity in the co culture recovered from 0.45 ± 0.24 Hz to 1.67 ± 0.24 Hz (control 1.79 + 0.22 Hz) . Muscle force remained stable over the next days. The VX-blocked muscle force of diaphragm was restored to 69.9 ± 21.3 % by obidoxime compared to control. Muscle force production after indirect stimulation was stable for 6 hours only. Our results suggest that the organotypic nerve-muscle co-cultures may be an appropriate tool for the screening of new therapeutic approaches in restoration of blocked neuromuscular transmission. Moreover, the model offers an additional advantage as long-term experiments may be performed and pre-and postsynaptic effects may be assessed directly. Additionally, the number of experimental animals could be reduced. The modulation of gene expression by the transcription factor CREM (cAMP responsiveelement modulator) represents a fundamental mechanism of gene control in response to elevation of intracellular cAMP levels. In vascular smooth muscle cells (VSMCs) CREM is involved in the regulation of cell proliferation and apoptosis supporting its relevance for vascular proliferative diseases. Mice with a global inactivation of CREM (CKO) showed a significant increase in neointima formation after ligation of the carotid artery and an increase of atherosclerotic plaque formation after high fat diet on an ApoE knockout background compared to wildtype controls (WT). On the cellular level a CREM deficiency was associated with a 1.3 fold increased proliferation rate of primary VSMCs after stimulation with the platelet-derived growth factor (PDGF; n=10 from 6 isolations). Microarray analysis and subsequent realtime-RT-PCR validation revealed that the alpha-type platelet-derived growth factor receptor (Pdgfra) the regulator of G-protein signaling 5 (Rgs5) and peptidylprolyl isomerase A (Ppia) were 1.5-2.5 fold upregulated in PDGF treated CKO VSMCs compared to WT VSMCs (n=6). Transcripts of Rgs5 (2.6 fold, ) and Ppia (1.8 fold, were also upregulated in the carotid artery of CKO mice in comparison to WT mice (n=10-12). We conclude that CREM deficiency is associated with transcriptional changes of Rgs5, Pdgfra, Ppia, which might explain the increased proliferation rate in CKO VSMCs and the increased responsiveness to pathophysiological conditions. (Supported by the IZKF Münster).

The role of non-catalytic p101 and p87 subunits in regulating phosphoinositide 3kinase γ by Gβγ and H-Ras Shymanets A. 1 Phosphoinositide 3-kinase γ (PI3Kγ) controls a plethora of cellular responses. PI3Kγ, a heterodimer formed by non-catalytic p101 or p87 and catalytic p110γ subunits, is regulated by Gβγ and Ras. Earlier we speculated that p101 binds to Gβγ to translocate cytosolic PI3Kγ to the plasma membrane, enabling direct activation of p110γ (Brock et al., J. Cell Biol. 2003) . However, the p87 subunit does not function as Gβγ adapter (Kurig et al., PNAS 2009) . Since the impact of each non-catalytic subunit in regulating p110γ by Gβγ and Ras still remains elusive, we studied their role in detail. Gβ1γ2 variants harbouring mutations in positions involved in interaction with Gα subunit were purified from Sf9 cells and tested for their ability to activate PI3Kγ. We observed that p101, but not p87, was able to rescue the stimulatory activity of Gβ1γ2 mutants incapable to activate p110γ (Shymanets et al., Biochem. J. doi:10 .1042/BJ20111664). To further study the functional impact of the non-catalytic subunits on PI3Kγ regulation, we have designed phospholipid vesicles containing similar amounts of recombinant PI3Kγ variants. Although p87/p110γ exhibited stronger sensitivity to Gβ1γ2 than p110γ, the activity of vesicles-associated p101/p110γ was significantly higher as compared to vesicles-associated p87/p110γ or p110γ in the absence and in the presence of Gβ1γ2.

To study an effect of Ras proteins on PI3Kγ variants, recombinantly expressed H-Ras was purified from Sf9 cells. The posttranslational processing and lipidation of the protein was verified by mass spectrometry analysis. The impact of H-Ras on regulation of p87/p110γ and p101/p110γ differed, which may explain integration of PI3Kγ variants in different signalling pathways. Taken together, p87 and p101 subunits implement discrete functions in respect to (i) membrane recruitment of PI3Kγ and (ii) regulation of enzymatic activity by Gβγ and H-Ras.

Preparation of consolidated exposure scenarios for mixtures under REACh Sica M., Dorn S., Mostert V. DR. KNOELL CONSULT GmbH, Marie-Curie-Str. 8, 51377 Leverkusen, Germany Under REACh, formulators of mixtures need to include substance-related information into extended safety data sheets (eSDS), if mixtures are classified as dangerous according to the Dangerous Preparation Directive (Directive 1999/45/EC). One way to add information on substances into eSDS of mixtures is to generate exposure scenarios (ESs) for mixtures. In order to fulfil this task, two approaches have been developed for the identification of the risk-determining substances (lead substances) in the mixtures: The critical component approach (CCA) relies on DNELs and PNECs for all substances, their concentrations in the mixtures as well as substance and use-specific availability parameters (ECHA, 2008) . In contrast, the DPD+ method is based on the legislation for classification of preparations (Directive 1999/45/EC). The DPD+ method defines a lead substance for each route of human exposure and for the aquatic environment (CEFIC, 2010) . However, each of these methods has certain limitations. The aim of the present work is to improve the preparation of consolidated ESs for a number of mixtures and provide information about their safe use. To this end, we first adopted information on risk management measures (RMMs) and operational conditions (OCs) of the lead substances using the DPD+ methodology. At the same time, we considered the specific conditions of use of the mixtures (e.g. spraying, brush painting). We then conducted risk assessments by deriving DNELs for the mixtures and using exposure modelling tools recommended under REACh (e.g., ECETOC TRA, RISKOFDERM, ART). We compared the outcome of these assessments with results obtained from the application of the DPD+ methodology. The work presents the results of application of DPD+ approach and the CCA and indicates possible improvements for the risk assessment of mixtures. To check for seasonal and weather dependent influences in the prescription rate of drugs used to treat cardiovascular and respiratory diseases (ATC codes C and R) a survey covering all prescriptions of a specimen German general regional health insurance (AOK plus, data for Saxony, largest health insurance service, approx. 50 % of all Saxonian citizens are inscribed to this service) for the years 2005 to 2007 was analysed on a monthly basis. The number of prescriptions for cardiovascular drugs changed approximately +/-20 % around the mean for the different month without a clear seasonal pattern. For respiratory drugs only the systemic anticholinergics and drugs used to treat obstructive lung disease displayed a distinct seasonal pattern with a 50 -70 % above average prescription figure during spring time (February to May) and a 25 to 40 % trough in late summer/autumn (July to October). The data have to be analysed for further cofounders (e.g. influenza prevalence, environmental conditions etc.) to fully understand the fluctuations observed. The prescription rate for cardiovascular drugs and respiratory drugs seems to be influenced by multiple factors aside seasonal influences.

Pasteurella multocida toxin prevents osteoblast differentiation by activation of heterotrimeric G proteins Siegert P., Aktories K., Orth J. Institut für Experimentelle und Klinische Pharmakologie und Toxikologie Abt. I, Albertstr. 25, 79104 Freiburg i. Br., Germany Pasteurella multocida toxin (PMT) is a major virulence factor of Pasteurella multocida causing pasteurellosis in man and animals and is responsible for atrophic rhinitis in pigs. The toxin modulates various signaling pathways by acting on the heterotrimeric G proteins Gαq, Gα12/13 and Gαi. PMT activates Gq to increase inositol phosphate production via phospholipase Cβ and alteration of gene expression via the JAK/STAT pathway. The toxin also activates RhoA via Gαq and Gα12/13 family proteins. We showed that the underlying mechanism of the activation of heterotrimeric G proteins is a deamidation of an essential glutamine residue leading to a constitutive activation of the G protein.

Because PMT is the causative agent to induce progressive atrophic rhinitis in pigs, which is characterized by loss of nasal turbinate bones leading to a twisting and/or shortening of the snout, we studied the effect of PMT on bone cells. Here we studied the effect of the toxin on osteoblast differentiation in ST-2 cells and in primary osteoblasts from rat calvaria. ST-2 cells are stromal derived cells, which can be differentiated into osteoblasts or adipocytes. The toxin inhibits the differentiation of ST-2 cells into osteoblasts studied by determination of specific osteoblast markers. Additionally, PMT represses the induction of transcription factors essential for osteoblast differentiation. Moreover, the principal pathways activated by PMT to induce these effects were investigated.

Ventilator-induced lung injury (VILI) is a serious problem in intensive care medicine. Its mechanisms are only incompletely understood, although it is widely accepted that ventilation-induced inflammation (biotrauma) makes an important contribution. The isolated perfused mouse lung (IPL) is a valuable tool to investigate the mechanisms of VILI. Several studies have shown considerable differences between various mouse strains with respect to lung mechanics and inflammatory responses. Therefore, we hypothesized that the pulmonary responses to mechanical ventilation differ between C57Bl/6 and BALB/c mice. In addition, this study introduces the novel half lung technique that allows to obtain lung tissue from the same mouse at two different time points.

Isolated perfused mouse lungs from C57Bl/6 or BALB/c were subjected to high (25cmH 2O) or low pressure (8cmH2O) ventilation for 240 minutes. After 180 minutes the left lung was removed and used for Western Blot analysis. The right lung was ventilated for another 60 minutes. By the end of experiment the right lung was removed and qRT-PCR performed. During the whole experiment perfusate sample were taken from the venous catheter and used for protein quantification by ELISA.

It was possible to remove half of the lung and to further ventilate the other half without acute changes in lung mechanics. In both strains high pressure ventilation elicited a significantly higher cytokine release than low pressure ventilation. C57Bl/6 mice showed higher TNF, IL-1β and amphiregulin levels after high pressure ventilation, whereas BALB/c exhibited increased production of CXC chemokines (Cxcl-1, Cxcl-2) and IL-6. Kinase activities (JNK, Akt, ERK1/2, p38 MAP kinase) were increased in high pressure ventilated animals, but were strain independent.

The novel half lung technique builds on the well established IPL method. It permits to separately analyze the left and the right lung at different time points during continual ventilation. This method reduces animal numbers by 50% and allows statistical within subject analysis. Using this method, the present study showed that inflammatory response to mechanical ventilation differ between C57Bl/6 and BALB/c. These findings show that the biotrauma response in mice depends on the strain that is studied. Macrophages play an important role as an integral part of the first line of immune defense. Two different macrophage populations have been described. M1 macrophages produce proinflammatory cytokines and are involved in inflammatory processes. By contrast, M2 macrophages release anti-inflammatory cytokines and extracellular matrix components. They can enhance wound repair and angiogenesis, but they can also promote tumor progression. Recently, industrial nanoparticles have raised concerns because of their putative toxic effects. On the other hand, specifically designed nanoparticles can be used as clinical diagnostics and as drug carriers for pharmacotherapy. Thus, investigations on the interactions of engineered nanoparticles with living cells and organisms are of great importance. Macrophages as phagocytosing cells scavenge nanoparticles circulating in the bloodstream. Therefore, we analyzed how nanoparticles with different surface functionalization might affect functions of human macrophages. Monocytes were isolated from buffy coats and differentiated to macrophages with macrophage colony-stimulating factor. Carboxy-(PS-COOH) and amino-(PS-NH 2) functionalized polystyrene nanoparticles were produced by the miniemulsion polymerization process and the average particle size, the polydispersity index and their zeta potential were determined by dynamic light scattering. The macrophages were cultured in the presence or absence of different concentrations of PS-COOH and PS-NH2 nanoparticles for up to 6 days. Analysis of cell viability revealed that PS-NH2 but not PS-COOH concentration-and time-dependently reduced the macrophage viability. By annexin V/propidium iodide double staining we could show that PS-NH2 trigger apoptosis in macrophages. We further polarized macrophages to either M1 or M2 using IFN-γ and LPS or IL-4, respectively. These macrophage populations were characterized by their expression of extracellular markers by flow cytometry and their production of cytokines by ELISA. The effects of functionalized polysterene nanoparticles on the cytokine production and surface marker expression of M1 and M2 macrophages were analyzed. Our data indicate that surface functionalization is a critical parameter in the nanoparticle-induced toxicity in human macrophages. This work was supported by the DFG SPP1313.

Loos C., Lunov O., Syrovets T., Simmet T. Ulm University Institute of Pharmacology of Natural Products & Clinical Pharmacology, Helmholtzstr. 20, 89081 Ulm, Germany Nanoparticles are currently used for various medical applications including imaging, diagnosis and drug delivery. Due to particle size and surface area, their fundamental properties differ significantly from those of corresponding bulk materials. Nanoparticles circulating in the blood are mainly sequestrated by the reticuloendothelial system that consists predominantly of phagocytic macrophages. Macrophages express a variety of cellular receptors for sensing and internalizing particular material like viruses, microorganisms, and foreign particulate matter including nanoparticles. Therefore, a detailed understanding of the intracellular fate and processing of the nanoparticles by macrophages is indispensable for controlled biomedical applications of nanoparticles. Introducing distinct surface modifications, one might control nanoparticle uptake by different cell types and thereby target specific tissues and cellular compartments. Tumor cell lines are frequently used as models for primary cells to analyze the effect of nanoparticles on cells. Here we show that carboxy-(PS-COOH) and aminofunctionalized (PS-NH 2) polystyrene nanoparticles of ~100 nm in diameter are internalized by human macrophages, THP-1 monocytic leukemia cells, and by PMAdifferentiated THP-1 cells via different mechanisms. In buffer, macrophages and THP-1 rapidly internalize both types of nanoparticles, yet, the carboxy-functionalized particles were taken up to a higher extent. The uptake of both nanoparticles was drastically reduced in media containing serum. Using pharmacological and antisense in vitro knockdown approaches, we showed that the specific interaction between CD64 receptors and the particles determines the macrophage uptake of particles by phagocytosis, whereas particle internalization by THP-1 cells occurred via dynamin IIdependent endocytosis. By contrast, PMA-differentiated THP-1 cells took up the particles via macropinocytosis. In line with the in vitro data, more intravenously applied PS-COOH particles accumulated in liver tissue, whereas PS-NH 2 were preferentially targeted to tumor tissue. These data show that the amount of particle internalization, the uptake mechanisms, and kinetics differ significantly among primary cells and model tumor cells, whether differentiated or not, and that they are further critically dependent on the particle opsonisation by serum proteins. This work was supported by the DFG SPP1313. Specifically designed and functionalized nanoparticles hold great promise for a variety of biomedical applications. To ensure their safe application, such particles require a rigorous analysis of their effects on cell functions. Here we demonstrate that aminofunctionalized polystyrene nanoparticles (PS-NH2) of ~100 nm in diameter in contrast to carboxy-(PS-COOH) and nonfunctionalized (PS) particles induce an NLRP3 inflammasome activation and the subsequent release of IL-1β in human macrophages. Amino-functionalized PS nanoparticles induced time-dependent lysosomal destabilization followed by release of lysosomal enzymes. This resulted in mitochondrial damage and formation of reactive oxygen species. Accumulation of mitochondrial reactive oxygen species was accompanied by oxidation of thioredoxin, a protein playing a central role in maintaining the cellular redox balance. Upon oxidation, thioredoxin dissociated from the thioredoxin-interacting protein (TXNIP). Liberated TXNIP, in turn, interacted with the NLRP3 protein resulting in a conformational change of the pyrin domain of the NLRP3 protein as predicted by molecular modeling. TXNIP interaction with NLRP3 led to assembly of the NLRP3 inflammasome complex, to caspase-1 activation, and release of IL-1β. Using an in vitro knockdown approach, we showed that PS-NH 2 induced activation exclusively of NLRP3, whereas other inflammasomes remained unaffected. Treatment of macrophages with N-acetyl-L-cysteine, a scavenger of reactive oxygen species, abolished both, the caspase-1 activation and the subsequent release of IL-1β caused by PS-NH2 nanoparticles. These data reveal a novel mechanism of the NLRP3 activation induced by amino-functionalized nanoparticles and provide a strategy as to how such an effect can be functionally antagonized by supplementation with a radical scavenger. This work was supported by the DFG SPP1313. The semi-permeable barrier of the endothelial cell lining of the blood vessels has important synthetic and metabolic functions including transport of cells and biomolecules, regulation of vascular smooth muscle tone, and control of hemostasis. Plasmin is a serine protease, which is generated from its zymogen plasminogen under physiological and pathological conditions. Small amounts of plasmin are produced in the context of contact activation during inflammation. Consistently, increased generation of plasmin has been reported during atherosclerosis. We have shown previously that plasmin, in addition to its role in fibrinolysis, could induce proinflammatory activation of various cells including monocytes, macrophages, and dendritic cells. Therefore, we analyzed how plasmin might affect the functions of endothelial cells, which could be relevant during inflammation and atherosclerosis. Using flow cytometry, Western immunoblotting and fluorescent microscopy, we show that endothelial cells of different origin express the plasmin receptor complex composed of annexin A2 and S100A10. Addition of plasmin to human umbilical vein endothelial cells (HUVEC) induced timeand concentration-dependent cytotoxic effects in the cells. In addition, within 30 min plasmin triggered a rapid and prolonged expression of free radical oxygen species (ROS) in endothelial cells as analyzed by microscopy and fluorometry using the ROSsensitive dye carboxy-H 2DCFDA. The ROS production in endothelial cells was accompanied by cell detachment. Fluorometric and Western blot analysis of caspase 3 activation in the cells treated with plasmin showed that plasmin induced apoptotic cell death in endothelial cells, which was evident already several hours after exposure to plasmin. Thus, plasmin might induce production of ROS in endothelial cells, their detachment and apoptosis, events which might be relevant for the development of atherosclerosis. This work was supported by the DFG. Sesquiterpene lactones (STL) comprise a large group of secondary plant metabolites that constitute the active principle of a number of traditional anti-inflammatory phytomedicines. Specifically helenalin and parthenolide have recently gained considerable attention as lead compounds or putative therapeutics for the treatment of inflammation and possibly cancer. Both compounds have been shown to interfere with the signal transduction through inhibition of the nuclear factor κB (NF-κB). Whereas the inhibitory effects of the STL on NF-κB are undisputed, their molecular mechanism of action remains a matter of debate. Surface plasmon resonance (SPR) analysis allows label-free measurement of molecular interactions. Yet, analysis of the interaction of immobilized recombinant proteins with small molecular ligands remains a technically challenging task. In the present study we used SPR technology to investigate the molecular interaction of the STL helenalin with putative intracellular target proteins such as the NF-κB protein p65/RelA, the catalytic subunits of the IKK complex, namely IKKα and IKKβ, and the intracellular antioxidant glutathione (GSH). At physiological pH 7.4, helenalin interacts with RelA (K D = 4.8 µM), yet it failed to bind either IKKα or IKKβ. Hence, when DNA with NF-κB binding consensus sequence was immobilized on sensor chips, the binding of RelA was inhibited by helenalin with an IC50 of 5.0 µM. Moreover, we provided several lines of evidence that STL may modify RelA on cysteine 38 by a Michael-type addition. This interaction was confirmed by molecular docking that identified the best matching interaction between RelA and helenalin with predicted hydrogen bonding interactions between helenalin and residues Arg35, Lys37, Gly44 and Ile118 of RelA. Consistent with our hypothesis that helenalin interacts with sulfhydryl groups at pH 8.0, helenalin was also able to interact with reduced, but not oxidized, glutathione with a KD of 24 µM, though no significant interaction was observed at pH 7.4. Thus, we showed that the sesquiterpene lactone helenalin interacts with the NF-κB protein RelA but not with IKKα or IKKβ. Moreover, at physiological pH, helenalin does not interact with glutathione to any significant extent. Direct interaction of helenalin with RelA leading to inhibition of RelA-DNA binding and transactivation might present the molecular mechanisms underlying the anti-inflammatory effects of STLs. Although nanosized materials are quickly taken up by macrophages, our understanding of the involved processes is still rather limited. Therefore, we analyzed the uptake of diagnostically used carboxydextran-coated iron oxide nanoparticles of two different sizes, superparamagnetic iron oxide nanoparticles of 60 nm (SPIO) and ultrasmall superparamagnetic iron oxide nanoparticles of 20 nm (USPIO), by human macrophages. By pharmacological and in vitro knockdown approaches, the principal uptake mechanism of macrophages for both particles was identified as clathrin-mediated, scavenger receptor A-dependent endocytosis. Further, we created a mathematical model of the nanoparticle uptake by macrophages that permitted determination of key parameters of endocytotic process, such as the uptake rate, the mean uptake time, the number of particles taken up by a cell, and the correlation between the number of internalized particles and their extracellular concentration. The model also provided information on the individual and collective wrapping time of the nanoparticles and described the relation between biophysical parameters such as cytoskeletal forces, membrane elasticity, and the uptake time. Finally, we gained information on the minimal linear spacing between simultaneously acting neighboring endocytotic pits that contain single nanoparticles and govern the collective uptake process. The calculated parameters were further confirmed experimentally using spinning disc confocal microscopy. Thus, the new model provides important insights into the biophysical processes involved in endocytosis of nanoparticles by human macrophages. This work was supported by the DFG SPP1313. 

Prostaglandins (PG) are hormones which are formed during inflammatory processes from arachidonic acid by cyclooxygenases and prostaglandin synthases [4] . In the subsequent metabolism, in which the five-membered ring is dehydrated, α,β-unsaturated carbonyl compounds are generated [2, 3] . These come along with mercapto groups of amino acids in a Michael addition reaction associated with activation of cellular enzyme cascades [1] that potentially contribute to their possessed antiinflammatory, antineoplastic and antiviral effects [5] . However little is known so far about possible adverse health effects.We addressed the question whether selected cyclopentenone prostaglandins (cyPG) exhibit potential mutagenic and genotoxic properties in the hamster lung fibroblast cell line V79.

Induction of DNA damage was investigated by single cell gel electrophoresis assay (SCGE). The impact of cyPG on cellular redox status was detected by total glutathione (tGSH) assay. The induction of micronuclei and apoptosis was determined by staining with 4',6-diamidino-2-phenylindole (DAPI). Furthermore the hypo-xanthine-guanine phosphoribosyltransferase (hprt) assay was used for mutagenicity testing. , followed by prostaglandin A2 (PGA2), showed the most distinctive genotoxicity, i.e., induction of micronuclei, and apoptotic effects. Furthermore, the 15dPGJ2 and PGA2 -induced significant decrease in the tGSH level in V79 may contribute to the observed increase in oxidative DNA-damage. However, none of the tested cyPG exhibited mutagenic properties in the hprt assay.

In conclusion, a potential in vitro genotoxicity of cyPG has been observed which may be involved in carcinogenesis associated with chronic inflammation.

Parabens and methylisothiazolinone are used as preservatives in personal care products. Sensitization to parabens and methylisothiazolinone is relatively rare considering their wide use in cosmetics, but only few quantitative or clinical data exist. Therefore, we have tested methyl-, ethyl-, propyl-, isopropyl-, butyl-, isobutyl-, pentyl-, phenyl-and benzylparaben , and methylisothiazolinone in the loose-fit coculture-based sensitization assay (LCSA) developed by our working group. The coculture of primary human keratinocytes and allogenic dendritic cell-related cells (DC-rc) in this assay emulates the in vivo situation of the human skin. Sensitization potential of the test substances was determined by flow cytometric analysis of the DC-rc maturation marker CD86. Determination of the concentration required to cause a half-maximal increase in CD86-expression (EC 50) allowed a quantitative evaluation. The irritative potential of the substances was assessed by 7-AAD (7-amino-actinomycin D)-staining. The concentration required to devitalize 50 % of the examined cells compared to a zero control was termed EC50%. Parabens exhibited weak (methyl-, ethyl-, propyl-and isopropylparaben) or strong (butyl-, isobutyl-, pentyl-and benzylparaben) sensitizing potential, phenylparaben was found to be a moderate sensitizer, with EC50-values ranging from 16.67 µmol/l (pentylparaben) to 325.36 µmol/l (methylparaben). Due to a pronounced cytotoxicity (EC50% = 70.94 µmol/l), we could not estimate an EC50-value for methylisothiazolinone. Sensitization potential of parabens correlated with side chain length. Parabens showed no (methyl-and ethylparaben) or weak irritative potential (propyl-, isopropyl-, butyl-, isobutyl-, phenyl-and benzylparaben) , only pentylparaben was rated to be irritative. Apart from phenyl-and benzylparaben, irritative potential also correlated with side chain length but did not correlate strictly with the sensitization potential. Overall, we were able to demonstrate and compare the sensitizing potential of parabens in this in vitro test. It was weak for methyl-and propylparaben, the most commonly used parabens. Furthermore, we showed an irritative potential for most of the perservatives. Thus the LCSA is a useful in vitro test to compare the sensitizing potential of xenobiotics.

Phosphorylation of the neurodegeneration-related Septin 4 by protein kinase DYRK1A at serine 107 affects protein stability Soppa U. 1 Septins are GTP-binding proteins forming heterooligomeric complexes and filaments by interactions of the 13 family members. These complexes have important functions by building scaffolds for proteins involved in cell cycle or cell polarity but their subcellular distribution as well as their regulation remain largely unclear. Septin 4 (SEPT4) was found in neurodegeneration related protein aggregates and is associated with migration of cortical neurons. Here we first describe a potential mechanism for regulation of SEPT4 by phosphorylation via Dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A). DYRK1A is overexpressed in Down syndrome and supposed to be involved in neurodevelopment and neurodegeneration. By site directed mutagenesis of Flag tagged mouse SEPT4 and overexpression in HeLa cells we identified serine 107 as the major phosphorylation site of DYRK1A and generated a phosphospecific antibody. Transient coexpression of SEPT4 and DYRK1A in HeLa cells increased phosphorylation of serine 107 by 50% in relation to basal phosphorylation. In contrast, cotransfection of the kinase deficient DYRK1A mutants K188R and D287N did not increase serine 107 phosphorylation. Moreover we could show, that inhibition of kinase activity by the DYRK1A inhibitor harmine reduced phosphorylation of exogenous SEPT4 at serine 107 about 25% in HeLa cells. Furthermore, down regulation of DYRK1A by RNA interference lead to decreased phosphorylated serine 107. These results indicate that endogenous DYRK1A contributes to SEPT4 phosphorylation in HeLa cells. Finally we analyzed protein stability of wild type SEPT4 compared to the phosphorylation resistant S107A mutant in HeLa cells by inhibition of translation with cycloheximide. We found that in living cells the SEPT4 S107A mutant is more stable than wild type SEPT4. In summary, our results suggest phosphorylation at serine 107 by DYRK1A as a novel mechanism to regulate SEPT4 stability and indicate a possible link of these proteins in cellular processes.

Is formaldehyde a good example for a "genotoxic carcinogen" with a threshold mode of action? Speit G. Institut für Humangenetik, Universität Ulm, 89069 Ulm, Germany Formaldehyde (FA) induces toxic and genotoxic effects in directly exposed cells (site of contact). Several studies in which FA was administered to rats by inhalation showed evidence of tumor induction in nasal epithelium. There is also some epidemiological evidence that FA causes nasopharyngeal cancer in humans. Although FA is a known mutagen, it is still a matter of discussion whether carcinogenesis is primarily mediated via a mutagenic mode of action. There is evidence that cytotoxicity and induced proliferation are the main causes for tumor formation. However, a decisive role of mutagenesis cannot be excluded and a mutagenic mode of action has to be considered for risk estimation. The basic assumption is that mutagens have a non-threshold mode of action. A threshold mode of action for a chemical is likely when a substance with a known mutagenic potential does not induce mutations at low concentrations due to a specific type of reaction with the genetic material and / or physiological protective mechanisms. Because FA is a directly acting DNA-reactive substance, a threshold mode of action may only be considered because of physiological protective mechanisms. After inhalation, pre-lesion protection occurs by unspecific binding to mucus, cellular proteins and glutathione. Furthermore, FA is efficiently inactivated by enzymatic pathways. If FA reaches and damages the nuclear DNA, DNA repair mechanisms act as efficient postlesion protection mechanisms. In vivo inhalation studies with rats indicated that FA induces primary DNA damage in the nasal epithelium but increased mutation frequencies were not measured. Data are now available to show the relative distribution of endogenous versus exogenous DNA adducts in different locations of the nose and other organs. Considering the FA concentrations present in every living cell and the background levels of endogenous DNA adducts, appropriate risk assessment and the identification of practical thresholds for FA-induced genotoxicity become feasible.

Fraud and misconduct in clinical trials Steffen C. formerly Federal Institute for Drugs and Medical Devices Clinical Trials Unit, Kurt-Georg-Kiesinger-Allee 3, 53175 Bonn, Germany Plagiarism in scientific publications has been the subject of public ("Guttenplag") and scientific debate. Plagiarism violates, however, "only" the intellectual property of its authors. In clinical trials, misconduct may also endager the health of patients. The suppression or falsification of data in clinical trials may mislead patients and doctors to use worthless treatments. Clinicians may be provoked to repeat these trials, thereby wasting time and money. In clinical trials, misconduct includes everything from suppression or their repeated publication, the "correction" of unwanted results to the complete invention of the data, their intentional or negligent misinterpretation leading to the publication of biased conclusions from otherwise correctly performed clinical studies. The peer review system cannot protect against fraud, as few reviewers will have the means and the time to reevaluate original data. They will have to rely on these data, the calculations and statistics that are presented to them. Some kinds of fraudulent behavior, such as double publications, can be found in the review process, but this is also time-consuming and depends on a helpful librarian. Other kind of fraud, as the suppression of patient data in clinical trials (the deletion of "non-responders", according to Cinderella: The good ones go into the pot, the bad ones go into your crop) can only be detected by the National Competent Authorities performing an inspection according to Good Clinical Practice. Even if the results of such an inspection become public as in the case of Ukrain (Gansauge et al. 2002) , there is no institution that will further analyze and publish the misconduct or initiate the retraction of the incriminated paper. A German agency similar to the US Office of Scientific Integrity could foster Good Clinical Practice in Germany. Although it is sometimes very difficult to decide whether improper results arise from fraud or error, a bias for the source of funding is obvious. Trials funded by for-profit organizations were significantly more likely to recommend the experimental drug as treatment of choice (Als-Nielsen et al. 2003) . Medicinal products with disputed efficacy such as orally applied enzymes for systemic action, bacterial preparations for irritable bowel syndrome and food supplements are to be reviewed with special attention. Further examples from the author's experience will be presented. ]i favors further KCa opening, KCa may establish a feed-forward regulation of Ca 2+ influx. In the present study we analyzed whether KCa channels of SK4 and BK type play a role in sustaining Ca 2+ oscillations at G1-to S-phase transition of primary mouse tumor cells and in human breast cancer cells, aiming towards a better understanding how KCa modulate tumor cell proliferation. Methods: KCa expression was quantified by qPCR in human breast cancer biopsies, transgenic MMTV/c-neu + mouse mammary tumors and primary tumor cells derived thereof. The identity of the tumor cells was verified by gliolan staining. Proliferation of the primary mammary tumor cells in the presence or absence of BK and SK4 modulators was tested using a real time cell monitoring system. The cellular DNA content as a measure for cell ploidity was determined by propidium iodide staining and flow cytometry. Changes in [Ca 2+ ]i oscillations and peak amplitude were determined using Ca 2+ indicator FURA-2AM. Results: SK4, but not BK, expression is detectable in human and mouse breast cancer biopsies and in primary tumor cells derived from the MMTV/c-neu + mouse model. SK4 inhibition by TRAM-34 dose-dependently (0,1 to 10 µM) inhibits the growth of primary mammary tumor cells probably by a G1 cell cycle arrest. Ca 2+ oscillations in proliferating MMTV/c-neu + tumor cells were ablated upon pharmacologic inhibition of SK4 channels.

-dependent cell cycle progression is dependent on SK4 activity. Blocking SK4 disrupts a feed-forward loop that coordinates Ca 2+ influx via TRP or CRAC channels in tumor cells. The consequences of SK4 inhibition in mammary tumors in vivo will be discussed.

Synthesis of a triphenylphosphonium substituted derivative of 5-hydroxymethyl-5-methylpyrroline N-oxide Stolze K. 1 ESR combined with spin trapping is a well-known analytical approach to detect free radicals formed in various biological systems, e.g. superoxide, hydroxyl and a series of carbon-centered free radicals, which are involved in oxidative stress. Our aim was to modify the established spin trap 5,5-dimethyl-pyrroline N-oxide (DMPO) with a functional side chain, which can be used further as anchor for moieties enabling the spin trap to penetrate mitochondrial membranes, such as the positively charged triphenylphosphonium substituent. Several synthetic routes were tested to introduce a 4-carboxybutyltriphenylphosphonium-substituent to the spin trap 5-hydroxymethyl-5-methylpyrroline Noxide (HMMPO). While the activation of the carboxy group via the corresponding chloride was not successful, the use of a mixed anhydride with acetic acid appeared to be a promising way, although the reaction is considerably slower. Preliminary spin trapping experiments have been performed with model systems generating superoxide, hydroxyl-, and carbon-centered radicals.

Othman E. M., Stopper H. Universität Würzburg Toxikologie, Versbacher Str. 9, 97078 Würzburg, Germany Type 2 diabetes mellitus (DM2) is a growing health problem affecting more than 150 million people worldwide. It is associated with severe acute and chronic complications that negatively influence both the quality of life and survival of affected individuals. Epidemiological studies clearly indicate that the risk of several types of cancer (including pancreas, liver, breast, colorectal, urinary tract and female reproductive organs) is increased in diabetic patients. Diabetic patients are exposed to oxidative stress which plays a pivotal role in the pathogenesis of both micro-and macro-vascular complications. This is due to a decreased antioxidant capacity and chronic exposure to increased levels of reactive oxygen species (ROS). Since the insulin resistance in DM2 leads to hyperinsulinemia we studied the cellular consequences of the elevated insulin level and showed that it generates superoxide anions (O 2-) and DNA damage by a NADPH oxidase dependent mechanism in cultured cells. In addition, we found elevated genomic damage in the lymphocytes of diabetic patients as well as oxidative stress and genomic damage in kidneys of diabetic rats. This effect of insulin may contribute to the pathogenesis and progression of DM2 complications including the elevated cancer risk. The classical transient receptor potential (TRPC) channel subfamily is regarded as nonselective, calcium permeable cation channels involved in a wide range of physiological events that require calcium signaling. Until now, the specific roles of TRPC channels in neuronal function are still elusive. Given that TRPC1 is able to form receptor-operated heterotetrameric channel complexes with other TRPC channel subunits, we investigated the role of TRPC1 for receptor-operated calcium influx in the heterologous expression system as well as in neurons. For this electrophysiological whole-cell measurements, fluorimetric calcium measurements, Mn 2+ quenching and qPCR analysis were applied. Furthermore, the effect of TRPC1 knock-down on neuronal migration was monitored performing scratch assays, videomicroscopy and G-actin/F-actin assays. Employing these techniques, we found that recombinant TRPC1 was not able to function as a homomeric channel. Instead, TRPC1 subunits formed functional receptor-operated heteromeric channel complexes with TRPC3, 4, 5, 6, and 7. Heteromers containing TRPC1 subunits showed significantly decreased calcium permeation in heterologous cell systems. Mutation of amino acids in the putative pore forming region of TRPC1 further reduced calcium permeability. In GnRH neurons endogenously expressing TRPC1, 2, 5, and 6, downregulation of TRPC1 by shRNA resulted in increased basal cytosolic calcium concentrations and elevated calcium permeability. TRPC1 was not involved in store-operated cation influx in GnRH neurons. Moreover, TRPC1 suppressed the migration of GnRH neurons without affecting cell proliferation. These findings suggest a novel regulatory mechanism relying on the expression of TRPC1 and the subsequent formation of heteromeric TRPC channel complexes with reduced calcium permeability, thereby fine-tuning neuronal migration. The transient receptor potential melastatin-3 (TRPM3) is a calcium permeable nonselective cation channel that can be activated by the neurosteroid pregnenolonesulfate (PregS) or heat. TRPM3 is expressed in various tissues, including insulin-secreting βcells and a subset of sensory neurons from dorsal root (DRG) and trigeminal ganglia. The ability of PregS to evoke TRPM3-like currents in pancreatic β-cells and to induce insulin secretion indicated its involvement in blood glucose regulation. However, TRPM3 -/mice show so far no metabolic deficits but further investigations are recommended to evaluate its function in insulin secretion. Further studies showed that TRPM3 is a nociceptor channel involved in sensing heat and inflammatory thermal hyperalgesia. We performed a calcium-based screening of a compound library (Spectrum Collection) that identified several natural compounds as TRPM3 blockers. The most potent blockers were the citrus fruit flavonoids hesperetin and naringenin as well as ononetin, a chalcon from ononis spinosa. The IC50 values of the substances are in the low micromoles ranges. Electrophysiological whole cell measurements as well as calcium measurements confirmed the potency of the TRPM3 blockers. Furthermore, we could show that these blockers are effective on endogenous TRPM3 in DRG neurons from mice and isolated β-cells. By drinking grapefruit juice naringenin could be consumed in concentrations that are sufficiently high enough to block TRPM3 activity in vivo. In sensory neurons, TRPM3 may exert similar functions as TRPV1. Thus, TRPM3 blocker could bear a therapeutic potential for analgesic treatment. XTT-based cell viability assay was used to determine the half-maximum effect concentration (EC50) for the investigated composite components in HGF. Following concentrations of substances were used to determine the induced double strand DNA breaks (DSBs): 1 /25× EC50, 1 /10× EC50 , 1 /3× EC50, and 1× EC50. Each experiment was performed at least four times. HGF were incubated with various concentrations of substances for a period of 6 hours. Induced DNA double-strand breaks (DSBs) were tested by the γH2AX focus assay, which is a direct marker for DSBs using anti γH2AX antibodies. For quantitative γH2AX analysis foci in cell nucleus were counted by eye down using a fluorescence microscope. Each experiment was performed at least four times.

In the XTT test following EC50 values of substances were found (mmol/L;mean +/-sem): TMP(EO)9TA a, b 0.087 ± 0.011; 1,6-HDDMA b, c 4.500 ± 0.700; ETMA a, c ; 12.000 ± 1.100; a significantly (p < 0.05) differently to 1,6-HDDMA, b significantly (p < 0.05) differently to ETMA, c significantly (p < 0.05) differently to TMP(EO)9TA.

After six hours of exposure with TMP(EO)9TA at 0.00348 mM there were induced 0.55 γ-H2AX foci-formations in HGFs, at 0.00870 mM 0.67 foci, at 0.02900 mM 0.86 foci and at 0.08700 mM 0.97 foci. After exposure with 1,6-HDDMA at 0.180 mM there were induced 0.45 γ-H2AX foci, at 0.450 mM 0.64 foci, at 1.500 mM 0.94 foci and at 4.500 mM 1.32 foci. After exposure with ETMA at 0.48 mM there were induced 0.43 γ-H2AX foci, at 1.2 mM 0.50 foci, at 4.0 mM 0.61 foci and at 12 mM 0.71 foci. The negative controls DMSO and medium cultures displayed 0.31 -0.34 γ-H2AX foci/cell. It was found that the induction of foci/cell were concentration-dependet for all xenobiotics in the order of: 1,6-HDDMA > TMP(EO)9TA > ETMA.

These results show that dental composite components can induce DSBs in primary oral cells and therefore these substances demonstrate a genotoxic potential.

Effects of antioxidants on the DNA-toxicity of dental (co)monomers in human gingival fibroblasts Styllou P. 1 , Scherthan H. Unreacted (co)monomers can be released from restorative dental materials and may show biologic activity after ingestion in the human organism. In previous studies the mutagenic/carcinogenic effect of dental monomers/co-monomers (e.g. methacrylates) on the human DNA was demonstrated. In this study the effects of the antioxidants vitamin C and N-acetylcysteine on the DNA toxicity of the (co)monomers triethylenglycol-dimethacrylate (TEGDMA) and 2-hydroxyethyl methacrylate (HEMA) was investigated. The induction of DNA double-strand breaks with (co)monomers alone and in combination with antioxidants was investigated in human gingival fibroblasts (HGF). HGF were incubated with substances without or with antioxidants for a period of 6 hours. Induced DNA double-strand breaks (DSBs) were tested by the γH2AX focus assay, which is a direct marker for DSBs using anti γH2AX antibodies. For quantitative analysis of the γ-H2AX test, foci were counted by the same investigator by eye down the fluorescence microscope. Each experiment was performed at least four times. The halfmaximum effect concentration EC 50 (mmol/l) of triethylenglykol dimethacrylat (TEGDMA) and 2-hydroxyethyl methacrylat (HEMA) was taken from of a previous study after using XTT-based cell viability assay.

TEGDMA induced significantly (p < 0.05) higher DSBs compared to HEMA (1.91 ± 0.04 vs 1.66 ± 0.02). The mean number of cells scored and the standard deviation (SD) were calculated.

When cells were exposed to TEGDMA in combination with the antioxidant vitamin C an increase of DSBs was observed (2.02 ± 0.06), compared to TEGDMA alone. When cells were exposed to HEMA in combination with vitamin C an increase of DSBs was observed (1.89 ± 0.07), compared to HEMA alone.

When cells were exposed to TEGDMA in combination with the antioxidant Nacetylcysteine a decrease of DSBs was observed (1.64 ± 0.04), compared to TEGDMA alone. When cells were exposed to HEMA in combination with N-acetylcysteine a decrease of DSBs was observed (0.76 ± 0.02), compared to HEMA alone.

These results show that dental (co)monomers can induce DSBs in primary oral cells. It also shows for the first time that the genotoxic potential may be reduced by the addition of the antioxidant N-acetylcysteine. Purpose: We aimed to investigate the role of superoxide and peroxynitrite generated by genetically destabilized eNOS for the development of endothelial dysfunction and vascular remodelling. Methods: A mutant of bovine eNOS in which Cys 101 was replaced by Ala (C101A) resulting in destabilization of eNOS has been generated (eNOS-C101A). Transgenic mice carrying C101A were generated on a C57Bl/6 background using the endotheliumspecific tie-2 promoter. By breeding these mice with eNOS knockouts (eNOS-KO), mice that express eNOS-C101A (eNOS-KO/eNOS-C101A-Tg) exclusively in the endothelium were obtained. Unilateral common carotid artery ligation experiments were performed in C57Bl/6, eNOS-KO, and eNOS-KO/ eNOS-C101A-Tg to study a role of destabilized eNOS for vascular lesion formation.

Results: Western blot analysis confirmed the expression of eNOS in eNOS-KO/eNOS-C101A-Tg in aorta (37.1±8.4%, n=9), skeletal muscle (45.4±5.3%, n=10) and myocardium (17.4±4.9%, n=7) and revealed an increased phosphorylation of eNOS on Ser1176/79 (470±47%) as compared to C57Bl/6 (p<0.05, n=8). Endothelium-specific overexpression of destabilized eNOS induced a large increase in superoxide and peroxynitrite formation in the aorta and the heart of eNOS-KO/eNOS-C101A-Tg (P<0.05, n=5-8), which was abolished by NOS-inhibitor L-nitroarginine (L-NA) suggesting eNOS-C101A as a source of elevated radical generation. Endothelium-specific introduction of eNOS-C101A at ~ 35% of C57Bl/6 level almost completely restored aortic endotheliumdependent relaxation. Experiments with L-NA, soluble guanylyl cyclase inhibitor ODQ, PEG-catalase and NO-scavenger Fe(DETC)2 indicated that endothelium-dependent relaxation in eNOS-KO/eNOS-C101A-Tg is NOS-and cGMP-dependent and NOmediated. Four weeks after the carotid artery ligation, neointima formation, media thickening and luminal narrowing were observed in the ligated arteries of all studied genotypes (P<0.05, n=4-7). Consistent with vasoprotective roles of eNOS, neointima formation was accelerated in eNOS-KO (n=4-7, P<0.05). Despite significantly higher vascular levels of nitrotyrosine and peroxynitrite, neointima formation in eNOS-KO/eNOS-C101A-Tg was substantially lower then in eNOS-KO and tended to be similar to C57Bl/6. Conclusions: Increased vascular superoxide and peroxynitrite formation caused by destabilization of eNOS does not induce endothelial dysfunction in healthy mice and has negligible effect on neointima formation.

Fenton reactivity as a determining parameter for the interaction of manganese oxide nanoparticles with lung epithelial cells Sydlik U. 1 , Bieschke C. Nanoparticles consisting of manganese oxide have been suggested for several innovative technological approaches, including the use in nanomedicine and diagnostics. Therefore, the interaction of such nanoparticles with human target cells is of particular interest for the success of nanomedical approaches but also with regard to unintended side effects. To address this problem, we tested different kinds of manganese nanoparticles (MnNP) in an in vitro system which we earlier evaluated for proliferative, apoptotic, and pro-inflammatory endpoints induced by carbon nanoparticles (CNP). MnNP were synthesized by hydrothermal treatment of manganese salt solutions. The particles were subsequently characterized by scanning electron microscopy and dynamic light scattering. Biological and toxic effects of the generated particles were studied in comparison to carbon nanoparticles (CNP) in experiments with rat and human lung epithelial cells (RLE-6TN and 16HBE14o-). Cytotoxicity was determined as measures of membrane damage (lactate dehydrogenase release) and metabolic activity (water soluble tetrazolium conversion). The oxidative capacity of the particles as well as the generation of intracellular oxidative stress was monitored using dichlorofluorescein diacetate in cell free experiments and flow cytometry assays (FACS), respectively. The particle-specific phosphorylation of src family kinases (SFK) and mitogen activated protein kinases Erk1/2 were investigated using Western Blot techniques. After physico-chemical characterization, a set of three MnNP consisting of Mn3O4 or MnO2 with significant differences in size and shape were selected. According to the different oxidation stages of manganese, the particles showed significant differences in Fenton reactivity in the cell free system. These data did not reflect the capacity of the particles to induce intracellular oxidative stress. The characteristic to trigger membranedependent signaling processes, however, was correlated to the intrinsic oxidative capacity of MnNP than to the ability to induce intracellular ROS. Furthermore, the metabolic activity (WST) was negatively correlated with intracellular ROS, indicating a link between mitochondrial activity and ROS generation. None of the particles had effects on the membrane integrity of the cells. The data demonstrate that MnNP, unlike other poorly soluble nanoparticles (e.g. CNP), mainly trigger adverse health effects through ROS production via the Fenton reaction.

Acute ozone induced airway inflammation does not effect resting human sympathetic nerve traffic Tank J. 1 Numerous mediators released in inflammatory and neuropathic pain states activate Gprotein-coupled receptors (GPCRs) and modulate nociception via activation of Gs, Gi/o, G12/13, or Gq/11 G proteins. Each of the G protein-coupled receptor pathways is involved in nociceptive modulation and pain processing, but the relative contribution of the individual signaling pathways in vivo has not yet been worked out. The Gq/11 signaling branch is of particular interest in pain research because it leads to the activation of phospholipase C, protein kinase C, and the release of calcium from intracellular stores. Using a conditional gene-targeting approach we generated double-deficient mice lacking Gaq and Ga11 selectively in nociceptors to investigate the contribution of the entire Gq/11signaling pathway in nociceptors towards the regulation of pain. We observed that mice lacking Gq/11 in nociceptive neurons show normal development of the nociceptive circuitry. The nociceptor-specific loss of Gq/11 results in reduced pain hypersensitivity following paw inflammation or spared nerve injury. Surprisingly, our behavioral and electrophysiological experiments also indicated defects in basal mechanical sensitivity in Gq/11 deficient mice, suggesting a novel function for Gq/11 in tonic modulation of acute nociception. Patch-clamp recordings revealed changes in voltagedependent tetrodotoxin-resistant and tetrodotoxin-sensitive sodium channels in nociceptors upon a loss of Gq/11, whereas potassium currents remained unchanged.

Our results indicate that the functional role of the Gq/11 branch of G-protein signaling in nociceptors in vivo not only spans sensitization mechanisms in pathological pain states, but is also operational in tonic modulation of basal nociception and acute pain.

Provocation of arrhythmic events in single primary isolated adult mouse ventricular cardiomyocytes Tekook M., Fehrmann E., Schulte J. S., Schmitz W., Müller F. U. Westfälische Wilhelms-Universität Institut für Pharmakologie und Toxikologie, Domagkstraße 12, 48149 Münster, Germany

AP duration and Ca 2+ cycling are altered in cardiomyocytes of different genetic mouse models. Here, we systematically tested various protocols to study the inducibility of arrhythmic events in mouse cardiomyocytes.

Adult ventricular cardiomyocytes were isolated from wildtype (WT) mice by enzymatic digestion and subsequently tested to trigger arrhythmic events within 6 hours after isolation. In patch clamp experiments (perforated patch, whole cell current clamp) APs were stimulated for 1 second (5Hz; 10Hz; 5Hz + S2-stimulus after 50-80ms) followed by a 4s rest period. The resting-membrane-potential (RMP) was observed over 30 cycles. We observed RMP-fluctuations of different length (amplitude <5 mV; % of 13 observed cardiomyocytes, mean events/cell; <1s: 100%, 17.1; <3s: 92%, 11.5; >3s: 69%, 5.8), spontaneous depolarizations (>5 mV; 92%, 5.1) and spontaneous APs (62%, 1.9). Intracellular Ca 2+ transients and sarcomere shortening were measured after loading cardiomyocytes with indo-1/AM. After preconditioning (10 min/1 Hz) cells were measured under basal and continuous isoprenaline (10 -6 M) stimulation (ISO). A 4 min pacing period was followed by a 1 min interval of no pacing. Pacing frequency was reduced after each cycle (1 Hz, 0.5 Hz, 0.25 

Arrhythmic events were provocable with stimulation/rest protocols both by field stimulation and direct stimulation via patch pipette. However, low stimulation frequencies seem to lead to distinct destabilization of cardiomyocytes probably due to Ca 2+ overload. We conclude that the tested stimulation protocols are able to provoke arrhythmic events even in WT single adult mouse ventricular cardiomyocytes and may serve as a tool to test for the relevance of potential proarrhythmic substrates in mouse models. Ruhr-Universität Bochum Pharmakologie MA Nord1, Bochum, Germany cGMP is a second messenger involved in many (patho-)physiological processes such as smooth muscle relaxation, platelet inhibition, and the development and plasticity of the nervous system. However, it is not fully understood how cGMP regulates these and other processes on a mechanistic level. In particular, the existence and functional relevance of global and local cGMP signaling domains is not clear. Recently, highly specific genetically-encoded optical biosensors for cGMP have been developed. These cGMP indicators are either based on fluorescence resonance energy transfer (FRET), with cGMP-binding domains sandwiched between fluorescent proteins with overlapping spectra, or they consist of a single fluorescent protein fused to cGMP-binding domains.

With these cGMP indicators, the spatiotemporal dynamics of cGMP signals, which result from the interplay between cGMP-producing guanylyl cyclases, cGMP-binding effectors, and cGMP-degrading phosphodiesterases (PDEs), can be monitored in living cells.

Here, we report the generation of transgenic mice expressing the FRET-based cGMP indicators cGi500 and cGi6000 with apparent cGMP affinities of 500 nM and 6000 nM, respectively. One mouse line expresses cGi6000 driven by a CMV promoter in neural cells. FRET experiments were performed with isolated cerebellar granule neurons, hippocampal neurons, and astrocytes. We observed nitric oxide (NO)-induced cGMP transients and analyzed the capability of other agents (natriuretic peptides, glutamate) to induce cGMP responses. In another mouse line, the SM22alpha promoter directs cGi500 expression specifically to smooth muscle cells (SMCs). FRET experiments have been performed with SMCs isolated from aorta, bladder and colon, as well as with intact vessels in the retina and cremaster muscle of transgenic animals. In primary SMCs we studied responses to NO, atrial and C-type natriuretic peptide (ANP,CNP). In different SMC types we observed differences in the overall ability to react to these stimuli and in the kinetics of the induced cGMP transients. We also studied the effects of PDE inhibitors on the NO-, ANP-, and CNP-induced cGMP signals. Importantly, we were able to detect cGMP transients upon NO stimulation in intact vessels of the retina and cremaster. We conclude that the cGi transgenic mouse lines are valuable tools to visualize cGMP signals in living cells in vitro and, possibly, also in vivo in the intact animal under physiological and pathophysiological conditions. Current research data dealing with pharmacotherapy of α-AMA intoxication shows a particularly high variability regarding the protective effect of silibinin. The aim of this study was therefore to evaluate the influence of the frequently used clinical antidotes benzylpenicillin, silibinin and their combination in human hepatocyte culture intoxicated with α-AMA. Cytotoxicity and apoptosis testing were performed after two and five days of simultaneously exposure to α-AMA and/ or tested antidotes. To quantify apoptosis, necrosis and cell viability, we used Cell Death Detection Elisa plus®, ToxiLight® Bioluminescence Assay and Cell Proliferation Kit II (XTT). Furthermore, we analysed the ways of apoptosis by using immunohistochemistry (differential detection of caspase 3, 8 and 9, activated caspase 3, and AIF). Exposure of hepatocytes to α-AMA at concentrations of 0,2 µM, 0,5 µM and 1 µM resulted in disorder of cell cultures, apoptosis and reduction in cell viability compared with unexposed hepatocytes.

In hepatocyte cultures treated with benzylpenicillin at concentrations of 30 µM and 1mM, silibinin at 50 µM and 100 µM and a combination of both (30 µM benzylpenicillin and 50 µM silibinin, 1mM benzylpenicillin and 100 µM silibinin), ToxiLight® values in the supernatant and XTT values were not significantly different from untreated cultures. Simultaneous exposure to α-AMA (at all tested concentrations) and benzylpenicillin, silibinin or combination of both showed higher cell viability and lower values of necrosis compared to the cultures exposed to α-AMA alone (exept 50 µM silibinin at 0,2 µM α-AMA); however, in both groups dosed with benzylpenicillin the highest hepatocyte viabilitiy was observed. This protective effect was particularly revealed at high α-AMA concentrations (0,5 µM and 1 µM).

In conclusion, our data suggest that benzylpenicillin in monotherapy is more effective than in combination with silibinin or silibinin alone. Glucocorticoids (GCs) are important hormones in the regulation of metabolic homeostasis. Synthetic GCs, such as dexamethasone (Dex), play a fundamental role in the treatment of inflammatory diseases. There are numerous side effects of a Dex therapy, e.g. the development of hypertension. In the pathogenesis of hypertension oxidative stress is a crucial factor. Glucocorticoid-induced hypertension has been shown to be associated with an imbalance between nitric oxide (NO) and superoxide. However, the source of this elevated superoxide production is unknown. We hypothesize that an uncoupling of the NO synthase (eNOS), a key mediator of vascular homeostasis, may contribute to Dex-induced oxidative stress. Incubation of human endothelial cells (EA.hy 926) with dexamethasone led to a decrease in eNOS expression at mRNA and protein levels. This effect of Dex was timeand concentration-dependent. Since the major cause of eNOS uncoupling is a deficiency of its co-factor tetrahydrobiopterin (BH 4), we analyzed the amount of BH4 in EA.hy 926 by HPLC. A concentration-dependent reduction of BH4 and also BH2 (dihydrobiopterin) could be demonstrated in response to treatment with dexamethasone. BH4 can be synthesized endogenously by two different pathways -the de novo pathway (from GTP with GTP cyclohydrolase I, GCH1, acting as the rate-limiting enzyme) and the salvage pathway (conversion of sepiapterin to BH4 involving dihydrofolate reductase, DHFR). Treatment of EA.hy 926 cells with Dex decreased mRNA and protein expression of both GCH1 and DHFR. Because BH4 is the major "coupling switch", an eNOS uncoupling is likely to occur in Dex-treated cells.

In summary, we showed that Dex treatment led to a reduced availability of the important co-factor BH4 which could lead to eNOS uncoupling. The uncoupled eNOS may possibly contribute to glucocorticoid-induced vascular oxidative stress. The cellular oncoprotein c-Fos is a major component of the heterodimeric transcription factor AP-1 and has been commonly found over-expressed in tumors and cancer cells of different origin. Previous work showed that mouse cells lacking the immediate-early gene c-fos are hypersensitive to ultraviolet (UVC) light. Here we demonstrate that in human telomerase-immortalized VH10tert foreskin fibroblasts (behaving like primary cells) and SV40-immortalized GM637 fibroblasts, UVC-triggered induction of c-Fos protein is a delayed and long-lasting event. Sustained up-regulation of c-Fos went along with transcriptional stimulation of the nucleotide excision repair (NER) gene xpf, carrying an AP-1 binding site in the promoter. c-fos mRNA was induced in a biphasic manner. An immediate c-fos mRNA expression (30-90 min after exposure) was not translated into the protein, the second wave of transcription (4-24h after UVC exposure) resulted in c-Fos protein expression, 18-48h post-UV. The stress-activated/mitogen-activated protein kinases (JNK, p38K and ERKs) were immediately induced upon UVC exposure and stayed active for at least 24h. Inhibitor experiments revealed that c-Fos was phosphorylated by ERKs and JNK. The activation of c-Fos preceded re-synthesis and the induction of xpf mRNA, which was observed 24-40h post-UVC, resulting in the increased expression of the XPF protein. Cells over-expressing c-Fos showed an accelerated induction of xpf mRNA, and consequently a faster repair of cyclobutane pyrimidine dimers (CPDs). siRNA-mediated silencing of c-Fos (transient c-Fos knockdown) resulted in abrogated UVC-triggered induction of XPF, attenuated repair of CPDs and increased apoptosis. Finally, we observed that the removal of CPDs but not of photoproducts was significantly faster when cells were pre-exposed to a low UVC dose, indicative of an adaptive response to DNA damage. The work was financed by Deutsche Forschungsgemeinschaft (DFG CH 665/2-1). The addition of clopidogrel to aspirin reduces ischemic events in patients with acute coronary syndrome and in those undergoing percutaneous coronary intervention (PCI). However, recurrent ischemic event occurrence during dual antiplatelet therapy remains a major concern. Variability in the pharmacodynamic response to clopidogrel is well recognized, and patients with higher platelet reactivity while receiving clopidogrel are at increased risk of ischemic cardiovascular events. Clopidogrel is an inactive prodrug requiring biotransformation to form the platelet inhibiting metabolite. Interindividual differences in clopidogrel metabolism are the major source of variability in antiplatelet response. Polymorphically expressed cytochrome P450 (CYP) enzymes play a critical role in the metabolism of clopidogrel.

These findings gave rise to the concept of personalized antiplatelet therapy -i.e. individual platelet function testing and correction of insufficient platelet inhibition to reduce ischemic events in patients with high on-clopidogrel platelet reactivity (HCPR). GRAVITAS was the first study to test this concept by comparing double-dose clopidogrel to standard-dose clopidogrel in patients with HCPR. GRAVITAS failed to correct HCPR consistently in the study arm, which coupled with a low overall event rate precluded demonstrating a substantial benefit from improved platelet inhibition. The TRIGGER-PCI trial tested the effectiveness of the more potent thienopyridine prasugrel versus clopidogrel in patients with HCPR after elective PCI with implantation of drug-eluting stents (DES). Switching from clopidogrel to prasugrel in patients with HCPR afforded effective platelet inhibition. However, given the low rate of adverse ischemic effects using contemporary DES after PCI in stable ischemic heart disease, the clinical utility of this strategy could not be demonstrated and the study was terminated prematurely for futility.

Multiple studies have shown that both heterozygotes and homozygotes for loss-offunction CYP2C19 alleles have higher rates of adverse cardiovascular events as compared with noncarriers on approved maintenance dosing of clopidogrel (75mg QD), albeit carriage of CYP2C19 loss-of-function alleles accounted for only a minor proportion of the variability in on-clopidogrel platelet reactivity. Results of ongoing studies with antiplatelet treatment stratified by CYP2C19 genotyping are awaited to assess the clinical benefit of this approach. The organic cation transporter novel type 2 (OCTN2/SLC22A5) represents a high affinity uptake system for carnitine. Besides metabolic disease like severe system carnitine deficiency, genetic variants within the SLC22A5 gene have been associated with inflammatory diseases like colitis ulcerosa. Against this background, we characterized OCTN2 expression in peripheral blood cells thereby identifying its expression in all cell types. In the present work we studied OCTN2 expression in monocytes and THP-1 cells as an in vitro model for this cell type. In addition we examined transcriptional regulation of the carnitine transporter in LPS activated THP-1 and investigated the effect of carnitine and its analog mildronate on the respective cytokine response. OCTN2 expression was characterized on monocytes and THP-1 cells on mRNA and protein level. Transporter mRNA expression could be shown in both cell types by realtime PCR. However, the protein expression was analyzed by western blot, flow cytometry and immunofluorescence microscopy demonstrating OCTN2 specific signals as well as a localization in the plasma membrane. Following THP-1 cells were activated using LPS (10ng/ml) for up to 6h, thereby indicating the expected cytokine response as demonstrated by increased TNFα (24fold induction) and IL-1β (37fold induction) mRNA levels. In addition, OCTN2 expression was analyzed identifying an initial reduction of around 60% compared to untreated cells. In parallel activated THP-1 cells were coincubated with increasing concentrations of the OCTN2 substrate carnitine or its analog resulting in reduced cytokine release as shown by ELISA for TNFα. Here, the TNFα effect was diminished by 64% in the presence of 50mM carnitine. This effect does not rely on a direct neutralization of LPS by carnitine since it was also present in cells only preincubated with carnitine.

In the present work we could show that THP-1 cells represent a useful model to study OCTN2 expression and function. In addition, we demonstrate immunosuppressive effects of OCTN2 substrates like carnitine. Further experiments will be necessary to identify the underlying mechanism of this observation. Castor oil has been used for more than 3000 years for its laxative effects and also to induce labor in pregnant women. Despite its wide-spread use, the mechanism of action remained unknown. The active metabolite of castor oil is ricinoleic acid which is released from castor oil by intestinal lipases. We have found that exposure of MEG-01 cells to ricinoleic acid caused an increase in [Ca 2+ ]i, an effect which was dose-dependent and abolished by pretreatment of cells with pertussis toxin, suggesting the involvement of a G-protein coupled receptor. To search for a putative receptor, we determined ricinoleic acid-induced [Ca 2+ ]i increases in cells transfected with a siRNA library directed against human GPCRs. In this way, we identified prostaglandin E2 receptors EP3 and EP4 as mediators of ricinoleic acid-induced effects. To test if EP3 and EP4 receptors mediate pharmacological effects of castor oil in vivo, we analyzed laxative effects induced by castor oil in wild-type (WT) mice, EP3-deficient (EP3 -/-) or EP4-deficient mice (EP4 -/-). While EP4 -/mice responded similarly to the WT mice, EP3 -/animals were totally insensitive to castor oil-induced laxation. Moreover, mice lacking the EP3 receptor only in the smooth muscle cells did not respond to castor oil, in contrast to mice which lack EP3 receptor only in epithelial cells of the intestinal mucosa. Similarly, ricinoleic acidinduced contractions of isolated ileal segments were absent in segments lacking EP3, consistent with a preferential expression of the EP3 receptor in the longitudinal muscle layer of the intestine. Also, ricinoleic acid-induced contractions of isolated uteri were dependent on the expression of EP3 receptor in the myometrium. These findings identify the cellular and molecular mechanism underlying the effects of castor oil and indicate a role of the EP3 receptor as a pharmacological target to induce laxative effects. Introduction: Patients seek health information from various sources. They are facing the challenge to differentiate between reliable and untrustworthy sources and at the same time identify the best drug therapy for them. Furthermore generalised health information confuses more than they benefit or rather unsettle. Patients are not necessarily qualified to assess the evidence of statements properly. There is thus a need for providing competent drug information, which is offered by the independent drug information service at the Institute of Clinical Pharmacology in Dresden, Germany.

For the present descriptive evaluation we selected 5 drugs (Arimidex ® , Cipralex ® Pentalong ® , Onbrez ® and Pradaxa ® ), that were affected by new referenceprice formation, generic registration, warnings or directions in 2011. In specified time frames we assessed the increase in and the cause of enquiries. Deductively we draw conclusions for a perspicuous presentation of patient information.

Since generic registrations of the aromatase inhibitor Arimidex ® enquiries on side effects of this drug were stable, but 17 additional consultations were held on generic changeovers. The antidepressant Cipralex ® as well as the long-acting β-agonist Onbrez ® were assigned to reference-price groups, which resulted in an 8-times (Cipralex ® : 5 → 40) and 5-times (Onbrez ® : 2 → 10), respectively, increase in enquiries.

Main aspect was to give background information on reference prices and point out therapeutic alternatives (Cipralex ® : 34 of 40; Onbrez ® : 10 of 10). An additional amount of 22 conversations were carried on the fictive registered drug Pentalong ® after health insurance companies advised practitioners to avoid recourse by not prescribing this organic nitrate. Notable insecurity was aroused by media reporting on lethal bleeding after taking Pradaxa ® for anticoagulation. Every tenth enquiry in the evaluation period was focussing on these instigative reports (21 of 227).

Patients are confronted by current changes, but often do not get enough background information from their health care providers to become acquainted with the tidings. Health seekers may find eligible data from media coverage. However the individual assessment as well as the risk-benefit-relation may not be feasible for them. The drug information service for patients is a convenient helpline to reduce lack of knowledge and uncertainties and therefore support shared decision making.

Insulin effects on hyaluronan production -a possible link between diabetes and cancer? Twarock S., Fischer J. W. Institut für Pharmakologie und Klinische Pharmakologie, Universitätsklinikum der Heinrich-Heine-Universität Düsseldorf, Moorenstraße 5, 40225 Düsseldorf, Germany Background: Epidemiological studies have shown an elevated incidence of certain tumor entities in diabetes type 1 and type 2 patients. To reveal the underlying mechanisms we focused on the effects of increased glucose uptake in cancer cells with respect to matrix production. Abundant production of hyaluronic acid (HA) in the vicinity of gastrointestinal cancer cells is a hallmark in tumor development. Esophageal cancer is a rare but severe kind of gastrointestinal cancer which is differentiated in adenocarcinoma and squamous cell carcinoma (SCC) of the esophagus.

We studied the effects of increased glucose levels on HA production in an SCC cell line (OSC1). In starving and full media, elevated glucose concentrations increased the production of HA secreted to the medium in 24h as measured by an HA-binding protein linked assay (starved, 0g/L glucose: 100±4.7%; 1g/L: 185.7±44.8%; 4g/L: 362.6±58.3%; full medium 100±15.2%; 155.3±32.3%; 422.7±32.0%). Surprisingly, total HA concentrations were about 2.2-2.8 fold higher under starved conditions. To investigate whether this effect might be due to insulin actions, starved cells were treated with 10 mg/L insulin for 24h. We observed a dosedependent decrease in HA production following insulin treatment (control vs insulin, 1g/L glucose: 100±6.5% vs 67.83±4.4%; 4g/L: 100±2.2% vs 71.8±6.6%). This finding might suggest that insulin directs glucose usage to the glycolytic pathway thereby diminishing HA synthesis. A premise to this assumption is the ability of OSC1 cells for insulin independent glucose uptake. To verify this thesis, mRNA expression levels of insulinindependent and insulin-dependent glucose transporters (GLUT1, GLUT4) were analyzed by qRT-PCR. The relative abundance was 24.32±3.49 in favor of GLUT1 indicating the presence of insulin-independent glucose transport. Conclusion: In OSC1 the absence of insulin actions caused increased HA production which might be due to diminished insulin driven glycolysis, thus leading to the use of early glucose metabolites for HA production instead of energy gain. This finding could be important in the context of diabetes type 2, where insulin actions are also diminished because of insulin resistance. Since increased HA production is of critical importance for cancer growth and spread, the cellular shift in glucose usage from glucose catabolism to HA anabolism could therefore indicate a possible link between diabetes type 2 and cancer progression.

Schwarz M., Unterberger E. Universtität Tübingen, Institut für klinische und experimentelle Pharmakologie und Toxikologie Abteilung Toxikologie, Wilhelmstraße 56, 72074 Tübingen, Germany Chemical hepatocarcinogenesis is a multi-stage process triggered by an intitiating mutation in a gene encoding an important cell-regulatory protein. Tumour initiation may be caused by genotoxic substances which directly interact with the DNA, causing mutations. Cells carrying permanent mutations experience clonal expansion which may be accelerated by exposure of the experimental animals to tumour promoters during the following step of tumour promotion. It has been shown that substances which constantly activate certain nuclear receptors act as tumour promoters in rodent liver, such as the model tumour promoter phenobarbital which, amongst others, activates constitutive androstane receptor (CAR). Since these tumour promoters do not seem to directly interact with DNA causing mutations they can be regarded as non-genotoxic carcinogens. However, the molecular mechanisms of non-genotoxic carcinogenesis are still widely unknown which also poses a major problem in preclinical drug-development. The aim of the MARCAR (BioMARkers and Molecular Tumour Classification for Nongenotoxic CARcinogenesis) project is to establish early biomarkers for non-genotoxic carcinogenesis by creating a comprehensive molecular profile of tumours generated by a regimen including model tumour promoters such as phenobarbital. The ultimate aim is to differentiate spontaneous liver tumours from tumours generated by non-genotoxic carcinogens. This molecular profile includes mutational analyses, immunostaining for known tumour-specific markers, phospho-proteome analyses, genome wide and promoter-specific DNA methylation analyses, as well as miRNA analyses. Mutation analyses were carried out with mouse and rat tissue from phenobarbital promoted liver tumours to identify mutations which phenobarbital provides a growth advantage for. Furthermore, real time PCR measurements show that the expression of a particular non-coding RNA and miRNA precursor is up-regulated in tissue isolated from phenobarbital promoted mouse liver tumours. Additional in-situ-hybridisation experiments demonstrated the localisation of this transcript in Ctnnb1-mutated tumours.

Larch-derived diterpenes are potent and selective TRPC6 blockers Urban N. 1 , Kübler W. The transient receptor potential channel TRPC6 is a poorly Ca 2+ -selective cation channel that is activated by the membrane-resident second messenger diacylglycerol (DAG). Consistent with the major sites of TRPC6 expression, its activation has been implicated in pulmonary and renal diseases, such as pulmonary hypertension, lung edema, chronic obstructive lung disease, allergic airway disease, and focal segmental glomerulosclerosis. Amongst various plant extracts, conifer oils and resins are traditionally used to treat pulmonary ailments. Therefore, we reasoned that they may contain constituents with a biological activity to modulate TRPC6 activity. The true turpentines, oils and resins of various coniferous genera were tested with respect to a possible inhibition of DAG-or receptor-induced activation of TRPC6 and TRPC3. Indeed, turpentines and resins, but not coniphere oils blocked TRPC6 and TRPC3 in a concentration-dependent manner. Interestingly, the larch-derived turpentine exerted a TRPC6-prevalent inhibition. We identified larixol and its mono-and diacetates as the specific compounds that are contained in larch resin and give rise to a TRPC6-selective block. Larixol acetates displayed an IC50 towards the DAG-or receptor-stimulated TRPC6 activity of about 0.3-1 µM, but did not strongly inhibit a number of other TRP channels, including TRPV1, TRPM2, TRPM3, TRPM8, or TRPA1. Selectivity for TRPC6 compared to its closest relative, TRPC3, was about 30-fold. Unlike conipherous oils, which contain toxic pinenes, the resin constituent larixol ant its acetates exerted no significant cellular toxicity at concentrations that are required to block TRPC6. Electrophysiological analysis confirmed the highly potent block, which was voltageindependent and reversible. In a murine hypoxia-induced pulmonary vasoconstriction (HPV) model, larixol acetate abrogated the Euler-Liljestrand mechanism and, thus, mimicked the phenotype of TRPC6 -/mice. We conclude that TRPC6 blockers and, more specifically, larixol-related derivatives may provide novel therapeutic strategies to treat or prevent pulmonary diseases.

Dioxin is an environmental contaminant, believed to affect basic biological equilibria such as calcium and iron homeostasis. However, the molecular mechanisms underlying these effects are still largely unknown. This strongly hampers the estimation of the hazard to humans associated with dioxin exposure and necessitates further studies aimed at the clarification of these mechanisms. It has been suggested that nearly all biological and biochemical processes are mediated by protein complexes. The most commonly used technology for monitoring changes in the expression of complex protein mixtures is still 2D gel electrophoresis, but this method suffers from poor expression of low or moderately abundant proteins. Blue native PAGE and subcellular fractionation form an ideal partnership when it comes to enrichment and analysis of intracellular organelles and low abundant multiprotein complexes. The aim of the study is to identify and characterize multiprotein complexes by Blue native PAGE to elucidate the network of protein-protein interactions that regulate protein function after dioxin exposure.

Sample preparation and subcellular fractionation RT4 cells were cultured in McCoy's 5A medium. Cells at confluence were harvested and fractionated into cytosolic, membrane/organelle and nuclear fraction by using the proteoextract subcellular proteome extraction kit. First dimension (BN-PAGE) 50 mg of protein sample was mixed with 5% of Coomassie blue G-250 (CBB G-250) and loaded in each lane of 4-15% polyacrylamide native gradient gels.

The lanes from the first dimension were cut into individual strips and were placed into a 12% SDS gel. The gels were stained with Coomassie and the spots were picked up for mass spectrometry.

BN/SDS-PAGE combined with MS led to the identification of proteins involved in the regulation of both calcium and iron homeostasis in dioxin-exposed cells. These results demonstrate for the first time that dioxin exposure simultaneously affects calcium and iron metabolism. Since important iron and calcium requirement changes occur during the regulation of cell growth, the protein expression changes observed in our study may be associated with dioxin-dependent cell-fate decisions.

The Murine Protease Inhibitor Serpina3n Inhibits Mechanical Allodynia in a Model of Neuropathic Pain Vicuna L. 1, 2 , Simonetti M. Several chronic diseases are accompanied by strong, long-lasting pain. A majority of chronic pain diseases are not well understood yet and cannot be controlled by conventional analgesics or non-pharmacological approaches. Therefore, there is a major need to develop novel therapeutic principles. Using a genetic screen, we identified Serpina3n, a serine protease inhibitor, which is homologous to human a1-antichymotrypsin, to be a determinant of low neuropathic pain. We found that Serpina3n is expressed in the dorsal root ganglia (DRG) and spinal cord and that it is upregulated in these tissues in mice developing neuropathic pain. Importantly, we observed that spinal delivery of recombinant Serpina3n inhibits mechanical allodynia in a mouse model of neuropathic pain. We identified a novel serine protease substrate for Serpina3n, which is upregulated in the spinal cord in mice undergoing neuropathic pain ('Enzyme E'). Recombinant Enzyme E delivered intrathecally to the spinal cord of mice elicited rapid and long-lasting allodynia, which was fully blocked by concomitant administration of Serpina3n. Our results suggest that serine protease-serpin signaling modulates spinal neuronal and glial cell networks involved in processing pain and that activity-induced spinal release of Serpina3n constitutes an endogenous defence mechanism against establishing chronic pain hypersensitivity. These data have important implications for the pathophysiology of pathological pain and potentially hold therapeutic relevance.

Gβγ subunits are involved in β-adrenergic receptor induced cardiac hypertrophy Vidal M., Lohse M. J., Lorenz K. Institut für Pharmakologie und Toxikologie Pharmakologie, Versbacher Str. 9, 97078 Würzburg, Germany

Introduction. Activated β1-adrenergic receptors and their G protein Gαs induce the development of cardiac hypertrophy. However, the hypertropic effects of direct activation of downstream effectors, such as adenylyl cyclase, cAMP or PKA, are controversely discussed. Recently, a hypertrophic pathway involving a G protein βγ subunit induced phosphorylation of the mitogenic kinases Erk1/2 at threonine 188 (Erk Thr188phosphorylation) has been described to mediate Erk-induced hypertrophy. This study aims to investigate whether Erk Thr188 -phosphorylation is involved in cardiac hypertrophy triggered by β-adrenergic receptors.

-phosphorylation was detected in HEK cells overexpressing β1-receptors, murine hearts and neonatal rat cardiomyocytes after isoprenaline treatment. We performed [ 3 H]-isoleucine incorporation assays to assess cardiomyocyte hypertrophy in vitro. Neonatal rat cardiomyocytes (NRCMs) overexpressing wild-type Erk2 showed a significant increase in [ 3 H]-isoleucine incorporation after isoprenaline treatment. In contrast, NRCMs transfected with Erk Thr188 -phosphorylation deficient mutants (Erk2 T188A and Erk2 T188S ) or pretreatment with the Erk inhibitor, PD98059, significantly attenuated cardiomyocyte hypertrophy. For in vivo studies, isoprenaline was given subcutaneously for 14 days to wild-type mice and transgenic mice overexpressing either wild-type Erk2 T188T or Erk2 T188S . Echocardiography and histological analyses revealed that Erk T188S mice developed less left ventricular hypertrophy than control mice. Hypertrophic target proteins of Erk (e.g. Elk1) are located in the nucleus. Western blot and confocal microscopy analyses showed that overexpressed Erk2 T188A or Erk2 T188S are retained in the cytosol and prevented Elk1-phosphorylation after isoprenaline stimulation. Co-immunopreciptation assays in HEK cells and NRCMs underlined the direct involvement of G protein βγ/Erk interaction upon isoprenaline stimulation. In line with this finding, direct activation of adenylyl cyclase by forskolin did not lead to Gβγ induced Erk Thr188 -phosphorylation. Conclusion. Taken together, Gβγ-subunits participate in β1-adrenergic receptor mediated hypertrophy by enhancing Erk Thr188 -phosphorylation. These findings add important insight to the molecular signaling of G proteins in cardiac hypertrophy.

The protein tyrosine kinase Src and its role upon alpha-toxin stimulation of human platelets Vogel K. 1 , Burke M. Introduction: Alpha-toxin, a 34 kDa calcium pore forming exotoxin, is a major virulence factor in the pathogenesis of Staphylococcus aureus infections. Alpha-toxin affects human blood cells such as platelets and induces aggregation that is accompanied by multiple changes in platelet protein tyrosine phosphorylation and dephosphorylation (1).

In the present paper, we focused our interest on the protein tyrosine kinase Src, the most abundant member of the Src-family kinases present in platelets (2) . By the use of various inhibitors, we studied Src and its role in α-toxin-induced platelet aggregation.

Methods: Isolated human platelets from healthy volunteers were stimulated with α-toxin in the presence or absence of the Src-family member inhibitors PP1, PP2 or SU6654 (referred as Src inhibitors). Src and autophosphorylation of Src were analyzed by SDS-PAGE and Western blotting using specific antibodies against Src and Tyr-416-phospho-Src from Calbiochem and Cell Signaling, respectively (3). Furthermore, calpeptin, an inhibitor of the calcium-dependent protease calpain, was used. Platelet aggregation was measured by the method of Born.

Staphylococcal α-toxin induced platelet aggregation in a concentration-dependent manner (0.18 -3.0 µg/ml of toxin). Pre-incubation with 3 Src inhibitors (PP1, PP2 or SU6654) reduced α-toxin-induced platelet aggregation by about 50%. Similar inhibitory effects have been observed by the use of calpeptin that acts as an inhibitor of the Src degrading protease calpain. With respect to Src itself, a-toxin induced autophosphorylation at Tyr-416 followed by a fast and complete dephosphorylation within 10 min. While calpeptin modified the time course of dephosphorylation, only little effect of the Src inhibitors has been seen on Tyr-416 phosphorylation/dephosphorylation.

The typical calpain-dependent degradation of Src can be blocked by calpeptin (1µM), but also by depletion of extracellular calcium indicating that it is a calcium-dependent process.

Conclusion: Taken together, our data demonstrate that α-toxin of Staphylococcus aureus induces platelet aggregation accompanied by Src degradation and autophosphorylation at Tyr-416 typically observed in activated platelets. Inhibition of the cellular tyrosine kinase Src as well as the protease calpeptin reduces aggregation indicating an important role of Src and/or other Src-family members in α-toxin-induced platelet stimulation. The five subtypes of muscarinic acetylcholine receptors belong to the superfamily of Gprotein coupled receptors. The even-numbered subtypes M2 and M4 prefer coupling to Gi proteins, whereas the odd-numbered receptors M1, M3 and M5 prefer coupling to Gq proteins.

With respect to ligand binding and M2 receptor activation, the conserved epitope Trp 7.35 at the beginning of TM7 displays remarkable functional features. It is located at the junction between the orthosteric and the allosteric binding site of the M2 receptor [1] . In the inactive M2 receptor, it provides subtype-independent baseline affinity for allosteric antagonists [1] . In the active receptor, M2 Trp 7.35 affords binding affinity for the full agonist acetylcholine and intrinsic efficacy for the partial agonist pilocarpine [2] .

To study the role of Trp 7.35 for M3 receptor activation, agonist-induced formation of Dmyo-inositol-monophosphate was measured in CHO-cells transfected with the respective human receptor-cDNA. Surface receptor expression measured by radioligand binding was similar in hM3 wild-type-cells and hM3 Trp 7.35→Ala-cells, amounting to 0.07 and 0.11x10 6 receptors per cell, respectively. The intrinsic efficacy of acetylcholine was not influenced at M3 Trp 7.35→Ala relative to M3 wild-type, whereas potency was reduced about tenfold. These findings resemble those made previously in M2 and the corresponding mutant.

In the case of pilocarpine, replacement of Trp 7.35 by alanine in M3 did not reduce intrinsic efficacy. This finding is in contrast to M2, where the corresponding mutation induced a loss of pilocarpine's intrinsic efficacy. The potency of pilocarpine was diminished about tenfold at the M3 Trp 7.35→Ala mutant relative to M3 wild-type. This finding is also in contrast to M2, at which pilocarpine's potency was not sensitive to the Trp 7.35→Ala mutation. Taken together, the diverging sensitivity of pilocarpine to the Trp 7.35→Ala mutation between the M3 and the M2 receptor suggests that the role of this epitope for receptor function may differ between even-and odd-numbered muscarinic acetylcholine receptors. In vivo experiments for inhalation toxicity are time and animal consuming. Thus several in vitro methods aim to replace or reduce and refine the in vivo experiments. Human 3Dtissue models are commercially available reconstructed from different donors (normal, smokers, chronic obstructive pulmonary diseases), which show a normal human bronchiole tissue that reveals a pseudostratified epithelial structure, numerous microvilli and cilia on the apical surface of the cultures. The presence of tight junctions and mucus secretion has also been confirmed comparable to the in vivo situation. These 3D-models are cultured on a porous membrane as air-liquid interface. Test substances can be applied apically, either as solution or with an aerosol-inducer.

In our in house validation to test the strengths, handling and reproducibility of such 3Dmodel systems as well as determining the correlation between in vivo inhalation data, we have assessed the EpiAirway TM model from MatTek, USA. A set of 20 substances were selected with known in vivo toxicity data and mode of action. The substances were tested in the EpiAirway model an in parallel, in 3T3 and A549 cell lines to assess putative unspecific cytotoxic effects of the test substances.

A comparison of toxicity data from the 3D-model and the in vivo data revealed, that the model is only predictive of respiratory toxicity in vivo for a subset of substances with specific modes of action. The EpiAirway TM model has proven to be robust, showing high reproducibility between pre-and main-tests as well as in the concurrent controls but it will need a strict definition of its applicability domain or further development of the test protocol to achieve a wider applicability.

Remodeling of intracellular Ca 2+ handling and cyclic AMP-dependent signaling in atrial myocytes from patients with chronic atrial fibrillation. Voigt N. 1 Background: In atrial myocytes Ca 2+ entry through L-Type Ca 2+ channels (ICa,L) triggers a larger Ca 2+ release (Ca 2+ transient,CaT) from the sarcoplasmic reticulum activating contractile myofilaments. Reduced ICa,L is a hallmark of atrial remodeling in chronic atrial fibrillation (cAF) and is supposed to contribute to action potential shortening and contractile dysfunction. However, the coupling efficiency between ICa,L and CaT and its regulation by cAMP-dependent signaling in cAF patients are unexplored. Methods: ICa,L (voltage-clamp) and CaT (Fluo-3) were measured simultaneously in rightatrial myocytes from sinus-rhythm (Ctl) or cAF patients. A saturating concentration of the non-selective β-adrenoceptor (AR) agonist isoprenaline (ISO, 1µM) and the nonselective phosphodiasterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX, 10µM) were used to increase cellular cAMP content. cAMP content was assessed with immunoassay. Results: In cAF amplitudes of ICa,L (3.3±0.3pA/pF, n=12/6 [myocytes/patients] vs 6.2±0.8 pA/pF, n=15/10, p<0.01) and CaT (182.2±26.8nM vs 307.5±30.9nM, p<0.01) were lower than in Ctl myocytes, whereas diastolic [Ca 2+ ]i levels were unchanged (cAF, 312.3±56.7nM; Ctl, 305.3±43.6nM). The coupling efficiency between ICa,L and CaT was similar in Ctl and cAF. Application of ISO increased ICa,L amplitude to 10.4±2.0pA/pF (n=7/4) in cAF and to 14.3±1.7pA/pF (n=9/7) in Ctl. The corresponding CaTs increased to 493.0±132.3nM in cAF and to 545.0±79.0nM in Ctl. Although the amplitudes of ICa,L and CaT also increased after PDE inhibition with IBMX, the magnitude of these increases was smaller than the ISO-induced enhancements. Both ISO and IBMX had no effect on diastolic [Ca 2+ ]I and coupling efficiency. However, the relative ISO-induced increases in ICa,L (cAF, +202.3±47.3% vs Ctl, +98.4±16.1%, p<0.05) and CaT (cAF, +215.4±57.8% vs Ctl, +101.9±20.3%, p=0.06) were significantly higher in cAF compared to Ctl myocytes and a similar tendency was found for IBMX. Basal cAMP levels were higher in cAF compared to Ctl (cAF, 9.9±1.5pmol/mg, n=6 vs Ctl, 5.0±0.6pmol/mg, n=7, p<0.05), pointing to an increased cAMP-dependent signaling in cAF patients. Conclusions: These data point to remodeling of cAMP-dependent signaling in cAF patients which likely contributes to the stronger relative increases of ICa,L and CaT amplitudes after β-AR stimulation and PDE inhibition. Remodeling of cAMP-dependent signaling might be a novel contributor to AF pathophysiology.

Direct visualisation of G-protein-coupled receptors and heterotrimeric G-proteins using single-molecule microscopy Wagner J. 1 G-protein-coupled receptors (GPCRs) form the largest family of membrane-bound receptors and mediate the effects of several extracellular stimuli. Although the basic mechanisms of GPCR signalling have been extensively studied, a full characterization of the involved protein-protein interaction is still missing, largely due to technical limitations. In this study, we developed new methods for labelling GPCRs and G-protein subunits based on SNAP-and CLIP-tags and visualise them with single-molecule sensitivity. The SNAP-tag is a mutant of the DNA repair protein O 6 -alkylguanine-DNA alkyltransferase that reacts with fluorescent benzylguanine derivatives, whereas the CLIP-tag is reacting specifically with O 2 -benzylcytosine derivatives. These tags allow labelling proteins directly in living cells with very high specificity and low background. SNAP/CLIP-tagged receptors and G-proteins were covalently labelled with small organic fluorophores and visualised by total internal reflection fluorescence microscopy, which allows to selectively illuminate only fluorescent molecules located on or immediately underneath the cell surface. Particles were automatically analysed with previously published as well as newly developed algorithms. The results indicated that both receptors and Gproteins, although diffusing with high speed on the cell surface (diffusion coefficients: receptors ~ 0.05 mm 2 /s, G-proteins ~ 0.1mm 2 /s), can be visualised and correctly tracked. A variable fraction of receptors and G-proteins are immobile or show hop movements, possibly suggesting their interaction with cytoskeletal or other membranebound proteins. Our data also suggest the feasibility of performing two-colour analyses with SNAP-and CLIP-tagged proteins aimed at directly visualizing transient interactions between receptors and G-proteins or among G-protein subunits. In-vitro screening systems are particularly well suited to preclinical toxicology testing at an early stage of drug development as they have the advantage of being fast and requiring only a small amount of test substance. The demands for in-vitro screening assays for systemic toxicity are multiple and include the need of organ specific cell systems, the use of optimal cell numbers, cell passages and incubation times. Even minimal changes in the conditions of the test system may lead to significant changes of the biological system. Therefore a reliable normalization compensating biological variability is crucial prior to any interpretation of results generated from a biological system. BASF has developed an in-vitro metabolite profiling assay and a subsequently tuned normalization strategy allowing the prediction of specific organ toxicity. The in-vitro assay consist of exposing cells lines to test substances and to determine the metabolite profile using chromatography coupled to mass spectrometry systems. Herein, we compare five different normalization strategies referring to their suitability in the application to in-vitro metabolite profiling data. The strategies comprise statistical approaches, approaches referring to reference values from each individual sample or samples generated in dependent batches. Best results were achieved by an individual strategy using a new reference value correlating well over a large range of cell counts previously used for generating corresponding cell extracts. Statistical analysis revealed the normalization based on the new reference value greatly improved the quality of the results compared to non-normalized samples as well as to all remaining strategies. Generation and application of this new reference value and the corresponding normalization strategy will be presented the first time. Validation will be featured on the basis of extracts of the human hepatocellular carcinoma cell line Hep G2.

Molecular mechanisms of the inhibitory function of RhoH in phospholipase Cmediated signalling Walliser C., Löschmann Y., Ziegler V., Kühne E., Schilling P., Rasonabe Z., Bühler A., Vatter P., Gierschik P. Universitätsklinikum Ulm Institut für Pharmakologie und Toxikologie, Albert-Einstein-Allee 11, 89081 Ulm, Germany Rho GTPases are a subfamily of Ras GTPases regulating diverse signalling pathways, for example those regulating the reorganisation of the actin cytoskeleton. Among them, Rac2 and RhoH show an expression restricted to the hematopoietic lineage. RhoH is constitutively active, because it carries mutations in two positions (S13 and N62) known to be important for GTP hydrolysis. Hence, RhoH is controlled on the level of protein expression and, possibly, by tyrosine phosphorylation. RhoH has been implicated in human malignancies, since the gene is subject to somatic hypermutation in its noncoding regions and to translocation to the gene encoding LAZ3/BCL6 or to other genes in human B-cell lymphomas. Furthermore, RhoH is overexpressed in primary human chronic lymphocytic leukemia (CLL) cells. These findings suggested that RhoH is involved in the initiation and/or progression of CLL. We previously showed that RhoH acts as a potent inhibitor of both Rac2-mediated phospholipase C-β 2 (PLCβ2) and PLCγ2 activation in intact cells. The aim of this study was to elucidate the molecular mechanisms of the inhibitory effect of RhoH on PLC activity. The results showed that RhoH directly inhibited the activity of constitutively active variants of PLCγ2, PLCβ2, and PLCδ1, but that it had little or no effect on the activity of PLCγ1 and PLCε. The amino acid residues S13 and N62, likely to be the cause for the GTPase-deficiency of RhoH, are not required for the inhibitory function of RhoH. Furthermore, the switch-I and switch-II regions of RhoH are not necessary for the inhibitory effect of RhoH, since RhoH mutants carrying switch-I or switch-II regions of Rac2 caused inhibitory effects on Rac2-mediated PLCβ2 and PLCγ2 stimulation indistinguishable from wild-type. Interestingly, RhoH seems to interact with regions of PLCγ2 distinct from those which are necessary for Rac2 interaction, as the split pleckstrin homology domain of PLCγ2, which is essential for its interaction with activated Rac2, is dispensable for the inhibitory effect of RhoH. In summary, our results indicate, that RhoH acts as a PLC-isozyme-specific negative regulator of the activity of PLCβ2 and PLCγ2, both of which are specifically expressed in hematopoietic cells. These findings suggest a novel mechanism of PLC isozyme regulation by RhoH. The results also suggest that RhoH plays an important role in B cell maturation, function, and leukemogenesis by modulating B-cell-receptor-mediated PLCγ2 activation.

Effect of Rac1 inhibition on doxorubicin mediated cell response Wartlick F., Fritz G. Heinrich-Heine-Universität Düsseldorf Institut für toxikologie, Universitätsstrasse 1, 40225 Düsseldorf, Germany

Background: The small GTPase Rac1 is a well characterized member of the Rashomologous (Rho) family. Rac1 is not only a key regulator of the actin cytoskeleton but also regulates the activity of NADPH oxidase, stress kinases and transcription factors (e.g. NF-κB, AP1). Furthermore, Rac1 can translocate into the nucleus and interacts with topoisomerase type II (Topo II). Yet the general nuclear function of Rac1 is still unclear. Here, we address the question how Rac1 influences the genotoxicity of the Topo II poisons doxorubicin and etoposide. Methods: To study the function of Rac1, human hepatoma cells were pretreated with the Rac1-inhibitor EHT 1864 before they were exposed to doxorubicin, etoposide or, for control, ionizing radiation (IR).

To check the influence of Rac1 inhibition on the outcome of genotoxin treatments, cell viability and cellular stress response were analyzed by the WST-assay, Western blot (WB), co-immunoprecipitation experiments, FACS-analysis and the alkaline comet-assay. Results: As compared to the control, cells that have been pretreated with the Rac1 inhibitor showed a higher viability, less phosphorylation of H2AX (S139) and a reduced DNA damage formation (measured by alkaline comet-assay) after treatment with doxorubicin and etoposide but not after treatment with IR. Furthermore inhibition of Rac1 resulted in a reduced phosphorylation of Topo IIα (S1106) and an increased interaction of Topo IIα with Hsp90 in doxorubicin treated cells.

The data indicate that inhibition of Rac1 protects human hepatoma cells against Topo II poisons due to interference with Topo IIα function. The presence of drugs or other potential toxic substances in milk has enormous toxicological and nutritional consequences for consumers of dairy products. The ATPbinding cassette (ABC) transport protein Breast Cancer Resistance Protein (BCRP; ABCG2) is expressed in alveolar epithelial cells of the mammary gland in cows, sheeps and goats. BCRP is known to play a major role in the active secretion of a variety of xenobiotics into human milk. So far there is little information about the transport activity and substrate specificity of dairy BCRP. Therefore we aimed to establish a MDCK cell in vitro model expressing BCRP of dairy animals. BCRP mRNA was isolated from bovine, caprine and ovine mammary gland. Full-length clones were generated using RACE (rapid amplification of cDNA ends) PCR. The final full-length bovine, ovine and caprine ABCG2 cDNA-clone sequences were submitted to the NCBI genebank (EU570105, GQ141082 and GQ241418). Stable transfection of BCRP in MDCK cells was performed and the subcellular localization of BCRP at the apical plasma membrane was identified by confocal laser scanning microscopy. BCRP-mediated transport of the substrate Hoechst 33342 was measured and the selectivity was determined by the BCRP inhibitor Ko143. Inhibition studies using Hoechst 33342 identified various drugs including the antibiotic enrofloxacin or anthelmintic agents like oxfendazole as substrates of bovine, caprine and ovine BCRP. To further characterize BCRP carrier activity, bidirectional transport studies were performed with Transwell® filter inserts that allow studying drug transport between an apical and basolateral compartment. Cell monolayer integrity was checked by measuring TEER values as well as by measuring the paracellular flux marker atenolol by LC/MS. Bidirectional transport studies with Enrofloxacin were performed to characterize the BCRP transporter activity. Our results may contribute to increase the understanding of carrier associated drug transport into the milk of dairy cattle and therefore enlarge consumer protection.

Acrolein and acrylamide: excretion of mercapturic acids after consumption of potato chips Watzek N., Scherbl D., Berger F., Feld J., Eisenbrand G., Richling E. Technische Universität Kaiserslautern Fachbereich Chemie; Fachrichtung Lebensmittelchemie & Toxikologie, Erwin-Schrödinger-Str. 52, 67663 Kaiserslautern, Germany Acrolein (AC) and acrylamide (AA) may be formed from food constituents during heating of food. AC is supposed to be generated via heat induced formation from glycerides/glycerol, AA is known to arise during the Maillard reaction from asparagine and reducing carbohydrates. AC also has also been suggested to be formed by endogenous metabolism as a side product of carbohydrate and/or amino acid turnover or by oxidative desamination of polyamines [1] . As an α,b-unsaturated aldehyde, AC forms 1,4-Michael-adducts with biomolecular nucleophiles, such as sulfhydryl and amino groups. In the organism, AC and AA are preferentially conjugated to glutathione and are excreted as mercapturic acids (MA), N-acetyl-S-(3-hydroxypropyl)-cysteine , N-acetyl-S-(carboxyethyl)-cysteine (CEMA), (N-acetyl-S-(2-carbamoylethyl)-cysteine (AAMA), and (N-acetyl-S-(2-hydroxy-2-carbamoylethyl)-cysteine (GAMA). Data on human exposure to AC and its occurrence in the diet are scarce. In general, contents in heat treated foods are considered to be in the low ppb range (µg/kg) [2] . Nevertheless, in a pilot study in humans urinary 3-HPMA excretion of 14 non-smokers was reported to be about three fold higher, as compared to AAMA [3] . In the present human intervention study we monitored the excretion of MAs in five healthy volunteers (male) after ingestion of commercially available potato chips (175 g), equivalent to an uptake of 44 µg AA (absolute amount), together with an as yet unknown amount of acrolein [4] . Urinary MA contents were monitored by HPLC-MS/MS following solid phase clean-up of urine for up to 24 h after test meal uptake. The results demonstrated kinetics of 3-HPMA and CEMA excretion in human urine to be clearly related to ingestion of the potato chip meal. On the basis of AUC values, total excretion of 3-HPMA plus CEMA exceeded that of AAMA plus GAMA by a factor of about four. The results confirm earlier findings on urinary MAs, suggesting markedly higher human exposure to dietary AC / potential AC precursors than to AA. It is an as yet unresolved question, whether and to what extent concomitant substantial AC exposure may influence toxicology of such dietary heat-induced toxicants. [1] Stevens, J.F. and Maier, C.S. (2008) Molecular Nutrition & Food Research 52; 7 [2] Osorio, V. M. and de Lourdes Cardeal, Z., (2011) Journal of Chromatography A 1218; 3332 [3] Schettgen, T., Musiol, A., and Kraus, T., (2008) Rapid Communications in Mass Spectrometry 22; 2629 [4] Ewert, A., Granvogl, M., and Schieberle, P., (2011) Lebensmittelchemikertag 2011 450 Protective effects of increased NAD + levels in human peripheral blood mononuclear cells exposed to DNA damaging agents Weidele K., Beneke S., Bürkle A. University of Konstanz Molecular Toxicology Group, Department of Biology, Jacob-Burckhardt-Str.31, 78457 Konstanz, Germany

The DNA damage-activated enzyme poly(ADP-ribose) polymerase 1 (PARP-1) acts as a nick sensor and modifies target proteins by covalent attachment of poly(ADP-ribose) [PAR] using NAD + as substrate. The intracellular levels of PAR and NAD + are important parameters for biological responses to genotoxic stress and influence diverse cellular functions including DNA repair or maintenance of genomic stability. Notably, loss of genomic stability is a hallmark of both carcinogenesis and the ageing process. Here we analysed the impact of elevated NAD + levels in human blood peripheral mononuclear cells (PBMC) with regard to (i) poly(ADP-ribose) formation, (ii) cell death, (iii) initial DNA damage and subsequent repair, as well as the influence on (iv) genomic stability under genotoxic stress. After ex vivo supplementation of PBMC with low concentration of NAD + precursor nicotinic acid (NA) intracellular NAD + level significantly increased up to 2 fold in unstimulated [1] and 1.5 fold in mitogen-stimulated cells. After DNA damage infliction, PARP activity was dramatically increased in supplemented cells, necrotic cell death was reduced and DNA strand break repair was significantly affected. Furthermore the frequency of micronuclei decreased significantly after irradiation damage, emphasizing the fundamental role of adequate NAD + levels in maintaining genomic integrity. The cyclic purine nucleotides adenosine 3':5' monophosphate (cAMP) and guanosine 3':5' monophosphate (cGMP) are well-examined second messengers with many proven biological functions. In a recent study, using a highly sensitive and specific mass spectrometry method, we have shown that cyclic 3':5' cytidine monophosphate (cCMP), a pyrimidine nucleotide, is naturally occurring in several mammalian cells [1] . cCMP activates both cAMP-and cGMP-dependent protein kinases with low potency [2] but the physiological function of cCMP is still very poorly understood. In an effort to delineate the function of cCMP, we analyzed expression of the early response gene EGR1. We chose this gene because it is regulated by numerous stimuli including cAMP [3] . In our first study, we showed that dibutyryl-cCMP and cCMP failed to increase EGR1 gene expression levels after stimulation of KB cells under various experimental conditions using real-time PCR (TaqMan®). We have now changed the experimental set-up using HeLa cells and the new cCMP analogue, cCMP-acetoxymethyl ester (cCMP-AM), still focusing on gene expression of EGR1. Esterification of the negatively charged cyclic phosphate of cCMP allows better transport of the nucleotide across the cell membrane, thus augmenting possible intracellular effects. HeLa cells were stimulated in cell culture medium with extracellularly applied cCMP-AM (3, 10, 33, 100 µM) over 15 to 240 min 24 h after seeding. Analysis of real time PCR (TaqMan®) experiments, using β-actin as a housekeeping gene, showed a significant increase of EGR1 expression in a time and concentration dependent manner. These effects were specific for stimulation with cCMP-AM but not the control phosphate trisacetoxymethyl ester. HeLa cells were also cultured in serum free resting medium (MCDB 153, Sigma) that induces growth arrest, one to eight hours prior to stimulation. Here, even higher EGR1 expression levels through cCMP-AM stimulation could be seen. These results suggest that cCMP could function as a second messenger just as cAMP and cGMP do. Studies are in progress to further examine the mechanisms of the cCMP-AM effects on EGR1 expression in HeLa cells.

Methyl-CpG-binding protein 2 (MeCP2) recognizes methylated DNA, it is involved in chromatin remodeling and it acts as a transcriptional repressor or activator. We have previously shown that expression of MeCP2 is diminished in murine and human heart failure. Prevention of MeCP2 downregulation in transgenic mouse models aggravated cardiac hallmarks of heart failure. In patients with Rett syndrome, which is caused by mutations in the MeCP2 gene, mitochondrial function was found to be altered in the central nervous system. As the impact of MeCP2 on mitochondrial function in the heart is unknown, the aim of the present study was to characterize the significance of MeCP2 of cardiac mitochondria in mouse models with cardiac myocyte-specific expression or ablation of MeCP2.

In order to investigate the cardiac function of MeCP2, two genetically modified mouse models were previously generated, including mice with inducible transgenic expression of MeCP2 in cardiac myocytes under control of the tetracycline-system (MeCP2-Tg) and mice with targeted ablation of MeCP2 in myocytes (MeCP2 MLCCre ). These mice were analyzed under basal conditions and after chronic transverse aortic constriction (TAC). At baseline, cardiac-specific overexpression of MeCP2 did not cause any difference in cardiac function as compared to control mice using Millar catheterization. Isolated interfibrillar mitochondria showed a decrease in citrate synthase activity. After chronic pressure overload, the decrease in cardiac MeCP2 expression could be completely prevented by the MeCP2 transgene. Cardiac contractility and relaxation were significantly decreased in MeCP2-Tg animals. Upon electron microscopical investigation, transgenic MeCP2 expression was associated with a significant reduction of interfibrillar mitochondria, clustering of mitochondria in the perinuclear region and smaller mitochondrial cross sections as compared with control specimens. In contrast, cardiac myocyte-specific ablation of MeCP2 caused a rightward shift in the size distribution of mitochondria as compared with MeCP2-Tg hearts.

Epigenetic processes, including the recognition of DNA methylation by MeCP2, may play an important role in the control of mitochondrial gene expression, structure, subcellular localization and function in the heart. Thus, precise control of MeCP2 expression and function is essential to prevent deterioration of metabolic function during chronic heart failure.

Normalisation of blood pressure does not prevent angiotensin II-induced DNA damage in kidney and heart of ren2 rats Weissenberger S., Hey V., Lau D., Schupp N. Universität Würzburg Institut für Pharmakologie und Toxikologie, Versbacher Strass 9, 97078 Würzburg, Germany Increased activity of the renin angiotensin system (RAS) with enhanced levels of angiotensin II (AngII) leads to oxidative stress with endothelial dysfunction, hypertension and atherosclerosis. Epidemiological studies revealed a higher cancer mortality and an increased kidney cancer incidence in hypertensive patients. We could show in vitro and in vivo that AngII causes structural DNA damage dose-dependently in kidney cells and in the kidney. Elevated AngII levels therefore might contribute to carcinogenesis of the kidney. In a model of high AngII organ levels, the transgenic ren2 rat, carrying an additional renin gene, DNA damage in the kidney was analysed in animals of 13 and 32 weeks. Untreated ren2 rats exhibit increased blood pressure from the age of 8 weeks on. Therefore, the line is kept on angiotensin I converting enzyme inhibitor therapy, which normalizes blood pressure and kidney function to values of control Sprague Dawley rats. Despite this normalized blood pressure of the ren2 animals, a significant higher superoxide production could be observed in kidneys already in 13 week old animals. Also a higher frequency of structural DNA damage and double strand breaks could be detected in the comet assay and with an antibody against the double strand break marker γ-H2AX in kidneys. Further, fittingly, an increased DNA repair activity exists in kidneys of ren2 rats compared to control rats. As another organ affected by hypertension the heart of the ren2 animals was analysed for oxidative DNA damage. Although only a marginal increase of superoxide production could be found, also in the heart a significant higher frequency of DNA double strand breaks and cells positive for DNA repair activity could be observed. Our data let us conclude that normalization of blood pressure in a state of activated RAS is not sufficient to prevent AngII-induced genotoxicity. This further implies that also patients with treated hypertension still might suffer from endorgan-damaging effects of elevated AngII levels.

The D541A pore mutation leads to complete inactivation of TRPV6 channels in epididymis Weißgerber P. 1 , Kriebs U. Replacement of aspartate residue 541 by alanine (D541A) in the pore of TRPV6 channels in mice disrupts Ca 2+ absorption by the epididymal epithelium resulting in abnormally high Ca 2+ concentrations in epididymal luminal fluid and in a dramatic but incomplete loss of sperm motility and fertilisation capacity raising the possibility of residual activity of channels formed by TRPV6 D541A proteins (Sci Signal 4, ra27, 2011) . It is known from other cation channels that introducing pore mutations even if they largely affect their conductivity and permeability can evoke considerably different phenotypes compared to the deletion of the corresponding protein. To gain insights whether the TRPV6 D541A pore mutant still contributes to residual channel activity and/or channelindependent functions in vivo, we compared important fertility-parameters between Trpv6 -/and Trpv6 D541A/D541A mice: The fertilization rate observed in permanent matings, the in vivo fertilization rate as judged by the rate of embryos isolated from plug positive females of matings with males homozygous for either Trpv6 mutation as well as the motility, in vitro fertilization capacity and viability of sperm were reduced to the same extent in both genotypes. Also, no differences were observed in copulatory behavior between Trpv6 -/and Trpv6 D541A/D541A males. The profound reduction in Ca 2+ uptake by the epididymal epithelium was identical in Trpv6 -/and Trpv6 D541A/D541A males. This direct comparison of these parameters indicate that deleting TRPV6 does not further aggravate the phenotype observed in Trpv6 D541A/D541A mice, and -in our opinion -allows the conclusion that the D541A pore mutant of the TRPV6 protein leads to complete inactivation of the TRPV6 channel activity or channel-independent scaffolding functions in epididymal epithelium.

Characterization of a naturally occurring C-terminal mutation (N996I) on hERG channel function in HEK293 cells Sellmaier V. 1 , Moretti A. Long-QT-syndromes (LQTS) are acquired or inherited disorders which predispose patients to cardiac arrhythmias and sudden death. In affected individuals, the electrocardiogram shows a prolongation in the QT interval, due to an unstable repolarization of the action potential. Acquired forms of LQTS are often the result of treatment with medications that block cardiac potassium channels, such as class III antiarrhythmic drugs or antihistamines. Inherited LQTS are caused by mutations in cardiac ion channels. Congenital long QT syndrome 2 (LQT2) is caused by loss-offunction mutations in the human ether-á-go-go-related gene HERG (also known as KCNH2 or Kv11.1). HERG encodes the pore-forming α-subunit of the rapid delayed rectifier potassium current I Kr, whose physiological role is to repolarize the late phase of cardiac action potentials. HERG channel α-subunits exist as 2 isoforms (1a and 1b) that are identical except for structurally divergent N termini. Native cardiac IKr channels are tetraheteromers containing 2 of each α-subunit types. A loss-of-function can be due to either defects in a) channel opening (gating), b) ion permeation or c) protein maturation and trafficking. We have identified a so far uncharacterized dominant missense mutation in the HERG1 gene (N996I) in a patient with LQT. Both hERG1a and hERG1b subunits were cloned from a human heart cDNA library and the specific N996I mutation introduced by site directed mutagenesis. HEK 293 cells were transiently transfected with equal amounts of mutated hERG1a and wild type (WT) hERG1b cDNAs and the resulting potassium current compared to hERG1a/b WT. Whole-cell patch-clamp analysis showed similar current densities for WT versus mutated channels. Also the voltage-dependence of activation was unchanged with a halfmaximal activation at -27 mV for WT and -29 mV for the mutated channel assembly. Differences were found for the deactivation and inactivation kinetics. The deactivation was faster in the mutated channels with t fast = 62 ms and tslow = 480 ms versus tfast = 90 ms and tfast = 620 ms in WT channels, determined from the tail current at -40 mV after a 5-second pulse to + 50 mV. The half-maximal steady-state inactivation of the tail current was shifted by 20 mV to the depolarizing direction in the mutated channel compared to the WT. A defect in channel gating appears to be the most likely explanation. Background: ABCC2 is adjacent to P-Glycoprotein the most important efflux transporter for various endogenous and exogenous compounds and is expressed at several compartment barriers. By increasing evidence it is shown that the ABCC2 polymorphisms are of clinical significance. The aim of our study is to analyze the epigenetic regulation of distinct ABCC2 haplotypes by the influence of microRNAs. Methods: ABCC2 cDNA clones containing five distinct haplotypes were generated by site-directed mutagenesis, cloned into pIRES-ZsGreen expression vectors and transfected into different cell lines for further functional analysis. One modified vector set contained a short 3'UTR sequence of ABCC2 whereas the other contained the full length (fl) sequence. miRNAs potentially interacting with ABCC2 were identified form an miRNA array after rifampicin stimulation of HepG2 cells.

Results: We could demonstrate that there is no difference in the basal protein expression level comparing the two vector types (fl 3'UTR vs. mod. 3'UTR) concerning the -24C/1249G/3972C (CGC) ABCC2 WT in HepG2 cells. Using the fl vector construct, the expression level of CAC haplotype protein was increased (136,15% ± 20%). Transfection assays with the miR-397, which was identified as a candidate miRNA targeting ABCC2 mRNA, and the two different vector constructs harboring the CAC or the CGC (WT) haplotype, confirmed that miR-379 was able to down regulate the ABCC2 protein expression. There was no significance in downregulation for one ABCC2 haplotype, respectively (modified 3'UTR: CGC 22-34%; CAC 20-40%, fl 3'UTR: CGC 20-25%; CAC 17-44% down regulation compared to miR-negative control). Discussion: Differences of ABCC2 protein expression level are due to the epigenetic regulation of ABCC2 haplotypes. To further characterize the effect of miR-379 on TCG, TGT and CGT ABCC2 haplotypes, transfection assays are currently performed using cell cultures as well human primary leukocyte cultures.

Sex differences affect the pathophysiology and pharmacology of leukotriene biosynthesis Werz O. Friedrich-Schiller-University Jena, Institute of Pharmacy, Philosophenweg 14, 07743 Jena, Germany Inflammatory diseases affect more females than males. Thus, women suffer more often from asthma, rheumatoide arthritis, Alzheimer´s Disease, and many autoimmune diseases than men. Of interest, sex differences also exist in drug responses, with respect to both pharmacodynamics and pharmacokinetics. We have recently discovered a sex bias in the biosynthesis of pro-inflammatory leukotrienes (LTs) due to testosterone, which may represent a molecular basis for gender differences in inflammation and asthma. Interestingly, testosterone downregulates LT biosynthesis and also causes a sex bias in the efficiency of LT synthesis inhibitors, which demands for the clinical evaluation of a gender-tailored therapy with anti-LTs. We found that certain inhibitors of LT biosynthesis were more efficiently in females than in males, and that androgens are responsible for these gender differences. In fact, the FLAP inhibitor MK886 effectively reduced LTB 4 pleural levels in female but not in male rats treated with carrageenan, and MK886 increased survival only of female mice in the LT-related disease model of PAF-induced lethal shock. Administration of testosterone to female mice abolished the protective effects of MK886. In view of the current active development of LT synthesis inhibitors as therapeutics in respiratory and cardiovascular diseases, our data prompt for consideration of gender issues in the development and use of such drugs, in order to optimize medical therapy both for men and women. Considering the complexity of deposition and kinetics of air-borne nanomaterials in the lung, potential pulmonary toxicity of biopersistent nanomaterials should be evaluated by inhalation studies. Those studies demand special equipment and large quantities of test material. Intratracheal instillation appears as a simple and low substance-consuming alternative, although bolus dosing and the more central distribution of the particles in the lung are a well known trade-off. We compared the inflammatory response of the lung to amorphous silica (AS) after instillation and inhalation. For inhalation the established short-term protocol for nanomaterials was employed (Ma-Hock et al. Inhalation Toxicology, 21:102, 2009 ): Male Wistar rats were exposed to the test items for 6 h/day on five consecutive days. The lungs were evaluated by analysis of bronchoalveolar lavage fluid (BALF) and by histopathology three days and three weeks after the end of the exposure. In a parallel study, rats were intratracheally instilled and equally evaluated three days after instillation. Assuming a deposition rate of 10%, the instilled dose corresponded to the aerosol concentration of 10 mg/m 3 used for inhalation. Results show that inhalation and instillation of nominally equal mounts of amourphous silica elicit different results in the lung with inhalative treatment being less harmfull. This difference may be due to the bolus effect inevitable linked to instillation. Instillation stuldies with amorphous silica may, therefore, be of limited value with respect to doseresponse assessment. Sunscreen products containing UV filters protect consumers from the harmful effects of UV exposure. Pigmentary grades of metal oxides like ZnO result in an opaque whiteness as a result of scattering visible light, whereasnanoparticles result in transparent products for better consumer acceptance and thus improved protection of human skin against UV-induced damage. In addition scatter UV light is most efficiently reflected at a nanosize of 60-120 nm. In the last 2 years the toxicological properties of nanoZnO in comparison with pigmentary ZnO were examined, the results of these comprehensive studies are presented. All tests were performed according to OECD guidelines, which were modified, especially in regard of substance preparation where appropriate. Nanosized ZnO showed no acute toxicity after dermal application, in the BCOP assay as well as in the Epiderm assay it showed no corrosion / irritation potential. Nanosized ZnO does not penetrate the intact as well as the sunburned skin. A dermal absorption test in rats (OECD 427) with 65 Zn-labelled test item as well as penetration tests in weanling pigs after UV radiation did not show a penetration of the ZnO nanoparticles through the skin. Genotoxicity was tested in vitro in the Ames Test, in the Chromosomal Aberration Test in V79 cells, both showing negative results whereas the Mouse lymphoma mutation test / L5178Y/TK+/-cells was positive. In vivo no mutagenic effect was detected in two Mouse Micronucleus tests, on with intraperitoneally application and another after repeated inhalation. Nanosized ZnO was tested in 5-days, 14-days and 90-days inhalation studies, in all studies the predominant effects were reversible local inflammatory changes in the nasal cavity and lungs, with a NOAEC of 1.5 mg/m3 in the 90-day study. In a Prenatal Developmental Toxicity Study according to OECD TG 414, with repeated inhalation exposure to female Wistar rats from gestation day 6 through 19, maternal toxicity was observed by increase lung weights and inflammations in the lungs. But no substance-related effects on reproductive parameters (conception rate, corpora lutea, implantation sites, preimplantation loss, postimplantation loss, resorptions, dead fetuses) and no increase in external and soft tissue malformations and variations could be detected. The overall result of all the toxicological studies with nanosized and pigmentary ZnO is that the toxicological profile of both is very similar.

Studies were sponsored partly by CEFCI LRI and partly by BASF SE.

Use of REACh Registration Data for Improving Thresholds of Toxicological Concern (TTC) Wieneke N., Dorn S., Jakupoglu C., Schäfer C., Sica M., Wiegand C., Mostert V. DR. KNOELL CONSULT GmbH, Marie-Curie-Str. 8, 51377 Leverkusen, Germany

The Threshold of Toxicological Concern (TTC) concept is utilised to identify human exposures that are so low that in-depth toxicological investigations are expendable. This is called "exposure-based waiving". Exposure-based waiving serves to focus available resources on substances with relevant human exposure potential. Important work into establishing TTC values has been published by Munro et al. (1996) . The initial report used a database of 613 organic substances compiled from publicly available sources. In total, 2941 NOELs were collected in this fashion. The Munro concept used the Cramer classification to categorise substances according to their hazard potential. We broadened the TTC database by including NOAELs published on the EChA website as per 3 November 2011, containing data for more than 3900 registrations. Only nongaseous mono-constituent substances with oral NOAELs were included in the TTC database. Organophosphates and genotoxic substances were excluded from the database as well as NOAELs obtained for surrogate substances. NOAELs for all systemic endpoints (general toxicity, developmental toxicity, fertility, neoplasia) were taken into account. Where appropriate, default assessment factors of up to 6 were used to establish chronic NOAELs for each substance. For every eligible substance, we collected the published CLP category for acute oral toxicity as a potential predictor of overall hazard potential. This gives rise to five categories of acute oral toxicity. A TTC is calculated from the 5 th percentile of NOAELs in each of these categories using the REACh rules for establishing DNELs for workers and the general population. This poster presents the preliminary results for more than 1000 substances. The results indicate that the TTC concept becomes more robust when using the very broad EChA database. It also suggests that acute oral toxicity categories can be used as a predictor for the overall hazard potential of a substance.

Comparison of different in-vitro models for inhalation toxicology with respect to the effects of cigarette smoke total particulate matter Wiese J. 1 , Schumann B. B-and L-MOC can be cultivated up to 70 days without loss of viability, as determined by resazurin-assay. Viability of cell cultures was determined by MTT-assay after incubation with increasing doses of TPM. Exposure of H322 to TPM (30 mg/l) reduced viability to 95% or 83% after 24 or 72h, respectively. In A549 viability was 67% after 72h with TPM (30 mg/l). The same dose of TPM lead to a decrease in viability to 82% (24h) or 25% (72h) in NHBEC and to 74% (24h) or 28% (72h) in PLC. As a marker of oxidative stress the level of intra-cellular glutathione (GSH) was determined by HPLC. In both the tumor cell lines analysed GSH-level was increased by TPM (5 mg/l). In H322 the induction was 1.6 and 1.2 fold after 24 or 72h, respectively. While in A549 it was 1.3 (24h) and 1.4 fold (72h). In NHEC and PLC TPM (5 mg/l) did not have a significant effect on GSH-levels after 72h. In N-MOC TPM (5 mg/l) also did not modulate GSH after 24h, but it diminished GSH-level by 0.5 fold upon prolonged contact (72h). In B-MOC GSH concentrations also decreased to 0.6 or 0.8 fold the level of controls after 24 or 72h incubation.

The results presented show that in-vitro models of varying complexity and origin within the respiratory tract clearly differ in their response to TPM, which was used a model inhalative toxicant. The tumor cell lines used seem to be better adapted to chemical stress, while the models closer to the in-vivo situation are more vulnerable.

The nongenomic effects of the mineralocorticoid receptor in transgenic mouse heart Winter S. 1 , Schreier B. Within the renin-angiotensin-aldosterone system (RAAS), the mineralocorticoid receptor (MR) is important for the regulation of fluid and electrolyte balance in the kidney, salivary glands, sweat glands and colon. However, survival of patients with severe heart failure is increased when MR blockage is combined with standard therapy suggesting aldosterone, the MR ligand, as a key factor in the development of cardiovascular diseases, but the mechanism is not yet fully understood. In recent years, evidence accumulated that besides its function as a hormone-activated transcription factor the MR also functions via nongenomic pathways.

To investigate the function of the nongenomic effects of the MR in cardiovascular dysfunction, we generated a transgenic (TG) mouse model expressing a truncated human MR lacking the DNA-binding site (hMR DEF ) under control of the cardiac specific α myosin heavy chain promoter (αMHC), a model for nongenomic effects of the MR in the heart. In this mouse model no enhanced mortality could be observed. Body weight (BW), heart weight and relative heart weight were not different compared to wild type (WT) while left atrial weight/BW was increased by 20 % (WT 0,25 ± 0,01 mg/g vs. TG 0,30 ± 0,02 mg/g, p<0.05, n=12). Compared to WT mice neither surface electrocardiographic experiments nor echocardiographic experiments revealed modified parameters for TG mice under basal (i.e. unstimulated) conditons as well as under β-adrenergic stimulation by isoproterenol (ISO, 100 µl 1 mM ISO intraperitoneally applied).

To uncover the role of aldosterone in the development of cardiovascular diseases treatment with aldosterone and high-salt diet (1%) was performed. After 28 days cardiac function and heart dimensions were analyzed, surface electrocardiographie uncovered increased p duration (16 ± 0.5 ms vs. 14 ± 0.7 ms, p<0.05) and QTc interval (61 ± 2 ms vs. 50 ± 3 ms, p<0.05) in TG (n=7) compared to WT (n=5) animals. These findings probably indicate more sensitive conduction pathways to aldosterone in TG mice.

Oligomerization is important for regulation of phospholamban activity Wittmann T., Lohse M. J., Schmitt J. P. Institut für Pharmakologie und Toxikologie, Versbacher Straße 9, 97078 Würzburg, Germany Phospholamban (PLN) is a heart specific protein located in the membrane of the sarcoplasmic reticulum. It inhibits the Ca 2+ -ATPase SERCA2a, thereby decelerating cytosolic Ca 2+ clearance during diastole of the cardiac cycle. Upon phosphorylation the inhibitory activity of PLN on myocyte Ca 2+ transport is attenuated. Further, it is believed that phosphorylation favors the formation of (rather inactive) pentamers and that PLN pentamers itselves were an inferior substrate for phosphorylation compared to monomers. This would suggest an important role of PLN oligomerization in the regulation of PLN activity. To prove this hypothesis, we are investigating the patterns and kinetics of PLN phosphorylation in the context of alterations in PLN structure. The introduction of specific point mutations into the transmembrane region of PLN yielded mutants that are purely monomeric (L37A, I40A and C41F) or favor pentamer formation (I45A, V49A). Transfected HEK293 cells expressing these mutants or wildtype PLN were stimulated with forskolin to induce PLN phosphorylation before lysis of cells and Western blot analysis using antibodies directed against phosphorylated PLN. Surprisingly, phosphorylation was increased for both monomeric and pentameric PLN after stimulation with 0.25µM forskolin for less than one minute. At increasing forskolin concentrations phosphorylation signals increased in parallel for monomers and pentamers. For measurement of phosphorylation kinetics stimulation of cells with 2.5µM forskolin was stopped at different time points. We found phosphorylation of both PLN monomers and pentamers within seconds of stimulation. Differences in phosphorylation patterns became more pronounced when assays were performed at low temperature (14°C). Intriguingly, preliminary analyses suggest that PKA dependent phosphorylation occurs first in pentamers and that phosphorylation of monomers may catch up only after pentamer phosphorylation is almost complete. Our data suggest that both PLN pentamers and monomers are suitable substrates for PKA dependent PLN phosphorylation. Unlike the prevalent assumption, kinetics of pentamer phosphorylation seem to be at least as fast as that of PLN monomers in transfected HEK293 cells suggesting an important role of PLN pentamers in the regulation of PLN activity.

Regulation of cardiac contractility by nucleoside diphosphate kinases in zebrafish Wolf N. M. 1, 2 , Abu-Taha I. In the heart, nucleoside diphosphate kinases (NDPKs) can interact with heterotrimeric G proteins, thus regulating cAMP synthesis in a receptor independent manner and thereby influencing contractility in cardiomyocytes. We further investigated the interaction of NDPK isoforms with heterotrimeric G proteins in the heart in vivo and in vitro using zebrafish embryos and embryonic fibroblast from NDPK A/B double knockout mice (NDPK A/B KO MEFs). In zebrafish the morpholino-mediated knockdown of NDPK A did not lead to an obvious phenotype, although the total NDPK activity was reduced. Depletion of NDPK B caused a cardiac phenotype characterized by severely impaired atrial and ventricular contractility and insufficient blood flow. The depletion of NDPK B was associated with a significant decrease of protein levels of the heterotrimeric G protein subunits Gβγ, Gα s and Gαi. The knockdown of NDPK C led to a more restricted cardiac phenotype with markedly reduced pumping function of the ventricle, while the atrium was unaffected. In accordance to the reduced cardiac pumping function, cAMP levels were significantly diminished in the NDPK B and NDPK C morphants. Similar findings were obtained in NDPK A/B KO MEFs. The absence of NDPK A and B resulted in a decrease of the plasma membrane content of Gβ and Gαs and a significant reduction in cAMP synthesis. The protein expression of the isoform NDPK C was also significantly reduced in the NDPK A/B KO MEFs. The re-expression of NDPK B but not NDPK A rescued the basal cAMP production and the membrane content of G proteins. Interestingly, the overexpression of NDPK C led to a 5-fold enhancement of the cAMP level and a significant increase of the membrane content of Gβ and Gα, and thus rescued the knockout phenotype. Our data indicate, that the NDPK isoforms B and C are essential for cardiac contractility, most likely by forming a signaling complex at the plasma membrane including NDPK B, NDPK C and heterotrimeric G proteins. The isoform NDPK C, with its N-terminal hydrophobic region, might serve as a membrane anchor for the NDPK/G protein complex.

Induction of apoptosis via PKA-dependent and PKA-independent pathways by cyclic purine and pyrimidine nucleotides in mouse lymphoma cell lines Wolter S. 1 cAMP is a second messenger that plays an important role in intracellular signal transduction of various hormones and neurotransmitters. A major function of cAMP in eukaryotes is the activation of cAMP-dependent protein kinase A (PKA). PKA is involved in the control of a variety of cellular processes. PKA exists as an inactive tetramer of a dimeric regulatory (R2) and two catalytic (C) subunits that releases the active C-subunits upon binding of cAMP. Stimulation of the mouse T-lymphoma cell line S49 wild-type (wt) with dibutyryl (DB)-cAMP induces apoptosis by an intrinsic, mitochondria-dependent mechanism. Apoptosis induced by DB-cAMP occurs via a PKA-dependent mechanism, since S49 kincells lacking the catalytic subunit of PKA are resistant to DB-cAMP-mediated cell death. DB-cAMP is cleaved by esterases into the biologically active compound N 6 -MB-cAMP and into 2'-O-MB-cAMP.

Other cyclic nucleotides (cNMPs) in addition to cAMP, like cCMP and cUMP can also function as second messengers and activate PKA and cGMP-dependent protein kinase (PKG) 1 . Therefore, we investigated the effects of a series of membrane-permeable analogues of cAMP, cGMP, cCMP and cUMP in S49 wt und S49 kincells on apoptosis.

Stimulation with DB-cCMP or DB-cGMP induced neither apoptosis in S49 wt nor in S49 kincells. Interestingly, we observed apoptosis in S49 wt and S49 kin cells after incubation with membrane-permeable nucleotide acetoxymethyl ester (AM)-analogues of cGMP, cCMP, cUMP and also cAMP. Induction of apoptosis occurs via PKAdependent and also PKA-independent pathways. A potential role of PKG and of the Exchange protein activated by cAMP (Epac) in the induction of apoptosis is unsolved and will be explored by specific PKG-and Epac-activators in this system. Investigations are on the way to identify the targets, the involved signal transduction pathways and the mechanisms of pro-apoptotic actions mediated by cNMP-AMs.

(1) Wolter S, Golombek M, Seifert R (2011) Differential activation of cAMP-and cGMPdependent protein kinases by cyclic purine and pyrimidine nucleotides. Biochem Biophys Res Commun. In press Apoptosis in S49 cells : Induction of apoptosis in S49 wt and in S49 cells lacking the catalytic subunit of PKA (S49 kin-) with A) DB-cNMPs and B) cNMP-AMs for 72h.

Nebivolol reduces vascular inflammation in spontaneously hypertensive rats Wu Z., Xia N., Förstermann U., Li H. Institut für Pharmakologie, Obere Zahlbacher Str. 67, 55131 Mainz, Germany Nebivolol is a third generation β1 receptor blocker with additional effects on endothelial nitric oxide production. The aim of the present study is to investigate the antiinflammatory effects of Nebivolol in vivo. 48 spontaneously hypertensive rats (SHR) were divided into two groups: control or nebivolol treatment group. Nebivolol treatment (5 mg/kg/day for 10 days) significantly reduced the blood pressure and the heart rate in SHR. The drug had no effect on coagulation. Aorta from nebivolol-treated rats showed significantly improved endothelial function. Nebivolol did not change the expression levels of aortic NF-kB, but significantly reduced its DNA binding activity. Furthermore, nebivolol decreased the expression of adhesion molecules (e.g. ICAM-1, VCAM-1) and pro-inflammatory cytokines (e.g. IL-6). In conclusion, nebivolol reduces vascular inflammation in experimental hypertension. It inhibits NF-kB activity, decreases the expression of adhesion molecules and pro-inflammatory cytokine, and improves endothelial function.

Characterization of the cellular activity of PDE4 inhibitors using two novel PDE4 reporter cell lines Wunder F., Quednau R., Barg M., Tersteegen A. Bayer Pharma AG Lead Discovery Wuppertal, Aprather Weg 18a, 42096 Wuppertal, Germany Cyclic nucleotide-specific phosphodiesterases (PDEs) play an essential role in cellular signal transduction by regulating the intracellular levels of cAMP and cGMP and, therefore, are important pharmacological targets. We report here the generation and pharmacological characterization of two novel PDE4 reporter cell lines. Plasmid constructs encoding human PDE4B1 or PDE4D3 were stably co-transfected with the beta1-adrenoceptor in a parental cAMP reporter cell line expressing a cyclic nucleotide-gated (CNG) cation channel, acting as the biosensor for intracellular cAMP. In this reporter cell line, cAMP levels can be monitored in real-time via aequorin luminescence stimulated by calcium influx through the CNG channel. By using different PDE4 and non-PDE4 inhibitors, we could show that our novel PDE4B1 and PDE4D3 reporter cell lines specifically monitor PDE4 inhibition with high sensitivity. PDE4-selective inhibitors alone did not increase basal luminescence levels in this experimental setting. However, these inhibitors induced concentration-dependent luminescence signals in combination with the adrenoceptor agonist isoproterenol. In contrast, in a stable beta1-adrenoceptor reporter cell line with no recombinant PDE4 expression, PDE4 inhibitors had no effect on isoproterenol-stimulated luminescence signals. We compared the cellular activity of different PDE4 inhibitors with the in vitro inhibition of full-length and truncated (catalytic domain) PDE4D3 from cell lysates. Two different groups of PDE4 inhibitors could be identified. The first group, including the allosteric inhibitors PMNPQ and D159153, showed high cellular activity and much better inhibition of full-length versus truncated PDE4D3. The second inhibitor group, including classical competitive inhibitors like roflumilast, cilomilast and piclamilast, showed comparably lower cellular activity and similar inhibitory activity on full-length and truncated PDE4D3. The results imply that these novel PDE4 reporter cell lines are well-suited for the characterization of the cellular activity of PDE4 inhibitors and may also support a better understanding of the complex PDE4 pharmacology.

Plexin-B2 is required for kidney regeneration after acute renal failure Xia J. 1 , Gröne H Acute renal failure is a common clinical problem with unsatisfactory therapeutic options and high mortality in humans. Therefore, unraveling the mechanisms that promote kidney regeneration and repair may provide new therapeutic strategies for acute renal injury. Plexin-B2 belongs to a family of transmembrane receptors which mediate the cellular effects of semaphorins. While plexins have first been described in the context of axon guidance, several recent studies have established them as key regulators of organogenesis, the immune system and cancer. We have recently found that Plexin-B2 is highly expressed in the adult kidney, particularly in tubular epithelial cells which are most sensitive to acute ischemic injury. To study the role of Plexin-B2 during kidney regeneration we generated mice lacking Plexin-B2 specifically in tubular epithelial cells. Under physiological conditions, these mice displayed normal kidney morphology and function. In contrast, following ischemia/reperfusion injury, Plexin-B2 conditional knockout mice exhibited severely impaired kidney regeneration. While the renal function of control mice fully recovered within 3 weeks after injury, Plexin-B2 knockout mice had strongly elevated serum creatinine and urea levels associated with increased morbidity and mortality. This was accompanied by hyperproliferation of tubular epithelial cells and obstruction of tubular lumina. We conclude that Plexin-B2 is required for regeneration after acute ischemic renal injury and that pharmacological interventions activating Plexin-B2 might represent a new therapeutic strategy in acute renal failure. The NADPH oxidase enzyme complex consists of two membrane-bound catalytic subunits (a Nox protein and p22phox) and several cytosolic regulatory components including p47phox, p67phox, p40phox and the small GTPase Rac1. We have previously shown that treatment of apolipoprotein E knockout mice with resveratrol led to a downregulation of Nox2 and Nox4 in the heart. Our recent data demonstrated that resveratrol also reduced the enzymatic activity of cardiac NADPH oxidase. Because activation of NADPH oxidase enzyme complex is induced by translocation of the regulatory subunits, we studied whether the reduced enzymatic activity is due to an inhibition of such a translocation. Indeed, resveratrol treatment prevented Rac1 membrane translocation from cytosol. Resveratrol is known as an activator and expression enhancer of the longevity gene sirtuin 1 (SIRT1). We then wanted to find out whether the effect of resveratrol on Rac1 was mediated by SIRT1. SIRT1 is a histone/protein deacetylase. In vitro incubation of Rac1 with SIRT1 led to a reduction of lysine acetylation. Deacetylation of Rac1 on lysine 166 could be identified by Mass spectrometry analyses. The lysine 166 lies within the p67phox-binding region of Rac1. Consistently, in vitro incubation of Rac1 with SIRT1 markedly reduced its binding activity to p67phox. In conclusion, we provide evidence that Rac1 is a direct target molecule of SIRT1. SIRT1 deacetylates Rac1 on lysine 166 and thereby inhibits its interaction with p67phox. This is a novel mechanism of NADPH oxidase inhibition by SIRT1/resveratrol.

Mutational analysis of the effects of the cardioprotective drug dexrazoxane on Topoisomerase II beta in vitro Yan T., Deng S., Frensch I., Gödtel-Armbrust U., Wojnowski L. UNIVERSITÄTSMEDIZIN der Johannes Gutenberg-Universität Mainz Institut für Pharmakologie, Obere Zahlbacher Straße 67, 55101 Mainz, Germany Dexrazoxane (DRZ, ICRF-187) is the only approved drug shown to protect against anthracycline-induced heart failure. The protection is usually ascribed to the ironchelation by DRZ, which is thought to reduce anthracycline-induced oxidative stress. However, similarly to anthracyclines, DRZ is also an inhibitor of the anthracycline target Topoisomerase II (TOP2). We hypothesized that the cardioprotective effects of DRZ are mediated by the prevention of the anthracycline-induced DNA damage mediated by TOP2B, the dominant cardiac TOP2 isoform. This was investigated using TOP2B mutants resistant to DRZ, which were expressed in cells depleted of wild-type TOP2 isoforms. TOP2B-mediated double-strand DNA breaks were assessed as γ-H2AX. The levesl of DSB generated by the TOP2B mutants in response to the anthracycline doxorubicine (DOX) was indistinguishable from that mediated by a wild-type TOP2B. Preincubation with DRZ depleted wild-type TOP2B and this was accompanied by a decrease in the DNA damage following a subsequent exposure to DOX. In contrast, neither TOP2B depletion nor the reduction of DSB by DRZ was seen in DRZ-resistant TOP2B mutants. Furthermore, the cardially ineffective DRZ analog ICRF-161, capable of iron chelation but not of TOP2 binding, affected neither the stability of TOP2B, nor the DOX-induced DNA damage mediated by this enzyme. These results indicate that DRZ may exert its cardioprotective effects by reducing the DNA damage mediated by DOXpoisoned TOP2B rather than by iron chelation. They also suggest a cardioprotective function of TOP2B, which is currently under investigation using cardiomyocyte-specific TOP2B mouse knockouts. Aminoglycosides are important antibiotics in the treatment of life-threatening infections, especially those caused by Gram-negative bacteria. Their nephrotoxic and ototoxic potential is well-known, but little is known about the effects of aminoglycosides on the male reproductive system. We studied the effects of four aminoglycosides on Sertolicells in vitro. Rat Sertoli-cells from the cell line SerW3 were cultivated for 3, 6, and 9 days in DMEM supplemented with three different concentrations of amikacin, streptomycin (30 mg/l, 100 mg/l, 300 mg/l), gentamicin or tobramycin (10 mg/l, 30 mg/l, 100 mg/l). We determined the expression of two junctional proteins (connexin 43, Ncadherin) and one protein of the cytoskeleton (vimentin) by Western blot. Cells were solubilized in lysis buffer. Lysates were separated by SDS-PAGE and electroblotted on a PVDF-membrane. After incubation with primary antibodies overnight and horseradish peroxidase-conjugated secondary antibody the visualization was achieved by a chemiluminescence-detection system. In addition, proteins were detected by immunohistochemistry.

After three days in culture amikacin caused the most pronounced effect. At the lowest concentration tested (30 mg/l) connexin 43 and N-cadherin were reduced to 55±19% and 92±10% of the controls (n=6). No change was recognized for vimentin (102±16%). Effects obtained with streptomycin were less pronounced for these these proteins (68±16%, 96±7%, and 102±13%, respectively). Similar, but less pronounced effects were observed with gentamicin and tobramycin at a concentration of 10 mg/l (connexin 43: 88±15% and 81±15%; N-cadherin: 95±18% and 103±11%; vimentin: 81±12% and 102±19%) and 30 mg/l (connexin-43: 84±19% and 78±18%; N-cadherin: 98±19% and 103±13%; vimentin: 102±8% and 115±15%). After incubation for 6 and 9 days the effects occurred in the same range. The substances showed no influence on the viability of SerW3 Sertoli-cells up to 300 mg/l in the MTT assay. By immunohistochemistry we showed that the localisation of the proteins -connexin 43 and N-cadherin at the cell membrane and vimentin in the cytoplasm -was not influenced by the aminoglycosides.

Large conductance calcium-and voltage-gated potassium (BK) channels play an important role in controlling membrane potential and calcium influx, and are strongly modulated by protein kinases at multiple sites. The stress-regulated exon (STREX) adds to the BK channel C terminus a cysteine-rich insert of 59 amino acids that inverts the channel regulation by protein kinase A (PKA) from excitatory to inhibitory. Here we investigated the mechanisms by which the STREX insert influences BK channel regulation by protein kinase C (PKC). Activity of BK channels without STREX insert (BK-ZERO), transiently expressed in HEK cells, was inhibited by PKC in inside-out membrane patches (~ 50% inhibition). BK channels with STREX insert (BK-STREX), however, were insensitive to PKC. Phosphomimetic mutation of a PKC phosphorylation site (S700E) in BK-STREX, resulted in a ~50% reduction of basal channel activity, whereas the S700A mutant retained normal activity. To examine whether palmitoylation, and thus association of the STREX domain with the plasma membrane, prevents PKC inhibition of BK channel gating, palmitoylation was abolished by either site-directed mutagenesis (C12:13A) or by pharmacological inhibition of palmitoyl transferases with 2-bromopalmitate (2-BP). Both, mutation and pretreatment with 2-BP resulted in the expression of BK-STREX channels which were sensitive to PKC (~ 50% inhibition of channel activity). No inhibitory PKC effect was observed in patches of the BK-STREX S700A channel mutant pretreated with 2-BP. In a clonal rat somatomammotroph pituitary cell line (GH3B6), in which PCR products without (ZERO) and with the 174 bp STREX exon could be identified, the PKC activator PMA blocked channel activity by ~25 %. This inhibition was increased to over 50% when GH3B6 cells were pretreated with 2-BP, indicating that both channel isoforms were functionally active. In summary, the present study demonstrates that palmitoylation of STREX prevents BK channel regulation by PKC, which is mediated by phosphorylation of Ser700, probably by steric hindrance. Our results provide further evidence for a cross-talk between palmitoylation and phosphorylation as a crucial mechanism underlying the dynamic regulation of ion channels.

Human pleural mesothelial MeT-5A cells are a limited in vitro model system in determining potential asbestos-like genotoxic effects of multiwall carbon nanotubes Ziemann C. 1 , Reamon-Büttner S. Multiwall carbon nanotubes (MWCNT) are nanomaterials with important technological impact. Depending on their diameter, length, and biopersistence, however, some MWCNT seem to exhibit a fiber-like cytotoxic and genotoxic potential, similar to asbestos. Thus, a project funded by the German Federal Ministry of Education and Research (BMBF contract No. 03X0109A) focuses on potential adverse biological effects of different types of MWCNT to enlarge the knowledge base about toxicity determining parameters. This project comprises both in vitro (rat) and in vivo endpoints with long amosite asbestos as a positive control. As mesothelial cells are target cells for adverse effects of asbestos, in particular mesothelioma development, the human SV40transformed, non-malignant pleural mesothelial cell line MeT-5A was chosen as the main in vitro model in this project. In the present study part, MeT-5A cells were characterized concerning their usefulness as an in vitro model to study potential asbestos-like cytotoxic and genotoxic effects of different MWCNT varieties. Using an MWCNT-optimized lactate dehydrogenase liberation assay and proliferation parameters derived from cell counts, concentration-dependent cytotoxicity of long amosite asbestos (2, 10, and 20 µg/cm 2 ) was demonstrated in MeT-5A cells. Cells also showed asbestosinduced increase in DNA-strand breaks and oxidative DNA-damage in the hOGG1modified comet assay. Thus, MeT-5A cells were responsive to asbestos treatment. Owing to asbestos potential to induce aneugenic effects and spindle fiber damage, micronucleus induction, determination of numerical chromosome aberration, and altered meta-, ana-, and telophase morphology were planned as in vitro endpoints. MeT-5A cells were thus initially characterized in this regard and were found to exhibit highly variable chromosome numbers with lower than 10% cells exhibiting a normal diploid chromosome set, an up to twentyfold higher spontaneous micronucleus frequency, as compared to polychromatic bone marrow erythrocytes in rodents, and a profound number of aberrant meta-, ana-and telophases with bridges, lagging chromosomes and multipolar divisions. In conclusion, MeT-5A cells are of only limited value as an in vitro model system to study potential asbestos-like effects of MWCNT and also biopersistent fibers. The cells are indeed responsive to asbestos, but unfortunately demonstrate marked genomic instability and thus limited significance concerning genotoxic effects.

Waixenicin A inhibits cell proliferation through magnesium-dependent block of TRPM7 channels Zierler S. 1 Transient receptor potential melastatin 7 (TRPM7) channels represent the major magnesium-uptake mechanism in mammalian cells and are key regulators of cell growth and proliferation. They are abundantly expressed in a variety of human carcinoma cells controlling survival, growth and migration. These characteristics are the basis for recent interest in the channel as a target for cancer therapeutics. We screened a chemical library of marine organism-derived extracts and identified waixenicin A from the soft coral Sarcothelia edmondsoni as a strong inhibitor of overexpressed and native TRPM7. Waixenicin A activity was cytosolic and potentiated by intracellular free magnesium (Mg 2+ ) concentration. Mutating a Mg 2+ -sensitive site on the TRPM7 kinase domain reduced the potency of the compound, whereas kinase deletion enhanced its efficacy independent of Mg 2+ . Waixenicin A failed to inhibit the closely homologous TRPM6 channel and did not significantly affect TRPM2, TRPM4, and CRAC (calcium release activated calcium) channels. Therefore, waixenicin A represents the first potent and relatively specific inhibitor of TRPM7 ion channels. Consistent with TRPM7 inhibition, the compound blocked cell proliferation in human Jurkat T-cells and rat basophilic leukemia cells. Based on the compound's ability to inhibit cell proliferation through Mg 2+ -dependent block of TRPM7, waixenicin A or structural analogs may have cancer-specific therapeutic potential, particularly since certain cancers accumulate cytosolic Mg 2+ .

Release of metals from different sections of domestic drinking water installations Zietz B. P., Richter K., Laß J., Suchenwirth R., Huppmann R. Governmental Institute of Public Health of Lower Saxony Division of Environmental Medicine and Environmental Epidemiology, Roesebeckstraße 4-6, 30449 Hanover, Germany Different metals were used as important piping materials in the drinking water supply for a long time. Due to corrosion metals can leach into the tap water. Of special importance is the toxic element lead. However other heavy metals in drinking water such as copper, nickel and cadmium can also give reason for health concerns. In this study it was investigated in which amount relevant metals were released from different parts of domestic installations into the cold water. For the spatial allocation of the emission sources a sequential water sampling protocol was used after three hours of stagnation time representing the first 5 litre of the water column. After stagnation ten sample volumes were collected in series. Existing facilities of domestic installations constructed with different plumbing materials were examined predominantly from residential buildings. The elements Al, As, Cd, Cr, Cu, Fe, Mg, Mn, Ni, Pb, Sb, Se, U and Zn were detected by means of ICP-MS. In total 16 water pipe strands of 11 domestic installation systems were examined. They comprised 379 single water samples and 5306 single parameters. Depending upon the type of plumbing different courses and concentration ranges of the elements could be measured in the tap water samples. Terminal taps or installation parts were frequently responsible for a release of nickel and in several cases of cadmium. The concentration courses of the element zinc proved as a good indicator for the allocation of the emission source to a brass containing section of the installation (zinc as an alloy component of brass). One can conclude that an investigation by means of a sequential water sampling protocol and multi-element detection can be a valuable non-destructive method for drinking water-hygienic investigations of domestic installations.

Novel interaction partners of the murine TRPC4 protein Zimmermann J., Beck A., Flockerzi V. Universität des Saarlandes Experimentelle und Klinische Pharmakologie und Toxikologie, Kirrberger Str. 1, 66421 Homburg, Germany

In this work novel interaction partners of the murine protein Transient Receptor Potential Canonical 4 (mTRPC4) were identified. The TRPC4 protein is the major subunit of a cation channel, residing in the plasma membrane. It comprises six trans-membrane domains and cytosolic amino and carboxyl termini. Two major splice variants of the TRPC4 gene exist, TRPC4a (974 aa) and TRPC4b (890 aa), TRPC4b lacks aa 781 to 864 of the TRPC4a variant. Both TRPC4 variants are co-expressed in endothelial cells, intestinal smooth muscle and brain.

To identify TRPC4-interacting proteins a yeast two-hybrid system, CytoTrap®, which allows identification of protein-protein interactions within the cytosol was used. A premade mouse brain cDNA library was screened by the cytosolic amino and carboxyl terminal parts of mouse TRPC4a (aa 1 to 324; aa 622 to 974). For the carboxyl terminal part fourteen proteins were identified. To independently prove the interaction, the fulllength cDNAs of all fourteen proteins were cloned, fused to a FLAG-Tag and coexpressed with TRPC4 in HEK 293 cells. Co-immunoprecipitations were performed for all candidates using both the anti-FLAG-antibody and the antibody for TRPC4. In addition, changes of cytosolic calcium were monitored and TRPC4 currents were recorded in HEK 293 cells expressing the candidate cDNAs and stably expressing the TRPC4a or TRPC4b and the muscarinic receptor type 2 cDNAs. The Tarbp2 protein, one of the candidates shown to interact with TRPC4, changed calcium influx when coexpressed with TRPC4. In order to identify the domains of TRPC4 responsible for its interaction with the Tarbp2 protein, six TRPC4-GST-fusion proteins covering the carboxyl terminal 353 aa of TRPC4a were expressed in E. coli and used for pull-down experiments. By this approach two domains of TRPC4 could be identified to interact with Tarbp2. One of these domains is well conserved within the TRPC5 protein, corresponding to the result, that TRPC5 and Tarbp2 effectively co-immunoprecipitate, too. The Tarbp2 protein has been shown to be a component of the RISC loading complex, also known as the micro-RNA loading complex which is composed of DICER1, EIF2C2/AGO2 and Tarbp2 (Chendrimada et al. 2005) . By its interaction it may link TRPC4 to pre-microRNA processing.

Increased levels of Angiotensin II provoke DNA damage and have influence on DNA repair in mouse kidneys Zimnol A., Brand S., Schupp N. Universität Würzburg Institut für Toxikologie, Versbacherstr. 9, 97078 Würzburg, Germany

The renin-angiotensin system (RAS) plays a crucial role concerning the blood pressure, electrolyte balance and cardiovascular homeostasis. Angiotensin II (Ang II), the active hormone of the RAS, in higher concentrations leads to vasoconstriction, oxidative stress and hypertension. Hypertensive patients have an increased risk to develop cancer, especially kidney cancer. We have shown in vitro and in vivo, that Ang II is capable to cause an elevation of blood pressure as well as DNA damage dose-dependently.

To investigate whether the high blood pressure or the enhanced levels of Ang II are responsible for DNA damage, male C57BL/6-mice were equipped with osmotic minipumps, delivering Ang II in a concentration of 600ng/kg · min during 28 days. Additionally they were treated with ramipril, an angiotensin-converting-enzyme blocker, with the Ang II receptor antagonist candesartan, the vasodilator hydralazine, and the antioxidant tempol. DNA damage was analysed with the comet assay. We measured the base excision repair (BER)-related DNA repair in the kidney with a comet-based in vitro repair assay. Furthermore, the distribution and expression of the Ang II-type 1 (AT1) receptor in the kidney was analyzed by immunohistochemistry. Treatment with Ang II led to a significant increase of blood pressure, whereas the medication with candesartan decreased the systolic blood pressure. The intervention with hydralazine lowered the blood pressure only for a short time. The other substances had no effect at all on the blood pressure. Genomic damage, quantified with the comet assay, was augmented by Ang II and improved by all interventions, particularly by candesartan and tempol. Beyond that, Ang II showed a tendency to reduce DNA repair. Treatment with candesartan, hydralazine and tempol increased the repair capacity. Furthermore, Ang II tended to result in a downregulation of the AT1 receptor in kidney tubule cells. Candesartan and ramipril, especially were able to augment the expression of the AT1 receptor, whereas hydralazine achieved the opposite. These results demonstrate that Ang II leads to DNA damage in the kidney independent of blood pressure. Apparently elevated levels of Ang II affect DNA repair and expression of AT1 receptor. To confirm these findings we are going to examine more precisely the manifestation of other enzymes, which are implicated in DNA repair.

Regulation of HCN channel activity by Cyclic Cytidine 3´, 5´-Monophosphate Zong X., Krause S., Chen C. -C., Gruner C., Cao-Ehlker X., Fenske S., Wahl-Schott C., Biel M. LMU München, Department Pharmazie, Pharmakologie für Naturwissenschaften Center for Integrated Protein Science Munich (CIPSM), Butenandtstr. [5] [6] [7] [8] [9] [10] [11] [12] [13] 81377 München, Germany

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play a key role in controlling cardiac pacemaker activity and are essential for normal function of neuronal circuits. HCN channels are principally gated by voltage but are coactivated by the cyclic nucleotides cAMP and cGMP which directly bind to a C-terminal binding domain.

Recently, cyclic CMP (cCMP) was shown to be present in various cell lines and tissues at concentrations that are comparable to cellular cGMP levels. Moreover, there is recent evidence that cCMP can activate cAMP-and cGMP-dependent protein kinases in vivo.

Here, we examined whether cCMP exerts effects on HCN channels. To this end, we recorded HCN channel-mediated currents (I h) in HEK 293 cells that stably express HCN1, HCN2, HCN3 or HCN4, respectively. Currents were measured either with a standard patch-clamp setup or by employing the planar patch-clamp technology. In HCN2 and HCN4 channels, cCMP shifted the membrane potential for half maximal activation (V0.5) to more positive values. In addition, cCMP accelerated activation while it slowed down deactivation kinetics. The EC50 for cCMP was 30 µM which is about 5 times higher than the EC50 of cGMP. Cyclic CMP is a partial agonist of HCN channels since it activates only 65 % of the maximal current obtained with cAMP or cGMP. To identify in vivo effects of cCMP we recorded Ih of murine sinoatrial node (SAN) cells in the presence and absence of 1 mM cCMP. Like for heterologously expressed HCN channels, cCMP shifted V0.5 of Ih by about +5 mV. Importantly, the steepness of the diastolic depolarization of SAN pacemaker potentials which is mainly determined by the amplitude of Ih was profoundly increased by cCMP, compared to control conditions. As a consequence of the upregulation of Ih the frequency of SAN pacemaker potentials was increased by about 20 % in the presence of cCMP. Our results suggest that cCMP is a physiological regulator of HCN channel activity.

A 3D actinic keratosis like construct for assessment of innovative tumour therapeutics Zoschke C. 1 The incidence of Actinic Keratosis (AK) has increased dramatically in the last decades and it is considered the most frequent carcinoma in situ today. Especially immunosuppressed patients are at high risk to develop invasive squamous cell carcinoma (SCC) [1] which asks for most efficient and well-tolerated AK therapy. Yet, current measures do not fit with these demands. Nucleotide analogues, recently identified by molecular modelling [2, 3] , outperformed the current standard for the therapy of actinic keratosis, 5-fluoruracil, when tested in the tumour cell line SCC25, in normal human keratinocytes and fibroblasts [4] . As next step in pre-clinical drug assessment, we aimed to characterise the effect of the most selective nucleotide analogue OxBu in reconstructed human tumour skin. Based on the 3D construct of SCC developed by Höller and co-workers [5] for start we introduced several adaptations with respect to keratinocyte, fibroblast and SCC12 seeding to grow an AKlike construct with SCC12 cells forming nests in particular in the epidermis. In addition, first experiments with the OxBu on the AK like constructs showed promising results for an efficient treatment of actinic keratosis. Efficacy was derived from immunohistology (marker for proliferation: Ki-67, marker for SCC: cytokeratin-10, AxL, marker for invasion: MMP2, marker for apoptosis: caspase-7, nuclei were stained with DAPI) as well as the effects on the secretion of cytokeratin-18 and its caspase-induced cleavage product into the culture medium following drug exposure for up to 7 days. Efficacy of a 0.05% OxBu solution proved close to or even better when compared to both a 0.1% 5fluorouracil and 0.025% aphidicolin solution. The former being the gold standard of current AK therapy, the latter is a frequently used inhibitor of human polymerase alpha and delta, however, failed to be introduced into clinical use. -In fact, in monolayer cultures aphidicolin proved most toxic for normal human keratinocytes which was not true with OxBu [4] . -Therefore, these 3D tumour constructs offer a new approach to pre-clinical drug assessment and may be added to other 3D models of skin diseases currently gaining increased interest as test platforms.

IMA910, a multi-peptide cancer vaccine for advanced colorectal cancer, induces multiple CD8+ and CD4+ T-cell responses associated with improved survival Walter S. 1 , Kuttruff S. IMA910 is a novel peptide-based vaccine consisting of 10 HLA-A*02 and 3 HLA-DR binding synthetic peptides that were identified based on natural presentation on human colorectal cancer (CRC) samples. IMA910 was characterized in a phase I/II trial in advanced/metastatic CRC patients being at least clinically stable after 12 weeks of first-line oxaliplatin-based therapy. All patients received a single application of low-dose cyclophosphamide for immunomodulation. This was followed by repeated IMA910 vaccinations in combination with low-dose GM-CSF (cohort 1; n=66) or IMA910 / GM-CSF plus topically applied imiquimod (cohort 2; n=26). Before and post vaccination, patients were analyzed by HLA-multimer assay and intracellular cytokine (ICS) assay for CD8 + T-cell responses and by ICS assay for CD4 + Tcell responses. As immune status biomarkers, 6 phenotypically defined myeloid derived suppressor cell populations (MDSC1-6) were analyzed prior to immunotherapy. Tumor status of patients was monitored repeatedly by CT/MRI according to RECIST and corresponding tumor scans were reviewed centrally. Clinical assessment included disease control rate (DCR), time to progression (TTP), progression-free survival (PFS) and overall survival (OS). IMA910 overall was immunogenic in 73/81 (90%) evaluable patients, with 43% and 65% of patients mounting multiple CD8 + and CD4 + T-cell responses, respectively. Patients that received the immunomodulator imiquimod presented with significantly more multiple CD8 + cell responses as detected by ICS (p=0.016). Multiple CD8 + and multiple CD4 + responses were individually associated with significantly better clinical outcome. The association was most pronounced for patients with both multiple CD8 + and multiple CD4 + responses. These patients had significantly higher DCR at 6 months (p=0.002), improved TTP (p=0.006) and improved PFS (p=0.009) than other patients. Most importantly, a trend for prolonged OS was also observed in these patients (p=0.088, hazard ratio 0.53). In the study population, levels of 5 different MDSC phenotypes were significantly increased as compared to age/gender matched controls. High MDSC levels were associated with fewer immune responses and for MDSC4 and MDSC5 high frequencies were associated with shorter OS (p=0.007 and p=0.019, respectively). To summarize, both HLA-A*02 and HLA-DR restricted peptides in IMA910 were immunogenic. A significantly better clinical outcome of multi-TUMAP responders in comparison to patients with one/no TUMAP response strongly indicates clinical activity of IMA910. Acrylamide (AA), a genotoxic carcinogen (IARC class 2A) is formed in food by thermal treatment from different precursors. After oral ingestion, AA is metabolically epoxidized in the liver by CYP450 2E1 into glycidamide (GA). GA binds to DNA, forming covalent adducts, primarily at N7 of guanine (N7-GA-Gua). Both, AA and GA undergo conjugation to glutathione (GSH) to be excreted via urine as mercapturic acids (MA), namely Nacetyl-S-(2-carbamoylethyl)-cysteine (AAMA), and N-acetyl-S-(2-hydroxy-2carbamoylethyl)-cysteine (GAMA). In a dose response study, encompassing the dosage range from human dietary exposure levels up to 10 mg/kg bw, female Sprague Dawley (SD) rats on a diet devoid of detectable AA content were gavaged with single doses of AA. Formation of urinary MAs and of N7-GA-Gua DNA adducts in liver, kidney and lung was measured 16 h after application, a time point where cmax of N7-GA-Gua was reached. The untreated control group was found to excrete about 0.8 nmol (AAMA plus GAMA) in the urine (16 h), indicating a background of endogenous AA formation. Compared to untreated control, the lowest dosage of 0.1 µg AA/kg bw neither resulted in significantly enhanced MA excretion, nor in a detectable N7-GA-Gua adduct levels in any organ tested (limit of detection, LOD, 0.2 adducts/10 8 nucleotides). At the tenfold higher dose (1 µg/kg bw), adducts were found in kidney (about 1 adduct/10 8 nucleotides) and lung (< 1 adduct/10 8 nucleotides), but not in liver. At 10 and 100 µg/kg bw, adducts were found in all three organs, at levels not significantly different to those found at 1 µg AA/kg bw (about 1-2 adducts/10 8 nucleotides). The results of this in vivo study and of further recent research on AA toxicology will be discussed with respect to risk assessment. Exposure of rats to single doses of AA in the range of human dietary exposure (0.1-10 µg/kg bw ) leads to N7-GA-Gua adduct levels in the tissues monitored obviously not exceeding the range of steady state background DNA lesions associated with endogenous/exogenous exposure to various genotoxic electrophiles. Thus, the question of significant impact on human background DNA damage resulting from exposure to a given genotoxic carcinogen, and on potentially ensuing biological consequences may become a highly relevant issue in risk assessment.

Pharmaco-economic impact of price, volume and demographic development Böcking W., Kirch W. Institut für Klinische Pharmakologie, Medizinische Fakultät der TU Dresden, Fiedlerstr. 27, 01307 Dresden Health Insurance costs in Germany have grown by 3% p.a. over the last ten years and amount to approx. 280 bn EUR in 2009. While costs for stationary treatment as the largest cost category have been intensely analyzed over the past years, pharmaceutical expenses have been analyzed in less detail, mostly focusing on the Statutory Health Insurance side, even though pharmaceutical expenses have grown almost twice as much as costs for ambulant treatments. Therefore, the question was asked how pharmaceutical expenses in a large German private health insurance company are allocated with respect to age and indication groups, and how those have developed from 2007 to 2011. The data of a private health insurance company with more than 600.000 customers was split into price and volume effects per age group to understand if price or volume drives the cost development. Additionally, the two largest indication groups are analyzed in detail. As a result, both price and volume effects drive an overall cost increase. These effects are even stronger in older age groups. This cost increase is not sustainable for the German health insurance system over a longer period of time and will even further increase due to the ageing of the German population.

A novel animal replacement system for the detection of endocrine disruptive capabilities in sexual development Scheider J. 1, 2 , Winter P. Alternatives to animal testing for prediction of local toxicity and genotoxicity have been recently established. However, currently these methods are not suitable for measuring endocrine effects in developing organs such as e.g. embryonic gonads. Here we present a phenotypic anchoring of a comprehensive study on sex-specific gene expression analysis accompanied by histological analysis of endocrine disruption in chicken embryo gonads, having the potential for an animal replacing system for endocrine disruptive toxicologic and ecotoxicologic examinations of chemicals. Chicken embryos were inoculated with different amounts of tributyltin (TBT) and bisphenol-A (BPA). Embryos were incubated and their gonads analyzed histologically 2 d prior to hatching. From identically treated embryos right and left testes and ovaries were separated and genome-wide transcription profiles generated using SuperTag Digital Gene Expression (ST-DGE, SuperSAGE) profiling. Male and female gonadal tissues both revealed histological aberrations in response to TBT and BPA. Female gonads became masculinized in response to TBT and, viceversa, BPA-treated male gonads underwent feminization whereas in female gonads clearly visible structural aberrations occurred. In both chemicals mortality increased especially in the most affected sex (TBT: females, BPA: males). The expression profiles of more than 60 million mRNAs revealed massive effects of both chemicals, TBT and BPA, on important cellular signaling pathways. Gene expression differences were most pronounced in the phenotypically most affected sex. Our results demonstrate that endocrine disruptive chemicals exert their effects on several levels including but not restricted to known hormone-based pathways. Together with an ongoing study of gene expression differences in very young life stages and different chemicals these data will form the base for a blow-by-blow analysis of sexspecific gene expression of embryonic development. The project builds on already existing and further to generate data with the aim of the development of an in vitro method for testing chemicals at chicken eggs for 1) replacement of tests on juveniles and (sub-) adult rodents, 2) stages with impossibility of sensation of pain in the individuals, 3) highly sensitive prospects of modes of action of chemicals, which 4) might show consequences in the next generations.

3 channels are critical for oscillatory burst discharges in the reticular thalamus and absence epilepsy

Differential distribution of three members of a gene family encoding low voltage-activated (T-type) calcium channels

Hippocampal seizure resistance and reduced neuronal excitotoxicity in mice lacking the Cav 2.3 E/R-type voltage-gated calcium channel

Transcriptional upregulation of Cav3.2 mediates epileptogenesis in the pilocarpine model of epilepsy

Structure and functional expression of a member of the low voltage-activated calcium channel family

A molecular determinant of nickel inhibition in Cav3.2 T-type calcium channels

Histidine residues in the IS3-IS4 loop are critical for nickel-sensitive inhibition of the Cav2.3 calcium channel

Substrate recognition and translocation by polyspecific organic cation transporters

Proton Pump Inhibitors inhibit Metformin Uptake by Organic Cation Transporters (OCTs)

Structural determinants of inhibitor interaction with the human organic cation transporter OCT2 (SLC22A2)

Functional Characterization of the Human Organic Cation Transporter 2 Variant p.270Ala>Ser

Extra-adrenal glucocorticoid synthesis in the intestinal epithelium: more than a drop in the ocean?

Local glucocorticoid production in the mouse lung is induced by immune cell stimulation

Biomimetic materials in tissue engineering

Biomaterials offer cancer research the third dimension

Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering

Injectable self-assembling peptide nanofibers create intramyocardial microenvironments for endothelial cells

Directed growth of fibroblasts into three dimensional micropatterned geometries via selfassembling scaffolds

Novel PCL-based honeycomb scaffolds as drug delivery systems for rhBMP-2

Tissue engineering

Spatio-temporal VEGF and PDGF delivery patterns blood vessel formation and maturation

Presentation of RGDS epitopes on self-assembled nanofibers of branched peptide amphiphiles

Controlling Mammalian Cell Interactions on Patterned Polyelectrolyte Multilayer Surfaces Langmuir

avIntegrins as receptors for tumor targeting by circulating ligands

Heparin binding nanostructures to promote growth of blood vessels

Tirrell Endothelial cell adhesion to the fibronectin CS5 domain in artificial extracellular matrix proteins

Design and bioproduction of a recombinant multi(bio)functional elastin-like protein polymer containing cell adhesion sequences for tissue engineering purposes

Stimuli-Responsive Thin Coatings Using Elastin-Like Polymers for

Epac as a novel effector of airway smooth muscle relaxation

) cAMP inhibits airway smooth muscle phenotype modulation

Functional roles of Epac and PKA in human airway smooth muscle phenotype plasticity

Assessment of the Sensitizing and Irritative Potential of Preservatives by Loose-fit Coculture-based Sensitization Assay (LCSA) Sonnenburg A

NSC-631570 (Ukrain) in the palliative treatment of pancreatic cancer. Results of a phase II trial

Association of funding and conclusions in randomized drug trials: a reflection of treatment effect or adverse events

A general method for the covalent labeling of fusion proteins with small molecules in vivo

Robust single-particle tracking in live-cell time-lapse sequences

Correlation of Structural Class with No-Observed-Effect Levels: A Proposal for Establishing a Threshold of Concern

TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing

Index A

009, 019, 035

011, 014

003, 044

Objective: Hypertension and arterial stiffness is influenced by environmental and genetic factors. High plasma sodium concentration leads to mechanical stiffening of endothelial cells resulting in endothelial dysfunction and elevated blood pressure. Here we investigated whether endothelial cell stiffness of ex vivo preparations of human arteries is linked to plasma sodium concentrations and functional genetic variants of the mineralocorticoid receptor (NR3C2), rs2070951 modulating blood pressure, renin, and aldosterone levels, and rs5534, which alters a miRNA binding site. Design and methods: Twenty patients were enrolled after a vein stripping procedure and collateral arterial blood vessels were prepared for atomic force microscopy (AFM). Plasma sodium concentration was routinely determined and DNA for genotyping was extracted from EDTA blood samples. Sodium levels >140 mmol/L were defined as 'high'. After application of 5 µM amiloride, a specific blocker of the endothelial sodium channel (ENaC) changes in endothelial cell stiffness, were defined as 'weak' (≤10%), or 'strong' (>10%). Statistical analyzes were performed by ANOVA. Results: In ex vivo artery preparations of patients with high sodium levels (n=12), mechanical stiffness of endothelial cells was tend to increase (∆ amiloride) (p=0.06). Both NR3C2 variants were associated with a change >10% in endothelial stiffness after amiloride treatment. The rs2070951 C allele was significantly associated with a strong amiloride response (p=0.024), while the rs5534 A allele only showed a trend towards stronger amiloride effects (p=0.06). Conclusion: Our findings indicate that high plasma sodium concentration results in an increased endothelial amiloride response and thus influencing mechanical stiffness, modulated by functional NR3C2 variants. Our novel approach linking patients' sodium levels and genetic status to endothelial stiffness by AFM will be further evaluated in larger clinical settings.

Protein Expression Changes in BaP-Exposed Human Bladder Cancer Cells from Spliceosome Activation Towards Redistribution of the Cytoskeleton After Long-Term Exposure to Subacute Concentration Schmitz-Spanke S., Pink M., Jeske E., Stempelmann K., Rehn S., Verma N., Rettenmeier A. W. Universitätsklinikum Essen Institut für Hygiene und Arbeitsmedizin, Hufelandstr. 55, 45122 Essen, Germany Deregulation of the β-catenin signaling pathway plays an important role in the development of hepatocellular tumors. Activating mutations in Ctnnb1 (encoding β-Catenin) are frequently observed in murine and human liver tumors (e.g. human hepatoblastomas). Activation of β-Catenin signaling induces an overexpression of several cytochrome P450 (CYP) enzymes, including CYP2E1. Cytotoxicity of acetaminophen (AAP) is based on its CYP2E1-catalyzed metabolism to the electrophilic compound N-acetyl-p-benzo-quinone imine, which forms covalent adducts with cellular macromolecules if depletion of glutathione occurs. Treatment with AAP should therefore lead to a selective damage of CYP2E1-overexpressing Ctnnb1mutated hepatoma cells. Mice were injected with a single dose of the liver carcinogen N-Nitrosodiethylamine (DEN) and subsequently treated with the tumor promoter phenobarbital to select for Ctnnb1-mutated tumors. Administration of a single dose of AAP (300 mg/kg of body weight) followed the tumor promotion protocol. Two days after treatment immunohistological analysis of the livers showed about 90% necrotic tissue in the larger tumors which were positive for glutamine synthetase (GS), a marker for Ctnnb1-mutated tumor cells. By contrast, GS-negative tumors remained unaffected. At later time points we observed regeneration processes with infiltration of the necrotic tissue by inflammatory cells followed by fibrotic cells. Proliferation of normal hepatocytes surrounding the damaged areas could also be observed. However, repopulation of parts of the former tumor areas by remaining GS-positive tumor cells was also detected. These results suggest that treatment with AAP might serve as a future therapeutic possibility to selectively poison CYP2E1-overexpressing hepatoma.

Release of 5,6-Epoxyeicosatrienoic acid (5,6-EET) upon neuronal activity induces TRPA1-dependent mechanical pain hypersensitivity Sisignano M. 1 , Epoxyeicosatrienoic acids (EETs) are CYP-epoxygenase (CYP450) derived metabolites of arachidonic acid (AA) which act as endogenous signaling molecules in multiple biological systems. We investigated the specific contribution of 5,6-EET to Transient-Receptor potential-(TRP)-channel activation in nociceptor neurons, and its consequence for nociceptive processing. We found that during capsaicin-induced nociception 5,6-EET-levels increased in the DRG and it is released from activated sensory neurons in vitro. 5,6-EET potently induced a calcium flux [10 nM] in cultured DRG-neurons which was completely abolished when TRPA1 was deleted or inhibited. In spinal cord slices 5,6-EET dose-dependently enhanced the frequency, but not the amplitude of spontaneous excitatory postsynaptic currents (sEPSC) in lamina II neurons that also respond to mustard oil (AITC), indicating a presynaptic mechanism. Furthermore, 5,6-EET-induced enhancement of sEPSC frequency was abolished in TRPA1 null mice, suggesting that 5,6-EET pre-synaptically facilitates spinal cord synaptic transmission via TRPA1. Finally, intrathecal injection of 5,6-EET caused mechanical hyperagesia in wild type but not TRPA1 null mice. We conclude that 5,6-EET is synthesized upon acute activation of nociceptors and leads to mechanical hypersensitivity via TRPA1 at central afferent terminals in the spinal cord.

Sisnaiske J. 1 , Hardelauf H. 

Introduction: Neurite outgrowth and plasticity of neuronal networks are essential processes e.g. during brain development and learning. Thus, morphological readouts of neuronal connectivity are thought to be important endpoints to assess neurotoxic effects of environmental chemicals as well as when discovering new drugs. To analyze neurite outgrowth and connectivity level rapidly and easily in vitro we developed the Network Formation Assay (NFA) (PCT/EP2010/002811). This platform requires a spatially standardized hexagonal array for culturing neuronal networks with no need to fix or stain the cells to visualize neuritic processes. Methods: To demonstrate the feasibility of the NFA we performed experiments in which we disrupted mature neurite networks or inhibited generating networks of human SH-SY5Y cells with different concentrations of acrylamide (ACR). We also observed the counteracting effects of brain-derived neurotrophic factor (BDNF) and calpeptin in these systems. To create the hexagonal array we used a Poly(dimethylsulfoxide) bilayer stencil comprising through holes for adhesion spots and interconnecting tracks. Plasma stencilling a PEG-coated glass substrate produces adhesive nodes for the neurons and micron-scale-tracks for guiding neurite outgrowth and connectivity. Results: In both systems, the developing and mature network, we found not only a concentration dependant effect of ACR and BDNF but also a time dependant effect with a limited capability of the developing system to regenerate, even in the presence of ACR. The co-treatment of the cells showed that inhibition of calpains by calpeptin might reduce the effect of intracellular elevated Ca2+, a known neurotoxic mechanism of ACR. Moreover, the neurothrophin BDNF acts via TrkB receptors on pathways stimulating neurite outgrowth and thereby counteracting the adverse effect of ACR. Conclusion: With the NFA we provide a rapid and simple way to analyze neurite outgrowth and connection formation in real time. By spatially standardizing the array we provide assay coordinates to streamline the analysis process and bring it towards high throughput testing. Furthermore preliminary data showed that modification of the surface with biomolecules allows cell adhesion of other neuronal celltypes (e.g. primary mouse neurons) and extracellular matrix proteins (e.g. laminin) stimulate neurite outgrowth via integrins.

Transcriptional regulation of Nox4 by histone deacetylases Siuda D. 1, 2 , Zechner U. 3 , Prawitt D. 4 Nox4 is a member of the NADPH oxidase family, which represents a major source of reactive oxygen species (ROS) in the vascular wall. Nox4-mediated ROS production mainly depends on the expression levels of the enzyme. The present study is aimed to investigate the regulation mechanisms of Nox4 transcription by histone deacetylase (HDAC). In cultured human EA.hy 926 endothelial cells, treatment with the pan-HDAC inhibitors (scriptaid, trichostatin A, TSA, and suberoylanilide hydroxamic acid, SAHA) leads to a drastic decrease in Nox4 mRNA expression. A similar down-regulation of Nox4 mRNA expression can be achieved with siRNA-mediated knockdown of HDAC3. HDAC inhibition in endothelial cells is associated with enhanced histone acetylation in the human Nox4 promoter region, with no significant changes in DNA methylation. Consistently, scriptaid-treated cells show increased chromatin accessibility in Nox4 promoter. In addition, we provide evidence that c-jun plays an important role in controlling Nox4 transcription. Knockdown of c-jun with siRNA leads to a marked downregulation of Nox4 mRNA expression. In response to scriptaid treatment, the binding to c-jun to the Nox4 promoter region is reduced despite the open chromatin structure. In parallel, the binding of polymerase IIa to the Nox4 promoter is significantly inhibited as well, which may explain the reduction in Nox4 transcription. In conclusion, HDAC inhibition decreases Nox4 transcription in human endothelial cells by preventing the binding of transcription factor(s) and polymerase(s) to the Nox4 promoter. This is very likely because of a hyperacetylation-mediated steric inhibition.

Cyclopentenone prostaglandins induce oxidative DNA-damage in hamster lung fibroblast V79 cells Solecki G. M. 1