Báo cáo khoa học: Signal Transduction pot

Signal Transduction Abstracts Signal Transduction C1-1 Histone modifications regulate morphogenesis in C albicans C1-3 Tissue-specific regulation of RNT-1 function in C elegans D Hnisz and K Kuchler MFPL, Vienna, AUSTRIA J Shim, K Lee and J Lee Seoul National University, Seoul, REPUBLIC OF KOREA Candida albicans is an opportunistic human pathogen with remarkable phenotypic plasticity Its diploid cells can undergo an epigenetic phase-transition termed the white-opaque switching Whereas white cells have a round shape and are unable to mate, opaque cells have an oval shape and are mating-competent While the contribution of transcription factors to phase changes is widely investigated, little is known about epigenetic mechanisms underlying switching During the white-opaque transition, phasecommitment is dependent on a single master locus WOR1, but it includes changes in the expression levels of about 400 genes The major goal of our studies was to identify genes regulating white-opaque switching and analyze their regulatory mechanisms With bioinformatic tools we identified all histone-modifying genes of C albicans We created homozygous deletion mutants and assayed the influence of the deletions on the phase transitions, thus establishing functional categories for switching modulators Quantitative mating assays show that all the mutants maintain wild-type mating efficiencies arguing that the modulators have a effect specific rather than global effect We demonstrate with real-time PCR analyses that the expression of the modulators is independent of WOR1 Our results suggest a new model of the regulation of whiteopaque switching that includes at least another so far unidentified second master regulator that has a mutual transcriptional dependence on WOR1 to determine the phase outcome and the transcriptional feed-backs are mediated by histone-modifying cofactors Runx proteins are evolutionarily well-conserved transcription factors that are involved in essential aspects of the development of metazoan animals ranging from nematodes to humans We found that the expression of the nematode RUNX homolog, RNT-1, is tightly regulated in that it is expressed only in the intestine and hypodermis at specific developmental stages and that RNT-1 is almost absent in any tissue at the adult stage Ectopic expression of RNT-1 resulted in the over-proliferation of the hypodermal cells, indicating that tightly regulated attenuation of the RUNX protein is required for its proper function in vivo Recently, it was revealed that the C elegans genome contains one homolog of the Runx protein partner CBFbeta (BRO-1) bro-1 is expressed only in the hypodermis at all developmental stages Knockdown of bro-1 resulted in the up-regulation of RNT-1 in the hypodermis, but not in the intestine, suggesting that bro-1 acts as a co-repressor of RNT-1 We found that the nematode RNT-1 regulates its own transcription in the hypodermis, acting on its own cis-acting elements In situ hybridization experiments showed that the RNT-1 transcript is still present in the intestine at the adult stage, but not in the hypodermis, suggesting that RNT-1 might be regulated at the protein level Consistent with this, RNAi knockdown of ubq-1, a polyubiquitin gene, and uba-1, the E1 gene, resulted in higher stability of RNT-1 in the intestine and the treatment of MG132, a proteasome inhibitor, stabilized RNT-1 in the intestine Taken together, we conclude that RNT-1 expression is attenuated at the adult stage by two different mechanisms, transcriptional autoregulation in the hypodermis and proteasome-mediated degradation in the intestine C1-2 Glutathione regulates the transition from growth to development in Dictyostelium discoideum J Kim, C Choi, S Jeong, J Seo and S Kang Seoul National University, Seoul, REPUBLIC OF KOREA Glutathione is a ubiquitous tripeptide, found in most plants, microorganisms, and all mammalian tissues and most prevalent reducing thiol-containing compound in eukaryotic cells Glutathione serves as cellular thiol redox buffer to maintain a thiol/disulfide redox potential, and also known to participate in many cellular processes Disruption of GCS encoding c-glutamylcysteine resulted in glutathione auxotrophy and developmental defect in Dictyostelium discoideum And GCS-null cells showed different developmental progress, depending on the level of GSH To understand defective development, we investigated the development in suspension GCS-null cells depleted GSH did not aggregate and were still single cells in suspension with addition of cAMP Interestingly, the addition of 1mM GSH induced them to aggregate However, the defect in aggregation of them was not rescued by dithiothreitol, N-acetylcysteine and ascorbic acid GCS-null cells fail to decrease the expression of the growth-stage gene cprD, and not induce the expression of cAR1 (cAMP receptor), acaA (adenylyl cyclase A) and lagC (aggregation marker) that required for the earliest stages of development Dictyostelium cells that enter the development stage use G protein-coupled receptor signaling to direct chemotactic migration to a source of cAMP We overexpressed cAR1, the most important receptor for cAMP signal cascade, in GCS-null cells cAR1 overexpressing GCS-null cells still fail to aggregate in suspension These results suggest GCS-null cells are defective in production of the extracellular cAMP that serves as the extracellular chemoattractant and in cAMP signal cascade in D discoideum development ª 2007 The Authors Journal compilation ª 2007 FEBS C1-4 Differential cleavage of Dpp precursor modulates morphogen gradient in the Drosophila J Kunnapuu, I Bjorkgren and O Shimmi ă ă University of Helsinki, Helsinki, FINLAND We have previously shown that two different molecular forms of Decapentaplegic (Dpp), Drosophila BMP2/4 type ligand, are produced in the embryo (Shimmi et al, Cell 2005) As Dpp precursor contains two optimal furin recognition sites, we suspect that these two sites can be used for ligand maturation In order to understand how Dpp processing is regulated, and why two different forms of Dpp ligands are used for signaling, we mutated the optimal furin recognition sites to study their function The mutation of the first furin site drastically dropped the production of mature ligands in Drosophila S2 cells In contrast, the mutation of the second furin site did not affect the production rates or the signaling intensities of ligands, however, mature ligands were produced as a single form As previous reports indicated that vertebrate BMP4 was sequentially cleaved to help promote the long-range signaling, we focus on two different stages, the early embryo and the wing imaginal disc, in which Dpp works as a morphogen, to investigate the in vivo function of different forms of Dpp We will discuss how evolutionally conserved furin recognition sites of BMP2/4 type ligands contribute to regulate the long- or short-range signaling of these ligands during developmental stages 133 Abstracts C1-5 Phenotypic observation of Xenopus laevis MGP loss of function ˜ B S N Simoes, B Simoes1, A C Silva2, M Vitorino2, ˜ N Conceicao1, J A Belo2 and M L Cancela1 ¸ ˜ CCMAR, 2IBB, CBME – University of Algarve, Faro, PORTUGAL Matrix Gla protein (MGP) belongs to the family of vitamin K-dependent Gla proteins and is known to be involved in regulation of extracellular matrix calcification and maintenance of cartilage and soft tissue integrity during growth and development We have previously determined the spatial pattern of XlMGP expression during embryogenesis and showed that XlMGP transcripts are expressed during gastrulation in the dorsal mesoderm along Brachet’s cleft, as well as in the ventral mesoderm To understand the role of MGP in vertebrate development, zygotic knockdown of MGP messages was performed using morpholino oligos against XlMGP Specific and control morpholino oligos were injected into the blastomeres of Xenopus embryos at 4–8 cell stage and resulting phenotypes were analysed by the expression pattern of developmental marker genes Our data suggests that MGP knockdown induces changes in localization of different gene markers, thus affecting subsequent events in Xenopus early development Acknowledgements: NC is supported by a post-doctoral grant (SFRH/BPD/9451/2002), AS and MV by PhD grants (SFRH/BD/ 10035/2002 and SFRH/BD/24765/2005, respectively) and BS was a recipient of a technical fellowship This work is supported by project POCI /BIA-BCM/58677/2004 Signal Transduction C1-7 Cytokinine secondary hormone activates plasmatic membrane H+-ATPase which is important regulatory machine of the plant cell A N Sabitov1, K Musabekov1, M K Gilmanov2 and Z S Kudiyarova3 Chemistry Department, Chair of Colloid Chemistry, Catalysis and Oil Chemistry, Al-Faraby’s Kazakh National University, Almaty, KAZAKHSTAN, 2M.A Aitkhozhins Institute of Molecular Biology and Biochemistry, Almaty, KAZAKHSTAN, 3Biology Department, Al-Faraby’s Kazakh National University, Almaty, KAZAKHSTAN It is well known, that regulation of ionic transport the main feature of cell regulation However the mechanisms of regulation of ionic transport in plant cells are not enough investigated For this reason it is very important to investigate the effect of new powerful regulator – cytokinine secondary hormone (CSH) on plant cell ions transport In laboratory of structure and regulation of enzymes of M.A Aitkhozhins institute of molecular biology and biochemistry CSH was discovered and characterized CSH appears its physiological action at the concentration hundred times less than other phytohormones We have studied the effect of CSH on main ion transport enzyme - plasmatic membrane H+-ATPase from wheat grain It was shown that CSH activates plasmatic membrane H+-ATPase more than two times The one of the interesting features of studied H+-ATPase is the next The activity of this enzyme strong depends from Ca2+ and Mg2+ and this enzyme haven’t absolutely any activity with sodium and potassium ions We are suggested that regulatory effects of CSH first of all appear by activation of calcium ions transport through plasmatic membrane into the plant cells It is well known that calcium ions are main intracellular messenger which regulates many important cell functions CSH by this property is very close to fusicoccine – natural phytotoxin from Fusicoccum amygdali C1-8 Mathematical model of the Arabidopsis thaliana morphogenesis in a cellular automaton terms I R Akberdin, E A Ozonov, V V Mironova, D N Gorpinchenko, N A Omelyanchuk, V A Likhoshvai and N A Kolchanov Institute of Cytology and Genetics SB RAS, Novosibirsk, RUSSIAN FEDERATION C1-6 Lachesis restricts gametic cell fate in the female Development of organisms is a very complex process in which a gametophyte of Arabidopsis lot of gene networks of different cell types are integrated Develop- C Kagi1, C Moll1, L von Lyncker1, N Baumann1, ă U Grossniklaus2 and R Gross-Hardt1 ZMBP University of Tuăbingen, Tuăbingen, GERMANY, 2Institute of Plant Biology, Zuărich, SWITZERLAND In flowering plants, the egg and sperm cells form within haploid gametophytes The female gametophyte of Arabidopsis consists of two gametic cells, egg cell and central cell, that are flanked by five accessory cells We asked why some cells differentiate gametic cell fate whereas others become accessory cells In a screen for regulators of egg cell fate we isolated the lachesis (lis) mutant which forms supernumerary egg cells In lis mutants accessory cells differentiate gametic cell fate, indicating that Lis is involved in a mechanism that prevents accessory cells from adopting gametic cell fate The expression pattern of Lis suggests that this mechanism is generated in gametic cells, implying that lateral inhibition patterns the female gametophyte Lis is homologous to the yeast splicing factor PRP4 The puzzling link between the regulation of gametic cell fate and the splicing apparatus is corroborated by the finding that defects in a second splicing factor, CLOTHO, also result in the formation of supernumerary egg cells Possible implications will be discussed 134 ment of a cellular automaton that models the morphodynamics of different cell types is the first step in understanding and analysis of the regulatory mechanisms underlying the functioning of developmental gene networks A model of a cellular automaton has been developed, which simulates the embryonic development of shoot meristem in Arabidopsis thaliana The model adequately describes the basic stages in development of this organ in wild and mutant types Visualization of the cellular automaton was created that allows simulating of the process of development The visualization of cellular automaton model allows estimating distribution of three biologically meaningful signals, which unambiguously simulate the morphodynamics of the cell tissues The cellular automaton model introduced here to investigate the development of primary shoot meristem of the Arabidopsis thaliana in embryogenesis under different initial parameters of the model It allows recognizing of significant parameters, which greatly influence on behaviour of dynamic system and determining the stable state of this biological system by variation parameters In this visualization of cellular automaton you can remove some cells at discretion and continue calculation that allows analyzing and reproducing such experiment as laser excision ª 2007 The Authors Journal compilation ª 2007 FEBS Signal Transduction C1-9 The highly conserved mitochondrial protein GCP1 is essential for embryo development in Arabidopsis thaliana P F Huesgen1, K Haußuhl2, P Dessi3, J Adamski4, E Glaser3 ¨ and I Adamska1 Fachbereich Biologie, Universita¨t Konstanz, Konstanz, GERMANY, Qiagen AG, Hilden, GERMANY, 3Department of Biochemistry and Biophysics, Stockholm University, Stockholm, SWEDEN, 4GSFForschungszentrum fu Umwelt und Gesundheit, Neuherberg, ăr GERMANY Glycoproteases (GCP) are a family of putative Zn-metalloendopeptidases that are found highly conserved in taxonomically diverse species Our phylogenetic analysis revealed that all prokaryotes contain only one GCP, either of the gcp1-type (Bacteria) or the gcp2-type (Archaea) Eukaryotic organisms contain two gcp genes, one of each type GCP1 is a predicted to target to the mitochondria, while has no recognizable signal sequence and is probably located in the nucleus and/or cytoplasm We cloned the gcp1 gene from Arabidopsis thaliana and raised a polyclonal antibody against the purified recombinant protein With this antiserum, we demonstrated that GCP1 is located in the inner membrane of plant mitochondria The antiserum also detected specific bands in murine and human protein extracts In Arabidopsis, we detected a high GCP1 level in developing leaves, roots, flowers and pods of mature plants, as compared with fully developed organs or mature seeds, where only traces of GCP1 were present Using immunocytochemistry we investigated the tissue specific expression of GCP1 and demonstrated that this protein is strongly expressed in axial meristems We demonstrate that homozygous GCP1 knock-out mutants are not viable Embryos of these mutants were arrested at the globular stage and failed to undergo the transition to heart stage Based on our data we propose that the mitochondrial GCP1 is essential for cell division and/or differentiation in plants, and suggest that GCP1 plays a similar role in other species C1-10 Phospholipase D1 regulates neurogenin1 expression via mTOR during bFGF-induced neurite outgrowth in H19–7cells J Koo, M Yoon and J Han Department of Biochemistry and Molecular Biology, Hanyang University, Seoul, REPUBLIC OF KOREA Phospholipase D (PLD) catalyzes the hydrolysis of phospholipids, mainly phosphatidylcoline (PC), resulting in formation of phosphatidic acid (PA) PA has been associated with many aspects of mammalian physiology, which include cell proliferation, survival, transformation, progression and differentiation Recently, PA has been identified as an activator of mTOR signaling pathway PA binds to mTOR to promote various signals We previously showed that PLD1 activity is upregulated during neurogenesis induced by basic fibroblast growth factor (bFGF) treatment in H19–7 cells Subsequently, we investigated the role of PLD1 in neurogenesis, specifically expression of neurogenin1 (Ngn1), bHLH family which plays critical role in regulation of neural stem cell differentiation To figure out the effect of PLD1 on Ngn1 expression through mTOR signal, we treated permeable PA to H19–7 cell under differentiation condition Permeable PA treatment upregulated mTOR and Ngn1 expressions Furthermore, inhibition of PLD1 activity by PLD1 siRNA showed that expressions of mTOR and Ngn1 were completely inhibited However, mTOR and Ngn1 expression levels were upregulated when PLD1 activity was increased by bFGF in H19–7 cells To further confirm the role of PLD1 on Ngn1 expression through mTOR, we treated rapamycin to inhibit mTOR signal Treatment of rapamycin showed completely inhibited Ngn1 expression and neurogenic morphological change These results suggest that PLD1 regulates Ngn1 expression through mTOR signaling pathway during neurogenesis in H19–7 cell ª 2007 The Authors Journal compilation ª 2007 FEBS Abstracts C1-11 Tyrosine phosphatase epsilon is a negative regulator of the signal transducer Shc J Kraut-Cohen and A Elson The Weizmann Institute of Science, Rehovot, ISRAEL Reversible phosphorylation of tyrosine residues in proteins is a crucial mechanism for regulating cellular and physiological functions Protein tyrosine phosphatases (PTPs) are known to be major regulators of this process in vivo This study examines the relationship between PTP epsilon (PTPe) and the adaptor and signal transducer molecule Shc Basal interactions were observed between the non-receptor form of PTPe (cyt-PTPe) and p52 or p66 Shc in Jurkat T-cells and in Ras transformed NIH3T3 fibroblasts This interaction is dependent on the N-terminal part of cyt-PTPe and on the PTB domain of Shc Complex formation is not dependent on tyrosine phosphorylation of Shc in signaling processes as nonphosphorylatable Y-to-F mutants of Shc can still bind cytPTPe Cyt-PTPe can down-regulate phosphorylation of endogenous Shc at tyrosines 239/240 and 317 In correlation, expression of cytPTPe leads to reduced association of Shc with Grb2 and to a subsequent decrease in ERK activation Interestingly, these effects occur readily when signaling is stimulated by Src, but not when initiated by Neu or by the EGF receptor This difference is most likely the result of Neu’s ability to compete against PTPe for binding the Shc PTB domain: the interaction of Neu with Shc prevents Shc’s dephosphorylation and downregulation of the ERK pathway We conclude that PTPe is an important negative regulator of Shc-mediated signaling that acts in a kinase-specific manner C1-12 TRKA induction leading to cell death via cell cycle alteration and cH2AX accumulation in cytosol D Kim and E Jung Gyeongsang National University School of Medicine, Jinju, REPUBLIC OF KOREA In response to NGF, TrkA tyrosine kinase is activated by autophosphorylation and plays an important role in neuronal cell survival, differentiation, and apoptosis We show here that TrkA overexpression by the Tet-On system in U2OS cells mimicked NGF-mediated activation pathway, leading to tyrosine phosphorylation of cellular proteins, even in the absence of ligand engagement Overexpressed TrkA appeared to be mainly accumulated in cytosol and plasma membrane TrkA overexpression in U2OS cells induced the morphological change to neuron-like cells and interrupted cell cycle progression, especially on a G1-S transition, which led to cell death at a time-dependent manner The cell death by TrkA was inhibited by its specific tyrosine kinase inhibitor, GW441756 p53 upregulation upon DNA damage was decreased by TrkA overexpression, whereas p21 was upregulated by TrkA in a p53-independent manner Interestingly, cH2AX was largely increased in TrkA-overexpressed cells Also, cH2AX and TrkA were colocalized in cytosol in the absence of DNA damage, indicating that two proteins might have a functional relation Moreover, TrkA overexpression altered nuclear localization of cH2AX by DNA damage to partly cytosol Here, we first suggest that cH2AX could be implicated in cell death signaling cascade by TrkA in the absence or presence of DNA damage 135 Abstracts Signal Transduction C1-13 Identification of SIVA and PDCD2 as novel XIAP interaction partners C1-15 VEGF induces a specific gene repertoire in endothelial cells U Resch and R deMartin Department of Vascular Biology and Thrombosis Research, Medical University, Vienna, AUSTRIA J Schultes, B Schweighofer, J Pomyje, M Bilban, H Mayer and E Hofer Medical University Vienna, Vienna, AUSTRIA The X-linked inhibitor of apoptosis (XIAP) is a specific inhibitor of proapoptotic caspases 3, and In addition, XIAP plays a role in intracellular signaling cascades involved in cellular response to stress or inflammation Recently we have shown that XIAP activates the NF-jB pathway, which is dependent on the ubiquitin-E3-ligase activity within the RING-domain of XIAP However, the underlying molecular mechanism for this activation is unclear Therefore, a yeast-two-hybrid screen with full-length as well as truncated versions of XIAP was performed We identified two novel XIAP-interacting proteins, CD-27 binding protein (SIVA) and programmed cell death-2 (PDCD2) The three BIR domains of XIAP were dispensable for this interaction Coimmunoprecipitation experiments in HEK293 cells verified these interactions with both wild type and its E3-ligase mutant of XIAP Furthermore, SIVA was found to be ubiquitinated when overexpressed along with XIAP but not with its E3-ligase mutant To test if these proteins influence the XIAP-induced NF-jB activity, reporter gene assays were performed We found that SIVA had a inhibitory effect on the XIAP-induced NF-jB activity, whereas PDCD2 had no effect This suggests that XIAP mediated ubiquitination of SIVA inhibits its inhibitory effect on NF-jB activation, whereas the functional consequences of the interaction with PDCD2 remain to be elucidated VEGF-A via triggering of VEGFR2 is the major initiator of angiogenesis Specific signals/genes induced by VEGF-A, but not by other growth factors and cytokines, have so far not been fully established We have analyzed here the induction of signals and genes by VEGF-A/VEGFR2 in comparison to EGF and IL-1 HUVEC were induced by VEGF-A, EGF or IL-1, followed by Affymetrix microarray analyses and RT-PCR Specific inhibitors of NFAT and EGR-1 were used to judge the contribution of these factors The data show that VEGF-A/VEGFR2 preferentially triggers signals via PLC-gamma, PKC and Ca++, whereas EGF is unable to trigger this pathway and IL-1 is a preferential inducer of NFkappaB Downstream of PKC and Ca++ the factors EGR-1 and NFAT are important regulators Gene activation via PLCgamma provides VEGF with the potency to induce a wide spectrum of genes, which includes a large proportion of genes also regulated by IL-1 This is caused by the presence of overlapping binding sites for NFAT and NFkappaB in these genes In addition, a smaller number of genes was found to be strongly induced by VEGF, but not by EGF or IL1 These include the transcription factors Nurr1, Egr3, Hlx1 and Mef2C We propose that both properties, the ability to induce a large number of genes in common with inflammatory mediators, and a small group of exclusively regulated genes, is important for the role of VEGF as primary physiological inducer of angiogenesis Acknowledgement: Supported by FWF-NFN94–3 and EC LSHC-CT-2005–518178 C1-14 NF1 and Smad proteins, are they partners? G Kollarovic1, P Barath1, K Luciakova1 and B D Nelson2 Cancer Research Institute, Bratislava, SLOVAKIA, 2Stockholm University, Stockholm, SWEDEN The linkage between mitochondrial oxidative phosporylation and cytosolic ATP utilization needs co-operation between molecular machine called ATPase and adenine nucleotide translocator (ANT) ANTs are antiporters of inner mitochondrial membrane that exchange cytosolic ADP for mitochondrial ATP synthesized by ATPase There are four ANT isoforms in human cells Expression of the ANT2 isoform is unique because it is growth dependent The promoter region of human ANT2 gene is composed of five main regulatory elements which bind two transcription factors Three bind Sp1 protein, two of which activate ANT2 expression and one of them represses ANT2 transcription The other two regions bind transcription factor NF1 and repress ANT2 transcription One of them (G0-R element) has been shown to be responsible for growth dependent regulation of ANT2 transcription The aim of our work is to describe the mechanism of ANT2 repression in quiescent cells mediated by NF1 and other proteins First we found that there are additional binding sites for Smad proteins near G0-R element Therefore, we performed co-immunoprecipitation experiments and showed presence of Smad and NF1 in one protein complex Interaction of Smad and NF1 was also confirmed by in vitro GST pull downs The results from ChIP assay suggested changes in protein complex composition on the ANT2 promoter during G0 phase of cell cycle Finally, the proteins of Smad family are involved in TGFb signalling pathway And indeed, TGFb decrease the level of ANT2 mRNA as demonstrated by RT-PCR We believe that our data strongly suggest novel role for NF1 transcription factor that cooperate with Smad proteins in growth dependent transcriptional repression 136 C1-16 Functional analysis of a homeobox gene upregulated by VEGF in endothelial cells J Schultes, B Schweighofer, J Pomyje, C Sturtzel and E Hofer Medical University Vienna, Vienna, AUSTRIA The human HLX1 (H2.0-like homeobox 1) gene is a diverged homeobox gene Microarray data have shown that it is upregulated selectively by VEGF, the main trigger and key regulator of angiogenesis, and not by the general growth factor EGF or the inflammatory cytokine IL-1 We investigated here the regulation of the HLX1 gene in endothelial cells and developed tools to study its potential function in angiogenesis We show that HLX1 mRNA is upregulated 10-fold by VEGF and analysed the HLX1 gene by bioinformatics tools This revealed a conserved potential enhancer region 2.9 kb upstream of the start codon, composed of binding sites for ATF and CREB An analysis of the protein sequence detected aside the conserved homeodomain three different potential functional motifs, an SH3 binding site, serine/threonine phosphorylation sites, and a kinase binding site To analyse the function of HLX1 in angiogenesis recombinant adenoviruses were prepared to achieve overexpression of HLX1 in endothelial cells High expression of HLX1 by the recombinant adenovirus was confirmed This was used to test the influence of HLX1 on the induction of certain genes by VEGF MYCN, a gene involved in proliferation, was found to be upregulated by HLX1 In contrast, the endogenous HLX1 was strongly downregulated suggesting a feedback inhibition of the HLX1 gene Further analyses of the effects of HLX1 overexpression and inhibition on angiogenesis models are underway Acknowledgement: Supported by FWF-NFN94–3 and EC LSHC-CT-2005–518178 ª 2007 The Authors Journal compilation ª 2007 FEBS Signal Transduction C1-17 Signaling events downstream of MuSK and their role during neuromuscular synapse formation V Nizhynska, R Neumueller and R Herbst Medical University Vienna, Vienna, AUSTRIA The formation of the neuromuscular synapse (NMS) is regulated by the nerve-derived heparan sulphate proteoglycan agrin and the muscle-specific kinase MuSK Agrin induces a signal transduction pathway via MuSK, which induces pre- as well as postsynaptic differentiation Most importantly, activation of MuSK leads to the phosphorylation and redistribution of acetylcholine receptors (AChRs) and other postsynaptic proteins to synaptic sites The accumulation of high densities of AChRs at postsynaptic regions represents a hallmark of NMS formation and is required for proper NMS function The steps that follow MuSK activation and that lead to AChR clustering are however largely unknown Using MuSK mutant muscle cells, we have shown that MuSK carrying mutations in a juxtamembrane tyrosine or in the activation loop tyrosines is unable to induce AChR clustering In particular, a 13 aa juxtamembrane region of MuSK is necessary and sufficient to regulate NMS development in vitro and in vivo Further, we have shown that phosphoinositide 3-kinase (PI3-K) represents a component of the agrin/MuSK signaling pathway Muscle cells treated with specific PI3-K inhibitors are unable to form full-size AChR clusters in response to agrin and AChR phosphorylation is reduced Moreover, agrin-induced activation of Rac and Cdc42 is abolished in the presence of PI3-K inhibitors These results put PI3-K downstream of MuSK as regulator of AChR phosphorylation and clustering Its role during agrin-stimulated Rac and Cdc42 activation suggests a critical function during cytoskeletal reorganizations, which lead to the redistribution of actin-anchored AChRs Abstracts C1-19 Canonical Wnt signalling and Groucho proteins affect left-right asymmetry B Bajoghli1, N Aghaallaei1, D Soroldoni1 and T Czerny1,2 University of Veterinary Medicine Vienna, Vienna, AUSTRIA, University of Applied Sciences, FH-Campus Wien, Vienna, AUSTRIA Groucho/Tle proteins constitute a family of highly conserved cofactors for transcription Interaction of these corepressor proteins with Tcf/Lef transcription factors leads to repression of target genes in absence of canonical Wnt signalling activity Expression of the short family member Aes interferes with this corepressor function of Groucho/Tle proteins We misexpressed Aes during embryonic development of medaka fish and found effects on opticand otic vesicle outgrowth In addition we observed laterality defects during heart development A closer inspection of left-right asymmetry revealed effects of both Groucho/Tle proteins and canonical Wnt signalling on asymmetrically expressed TGFbeta family members in the axial mesoderm Both Aes and Wnt1 ectopically activate Lefty and Spaw genes, resulting in bilateral expression and consequently leading to laterality defects in organ formation When we looked at earlier events during left-right assignment we however found different roles for the two pathways Blocking Groucho function strongly affected the formation of the Kupffer¢s vesicle (equivalent to the ciliated part of the mouse node) and expression of the cilia marker Lrd Interestingly at this stage canonical Wnt signalling neither affected Lrd expression, nor Kupffer¢s vesicle formation Therefore Groucho/Tle proteins regulate left-right patterning at two different levels of the pathway, whereas canonical Wnt signalling specifically acts during later stages of left-right development C1-18 Cell-specific control of Wnt target genes: role of epigenetic modifications and differential promoter binding by TCFs C1-20 PLA2 is important in phosphatidic acid-induced Bcl-2 expression through ERK1/2 MAPK The recurrent use of a limited number of signaling systems in activation A Hecht, S Woehrle and B Wallmen University of Freiburg, Freiburg, GERMANY embryonic development poses the fundamental question of how the same effector molecules can generate distinct tissue-specific responses Canonical Wnt signaling provides a highly attractive model system to address this problem It performs important and widespread functions during ontogenesis, yet, it engages only betacatenin and members of the TCF family of DNA-binding proteins to control a large variety of ubiquitous and tissue-specific target genes TCF proteins are considered to act as bimodal switches in both the activation and the repression of their target genes Accordingly, they are believed to remain constantly bound to target promoters However, constant promoter occupancy by TCF proteins does not readily explain how distinct groups of Wnt target genes can be differentially regulated in a cell-type specific and developmentally controlled manner Here, we report a systematic comparison of Wnt-responsiveness, TCF promoter occupancy and epigenetic status of known Wnt/beta-catenin targets in different cell types Analysis of DNA methylation patterns and histone modifications at promoter regions revealed that Wnt-inducibility correlates with DNA hypomethylation and active histone marks In contrast, non-responsive promoters showed hypermethylation and repressive histone modifications Moreover, Wnt-responsiveness correlates with differential promoter occupation by TCFs Notably, in contrast to current models, TCF factors are not present on promoter regions of non-responding genes We hypothesize that distinct promoter occupancy by TCF proteins and epigenetic control mechanisms form a multi-layered control system to achieve differential regulation of Wnt target gene expression ª 2007 The Authors Journal compilation ª 2007 FEBS H Choi and J Han Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, Seoul, REPUBLIC OF KOREA Phosphatidic acid (PA), the product of PLD-mediated reaction, is lipid second messenger that participates in various intracellular signaling events and regulates a growing list of signaling protein In this study, we tried to find out the mechanism of Bcl-2 up-regulation by diC8PA treatment in HeLa cell Treatment with diC8PA resulted in significantly increased expression of Bcl-2 in HeLa cell Moreover diC8PA-induced Bcl-2 expression was blocked by mepacrine, an inhibitor of phospholipase A2 (PLA2), whereas enhanced rather by propranolol, an inhibitor of diC8PA phospholydrolase (PAP) Treatment of 1,2-Dipalmitoryl-sn-Glycero-3-phosphate (DPPA) instead of diC8PA also increased Bcl-2 expression indicating that Bcl-2 expression is mediated through lysophosphatidic acid (LPA), not through arachidonic acid To investigate the relation between ERK1/2 MAPK pathway and Bcl-2 expression, we used MEK1/2 inhibitor, PD98059 Pretreatment with PD98059 decreased diC8PA-induced Bcl-2 expression in HeLa cell, indicating that ERK1/2 MAPK was closely related with Bcl-2 expression In addition, the ERK1/2 MAPK is also associated with LPA-induced Bcl-2 expression When treated with PD98059, ERK1/2 MAPK activation and LPA-induced Bcl-2 expression were inhibited Taken together, PLA2 is important in diC8PAinduced Bcl-2 expression by producing LPA which is involved in ERK1/2 MAPK activation 137 Abstracts C1-21 Inhibitors of protein kinase A, protein kinase C and MAP kinases enhance the IL-13-induced expression of IL-6 by nasal polyp fibroblasts S D Athanasiou1, T Stathas2, P Goumas2, S Naxakis2, E Giannopoulou3 and A J Aletras1 Laboratory of Biochemistry, Department of Chemistry, University of Patras, Patras, GREECE, 2Department of Otolaryngology, Medical School, University of Patras, Patras, GREECE, 3Department of Pharmacology, Medical School, University of Patras, Patras, GREECE Nasal polyposis is a chronic inflammatory disease of the nasal mucosa that is characterized by inflammatory cell infiltration, modifications of epithelial differentiation, basement membrane thickening, extracellular matrix accumulation, and oedema IL-13 is a cytokine, generated by Th2 cells, implicating in the pathogenesis of various diseases characterized by fibrosis It modulates the collagen homeostasis, enhances the TIMP-1 and inhibits the IL-1binduced MMP-1 and -3 production by skin fibroblasts, and upregulates the expression of b1 integrin, VCAM-1, IL-6 and MCP-1 in lung fibroblasts In this study the effect of IL-13 on the fibrotic factor IL-6 production by nasal polyp fibroblasts and he signal transduction pathway mediating this effect, are investigated Polyp fibroblasts in culture expressed IL-13 receptors ELISA and RT-PCR showed that IL-13 up-regulated the IL-6 expression in a dose and time-dependent manner and this effect was not mediated by TGF-b1 RT-PCR showed that IL-13 did not affect the expression of TGF-b1 and its receptors Using specific inhibitors it was found that the inhibitors of protein kinases A and C, and of ERKs and JNKs enhanced the stimulatory effect of IL-13, while the inhibitors of cyclooxygenases, tyrosin kinases and NF-jB activation strongly suppressed this effect Signal Transduction C1-23 Effect of growth factors on acetaminophen-induced liver injury T Nam, H Hwang and I Kim Pukyong National University, Busan, REPUBLIC OF KOREA The growth factors (IGF-I and EGF) are involved in protecting against chemotherapeutic drug-induced cell death in human hepatoma cells Acetaminophen (AAP) hepatotoxicity is a leading cause of liver failure and the prevention of AAP-induced cell death has been the focus of many studies We examined whether two growth factors, IGF-I and EGF, could protect against AAP-induced cell death and investigated the protective mechanism involved Based on the results of MTS assays, Hoechst 33342 cell staining, and DNA fragmentation experiments, AAP induced cell death in a dose-dependent manner According to Western blot analysis, treatment with AAP increased the level of poly (ADP-ribose) polymerase (PARP) fragments in cells compared to that in control cells, and caspase-3, a key signaling molecule in apoptosis, was activated after AAP treatment Combined treatment with AAP and IGF-I, or EGF inhibited caspase-3 activation and PARP cleavage, consistent with the ability of growth factors to restore the level of glutathione (GSH) and cell viability in GSH and MTS assays, respectively We investigated whether the protective effect of growth factors against AAP cytotoxicity was related to MAPK signaling, which was involved oxidative stress, and Fas signaling, detected growth factors inhibit AAP cytotoxicity through MAPK signaling Thus, MAPK is involved in the protective effect of growth factors against AAP-induced cell death C1-22 Endothelin-1 activates Glut1 transcription via both PKC’s and MAPK signaling pathways Y Kao and J C Fong National Yang-Ming University, Taipei, TAIWAN C1-24 Role of PKCe in Gelsolin expression by histone We have demonstrated previously that endothelin-1 (ET-1) may sti- deacetylase inhibitor apicidin in human cervix mulate GLUT1-mediated glucose transport in 3T3-L1 adipocytes cancer cells via both protein kinase C (PKC) - and p42/p44 mitogen-activated protein kinase (MAPK)-dependent pathways In the present study, we further explored the molecular mechanism involved Our results indicate that both novel PKCe- and MAPK-dependent pathways are involved in ET-1 activation of Glut1 transcription and there is no interaction between PKCe and MAPK at the kinase activity level By using deletion and mutation constructs of luciferase reporter driven by Glut1 promoter with enhancers and 2, we were able to identify NF-jB and Sp1 binding sites on enhancer as the ET-1 response elements In concord, chromatin immunoprecipitation and co-immunoprecipitation experiments demonstrated that in cells treated with ET-1, NF-jB and Sp1 may form a binding complex bound to enhancer While nuclear contents of both NF-jB and Sp1 were increased by ET-1, only the increase in Sp1 required de novo protein synthesis In addition, we provide evidence that ET-1induced Sp1 expression requires both PKCe- and MAPK/CREBdependent pathways, whereas activation of NF-jB by ET-1 is mediated by a PKCe/reactive oxygen species (ROS) cascade Taken together, our results strongly suggest that by activating NF-jB via PKCe/ROS cascade and increasing Sp1 expression through both PKCe- and MAPK/CREB-dependent pathways, ET-1 may activate Glut1 transcription by inducing interaction between nuclear NF-jB and Sp1 as well as their binding to enhancer 138 Y Jeon1,2, J You1,3, J Park1, W Choi4, H Lee2 and J Han1 Sungkyunkwan University, Suwon, REPUBLIC OF KOREA, 2Konyang University, Daejeon, REPUBLIC OF KOREA, 3Konkuk University, Chungju, REPUBLIC OF KOREA, 4College of Medicine and CBITRC, Konkuk University, Chungju, REPUBLIC OF KOREA Down-regulation of gelsolin expression is associated with cellular transformation and induction of gelsolin exerts antitumorigenic effects In this study, we show that protein kinase C (PKC) signaling pathway is required for the induction of gelsolin by the histone deacetylase inhibitor apicidin in HeLa cells Apicidin induces gelsolin mRNA independently of the de novo protein synthesis Inhibitor study has revealed that the PKC signaling pathway is involved in the gelsolin expression Furthermore, inhibition of PKCe by either siRNA or dominant-negative mutant completely abrogates the expression of gelsolin by apicidin, indicating that PKCe is the major isoform for this process In parallel, apicidin induction of gelsolin is antagonized by the inhibition of Sp1 using dominant-negative Sp1 or specific Sp1 inhibitor mithramycin, and inhibition of PKC leads to suppression of Sp1 promoter activity Our results provide mechanistic insights into molecular mechanisms of gelsolin induction by apicidin ª 2007 The Authors Journal compilation ª 2007 FEBS Signal Transduction Abstracts C1-25 C1-27 Prion protein at the interface of MAPK and TGF- Dynamics of S100A11 protein in human beta signaling myoblasts after stimulation of differentiation S Wurm and C Wechselberger Upper Austrian Research GmbH, Linz, AUSTRIA Members of the transforming growth factor-beta (TGF-beta) superfamily control a multitude of cellular processes, including cell growth, differentiation and apoptosis On the other hand, TGFbeta’s have also been shown to act as tumor-promoting cytokines, underscoring the complexity of this signaling network Conventional TGF-beta signaling involves a heteromeric transmembrane receptor complex which leads to the phosphorylation of intracellular Smad proteins and finally to the transcriptional regulation of target genes Auxiliary, MAPK cascades have been described to be activated by TGF-beta and furthermore to modulate the conventional Smad response Previous studies have shown that also GPI-anchored proteins can influence TGF-beta signaling Now we were able to reveal for the first time by co-immunoprecipitation in HEK293 cells that also prion protein (PrP), a GPI-linked protein whose physiological roles are still poorly defined, interacts directly with members of the TGF-beta receptor complex We have already shown that PrP modulates both, the conventional as well as the MAPK-pathways in TGF-beta activated mouse mammary epithelial cells Recent findings corroborate the assumption that PrP can block the p42/44 MAPK pathway, e.g stimulated by EGF, another key regulator of cell growth and differentiation In response, endogenous TGFbeta1 production is enhanced in cells over-expressing PrP As TGF-beta as well as EGF signaling is often disturbed in tumorigenesis, PrP could fulfill an unexpected task during cancer development C1-26 Supportive evidence of desmin’s role in TGF-ß signaling and early cardiomyogenesis C Fuchs, M Stary and G Weitzer Max F Perutz Laboratories/Medical University of Vienna, Vienna, AUSTRIA Desmin is a type III intermediate filament protein and contributes to the stability of the myocardium Desmin specifically supports fusion of myoblasts and the commitment and differentiation of cardiomyocytes in myogenesis and cardiomyogenesis Expression of brachyuri and nkx2.5 is modulated by desmin For our study, we used murine embryonic stem cell derived embryoid bodies (EBs) where cardiomyogenesis is faithfully recapitulated Constitutive expression of desmin in EBs results in an enhanced expression level of the TGF-ß family member nodal, and shows upregulation of islet-1, sparc and nkx2.5 Vice versa, des-/- EBs show a decreased expression level of nodal, islet-1, sparc and nkx2.5 compared to wild type We tested the influence of desmin in TGFß signaling by using an inhibitor of ALK4 receptor, SB 431542 In wild type and des-/- EBs, expression of brachyuri, islet-1 and nkx2.5 were downregulated by SB 431542 whereas desmin overexpression in SB 431542 treated EBs rescued expression of brachyuri, islet-1 and nkx2.5 These results strongly suggest a role of desmin in TGF-ß signaling and early cardiomyogenesis ª 2007 The Authors Journal compilation ª 2007 FEBS A Makarov, L Kovalyov, K Lisitskaya and S Shishkin Bakh Institute of Biochemistry, Moscow, RUSSIAN FEDERATION Satellite cells participate in muscle regeneration and hypertrophy Proliferation and differentiation of satellite cells are regulated by a number of growth factors, including TGF-beta In this study human myoblasts were cultured in the F-12 media containing sodium pyruvate, gentamicin and fetal calf serum Differentiation of the myoblasts was induced by incubation in differentiation media (containing 2% of horse serum) Using 2D-PAGE it was shown significantly decreasing of protein fraction with molecular weight 11 kDa and isoelectric point 6.1 By MALDI-TOF massspectrometry this protein has been identified as S100A11 calcium binding protein (calgizzarin).This protein has been described as one of the messengers involved in signal transduction from TGFbeta receptor (Sakaguchi et al, 2004) We believe down-regulation of this protein can be one of the factors dependable for decreasing of myoblast sensitivity to TGF-beta during myogenic differentiation C1-28 Studying the role of tissue transglutaminase in neutrophil granulocyte differentiation K Csomos, Z Balajthy, G Zahuczky and L Fesus University of Debrecen, Debrecen, HUNGARY Neutrophils begin their differentiation in bone marrow and become matured granulocytes in the circulation system The proliferating myeloid cells not contain tissue transglutaminase (TG2) but during their differentiation this enzyme is induced and large amount of this protein is present in matured cells So far, its exact role in neutrophil differentiation has remained unclarified All-trans retinoic acid treated NB4 promyelocyte cells provide an appropriate model system to study neutrophil differentiation Lentivirus based antiTG2 shRNA expression vector was used to establish stabile TG2 knockdown NB4 cell line The examination of the normal and TG2 knockdown NB4 differentiation revealed that the enzyme is involved in the regulation of several genes (i.e gp91 phox) which are related to neutrophil granulocyte function These results and the phenomenon that the enzyme translocates to the nucleus in suggest that TG2 modulates gene expression In order to find the genes which are modulated by TG2 total gene expression analysis was performed using DNA microarray In TG2 knockdown cells the expression of 171 genes decreased and 173 increased at least 2-fold level Among these identified genes there are ones involved in neutrophil granulocyte function, transcription factors and apoptosis related genes 139 Abstracts Signal Transduction C1-29 Role of protein L-isoaspartyl o-methyltransferase in neuronal differentiation of P19 embryonal carcinoma C1-31 Kidins220: A novel neuronal protein that is up-regulated during neuroblastoma differentiation S Hong, S Lee and S Hong Department of Genetic Engineering, Sungkyunkwan University, Suwon, REPUBLIC OF KOREA ´ ´ C Lopez-Menendez1, T Lofstedt2, M Ovenberger2, S Pahlman2 ă and T Iglesias1 Instituto de Investigaciones Biome´dicas, Madrid, SPAIN, University Hospital MAS, Malmo SWEDEN ă, Protein L-isoaspartyl-( D-aspartyl) o-methyltransferase (PIMT, EC 2.1.1.77) is a cytosolic enzyme that methylates the side chain carboxyl group of racemized L-aspartyl or L-isoaspartyl residues in proteinaceous substrates with S-adenosylmethionine (AdoMet) as a methyl donor This enzyme is expressed highly in the brain but the functions of PIMT in it are poorly understood P19 embryonic carcinoma cells are pluripotent cells that can undergo irreversible differentiation into derivatives of three germ layers And P19 cells can be induced to differentiate into neuron-like cells by treating all-trans retinoic acid (at-RA) The purpose of this study to investigate the relationship of PIMT to neurogenesis After treatment of P19 cells with atRA, PIMT mRNA level and activity were measured during neuronal differentiation After days of t-RA treatment, the PIMT mRNA levels increased and reached the highest level at day and maintained the level during the further differentiation The activity of PIMT increased by 20% after days of differentiation and showed similar level during further differentiation But at day of neuronal differentiation when P19 neurons become mature, PIMT activity increased by 79% compared to that of control These results suggest that PIMT might be related to neurogenesis and influenced by the retinoic acid receptor pathway Neuroblastoma is a childhood tumor derived from the developing synpathetic nervous system that retains many characteristics of immature neural cells Despite their tumoral origin, neuroblastoma cell lines can be induced to differentiate in vitro by several agents, including retinoic acid and phorbol esters In vitro differentiated neuroblastoma cells have a neuronal phenotype as judged by their morphology, extension of neurites and expression of biochemical and functional neuronal markers Therefore, neuroblastoma cells are considered a useful model to study the initial phases of neuronal differentiation Kidins220 (Kinase D interacting substrate of 220 kDa) is a novel trans-membrane protein with unique features and unknown function It was first cloned as a physiological substrate for protein kinase D1 and later as a substrate of neurotrophin and ephrin receptor Kidins220 is abundantly expressed in the nervous system The localization of Kidins220 at the tips of extending neurites suggests that it may be participating in processes such as neuritogenesis and/or neurogenesis We have cloned the promoter and first intron of kidins220 gene and identified several putative regulatory elements for retinoic acid receptors We have studied the gene expression pattern of kidins220 in different neuroblastoma cell lines under several stimuli that modulate changes in their phenotype, maturation and aggresiveness C1-30 Interaction between the neurotrophin receptor target kidins220 and the ERM membrane-cytoskeleton linker protein Moesin A M Higuero1, R M Jean-Mairet1, J Vandekerckhove2 and T Iglesias1 Instituto de Investigaciones Biomedicas Alberto Sols, Madrid, SPAIN, 2Department of Medical Protein Research, VIB, Ghent, BELGIUM C1-32 A novel role for hippocalcin in bFGF-induced Kinase-D interacting substrate of 220 kDa (Kidins220), also neurite outgrowth of H19–7 cells known as ARMS, is a protein predominantly expressed in developing brain that was originally identified as a protein kinase D (PKD) substrate and as a downstream target of the neurotrophin receptors PKD is a serine/threonine kinase related to the PKC superfamily that serves as a diacylglycerol receptor One of its bestcharacterized functions is its role in regulating the fission of transport carriers from the golgi to the plasma membrane On the other hand, neurotrophins are fundamental factors in the development of the nervous system influencing processes such as neuronal survival, differentiation, synaptic plasticity and axonal and dendritic ramifications In order gain insight into Kidins220’s function, we decided to identify its physiological binding partners Using a proteomics approach, we identified the cytolinker protein Moesin, which belongs to the Ezrin/Radixin/Moesin (ERM) family, as a Kidins220 interacting protein We confirmed this interaction by coimmunoprecipitation from hippocampal neurons and PC12 cell lysates, and by colocalization studies The ERM proteins are key factors in cytoskeletal processes underlying diverse cellular functions such as cell division, adhesion, migration, morphology and intracellular signal transduction In neurons, ERM proteins have been implicated in developmental growth, morphology and migration These results suggest that the ERMs link the plasma membrane protein Kidins220 to the neuronal cytoskeleton and that this interaction might be relevant for the neurotrophin-induced cytoskeletal remodeling 140 D Oh, J Cho, S Park and J Han Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, Seoul, REPUBLIC OF KOREA Hippocalcin is a Ca2+ binding protein which is expressed mainly in the pyramidal nerve cell of the hippocampus, but the mechanism and functions underlying hippocalcin in the brain remains unclear To elucidate a role of hippocalcin, we utilized a conditionally immortalized hippocampal cell line (H19–7) We show here that bFGF-induced hippocalcin expression is involved in neurite outgrowth of H19–7 cells Increased expression of hippocalcin dramatically elongated neurites induced by bFGF stimulation and was concurrent with the expression of basic helix-loop-helix (bHLH) transcription factor, NeuroD Hippocalcin suppression blocked bFGF-induced neurite outgrowth and NeuroD expression Stimulation of bFGF resulted in the activation of phospholipase C-c (PLC-c) and Ca2+ Hippocalcin expression by bFGF stimulation was fully blocked by both the PLC-c inhibitor, U73122 and a chelator of intracellular Ca2+, BAPTA-AM, suggesting that hippocalcin expression by bFGF stimulation is dependent on PLC-c and Ca2+ Moreover, both U73122 and BAPTA-AM completely blocked bFGF-induced neurite outgrowth and NeuroD expression Taken together, these results suggest for the first time that bFGF induces hippocalcin expression through PLC-c activation and Ca2+, which leads to neurite outgrowth in H19–7 cells ª 2007 The Authors Journal compilation ª 2007 FEBS Signal Transduction C1-33 IL6 inhibits RANKL-induced osteoclastogenesis by diverting cells into the macrophage lineage: implication of STAT3 V Trichet, L Duplomb, M Baud’Huin, C Charrier, F Blanchard and D Heymann INSERM ERI-7, Nantes, FRANCE Osteoclasts are bone-resorptive cells differentiated from hematopoă etic precursors upon RANKL activation Some studies demonstrated that IL6 indirectly up-modulates osteoclastogenesis through the production of RANKL by osteoblasts To investigate the direct effect of IL6 on osteoclast, we used the monocyte cell line RAW 264.7 which differentiates into osteoclast in presence of RANKL The addition of IL6 irreversibly inhibited RANKL-induced osteoclastogenesis in a dose-dependant manner Furthermore, IL6 decreased the expression of osteoclast markers but up modulated macrophage markers To understand this phenomenon, we focused on STAT3, the main signaling molecule activated by IL6 Any of two STAT3 inhibitors used affected the IL-6 effect However, these experiments revealed that STAT3 is mandatory for osteoclastogenesis Indeed both inhibitors completely abolished RANKL-induced osteoclastogenesis of RAW 264.7 We showed that a basal level of phospho-STAT3 on Serine727 associated to an absence of phosphoSTAT3 on Tyrosine705 is essential for osteoclastogenesis IL6 stimulation induced both phosphorylations, and consequently RAW 264.7 generated macrophages With AG490 a decrease in Serine727phosphorylation leaded to an inhibition of osteoclastogenesis Finally, we showed that IL6 inhibits osteoclasts differentiation of mouse bone marrow precursors and human PBMCs In conclusion, IL6 inhibits RANKL-induced osteoclastogenesis by diverting cells into the macrophage lineage, and the activated-STAT3 level and its form of phosphorylation control osteoclastogenesis Abstracts C1-35 Opioids in epilepsy: lessons from prodynorphin KO mice C Schwarzer1, S Loacker1, M Sayyah1 and H Herzog2 Medical University Innsbruck, Innsbruck, AUSTRIA, 2Garvan Institute for Medical Research, Sydney, AUSTRALIA Neuropsychiatric disorders are one of the main challenges of human medicine with epilepsy being one of the most common serious disorders of the brain Increasing evidence suggests that neuropeptides, particularly the opioids, play an important role in epilepsy However, little is known about the mechanism of the endogenous opioid system in epileptogenesis and epilepsy Therefore, we investigated prodynorphin-KO mice (Dyn-/-)in models of acute seizures, epileptogenesis and epilepsy Compared with wildtype littermates (Dyn+/+), Dyn-/- mice showed a significantly reduced seizure threshold as assessed by tailvein infusion of pentylenetetrazole (PTZ) This phenotype could be rescued entirely by the kappa opioid receptor specific agonist U50488, but not the mu opioid receptor specific agonist DAMGO The delta opioid receptor specific agonist SNC80 decreased seizure threshold in both genotypes Pre-treatment with the kappa selective antagonist GNTI completely blocked the rescue effect of U-50488 Consistent with the reduced seizure threshold, Dyn-/- mice showed faster seizure onset and a prolonged time of seizure activity after ic injection of kainic acid In the PTZ kindling model, Dyn-/- mice showed a significantly faster kindling progression Three weeks after local injection of kainic acid into the dorsal hippocampus, Dyn-/- mice displayed an increased extent of granule cell layer dispersion and neuronal loss along the rostro-caudal axis of the ipsiand partially the contralateral hippocampus Our data strongly support a critical role for dynorphin in the regulation of hippocampal excitability, indicating an anticonvulsant role of kappa opioid recepors C1-34 Expression and role of a6 integrin in the early embryo A Dimiropoulos, K Konstantopoulos, M Christopoulos and N Zagris Department of Biology, University of Patras, Patras, GREECE C1-36 The biological significance of novel CK1-mediated phosphorylation of tau protein The integrin family of transmembrane proteins composed of hetand its associated proteins in rat brain erodimers of a and b subunits transfer information from the extracellular environment to the interior of the cell and vice versa We studied the expression pattern of a6 subunit in the early chick embryo by RT-PCR and immunofluorescence a6 integrin mRNA presence was first detectable at the blastula stage (XIII) It was intriguing to detect the a6A and a6B mRNA splice variants during the gastrula stage (HH2) a6 immunoreactivity was first detectable in the epiblast and the hypoblast at the blastula stage, was intense in the cells ingressing through the primitive streak during the gastrula stage (HH3–4) and was weak during the early neurula stage (HH5) Later in development, immuno- reactivity was prominent in the brain and the neural tube The neural crest cells migrating to the pharyngeal arches and to the eye expressed a6 integrin strongly The expression of a6 was strong in lens, was strong in the myotome in the somites, intense in the myocardium and endocardium in the heart and in the walls of dorsal aorta and gut Inhibition of function of a6 integrin by blocking antibodies indicated that a6 mediates the guided migration of cells and participates in brain and heart morphogenesis in the early embryo Acknowledgements: Supported by grants from the European Social Fund (ESF), Operational Program for Educational and Vocational Training II (EPEAEK II) particularly the Program ‘PYTHAGORAS II’ and from the University of Patras (‘K Karatheodoris’ grant B 397) ª 2007 The Authors Journal compilation ª 2007 FEBS K Suzuki, H Sasaki, F Kawakami and K Ohtsuki Kitasato University, Sagamihara, JAPAN The purpose of this recently, we reported that casein kinase (CK1) phophorylates two functional basic proteins [myelin basic protein (MBP) and tau protein (TP)] in the presence of two sulfated lipids [sulfatide and cholesterol-3-sulfate (SCS)] in vitro However, the physiological significance of the CK1-mediated high phosphorylation of these two SCS-BPs at the high level of SCS in an aged brain remains to be elucidated Therefore, the present study has been carried out to characterize the SCS-dependent phosphorylation of TP and its associated proteins by CK1 in the TP fraction from rat brain By the obtained result it was found that (i) in the presence of SCS, CK1 phosphorylated TP and its associated proteins (p82 and p55) in the partially purified TP fraction from rat brain; (ii) both PKC and CK were detected in the TP fraction; and (iii) p82 and p55 was identified as eIF-4B and syndapin 1, respectively These results suggest that the accumulated high levels of SCS preferentially induces the CK1-mediated high phosphorylation of TP, eIF-4B and syndapin 1, which are involved in the mechanisms of various other neuronal diseases including Alzheimer’s disease, and selectively suppresses their phosphorylation by PKC and CK2 in the high aged rat brain 141 Abstracts C1-37 IL-1beta upregulates Smad7 via NFjB activation in human chondrocytes ´ C Bauge1, J Attia1, S Leclercq2, P Galera1 and K Boumediene1 Connective Tissue Biochemistry, Caen, FRANCE, 2Saint-Martin Private Clinic, Caen, FRANCE We have previously shown that Interleukin-1b (IL-1b), a key-cytokine in osteoarthritis (OA) pathology, impairs TGFb signaling, through TbRII down-regulation and Smad7 up-regulation This mechanism could account for the reduced responsiveness of OA chondrocytes to TGFb and the cartilage breakdown associated with this disease The aim of this present study was to investigate the molecular mechanism underlying the IL-1ß-induced stimulation of Smad7 in human articular chondrocytes (HAC) HAC were treated with IL-1ß in the presence of TGFß1, PDTC (a repressor of NFjB pathway) or cycloheximide (a translation inhibitor) Then, mRNA steady-state and protein levels were estimated by real-time RT-PCR and immunocytology Furthermore, transient transfections of p65 expression vector or siRNA targeted p65 were achieved to define its effect of this transcription factor on Smad7 expression We showed that TßRII overexpression restores TGFß response of HAC However, this effect was total only for short time incubation, suggesting the implication of a subsequent mechanism Moreover, IL-1b causes a late induction of the inhibitory Smads, Smad7 This effect is direct as it does not require de novo synthesis In addition, we established, by experiments of gain/loss function, that the up-regulation of Smad7 by IL-1ß is mediated through NFjB pathway and especially p65 subunit These findings enlighten the regulatory process of IL-1b on Smad7 expression Understanding the molecular basis for IL-1ß induction of Smad7 and reduction of chondrocytes-responsiveness to TGFß provides news insight into molecular mechanisms of OA and may facilitate identification of novel approaches for its treatment C1-39 Transcriptional regulation of the small GTPase RhoB gene by the transforming growth factor b signaling pathway E Vasilaki, E Papadimitriou, C Stournaras and D Kardassis University of Crete, Heraklion, GREECE Small GTPases of the rho family control key biological processes such as cell growth, apoptosis and actin cytoskeleton organization We have shown previously that transforming growth factor b (TGFb) induced a rapid, non-genomic, activation of RhoA and RhoB in Swiss 3T3 fibroblasts and that this activation was associated with actin cytoskeleton reorganization We now show that TGFb increases the steady state mRNA levels of RhoB but not of RhoA gene in HaCaT keratinocytes This activation was observed as early as h post-TGFb addition and remained for 24 h The early transcriptional activation of RhoB gene by TGFb was abolished using a specific MEK1 kinase inhibitor suggesting the involvement of the MEK/ERK pathway in this process Using adenovirus-mediated gene transfer, we observed RhoB gene induction by Smad2 and Smad3 TGF-b induced transcriptional up-regulation of the RhoB gene and actin polymerization were not observed in Smad3-/- cells but both phenotypes were rescued by adenoviral mediated exogenous expression of Smad3 Both the TGFb/Smad and the MEK/ ERK pathways activated the human RhoB promoter via distinct promoter regions but there was no evidence for functional cooperativity between these two pathways on RhoB gene transcription Over expression of RhoB was associated with a decrease in RhoB promoter activity suggesting a mechanism of auto inhibition operating in RhoB gene regulation In summary, our findings indicate that TGFb regulates the function and the expression of the small GTPase RhoB via non-genomic and genomic pathways and that this dual regulation is important for actin cytoskeleton organization and possibly for other RhoB-dependent responses 142 Signal Transduction C1-38 Neurosteroids protect neural-crest derived cells from apoptosis, tempospatially activating prosurvival kinases I Charalampopoulos1, C Tsatsanis2, B Vergou1, I Alexaki3, E Castanas3, A Margioris2 and A Gravanis1 Department of Pharmacology, School of Medicine, University of Crete, Heraklion, GREECE, 2Department of Clinical Chemistry, School of Medicine, University of Crete, Heraklion, GREECE, Department of Exp Endocrinology, School of Medicine, University of Crete, Heraklion, GREECE We have recently shown that neurosteroid dehydroepiandrosterone (DHEA) at nM protects from apoptosis neural crest-derived PC12 cells, via G protein-associated specific membrane binding sites and subsequent activation within minutes of prosurvival transcription factors CREB and NFjB, upstream effectors of the antiapoptotic Bcl-2 proteins (Charalampopoulos et al, PNAS 2004; FASEB J 2006) We now describe the signalling pathways, involved in the transduction of the neuroprotective effects of DHEA Specifically, we have the following data: (i) Wortmannin, PD98059 and PP2, inhibitors of prosurvival kinases PI3K, MEK1/ and Src respectively, completely blocked the anti-apoptotic effects of DHEA in serum-deprived PC12 cells; (ii) the three kinase inhibitors completely reversed the induction by DHEA of the antiapoptotic proteins Bcl-2 and Bcl-xL, as well as the activation of prosurvival transcription factors CREB and NFjB; (iii) DHEA at 10 nM induced within minutes the phosphorylation of ERK1/2/ MEK1/2, PI3K/Akt and Src kinases in serum-deprived PC12 cells; (iv) the effect of DHEA on prosurvival kinase activation was mimicked by non permeable DHEA-BSA conjugate and was reversed by Pertussis toxin and glucocorticoids and androgens, suggesting the involvement of recently described DHEA specific membrane binding sites These findings suggest a strong neuroprotective role for neurosteroids during brain development and ageing Acknowledgement: Supported by a grant from the GGETPENED03-ED372 C1-40 Expression and function of arylhydrocarbon receptor in growth plate chondrocytes M Widerak, K Tahiri, M Dumontier, M Corvol and J Savouret Inserm UMR-S 747 Universite´ Paris 5, Paris, FRANCE Articular (AR) and growth plate (GR) cartilage is in a physiological state of variable hypoxia, which may be altered by inflammation or trauma The Aryl hydrocarbon receptor (AhR) is activated by xenobiotic ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or benzo(a)pyrene (tobacco tar fraction) The expression and function of AhR in cartilage are not known and this is the aim of the present study Quantitative RT-PCR experiments show that AhR mRNA is present as non measurable traces in AR and GR chondrocytes in basal conditions Basal expression of AhR was robustly induced by Interleukin-1beta (IL-1B, 20h) in GR cells cultured in normoxia (21% oxygen) and to a minor extent in hypoxia (0.5% oxygen) IL-1B was inefficient on AR cells We used the endogenous gene cytochrome P450–1A1 (CYP1A1) to monitor the functionality of AhR in chondrocytes AR and GR chondrocytes did not respond to TCDD treatement (20 h) in basal conditions regardless of oxygen pressure In GR cells only, the increase in AhR expression by IL-1B stimulated the expression of CYP1A1 which was further increased after TCDD treatement, in normoxia but not in hypoxia These observations show that AhR expression and functionality is restricted to GR chondrocytes in normoxic conditions and cytokine-dependent Our results also suggest that AhR ligands may exert disruptive effects on growth cartilage, during development and/or inflammatory processes ª 2007 The Authors Journal compilation ª 2007 FEBS Abstracts G4: Ischemic Precondition and Ischemia-reperfusion (4x) After excised tissue specimens (100 mg) were finely minced, grinded with a pestle after covering with liquid nitrogen ml of HSP-70 extraction reagent + protease inhibitors (Roche) was added and the slurry was pulverized After centrifugation at 21 000 · g for 10 at +40C the supernatant was collected and quantified for total protein concentration (BCA ) and HSP-70 (ELISA,Stressgen) Results: All the flaps in G1 survived completely G2 demonstrated a necrotic area of 62% Ischemic precondition and ischemiareperfusion groups significantly reduced the percentage flap necrosis to 24% in G3 and to 21% in G4 Basal HSP-70 level was 29.7 mg/g prot The values of HSP-70 in G3 and G4 were 48.41 mg/g prot and 86.42 mg/g prot, respectively Conclusion: There was a great correlation between HSP-70 levels and flap viability in ischemia reperfusion injury Ischemic preconditioning before ischemia reperfusion injury was apparently increasing the level of HSP 70 C4-45 M-domains couple the ClpB threading motor with the DnaK chaperone activity T Haslberger1, J Weibezahn1, R Zahn1, S Lee2, F Tsai2, B Bukau1 and A Mogk1 ZMBH, Heidelberg, GERMANY, 2Baylor College of Medicine, Houston, TX, USA The AAA+ chaperone ClpB mediates the reactivation of aggregated proteins in co-operation with the DnaK chaperone system ClpB consists of two AAA domains that drive the ATP-dependent threading of substrates through a central translocation channel Its unique middle (M)-domain forms a coiled-coil structure that laterally protrudes from the ClpB ring and is essential for aggregate solubilization We demonstrated that the conserved helix of the M-domain is specifically required for the DnaK-dependent shuffling of aggregated proteins, but not of soluble denatured substrates, to the pore entrance of the ClpB translocation channel Helix exhibits nucleotide-driven conformational changes possibly involving a transition between folded and unfolded states This molecular switch controls the ClpB ATPase cycle by contacting the first ATPase domain and establishes the M-domain as a regulatory device that acts in the disaggregation process by coupling the threading motor of ClpB with the DnaK chaperone activity C4-46 Regulation of the hypertonic stress response in NFAT5-deficient cells ´ K Drews-Elger, C Lopez-Rodrı´ guez and J Aramburu Universitat Pompeu Fabra, Barcelona, SPAIN The Rel transcription factor NFAT5 regulates the expression of osmoprotective genes that are fundamental for the adaptation and survival of mammalian cells to hypertonic stress In this regard, NFAT5-deficient mice suffer renal failure and T cell dysfunction associated to an ineffective response to hypertonic conditions in vivo One critical aspect of hypertonicity is that it can be genotoxic, causing DNA breaks, inhibiting the repair of DNA damage, and interfering with the cellular response to other agents such as UV and ionizing radiation In this work, we have studied the role of NFAT5 in the response to hypertonic and genotoxic stress NFAT5-null cells display an exacerbated genotoxic stress response under hypertonic conditions, evidenced by the early accumulation of molecular markers associated to DNA damage responses, progressive cell cycle arrest, and increased sensitivity to ionizing radiation Our results identify novel features in the function of NFAT5 and highlight its importance in regulating genotoxic stress responses 206 Signal Transduction C4-47 Chronic treatment with resveratrol induces redox-stress and subsequent p53-dependent senescence in cancer cells E H Heiss, Y D C Schilder and V M Dirsch University of Vienna, Vienna, AUSTRIA Induction of senescence, an irreversible growth arrest, in cancer cells is regarded as a mean to halt cancer progression The phytoalexin resveratrol (RV) is known to possess a variety of cancer preventive, cancer therapeutic and chemosensitizing properties Repetitive treatment with RV in a subapoptotic concentration of 30 lmol/l reactivated the senescence program in HCT 116 colon carcinoma cells which was dependent on functional p53 and p21 proteins Interestingly and in contrast to the widely accepted antioxidant property of RV we demonstrate that one causative stimulus for senescence induction by chronic RV is an increased level of reactive oxygen species (ROS) including hydrogen peroxide and superoxide: ROS at least partly originate from mitochondria and are associated with the RV-mediated retardation of cells in the ROS-rich S-phase of the cell cycle Consistently, co-incubation with N-acetyl cysteine or mito Q (a general or mitochondria-directed antioxidant) and caffeine (an inhibitor of the S-phase arrest in this setting), respectively, interfered with RV-mediated elevation of ROS and induction of senescence This is one of the first reports of a chemopreventive compound eliciting senescence in cancer cells in response to initiated redox stress C4-48 The EGFR: A putative ROS sensor Y Posen, A Scherz and Y Salomon Weizmann Institute of Science, Rehovot, ISRAEL The epidermal growth factor receptor (EGFR) is a transmembranal protein pivotal in communicating information between the extracellular and intracellular environments, constantly exposed to modifications prompted by environmental oxidative challenge To analyze its responses to oxidizing surroundings, we employed a light-sensitive photosensitizer generating physiologically relevant levels of reactive oxygen species (ROS) upon illumination Strict dictation of the magnitude and nature of ROS exposure are controlled by sensitizer concentrations or sample illumination, whose duration and intensity are manipulable We utilized photoswitchbased ROS challenge (pROS) to characterize primary ROS targets functioning as cellular redox-stress sensors We demonstrate pROS-induced, covalent, nonreducible and stable crosslinking of the EGFR (X-EGFR), detectable for hours after formation The crosslinked product, likely involving extracellular residues, accumulates on the cell surface X-EGFR formation is limited by overall receptor density forming in a ligand-independent manner, but is promoted by EGF when receptor expression levels are low Furthermore, we demonstrate that the crosslinked EGFR is biologically active, undergoing autophosphorylation, consequentially leading to downstream ERK and NFjB activation In conclusion, the EGFR may represent a cellular redox-stress sensor, enabling the cell to survey its dynamic environments and react accordingly in a ligand-independent manner ª 2007 The Authors Journal compilation ª 2007 FEBS Signal Transduction C4-49 Activation of stress kinases (SAPK/JNK) by genotoxins J Damrot and G Fritz Institute of Toxicology, University of Mainz, Mainz, GERMANY Activation of stress-activated protein kinases/ c-Jun-N-terminal kinases (SAPK/JNK) is part of the cellular response to genotoxins, controlling gene expression and survival The present study aims at elucidating the contribution of DNA-damage independent and dependent mechanisms in genotoxin-induced signaling to SAPK/ JNK To this end, we (i) investigated the effect of pharmacological inhibition of Ras/Rho GTPases by lovastatin on stress signaling and apoptosis and (ii) analysed the response of cells that are impaired in DNA damage-recognition and repair The data obtained show that lovastatin attenuates various ionizing radiation (IR) and doxorubicin-stimulated stress responses of HUVEC, including activation of NF-κB, Chk-1, Akt and SAPK/ JNK as well as expression of p53, p21, Fas-L and Fas-R Furthermore, the statin protected HUVEC from IR- and doxorubicininduced cell death (1, 2) Analysis of a number of cell lines impeded in correct processing of DNA damage showed that specifically CSB and DNA-PKcs are required for late activation of SAPK/JNK by alkylating compounds (3) Late signaling to SAPK/ JNK provoked by DNA-cross-linking agents (UV-light, cisplatin, transplatin) was affected by DNA repair factors as well Here, the type of repair factor involved depends on the nature of the genotoxin used References Nuebel T., Damrot J et al Clin Cancer Res 2006; 12: 933–9 Damrot J et al British J Pharmacol 2006; 149: 988–97 Fritz G and Kaina B Mol Biol Cell 2006; 17: 851–61 Damrot J et al in preparation Abstracts C4-51 NDRG2 is a new HIF-1 target gene and necessary for the hypoxia-induced apoptosis in A549 cells L Yao, L Wang, N Liu and X Liu The Fourth Military Medical University, Xi’an, Shanxxi, CHINA The NDRG2 gene belongs to a family of N-MYC downstreamregulated genes (NDRG) and is expressed in many normal tissues NDRG1 gene expression has been shown to be regulated in the stress response of certain cells However, its function is not yet fully understood Many studies have demonstrated that hypoxia, one of the stress responses, induced apoptosis in several cell types In the current study, we investigated NDRG2 involvement in the hypoxia response and found that NDRG2 expression was markedly up-regulated, at both the mRNA and the protein level, in several tumor cell lines exposed to hypoxic conditions or similar stresses We also observed that the expression of NDRG2 was regulated by hypoxia induce factor (HIF-1) in tumor cells under hypoxia condition Three hypoxia-responsive elements (HRE) in the NDRG2 promoter were found HRE1 could directly bind HIF-1 in vivo Importantly, we found that silencing or enforcing the expression of NDRG2 could strongly inhibit or increase the apoptosis in tumor cells under hypoxic conditions The data showed that NDRG2 was able to translocate from the cytoplasm to the nucleus The segment from 101 to 178 amino acids of NDRG2 is responsible for its nuclear translocation Taken together, this study suggests that NDRG2 is a HIF-1 target gene and closely related with the hypoxia-induced apoptosis in A549 cells C4-50 A vital role of peroxisome-specific fatty aldehyde dehydrogenase in protecting against oxidative stress B Ashibe and K Motojima Meiji Pharmaceutical University, Tokyo, JAPAN C4-52 Secreted Hsp70 induces EGFR transactivation in Fatty aldehyde dehydrogenase (FALDH, ALDH3A2) is thought A431 cells to be involved in the degradation of phytanic acid, a saturated branched-chain fatty acid derived from chlorophyll However, the identity, subcellular distribution and physiological roles of FALDH are unclear because several variants produced by alternative splicing present in varying amounts at different subcellular locations Subcellular fractionation experiments not provide a clear-cut conclusion because of the incomplete separation of organelles We established human cell lines heterologously expressing mouse FALDH from each cDNA without tagging under the control of an inducible promoter and detected the variant FALDH proteins using a mouse FALDH-specific antibody One variant, FALDH-V, was exclusively detected in peroxisomal membranes Human FALDH-V with a N-terminal myc sequence also localizes to peroxisomes The most dominant form, FALDH-N, and other variants examined, however, were distributed in the endoplasmic reticulum A GC–MS-based analysis of metabolites in FALDHexpressing cells incubated with phytol or phytanic acid showed that FALDH-V not FALDH-N is the key aldehyde dehydrogenase in the degradation pathway and protects peroxisomes from oxidative stress In contrast, both FALDHs had a protective effect against oxidative stress induced by a model aldehyde for lipid peroxidation, dodecanal These results suggest that FALDH variants are produced by alternative splicing and share a vital role in protecting against oxidative stress in an organelle-specific manner ª 2007 The Authors Journal compilation ª 2007 FEBS A L Evdonin and N D Medvedeva Institute of Cytology RAS, St Petersburg, RUSSIAN FEDERATION Though initially Hsp70 was thought to be a typical intracellular protein, recent studies demonstrated that Hsp70 is being released into the blood or the conditional medium of cultured cells under stresss conditions The purpose of the present study was to define the functions of the secreted protein Previously we have shown that the initial steps of heat stress were characterized by ligand-independent EGFR (epidermal growth factor receptor) transactivation and concomitant secretion of Hsp70 EGFR transactivation occurred via an auto/paracrine pathway, since the conditional medium of heated cells also induce EGFR transactivotion The depletion of Hsp70 from conditioned medium of heated cells abolishes EGFR transactivation, indicating that the secreted protein is essential for EGFR transactivation Analysing the effect of pure Hsp70, which induced EGFR transactivation and activation of EGFR-dependent signaling pathways proved the finding Both heat stress and pure Hsp70 stimulate activation of TLR2/4 and their association with EGFR These results suggested that secreted Hsp70 mediates the cross-communication of TLR and EGFR signaling systems in A431 cells and provide new insight in the function of extracellular Hsp70 secreted from epithelial cells during heat stress 207 Abstracts Signal Transduction C4-53 Flavonoid and soft laser radiation effects on oxidative stress exposed mononuclear cells populations C4-55 Amino acid starvation of MCF7 cells induces the expression of PKCg in a 5¢UTR-dependent manner D Lixandru1, M Pislea1, E Panait1, T Seremet1, E Radu1, J Horvath2, E Tanos2, I Stoian1, V Atanasiu1 and E Katona1 ‘Carol Davila’ University of Medicine and Pharmacy, Bucharest, ROMANIA, 2LASEUROPA, Budapest, HUNGARY H Amit, M Shapira and E Livneh Department of Microbiology and Immunology, Faculty of Health Sciences and the Cancer Research Center, Ben Gurion University, Beer Sheva, ISRAEL Low power long wavelength laser irradiation healing effects are yet far from being explained, as there are the anti-oxidant, anti-inflammatory and anti-cancerous effects of bioflavonoids Targeting disclosure of partial mechanisms involved in soft lasers and flavonoids biological effects in normal and stress conditions, in present studies we monitored short and long term changes occurring in human mononuclear cell populations state and behaviour We used therapeutic lasers (680 nm/25 mW, and 830 nm/55 mW) to expose various cell suspensions (simple, mixed, cultured virtually alone or in co-culture, in presence/absence of cytokines/growth factors, presence/absence of various concentrations of quercetin or epigallocatechin gallate, and/or of various concentrations of H2O2 or CuCl2) to doses and irradiation regimes of therapeutic significance (dose densities up to 6J/cm2) Selecting appropriate surface antigen markers and intracellular molecular reporters (JC-1, DCFH-DA, PI, Hoechst, 7-AAD, AnnexinV-FITC,) mitochondrial membrane potential, intracellular ROS levels, cell viability, proliferation rate, cell cycle progression, and percentage of apoptotic and necrotic cells were assessed in various mononuclear cell populations The obtained data demonstrate significant photobiomodulation of flavonoid effects, level of oxidative stress, signal transduction and human mononuclear cells crosstalk Acknowledgment: Partial financial support of the Romanian Ministry of Education and Research (grant CNCSIS 924/2006 and grant CEEX 74/2006) is gratefully acknowledged Protein Kinase C is a family of phospholipid dependent serine/ threonine kinases that are involved in a wide variety of cellular processes, including membrane receptor signal transduction, cell proliferation and differentiation The members of the PKC family differ from one another in their primary structure, tissue distribution, subcellular localization and their activation signals The novel PKCg isoform is implicated in differentiation, proliferation, secretion and apoptosis We have recently reported that PKCg is localized in the golgi, ER and nuclear envelope and translocates to the nuclear envelope upon PMA activation and serum-starvation Here we report for the first time that the expression of PKCg is induced upon amino acids starvation in MCF7 cells The mRNA increase, measured using quantitative Real Time PCR, could only partially explain our data Thus, we explored the regulation of PKCg at the translational level Analysis of the human 5’UTR (untranslated region) of PKCg revealed that it is especially long, GC rich and contains two uORFs (upstream open reading frames) Since 5¢UTRs are known translational regulators we have examined whether the 5’UTR of PKCg plays a role in its induction following amino acids starvation We cloned the 5¢UTR of PKCg upstream to a luciferase reporter gene and demonstrated that its induction is 5¢UTR dependent Moreover, site-directed mutagenesis of each of the uATGs of the two uORF’s enhanced expression of a luciferase reporter gene, suggesting that the basal expression of PKCg is repressed due to its uATGs Our data demonstrate unique translational regulation of the PKCg isoform under stress induced by amino acids starvation C4-54 Photobiomodulation of flavonoids cellular effects, seen in human mononuclear cells C4-56 Translocation of PKCg to nuclear envelope upon etoposide induced DNA damage I Doaga1, T Seremet1, E Panait1, G Katona1, E Radu1, J Horvath2, E Tanos2, V Atanasiu1, L Katona1 and E Katona1 ‘Carol Davila’ University of Medicine and Pharmacy, Bucharest, ROMANIA, 2LASEUROPA, Budapest, HUNGARY A Tamarkin, A Maissel and E Livneh Ben-Gurion University of the Negev, Beer Sheva, ISRAEL Pursuing disclosure of molecular and cellular mechanisms involved in photobiomodulation, we focused on monitoring low level long wavelength laser irradiation effects in human T leukemia lymphoblasts and in peripheral blood derived adherent and non-adherent mononuclear cells cultured in presence/absence of various concentrations of quercetin or epigallocatechin gallate Therapeutic lasers were used to irradiate cell suspensions daily or every second day with total incident doses of 2–15 lJ per cell Peripheral lymphocytes and monocytes/dendritic cells, cultured virtually alone or in co-culture, in presence/absence of growth factors, were irradiated separately or together Appropriate molecular reporters were selected to track changes occurring in state of cell signaling key players, cell viability, and cell cycle progression The data obtained by phase contrast, fluorescence, and confocal microscopy, steady-state fluorimetry, and flow cytometry, document significant, flavonoid and radiation dose, irradiation regime, cell type, state, and microenvironment dependent photobiomodulation of flavonoid effects on mitochondrial network shape and size and cell signaling leading to survival/proliferation or apoptosis/necrosis Acknowledgment: Partial financial support of the Romanian Ministry of Education and Research (grant CNCSIS 924/2006 and grant CEEX 74/2006) is gratefully acknowledged 208 Protein kinase C (PKC) is a family of serin/threonine kinases, playing a central role in the regulation of cell growth, apoptosis and transformation Activation of PKCs is regulated by phosphorylations and intracellular localization, and their recruitment to membranes is a marker for activation The C-terminus phosphorylation sites, the hydrophobic and turn motifs, were shown to be important for the enzymes stability and function PKCg is a member of the novel subfamily We have recently reported that PKCg is localized in the Golgi, ER and nuclear envelope (NE) and translocates to the NE upon PMA activation and serum-starvation Moreover, association of PKCg with the cyclin E/Cdk2 complex was demonstrated at the perinuclear region upon serum-starvation Here we show, using confocal microscopy and chimeric GFPPKCg plasmids, that the anti-cancer drug etoposide induces translocation of PKCg to NE in MCF-7 and Hela cells The possible role of the C-terminus phosphorylations was investigated by using GFP-PKCg constructs mutated at the hydrophobic and turn motifs The hydrophobic site mutant S675E, mimicking the phosphorylated serine, translocated faster and more intensively to the NE following etoposide treatment The S675A mutant depicted the slowest translocation Our results suggest that etoposide induces activation of PKCg and that the phosphorylation at the hydrophobic site is important for it’s translocation to the NE This translocation could be important for PKCg functions in cell cycle regulation and resistance to anti-cancer drugs ª 2007 The Authors Journal compilation ª 2007 FEBS Signal Transduction Abstracts C4-57 Induction of uncoupling protein in neonatal cardiomyocytes in response to camptothecin C4-59 TGF-beta1 alters MAPK activation and bronchial epithelial cell proliferation in asthma ´ ´ E Valouskova1, J Vostalova1, P Jezek2 and M Modriansky1 Institute of Medical Chemistry and Biochemistry, Faculty of ´ Medicine and Dentistry, Palacky University, Olomouc, CZECH REPUBLIC, 2Institute of Physiology, Czech Academy of Science, Praha, CZECH REPUBLIC A Semlali1, E Jacques1,2 and J Chakir1 Laval University, Que´bec, PQ, CANADA, 2Centre de recherche de l’Hoˆpital Laval, Institut universitaire de cardiologie et de pneumologie, Que´bec, PQ, CANADA Uncoupling protein (UcP2) is present in minute amounts in the inner mitochondrial membrane, allowing mild uncoupling of mitochondrial respiration Uncoupling is inevitably accompanied by attenuation of superoxide formation, which appears to be a physiological function of Ucp2 UcP2 levels are up-regulated by thyroid hormones, TNFa, and by unspecified redox regulations due to oxidative stress We show that UcP2 expression in rat neonatal cardiomyocytes is induced by camptothecin, a DNA:topoisomerase I complex inhibitor lmol/l camptothecin induced UcP2 levels similarly to L-thyroxine after 24 h of treatment Comparing h and 24 h treatment, inductions were accompanied by a decrease in caspase-3 activity, whereas DNA damage increased Mitochondrial membrane potential in camptothecin-treated cardiomyocytes, assessed from JC-1 fluorescence in situ, was lower than in untreated control The difference is more apparent when comparing camptothecin-treated cardiomyocytes from UcP2 -/- and UcP2 +/+ mice Diethyldithiocarbamate, an NFkB inhibitor, had no effect on the camptothecin mediated induction, suggesting that NFkB pathway is not involved Also presence of phosphorylated JNK was not detected during camptothecin treatment, ruling out another possibility of induction mechanism Taken together our data provide further support to participation of UcP2 in antiapoptotic signalling triggered by DNA damage Acknowledgment: This research is supported by grants GACR 301/05/0221 and MSM6198959216 C4-58 Phosphorylation of small heat shock proteins and their interaction with 14-3-3 proteins I S Chernik Moscow State University, Moscow, RUSSIAN FEDERATION Small heat shock proteins (sHsp) form a large group of stress proteins playing important role in protection of the cell against different unfavourable conditions and regulation of cell cycle Many sHsp undergo phosphorylation that affects their quaternary structure and functioning Widely distributed 14-3-3 proteins interact with proteins, containing phosphorylated Ser/Thr residues and by these means regulate their activity In order to understand involvement of 14-3-3 in the regulation of mechanisms o sHsp activity we analysed effect of phosphorylation of sHsp on their interaction with two isoforms of 14-3-3 We found that neither human Hsp27, phosphorylated by MAPKAP2 kinase, no human Hsp22, phosphorylated by Erk1 and cAMP-dependent protein kinase, were able to interact with 14-3-3 However, by using different methods (size exclusion chromatography, chemical cross-linking and surface plasmon resonance) we found that human Hsp20 phosphorylated by cAMP-dependent protein kinase, forms tight complexes with gamma and zeta isoforms of 14-3-3.We suppose that in muscle, where concentration of both 14-3-3 and Hsp20 is very high, phosphorylated Hsp20 can effectively compete with different protein substrates of 14-3-3 and by this means affect multiple cellular processes Acknowledgment: This investigation was supported by the Russian Foundation for Basic Research ª 2007 The Authors Journal compilation ª 2007 FEBS Epithelial damage is a feature of asthma that results in release of growth factors that may affect epithelial cell proliferation The objective of our study is to evaluate the importance of TGF-b1 in regulating epithelial cell repair in asthma We evaluated the effect of TGF-b1 on EGF-induced proliferation and downstream signalling in epithelial cells obtained from asthmatic subjects compared to cells from healthy subjects Cell proliferation was evaluated by BrdU incorporation EGFR, MAPK, TGF-b receptors, Smads, SARA and cyclin-dependant kinase inhibitors were evaluated by Western Blot TGF-b1 and receptor expression were measured by RT-PCR and by ELISA Proliferation of epithelial cells at baseline and after EGF stimulation was significantly reduced in cells derived from asthmatic subjects compared to cells obtained from healthy controls EGF induced ERK1/2 phosphorylation was reduced in epithelial cells from asthmatics compared to cells from healthy controls This was paralleled with a reduced EGFR phosphorylation Addition of TGF-b1 significantly decreased EGFinduced cell proliferation TGF-b1 production was higher in asthmatic epithelial cells compared to normal cells This was supported by a high expression of pSmad and SARA in cells derived from asthmatics compared to normal subjects Inhibition of TGF-b1 signalling in asthmatic epithelial cells restored EGF-induced ERK1/2 phosphorylation and proliferation Our results suggest that there is a defect in EGF / TGF-b balance in bronchial epithelial cells obtained from asthmatic subjects C4-60 Resveratrol accelerates human endothelial cell senescence by activating NADPH-oxidase Y D C Schilder1, E H Heiss1, D Sorescu2 and V M Dirsch1 Pharmacy, Vienna, AUSTRIA, 2Emory University School of Medicine, Atlanta, GA, USA Resveratrol (RV), a red wine polyphenol, has cardiovascular protective properties, such as being anti-inflammatory or stimulating NO-production RV has been shown to increase life-span in simple organisms We hypothesized that RV may exert its vaso-protective effect by delaying endothelial cell senescence Human umbilical vein endothelial cells (HUVECs) were chronically exposed to 10 lmol/l RV until they entered senescence RVtreated cells hereby showed an overall shorter replicative life span (75 vs 90 days) with lower maximum cumulative population doublings (30 vs 60) compared to control cells Consistently, RV-treated cells showed more senescence-associated-b-galactosidase positive cells than controls at passage 19 (70 ± vs 27 ± 7%) Since oxidative stress has been implicated in senescence, we measured reactive oxygen species (ROS) Within 24 h, RV increased intracellular levels of ROS by 50% Mitochondria and NADPH-oxidases (Noxes) are the main sources of ROS in HUVECs We therefore made use of various mitochondrial (CCCP, MitoQ and rotenone) and Nox-inhibitors (apocynin and DPI) respectively, and found that only Nox-inhibitors reduced ROS to control levels We furthermore showed that senescence induced by RV and increased ROS levels were associated with cell cycle arrest in S-phase (130% vs control) Conclusion: RV accelerates senescence in HUVECs by causing cell cycle arrest in S-phase and oxidative stress via activation of Nox 209 Abstracts C4-61 Quercetin protects human mesothelial cells against exposure to peritoneal dialysis fluid A Riesenhuber, D C Kasper, R Vargha, M Endemann, K Kratochwill and C Aufricht Department of Pediatrics, Medical University of Vienna, Vienna, AUSTRIA Introduction: During peritoneal dialysis, mesothelial cells have been shown to undergo severe damage due to continuous exposure to peritoneal dialysis fluid (PDF) with cytotoxic physicochemical properties In this study we investigate the cytoprotective role of the bioflavonoid Quercetin in the in vitro model of peritoneal dialysis Methods: Immortalized human mesothelial cells (Met5A) were exposed either to regular growth medium or to standard acidic lactate-puffered PDF (Dianeal PD4Ò) or to a more biocompatible lactate–bicarbonate-buffered PDF (Physioneal 40Ò) Parallel cell cultures were supplemented with 200 lmol/l Quercetin Cytotoxicity was assessed qualitatively by morphologic assessment and quantitatively by the release of cytoplasmic lactate dehydrogenase and fluorescence-activated cell sorting (FACS) Results: PDF exposure with bioincompatible Dianeal PD4Ò resulted in severe disruption of cell cultures and in significantly increased LDH release (P = 0.0007 vs control) Addition of 200 lmol/l Quercetin significantly decreased the LDH release (P = 0.04 vs ‘pure’ Dianeal PD4Ò exposure), comparable to control exposure and to more biocompatible Physioneal 40Ò exposure (P = 0.37) and resulted in markedly preservation of cell culture monolayers and cellular viability as assessed by FACS Conclusion: Introduction of cytoprotective agents such as Quercetin may represent an alternate approach to protect mesothelial cells from cytotoxicity of frequently used PDFs, comparably effective to the introduction of novel more biocompatible PDFs C4-62 Proteomics analysis of the stress response in mesothelial cells after exposure to peritoneal dialysis fluids K Kratochwill1, M Endemann1, M Lechner2, C Siehs3, A Riesenhuber1, D C Kasper1, B Mayer3, K R Herkner1, A Rizzi2 and C Aufricht1 Department of Pediatrics, Medical University of Vienna, Vienna, AUSTRIA, 2Institute of Analytical Chemistry, University of Vienna, Vienna, AUSTRIA, 3Emergentec Biodevelopment GmbH, Vienna, AUSTRIA Peritoneal dialysis fluids (PDF) are known to cause injury of the mesothelial cell layer by their low biocompatibility The complex composition of these fluids has been shown to determine protein expression patterns of stress-related proteins in human mesothelial cells (Arbeiter et al., 2001) In order to investigate other proteins involved in the cellular response to stress caused by PDFs a proteomics approach was applied to the in vitro model of PD For usually non-lethal exposure times immortalised human mesothelial cells (Met5A) were subjected to either PDF or normal growth medium as control Both PDF-stressed and control cells were then allowed to recover for 24 h Cells were harvested and proteins were concentrated and desalted by methanol/chloroform-precipitation and separated by two-dimensional flatbed electrophoresis Protein patterns were visualized using Coomassie-staining and 2D-Western analysis The gel-images were analysed using the Delta2D software (Decodon) and significantly altered spots were identified by MALDITOF MS After optimising the workflow of sample preparation and staining techniques for the cells used, analysis of 36 gels (18 treated, 18 control) revealed a set of differentially expressed proteins based on normalisation techniques offered by the Delta2D software Bioinformatic exploration of the data indicates involvement of cytoskeletal reorganisation, glucose metabolism and expression of stress proteins as response of mesothelial cells to PDF exposure 210 Signal Transduction C4-63 CRF receptors form dynamic complexes at the plasma membrane but only type receptor increases lateral mobility after CRF binding L Milan-Lobo1, D Runzler2, E Gaubitzer2, G Kohler2, ¨ ¨ A Bonci3, M Freissmuth1 and H H Sitte1 Insitute of Pharmacology, Medical University of Vienna, Vienna, AUSTRIA, 2Max F Perutz Laboratories, University of Vienna, Vienna, AUSTRIA, 3The Ernest Gallo Research Institute, Emeryville, CA, USA Corticotropin releasing factor (CRF) is a neuropeptide that exerts its action within the hypothalamic-pituitary-adrenal axis CRF is involved in neuronal circuits that are relevant to mood, motivation, addiction and drug dependence CRF-receptors (CRF-R) form dynamic complexes to serve distinct functions like signal transduction, endocytosis, etc We visualized the dynamics of receptor complex formation by applying three fluorescence microscopy techniques in living cells: FRET, FRAP and fluorescence correlation spectroscopy (FCS) FRET documented that CRF-R existed as oligomers at the cell surface, an observation that is consistent with findings in other G-protein couple receptors At low occupancy by the agonist CRF, CRF-R preferentially interacted with and signaled via Gs to elevate cAMP However, at high concentrations of CRF (100 nmol/l), complexes were formed between CRF-R and Gi This observation indicated that high receptor occupancy favored alternative signaling pathways Receptor occupancy also affected mobility of CRF-R type since, interestingly, both FCS and FRAP showed that agonist binding increased receptor mobility This effect was agonist specific because it was blocked by a specific antagonist These real time measurements therefore indicate that, in vivo, receptor activation triggers a conformational change which enhances its mobility Our data can be best described by a model where the receptor is trapped in the inactive state in a large complex or signalosome and released upon agonist activation to become highly mobile C4-64 Interactions between cyclopentenone prostaglandins and Glutathione-S-Transferase in renal mesangial cells ´ ´ ´ F J Sanchez-Gomez, J Gayarre and D Perez-Sala Centro de Investigaciones Biologicas, CSIC, Madrid, SPAIN Cyclopentenone prostaglandins (cyPG) are naturally occurring arachidonate metabolites CyPG display anti-inflammatory, antiviral and antitumoral effects, due in part to their capacity to form covalent adducts with thiol groups These properties have elicited the study of the post-translational modification of proteins by cyPG Renal mesangial cells (RMC) are specialized smooth muscle cells that play an important role in glomerulonephritis through the generation of inflammatory mediators and the processing of extracellular matrix To identify protein targets for the addition of the cyPG 15-deoxy-D12,14-PGJ2 (15d-PGJ2) in RMC we have prepared a biotinylated analog that retains the reactive cyclopentenone moiety (15d-PGJ2-B) and mimics the effects of 15d-PGJ2, both on the inhibition of iNOS expression and in the induction of stress proteins, such as Hsp70 15d-PGJ2-B did not activate PPAR, thus supporting the involvement of PPAR-independent mechanisms in the antiinflammatory effects of cyPG Proteins covalently modified by 15dPGJ2-B were identified by chromatography on avidin beads and analysis of the eluates by Western blot Using this approach we identified glutathione S-transferase pi (GSTp), an enzyme involved in the detoxification of electrophilic compounds, maintenance of cellular redox status and cell viability Consistent with this, 15d-PGJ2 bound to and inhibited GST in vitro Binding of 15d-PGJ2-B to GSTp was blocked by glutathione or glutathione conjugates Overexpression of GST in RMC was associated with decreased PPAR activation by 15d-PGJ2 In this work, we explore the complex interactions between 15d-PGJ2 and GST both in cells and in vitro, and study their consequences for the inflammatory response in RMC ª 2007 The Authors Journal compilation ª 2007 FEBS Signal Transduction C4-65 Oxidative stress triggers the changes of mitochondrial reticulum morphology in HeLa cells O K Nepryakhina, K G Lyamzaev, O Y Pletjushkina and B V Chernyak Moscow State University, Moscow, RUSSIAN FEDERATION We studied the changes in mitochondrial morphology induced by oxidative stress in HeLa cells Addition of hydrogen peroxide caused the fission of mitochondria, and respiratory chain inhibitors piericidin and myxothiazol (affectors of Complex I or Complex III, respectively) accelerated this process Preincubation of cells with mitochondria-targeted antioxidant, 10-(6’-ubiquinolyl)decyltriphenilphosphonium (MitoQ) prevented fragmentation induced either by one agent or by combination of them We suggest that the process of mitochondrial fission depends on ROS, produced by mitochondria themselves To study the further steps of changes in mitochondrial morphology we used the uncoupler, FCCP In these experiments we observed the clustering of mitochondria taking place after mitochondrial fission In the presence of tubulin polymerization inhibitor nocodazole the gathering of mitochondria around the nucleus and formation of clusters happen much faster Our preliminary data allow suggesting that cytochalazin A, inhibitor of actin polymerization, delays this process We observed that mitochondrial clustering is the next step in changes of mitochondrial morphology after fragmentation induced by oxidative stress We suppose that this process plays an important role in protecting cell from reactive oxygen species burst and subsequent cell death C4-66 The concentration of circulating immune complexes (CIC) of Black Sea fish at natural and experimental conditions N S Kuzminova Institute of Biology of the Southern Seas, Sevastopol, UKRAINE Marine fish are very sensitive to environmental and anthropogenic impact led changes of their metabolism Study of the components of defense reactions, is important task for the evaluation of state of fish impacted different pollutants Dangerous and, sometimes, lethal physiological effects are resulted low xenobiotic concentrations, particular, in legal concentrations It can be connected with a long of impact time or delicate defence system Thus, the aim of the work was to study the level of blood serum circulating immune complexes (CIC) of Black Sea fish The Black sea fish species from Sevastopol bays (Ukraine) were investigated: Mediterranean scad, high-body pickerel, red mullet, shore Mediterranean rockling, stargazer, goby fish, sea scorpion, flounder Scorpion fish Scorpaena porcus L was used for estimation of influence of legal level of oil on CIC also It was noted that the level of CIC was highest for sea scorpion, red mullet and flounder which are bottom and near bottom species However these immune adaptive reaction such tendency show the more negative impact of pollution on bottom fish, that can lead to decreasing of its hunting The concentration of small immune complexes in fish serum which were treated of oil was increased It is interesting to note, that the differences between control and experimental values were elevated with the exposition time increasing in times in day Thus, without mortality effects, the natural conditions and oil in legal concentration led to negative physiological alterations That is why the determination of legal concentration of xenobiotics (in particular, oil) should be evaluated more carefully, taking into account the determination of defence mechanisms of organisms also ª 2007 The Authors Journal compilation ª 2007 FEBS Abstracts C4-67 Pre-treatment of irradiation protects kidneys from ischemia and reperfusion injury in mice K Park and J Kim Kyungpook National University School of Medicine, Daegu, REPUBLIC OF KOREA Irradiation simultaneously activates death and survival machineries in cells We hypothesized that previous irradiation would confer ischemia/reperfusion (I/R) injury resistance to kidneys Eight gray (Gy)-irradiation in a single dose using a cesium-137 source irradiator protected renal function and structure from 20, 28 and 32 of bilateral renal ischemia induced days after irradiation in mice Using and Gy in a single dose, or using fractionated doses totaling Gy (2 Gy · 3) and Gy (2 Gy · 4) did not protect kidneys from 28 of ischemia induced day after irradiation Additionally, Gy in a single dose significantly increased heat shock protein (HSP)-27 and -72 expression in kidneys, whereas and Gy in a single dose, and Gy in a fractionated dose did not The increased levels of HSP-27 were similar to those observed days after 15 of ischemic preconditioning (which resulted in partial kidney protection to subsequent I/R), however, these levels did not reach those attained after 30 of ischemic preconditioning (which completely protected kidneys from subsequent I/R) Post-ischemic activation of c-Jun N-terminal stress-activated protein kinase (JNK1/2) was significantly greater in the non-irradiated mice than in the Gy-irradiated mice, whereas activation of extracellular signal-regulated kinase 1/2 (ERK1/2) was significantly greater in irradiated mice In conclusion, Gy in a single dose protected kidneys from I/R injury and this protection was associated with the increased expression of HSP-27 and -72, and an increased ratio of ERK1/2 activation to JNK1/2 activation C4-68 Analysis of the impact of cadmium on oxidative stress in experimental rat model J Zidkova1, K Kontrova1, K Szabova1, K Vanova1, A Fucikova2, J Szakova2, P Tlustos2, I Sestakova3, V Zidek4 and M Pravenec4 Institute of Chemical Technology in Prague, Prague, CZECH REPUBLIC, 2Faculty of Agronomy, Czech University of Agriculture, Prague, CZECH REPUBLIC, 3Institute of Physical Chemistry, Academy of Sciences, Prague, CZECH REPUBLIC, Institute of Physiology, Academy of Sciences, Prague, CZECH REPUBLIC An increasing attention has been paid to contamination of the environment with heavy metals in soil Analysis of the impact of heavy metals, especially Cd and Pb individually and in combination in the form of inorganic salts corresponding to daily intake in experimental diets on oxidative stress in experimental animals was studied Metallothioneins (MT) are considered to be important in homeostatic control and their role is examined in connection with oxidative stress Relationship between the level of hepatic MT and the exposure of the rat to heavy metals has been shown as well as the influence of the genetic background of the animals Quantity of the rat MT was performed by differential pulse polarography Determination of the specific activity of oxidative stress enzymes was performed in heparinized plasma Peroxidase, superoxiddismutase, glutathione reductase, glutathione-S-transferase and catalase were selected for our studies In the case of glutathionreductase, the increase of enzymatic activity is considerable after intraperitoneal application of cadmium solution in comparison with cadmium application in diet or tap water Acknowledgment: Funding: MSM 6046137305 and GACR 523/ 06/0439 211 Abstracts C4-69 Effect of cadmium and zinc ions on protein synthesis and apoptotic activity in mouse organs I Staneviciene, I Sadauskiene, A Smalinskiene, L Ivanoviene, A Kasauskas and L Ivanov Kaunas University of Medicine, Kaunas, LITHUANIA The effects of weeks-term exposure to Cd and Zn ions on translational machinery and death of mouse liver cells have been evaluated in vivo We found that after intraperitoneal injections of CdCl2 solution the intensities of protein synthesis in the mice liver reduced by 34%, in kidney by 39%, and in heart by 43% as compared with norm Exposure of mice to Cd resulted in a 44% decrease of the acceptor activity of leucine tRNA and the activity of leucyl-tRNA synthetase was increased by 59% versus the control level The number of TUNEL+ cells was significantly higher in the liver of Cd-exposed mice than in control Zn ions did affect neither translation nor the apoptotic activity Examination of the common effect of Zn and Cd ions showed the protective effect of Zn on translation machinery and death of mouse liver cells Zn ions mitigated Cd induced inhibition of protein synthesis intensity in the mice liver, kidney and heart by 52%, 68%, and 50%, respectively and decreased a number of apoptotic cells The acceptor activity of leucine tRNA under action of both metal ions returned to the control level Comparing to control, Zn pre-treatment brought about an activation of leucyl-tRNA synthetase by 24% of the liver after Cd exposure Thus, Zn ions are capable to protect the translation machinery against inhibition and to normalize an increase in the apoptotic activity of liver cells during administration of Cd for weeks C4-70 Probing regulatory proteins for vascular contraction by DNA microarray J Kim, S Kim, D Yang, E Chung, M Kim, J Cho, W Park and I Kim Kyungpook National University School of Medicine, Daegu, REPUBLIC OF KOREA We hypothesized that a heat-shock response modulates contractility of vascular smooth muscles Human radial arteries strips were mounted in organ baths, exposed to 42°C for 45 min, and returned to equilibrate at 37°C This study examined gene expression changes associated with heat-shock response in radial arteries of patients with hyperlipidemia using a high-density microarray that contained 5763 human cDNA The results of a microarray hybridization experiment from four different radial arteries were analyzed and classified by the Cluster program Among these differentiallyexpressed genes, HSP70, HSP10 and HSP60 were significantly increased by the heat response and 15 genes also increased, 22 genes decreased in non-HSP genes Among these 37 genes, crystallin, alpha B (CRYAB) (up 1.92- fold), myosin light polypeptide kinase (MYLK) transcript variant 8.6 (up 1.70 -fold, up 1.68- fold ), catenin (cadherin-associated protein, alpha-like (CTNNAL1) ) (down-0.57 fold) and tropomyosin (TPM3) (down 0.68- fold) were thought to be related with the contraction In conclusion, our data showed the gene expression profile by heat shock and these results suggested that these genes may play a pivotal role in the augmentation of vascular contraction after heat shock 212 Signal Transduction C4-71 Heat shock augments myosin phosphatase target-subunit phosphorylation J Kim1,2, S Jeon1, I Baek1,2, Y Seok1,2, H Shin3 and I Kim1,2 Kyungpook National University, Daegu, REPUBLIC OF KOREA, BK21Project, Daegu, REPUBLIC OF KOREA, 3Dongguk University, Kyongju, REPUBLIC OF KOREA Our previous study demonstrated that heat shock augmented vascular contraction in response to KCl In the present study, we hypothesized that heat shock augments myosin phosphatase targetsubunit (MYPT1) phosphorylation resulting in augmented vascular contraction Endothelium-denuded rat aortic rings were mounted in organ baths, exposed to heat shock (42°C for 45 min), and subjected to contraction with 50 mmol/l KCl h after the heat shock followed by western blot analysis for phosphorylation of MLC20 (the 20 kDa light chains of myosin II) or MYPT1 The contractile responses in both control and heat shock-treated aorta were inhibited by Y27632, an inhibitor of Rho-kinase The level of the MLC20 and MYPT1Thr855 phosphorylation in response to KCl was higher in heat shock-treated aorta than that in timed-control The increased MYPT1Thr855 phosphorylation was inhibited by Y27632 (1.0 lmol/l) in parallel with inhibition of MLC20 phosphorylation and vascular contraction These results indicate that heat shock augments MYPT1 phosphorylation resulting in augmented vascular contraction C4-72 Protein Tyr-phosphorylation levels in human platelets stimulated with von Willebrand factor under shear stress R Deana, M Pavanetto, A Zarpellon, A Folda and A Donella-Deana University of Padova, Padova, ITALY Von Willebrand factor (VWF) is a large, multimeric glycoprotein present in blood plasma, which plays an important role in the primary haemostasis and platelet adhesion to subendothelial connective tissue VWF binds to the receptor GPIba of resting platelets evoking a complex intracellular signaling that culminates with the activation of integrin aIIbb3 which becomes competent to bind fibrinogen and other cytoadhesive proteins mediating aggregation In this study we monitored the protein Tyr-phosphorylation (TP) level in platelets stimulated by VWF under low shear rate The Tyr-phosphorylation pattern of ristocetin/vWF-stimulated platelets showed an increase in the phosphorylation of a protein band of about 72 kDa, (which likely consists of the TPK Syk), and a parallel dephosphorylation of a protein of about 58 kDa The dephosphorylation was strictly dependent on the VWF-mediated integrin aIIbb3 activation Employment of specific inhibitors demonstrated that PLC gamma and TPKs Src and Syk, but not PLA2 and COX, play an important role in this dephosphorylative event The use of the phosphatase inhibitor stibogluconate and immunoprecipitation experiments with specific anti-phosphatase antibodies suggested that PTP-1B, but not SHIP-1 and SHIP-2, is likely responsible for the VWF-induced p58-dephosphorylation Experiments aiming to identify the p58 protein are in progress ª 2007 The Authors Journal compilation ª 2007 FEBS Signal Transduction C4-73 Glycosaminiglycans as an instrument in work of molecular machine of connective tissue M V Knyazyeva1, A V Prokopyuk2, O I Babaeva2, S V Ivannikova2 and V A Knyazyev2 Kharkov National University, Kharkov, UKRAINE, 2NGO ‘New thinking in medicine’, Kharkov, UKRAINE The experiment with the use of 110 male white rats aged 3- and 12-months has been carried out to explain the reported changes in the compositional content of glycosaminoglycans (GAG) in the blood serum of patients having different diseases: aorta aneurism with rupture damage (AA) and ovarian cancer of III-IV stages (OC) after neoajuvant polychemotherapy (NChT).During the experiment we investigated the effect of stress factor- 30-day hypokinesia (HK) and readaptation to normal life(RA)- on the connective tissue (CT) of different localization Aorta, myocardium, fragment of femoral bone tissue were taken for the investigation It was found that collagen, non-collagen proteins and GAG markers were characterized by phase changes in the HK and RA dynamics.The phases of hydroxyproline, proline, tyrosine, hexosamine (HA), hexuronic acids (HX) changes in the aorta, myocardium and bone not coincide in time in response to one and the same stress factor The comparison of bone, myocardium, aorta tissue reactions on the 30-th day of HK and RA in the rats under investigation showed that aorta tissue was the most sensitive to the above stress Stable simultaneous reduction of the components of GAG- HA and HX in the aorta tissues in the group of older rats is in accordance with the changes of GAG level in blood in the animals under investigation and with the ones in patients having AA and OC after the use of NChT The experiment showed that the vessels (but not bone) tissues were the main source of CT-metabolites in blood The data allowed to consider GAG as an instrument in work of molecular machine of CT in different pathologies and stress Abstracts C4-75 Experimentally induced acute renal failure: determination of oxidative/antioxidative balance T P Cvetkovic, V Djordjevic, D Pavlovic, G Kocic, T Jevtovic, D Sokolovic and J Basic Institute of Biochemistry, Medical Faculty, Nis, SERBIA Acute renal failure (ARF) affects 5–20% of patients in the intensive care unit (ICU) in spite of different etiological causes (ARF) is followed by intensive oxidative stress Complex manifestations such as rapid depletion in cellular ATP, increase in cytosolic calcium, activation of phospholipases and proteases, infiltration of neutrophils as well as arachidonic acid metabolism may reflect changes in the steady-state concentration of pro/antioxidants resulting in oxidative stress All experiments were performed on male Sprague Dawley rats Ischemia was induced by ligation of A renalis with different time intervals of reperfusion (2 and 24 h) In myoglobinuric model of ARF, glycerol (50%, v/v) was injected im (8 ml/kg) Uremic ARF was induced by bilaterally ligated ureters MDA content as well as content of CRD were significantly elevated in kidney and plasma in all models of ARF compared to control values, (P < 0.01) In spite of the kidney exposition to oxidative stress, anti-oxidative factors remove free radicals protecting them These results indicate that the concentration of GSH was similar in RBC and kidney, but the activity of enzymes was significantly changed compared to the control groups The results of this experimental study show that oxidative stress is one of the most important pathogenetic mechanisms in ischemia/reperfusion, mioglobinuric as well as in obstructive acute renal failure (ARF) C4-74 Study of regulatory proteins of eIF2-alpha subunit phosphatase/protein phosphatase (PP1) during ischemic reperfusion L Garcia-Bonilla1, C Cid2, M Gomez-Calcerrada1, I Ayuso1, J Burda3, M Salinas1 and A Alcazar1 Hospital Ramon y Cajal, Madrid, SPAIN, 2Centro de Astrobiologia, CSIC-INTA, Torrejon de Ardoz, SPAIN, 3Institute of Neurobiology, Kosice, SLOVAKIA Phosphorylation of the eukaryotic translation initiation factor subunit alpha (eIF2a) plays an important role in the inhibition of translation, including the postischemic reperfusion Translation is strongly inhibited upon reperfusion after an episode of cerebral ischemia and has been related with the induced neuronal death The eIF2a dephosphorylation is in parallel to the restoration of the translation at later reperfusion Protein phosphatase (PP1) has been established as the physiological eIF2a phosphatase and compelling evidences indicate that PP1 contributes to the cellular recovery from stress by acting as an eIF2a phosphatase Dephosphorylation by PP1 is the result of the interaction of PP1 catalytic subunit (PP1c) with numerous regulatory proteins These proteins function as targeting subunits, inhibitors or modulators, and converts PP1c into different complexes that differ in their distinct substrate specificities and regulation Thus, we considered of interest to study PP1 complexes in the postischemic reperfusion in an animal model of cerebral ischemia We have studied the growth arrest and DNA damage-inducible protein 34 (GADD34), a PP1 regulator that induce eIF2a dephosphorylation, in addition to 78kDa glucose-regulated protein (GRP78), 71-kDa heat shock cognate protein (HSC70) and dopamine- and cAMP-regulated phosphoprotein 32 kDa (DARPP32) We show that associations of these regulatory proteins with PP1c changed during reperfusion and these changes correlated with phosphorylated eIF2a levels (FIS 05/0312 and 05/1099) ª 2007 The Authors Journal compilation ª 2007 FEBS C4-76 Therapeutically effects of Techirghiol Sapropelic Mudin oxidative stress at patients with osteoarthritis V Marin1, D Profir1, O Surdu1 and N Rosoiu1,2 Balneary And Rehabilitation Sanatorium, Techirghiol, ROMANIA, Universitatea ‘OVIDIUS’, Constanta, ROMANIA Articular cartilage degradation/regeneration mechanisms represent elements of high topicality in research and medical practice due to the ever-increasing incidence of articular degenerative disorders The oxidative stress is considered to play an important role in degeneration mechanism of articular cartilage The saprogenic mud is black deposit, rich in colloidal iron hidrosulphur, from the bottom of the salty lakes and seas It is formed under the action of microorganisms, from the inorganic substance of the soil, the flora and fauna of the aquatic basin, as the consequence of some biological and chemical transformations, during the biological epochs The active chemical components of the saprogenic mud and of its natural extracts spread in the body, either by passing through the skin barrier or by entering directly in the circulation according to the way of administration-and It releases local and general functional reactions, inhibiting or activating some enzymatic systems or intermediate metabolites We performed a clinical study on 30 patients with osteoarthritis with different localization To these patients we determined the values of glutathione-reductase, total antioxidant status (TAS), SOD, and GSH before and after mud applications in order to assess the oxidative stress of the body cells and relevance of value therapy with Techirghiol Sapropelic Mud of osteoarthritis 213 Abstracts C4-77 Assessment of DNA base oxidation and antioxidant defense in postmenopausal women under hormone replacement therapy T Akcay1, Y Dincer1, I Saygılı1, H Seyisoglu2 and ¸ E Ertungalp2 Department of Biochemistry, Cerrahpasa Medical Faculty, Istanbul University, Istanbul, TURKEY, 2Department of Gynecology and Obstetrics, Medical Faculty, Istanbul University, Istanbul, TURKEY Objectives: The aim of the present study is to evaluate oxidative stress by investigating oxidative DNA damage as Fpg-sensitive sites, glutathione peroxidase (GPx), superoxide dismutase (SOD) activities and reduced glutathione (GSH) level, nitrite level as stabile end -product of NO in women receiving hormone replacement therapy (HRT) Study Design: The present study has investigated the effects of HRT on nitrite level, GSH level, activity of SOD, GPx and oxidative damage to DNA in the comet assays, by measuring levels of Fpg-sensitive sites in controls and post menopausal women Results: Although no significant differences were found in the SOD activities, in all groups receiving HRT, increased DNA oxidation together with an increased GPx activity and nitrite level as well as a decreased GSH level as compared controls was observed Estrogen alone or estrogen in combination with progesterone and duration of use did not significantly alter the results Conclusions: HRT caused oxidative stress by looking at DNA oxidation C4-78 Effect of olive oil polyphenols on erythrocytes oxidative damage F Paiva-Martins1, J Fernandes2, A Santos-Silva3,4, L Belo5,4, F Borges6 and S Rocha5,4 CIQ, Dep Quı´mica, Faculdade de Cieˆncias, Universidade Porto, Porto, PORTUGAL, 2Faculdade de Cieˆncias Universidade Porto, Porto, PORTUGAL, 3Department of Bioquı´mica, Faculdade de ´cia, Universidade Porto, Porto, PORTUGAL, 4IBMC, Farma ´cia, Porto, PORTUGAL, 5Dep Bioquı´mica, Faculdade de Farma ´cia, Porto, PORPorto, PORTUGAL, 6IQFM, Faculdade de Farma TUGAL Healthy subjects are equipped with red blood cells (RBC) antioxidants, namely glutathione, tocopherol and ascorbate Ascorbate is highly susceptible to oxidation in plasma, being recycled from its oxidized form in RBCs However, if reactive oxygen species are overproduced or if the endogenous defenses are impaired, ‘oxidative stress’ will result, inducing damage on the erythrocyte membrane and production of powerful promoters of oxidative processes in the blood We studied the capacity of the most important olive oil PhC, namely hydroxytyrosol (Hy), 3,4-dihydroxyphenylethanol-elenolic acid (EA), 3,4-dihydroxyphenylethanol-elenolic acid dialdehyde (EDA) and oleuropein (Ol), to protect RBCs from injury induced by H2O2, and by 2,2’-Azobis(2-amidinopropane hydrochloride) (AAPH) All PhC protected RBCs from hemolysis induced by H2O2 and AAPH (P < 0.05) The order of activity against H2O2 was EDA > EA > Hy > Ol The same order was observed against AAPH, with exception for EA, which presented no protection All compounds reduced the membrane bound hemoglobin, with EDA showing the highest reduction Interactions of these PhC with membrane proteins were also observed in the protein, showing the existence of further mechanisms of protection besides the scavenging activity EDA, the most abundant PhC found in olive oil, may be of great importance regarding the protective effect of olive oil 214 Signal Transduction C4-79 Abstract withdrawn C4-80 Plasma adhesion and inflammation markers in subjects with impaired and diabetic glucose tolerance ă D Konukoglu1, S Fırtına1 and O Serin2 Department of Biochemistry, Cerrahpasa Medical Faculty, Istanbul University, Istanbul, TURKEY, 2Biochemistry Department, Taksim Education and Research Hospital, Istanbul, TURKEY The effects of acute hyperglycemia on endothelial dysfunction marker, asymmetrical dimethyl-L-arginine (ADMA), plasma soluble adhesion molecules , intercellular adhesion molecule (ICAM-1) and vascular adhesion molecule (VCAM-1) and oxidative and inflammatuar marker, Secretory phospholipase A2 (sPLA2) activities, were measured in subjects with normal glucose tolerance (NGT, n = 35), impaired glucose tolerance (IGT, n = 25) and diabetic glucose tolerance (DGT, n = 20) At baseline plasma ADMA, sICAM1 and sCRP concentrations and plasma sPLA2 activities were significantly higher in both IGT and DGT groups than in NGT group (each P < 0.01) DGT group have significantly higher plasma sICAM-1 and ADMA concentrations than IGT group (for each P < 0.01).Two hours after glucose loading, plasma ADMA and sCRP concentrations and sPLA2 activities were significantly elevated in all three groups when compared with baseline levels (for each, P < 0.01) Plasma sVCAM-1 and s ICAM concentrations were elevated from baseline levels after glucose loading in IGT and DGT group (P < 0.01, for each comparison) Correlation analysis indicated that there is a significant relationship between endothelial dysfunction markers and soluble adhesion and oxidative markers In conclusion, plasma concentrations of endothelial dysfunction, inflammation and oxidative markers were elevated in prediabetic state C4-81 Nitric oxide consumption by circulating neutrophils in sickle cell disease M Aslan1 and D Canatan2 Akdeniz University School of Medicine, Antalya, TURKEY, Suleyman Demirel University School of Medicine, Isparta, TURKEY Interaction of nitric oxide (NO) with enzymatic sources of reactive inflammatory mediators exerts modulatory actions on inflammatory signaling mechanisms Therefore, we aimed to measure NADPH oxidase and cylooxygenase (COX) activities and determine NO consumption in neutrophils isolated from sickle cell disease (SCD) patients and controls Functional assay of NADPH oxidase was performed by measuring superoxide release, which was similar in both groups at basal conditions and in response to 10 lmol/l fMLP stimulation Total COX activity was significantly increased in SCD neutrophils The increase in total COX activity observed in SCD neutrophils was due to enhanced activity of COX-2, differentiated by using the isoform-specific inhibitors DuP-697 and SC-560 Western blot analysis of COX-2 protein level in SCD and control neutrophils confirmed significantly increased enzyme activity in the diseased group Electrochemical measurement of NO consumption performed in the presence of an NO donor (10 lmol/l, PAPA NONOate) both under basal conditions and after 10 lmol/l fMLP stimulation, revealed a significant decrease in SCD neutrophils compared to controls The presented data suggests that decreased NO uptake by SCD neutrophils can alter redox signaling and impact on vascular inflammation associated with the disease Acknowledgment: This study was supported by a grant from TUBITAK No: SBAG-2797 ª 2007 The Authors Journal compilation ª 2007 FEBS Signal Transduction Abstracts C4-82 A disease-causing variant of short-chain acyl-CoA dehydrogenase promotes oxidative stress C4-84 IL-1b-induced matrix metalloproteinase-9 expression via transactivation of Src, PDGFR, and Akt pathways in rat brain astrocytes S P Schmidt1, T J Corydon2 and N Gregersen1 Research Unit for Molecular Medicine, University of Aarhus, Aarhus, DENMARK, 2Institute of Human Genetics, University of Aarhus, Aarhus, DENMARK C Wu and C Yang Chang Gung College Of Medicine And Technology, Taipei, TAIWAN Short-chain acyl-CoA dehydrogenase deficiency (SCADD) is a rare recessively inherited metabolic disorder, affecting the mitochondrial b-oxidation Patients are usually presenting neuromuscular features such as developmental delay and hypotonia To investigate the pathogenesis of the disease, astrocytic cells stably expressing five different variants of the SCAD gene, previously identified in SCAD patients, were developed by transduction In the cloning process, the viability of cells expressing two severe variant proteins was severely reduced, compared with cells expressing the wildtype (wt) protein One of these was the rare variation 319C > T (R83C), which is unable to assemble into catalytically active tetramers, as well as having aggregational tendencies in vitro To investigate whether this variation inflicts with the ability of the cell to overcome a stress-full situation, cells expressing the wt or the 319C > T variation was subjected to heat stress of 40°C, and the stress-response was followed over a time period of 24 h, monitored by selected stress response genes (Hsp70, Hsp60 and HO-1 (Hemeoxygenase-1)) The cell line expressing the R83C variant protein revealed an elevated production of HO-1 compared with the wt cells, indicating oxidative stress, elicited by the misfolded mitochondrial SCAD variant protein Mitochondria of the two cell-lines will be subjected to quantitative proteomics using the method Stable Isotope Labeling with Amino acids in Cell culture (SILAC), to observe differences in the mitochondrial proteomes C4-83 LPS induces VCAM-1 expression via Src/EGFR/ PI3-K/Akt pathway in human tracheal smooth muscle cells W Lin and C Yang Chang Gun University, Tao-Yuan, TAIWAN In our previous study, LPS has been shown to induce vascular cell adhesion molecule-1(VCAM-1) expression through MAPKs and NF-jB in human tracheal smooth muscle cells (HTSMC) In addition to these pathways, the non-receptor tyrosine kinases (Src), EGF receptor (EGFR), and phosphphatidylinositol 3-kinase (PI3K) have been implicated in linking to expression of several inflammatory target proteins Here, we further investigated whether these different components involved in LPS-induced VCAM-1 expression in HTSMC We initially observed that LPS-induced VCAM-1 promoter activity, protein and mRNA expression was attenuated by the inhibitors of Src (PP1), EGFR (AG1478), PI3-K (LY294002 and wortmannin), and Akt (SH-5), revealed by reporter gene assay, Western blotting, and RT-PCR analyses Furthermore, LPS-stimulated Src, EGFR, and Akt phosphorylation was inhibited by PP1, AG1478, and wortmannin, respectively Moreover, VCAM-1 expression was blocked by p300 inhibitor (curcumin) pretreatment We further confirmed that LPS-stimulated Akt activation which translocated into nucleus and associated with p300 and VCAM-1 promoter region revealed by immunofluorescence assay and chromatin immunoprecipitation This association of Akt and p300 to VCAM-1 promoter was inhibited by PP1, AG1478 and wortmannin LPS-induced up-regulation of VCAM-1 would enhance the adhesion of neutrophils onto HTSM monolayer, which was inhibited by PP1, AG1478, LY294002, wortmannin These results suggested that Akt phosphorylation mediated through transactivation of Src/EGFR promoted the transcriptional p300 activity and eventually led to VCAM-1 expression induced by LPS ª 2007 The Authors Journal compilation ª 2007 FEBS Matrix metalloproteinases (MMPs) have been shown to be responsible for degradation of extracellular matrix and associated with various inflammatory diseases IL-1b has been shown to transactivate Src, growth factor receptors, and phosphatidylinositol 3-kinase (PI3-K)/Akt Our results showed that IL-1b induced MMP-9 mRNA and protein expression determined by RT-PCR and zymographic analysis, respectively These results showed that IL-1b stimulated phopshorylation of Src, Pyk2, PDGFR and Akt was mediated by Src and PDGFR, because the inhibitors of Src (PP1) and PDGFR (AG1296) attenuated the IL-1b-induced responses Moreover, PP1, AG1296, and LY294002 inhibited IL-1b-induced MMP-9 mRNA expression In conclusion, our data demonstrated that in RBA-1 cells, IL-1b-stimulated MMP-9 expression was mediated, at least in part, through transactivation of c-Src, Pyk2, PDGFR, and PI3-K/Akt C4-85 Cigarette smoke-mediated necrosis: a consequence of two independent toxicological mechanisms A Csordas, Jr.1, G Wick1 and D Bernhard2 Division of Experimental Pathophysiology and Immunology, Biocenter, Innsbruck Medical University, Innsbruck, AUSTRIA, Cardiac Surgery Research Laboratory, Department of Cardiac Surgery, Innsbruck Medical University, Innsbruck, AUSTRIA Despite various preventive strategies implemented so far, cigarette smoke (CS) consumption remains a highly prevalent risk factor for the development of accelerated CVD However, the initial insult responsible for accelerated atherogenesis in smokers is unknown During the last couple of years, we investigated the ability of CS to act as a stress factor for endothelial cells Besides mediating pro-atherogenic morphological alterations of endothelial cells, CS causes an atypical mode of necrosis-like cell death In the present study, we embarked on an in-depth analysis of the impact of CS on the mitochondrial respiratory chain as related to cell death induction We found that cellular ATP levels are maintained throughout the entire process of cell demise Moreover, though mitochondria undergo oxidative attack, respiration appears unaffected by CS constituents Wash out of CS-extract of cells that have undergone oxidative damage resulted in prevention of cell death However, when CS-extract that has been deprived of its oxidative capacity was added to these cells instead of fresh medium, necrosis induction was again visible At the same time, the nonoxidizing CSE abrogated cellular protein synthesis In summary, these results question an adverse impact of CS consumption on mitochondria as a toxicology mechanism relevant for endothelial cell damage, and instead point to the role of oxidative damage coupled with impaired protein synthesis in CS-induced cell death 215 Abstracts C4-86 If the stressed cell could develop mutations resulted from DNA bases tautomer formation inside the cell? A review Y I C Cherepenko Institute of Molecular Biology and Genetics, Kyiv, UKRAINE Spontaneous mutations are a life feature The problem is raised how to control their endogenous sources especially in malignancies accumulating multiple mutations The wonder-drug Gleevec becomes inefficient because of rapid arising resistant mutations The sources are different One covers DNA damage with hydrolytic, oxidative stress factor action and H2O action in two G neighbors The other concerns the DNA transactions with errors in replication, repair, recombination and transpositions Changing the kinetics of DNA transactions has mutagenic effect Epigenetic alterations of DNA and histones lead to mutation induction The adaptive mutations or increasing mutation rate in prokaryotes under stress is a new insight to study cell potential to mutate In eukaryotes with the germline and soma isolated this could make no sense Immortal strand hypothesis (J Cairns) explains how the mutation rate could be slowed down Quantum-chemical calculations showed that not only Watson– Crick pairs but also rare non-canonic pairs (e.g A: C, G: T due to the proton ability to migrate from one to another atom forming base tautomer) could be inserted into DNA At cell homeostasis tautomer effect could be negligible Under stress conditions with DNA damaged the pool of dNTP inside the cell is 4-fold elevated compared to that of normal S-period Due to this elevaion without DNA damage cells mutate 3-fold more in comparison to wild type cells and 2-fold more when DNA is damaged (A Chabes et al.) This difference could be explained with tautomer effects seen in the former case and masked by translesion polymerases work in the latter case Control of the dNTP pool as overcrowding molecules able to tautomer transitions could be an effective tool to control the mutations induction C4-87 Kinetic properties of lignin peroxidase in pistachio nut extract K Khorsandi and E Keyhani University of Tehran, Tehran, ISLAMIC REPUBLIC OF IRAN Pistachio nuts, produced by the pistachio tree (Pistacia vera L.) are a rich source of essential nutrients, fiber and protein Although their nutrient composition has been well characterized, there is no reported research on the biochemistry of pistachio nuts Peroxidases are hemoproteins that fulfil a wide range of physiological functions while scavenging hydrogen peroxide produced by oxidative stress or as by-product of aerobic metabolism In this work, lignin peroxidase activity was assayed in pistachio nut extract The activity was measured by monitoring the H2O2-mediated disappearance of ferulic acid at 310 nm The pH activity profile showed a single peak at 5.0 Kinetics parameters were determined at optimum pH with Vmax and catalytic efficiency expressed per mg extract protein With ferulic acid as the varied substrate, Km, Vmax and catalytic efficiency were, respectively, 45 lmol/l, 63.5 lM/min and 1.4/min With H2O2 as the varied substrate, Km, Vmax and catalytic efficiency were, respectively, 145 lmol/l, 88 lM/min and 0.60/min IC50 was 43 lM for KCN and lM for NaN3 KCN exhibited competitive inhibition with both ferulic acid and H2O2 as the varied substrate, while NaN3 exhibited competitive inhibition with ferulic acid and noncompetitive inhibition with H2O2 as the varied substrate No loss of activity was recorded after 10 incubation of the extract at up to 55°C, but 10 at 75°C led to complete loss of activity Acknowledgment: Supported by the J and E Research Foundation, Tehran, Iran 216 Signal Transduction C4-88 Nitric oxide donor, SNAP stabilizes trans-active hypoxia-inducible factor-1a H Park University of Seoul, Seoul, REPUBLIC OF KOREA Using molecular oxygen, the proline (402 and 564) residues and asparagine (803) residue of HIF-1a are hydroxylated by Proline Hydroxylase Domain (PHD2) and Factor-Inhibiting HIF-1a (FIH-1), respectively The proline hydroxylation of HIF-1a leads to interaction with E3 ubiquitin ligase, a von Hippel-Lindau protein (VHL), while asparagine hydroxylation inhibits the interaction with its coactivator, CBP/p300 Therefore, HIF-1a can be activated when both hydroxylases are inhibited We found that a nitric oxide (NO) donor, (±)-S-nitroso-N-acetylpenicillamine (SNAP), stabilizes HIF-1a, and activates its target genes, vascular endothelial growth factor and carbonic anhydrase 9, in a cGMP-independent pathway We found that SNAP causes HIF-1a to avoid ubiquitination and interact with CBP However, SNAP fails to inhibit the hydroxylation activities of both purified PHD2 and purified FIH-1 in vitro, which suggests that SNAP activates HIF-1a by indirectly inhibiting both PHD2 and FIH-1 Acknowledgment: This study was supported by a grant (200401969) from the Neurobiology Research Program, Korea C5-1 Probing of proteasome-substrate interaction with force spectroscopy M Beuttler, S Breuer, W Susanne, R Guckenberger and W Baumeister Max Planck Institute of Biochemistry, Martinsried, GERMANY Atomic force microscopy (AFM) is an established method to investigate biological samples in their physiological environment We use AFM for imaging and in particular for force measurements to investigate the mechanism of translocation of substrate molecules into the 20S proteasomes The proteasome is a barrel-shaped proteolytic complex of about 15 nm height and 11 nm width exhibiting small openings at both ends Unfolded proteins to be degraded have to pass these orifices and wind their way through the proteasome to access the central chamber where the proteolytic action takes place To characterize the forces involved in this process unfolded proteins are bound to the AFM tip and are offered as substrate molecules to immobilized proteasomes As an AFM tip lined with protein is not suitable for imaging, immobilization of the proteasomes in a very dense formation is required Moreover, to guarantee successful interaction between the substrate molecules and the proteasomes leading to translocation and degradation, the proteasomes need to be oriented in an upright manner By extensive screening a procedure of sample preparation could be established fulfilling all this and enabling force measurements Here we present our first results of such force measurements that allows us to describe the mechanism of substrate translocation into the 20S proteasome and the involved forces on a single molecule level ª 2007 The Authors Journal compilation ª 2007 FEBS Signal Transduction C5-2 Studying the translocation of substrate molecules into the 20S proteasome on ensemble and single molecule level S Breuer, F Huth, K Felderer, R Guckenberger, W Baumeister and S Witt MPI for Biochemistry, Martinsried, GERMANY The 20S proteasome degrades unfolded substrate molecules in an ATP-independent manner It consists of four heptameric rings that form a cylindrical structure, which is traversed by a central channel, widening into three cavities Substrates to be degraded have to wind their way through the interior of the proteasome to the central chamber where proteolysis takes place Elucidation of the mechanism underlying substrate translocation is performed using complementary approaches on ensemble measurements as well as on single molecule level Firstly, we established the formation of ‘host-guest’ complexes Denatured proteins were offered to inhibited proteasomes for uptake via substrate translocation and subsequently trapped in the internal cavities of the proteasome by refolding These complexes were analysed by size-exclusion chromatography in combination with UV/vis and fluorescence spectroscopy, electron microscopy as well as electro spray ionization mass spectroscopy Secondly, we further confirmed and specified our results on a single molecule level using confocal fluorescence microscopy Inhibited as well as active proteasomes and substrate molecules were labeled with different fluorophores The set up of our confocal microscope enables the simultaneous visualization of different fluorophores as well as the determination of FRET effects Here we present our first successful attempts to visualize the process of substrate translocation in and through the 20S proteasome on a single molecule level Abstracts C5-4 Identification of novel complex between S6K1 and Roc1 G Panasyuk1, I Gout2 and V Filonenko1 Institute of Molecular Biology and Genetics, Kyiv, UKRAINE, University College London, London, UK Signaling via PI3K and mTOR pathways mediate the activation of S6K in response to various mitogenic stimuli Ribosomal S6 kinases (S6Ks) are principal players in the regulation of cell growth and energy metabolism via phosphoinositide 3-kinases (PI3K) and the mammalian target of rapamycin (mTOR) signaling pathways The yeast two-hybrid screen was used to isolate binding partners towards S6K1 As a result two clones corresponding to Roc1 (Rbx1) were identified The eukaryotic protein degradation pathway involves the ubiquitin (Ub) modification of substrates targeted for degradation by the 26S proteasome The addition of Ub, a process called ubiquitination, is mediated by enzymes including the E3 Ub ligases which transfer the Ub to targeted substrates Roc1 is a part of the SCF (Skp-Cullin-F-box) E3 ligase complex together with Skp1, Cul1/Cdc53 and an F-box protein The specificity of Roc1/S6K1 interaction was confirmed in yeast cells in maiting assay Furthermore, direct binding of Roc1 and S6K1 was shown in a pull-down assay and by co-immunoprecipitation of transienly expressed proteins In addition, we found that S6K1 is ubiqitinated in cells and its ubiquitination is partly dependent on Roc1 expression The physiological relevance of the identified interaction is currently under investigation C5-3 Hedging and nursing of the 20S proteasome: towards understanding assembly and substrate uptake S Witt, S Breuer, M Beuttler, F Huth, R Guckenberger and W Baumeister Max-Planck-Institute of Biochemistry, Martinsried, GERMANY While the structure and enzymatic mechanism of the 20S proteasome are understood in great detail, our knowledge of the assembly pathway and the mechanisms involved in the substrate translocation are still substantially incomplete Using the 20S proteasome from Rhodococcus erythropolis, a close homologue of the Mycobacterium tuberculosis proteasome, we attempt to come to a comprehensive understanding of the events occurring during proteasome biogenesis Based on a great deal of biochemical and structural data we propose a model in which the critical inter-subunit interactions driving the assembly and the maturation of the active sites are identified For the elucidation of the mechanism underlying the translocation of polypeptide chains into the central cavity of the proteasome and the precise role of the two antechambers we use archaeal 20S proteasomes as model systems Firstly, we perform force-spectroscopy measurements during the translocation of polypeptide chains into the interior of the 20S complex The substrates molecules are coupled to the tip of an atomic force microscope (AFM) and offered as bait to immobilized proteasomes Secondly, we use proteasome-substrate ‘host-guest’ complexes to characterize the state of the proteasome and of substrates ‘en route’ to the central cavity by the complementary use of electron microscopy and x-ray crystallography on an ensemble level as well as confocal fluorescence microscopy on a single molecule level ª 2007 The Authors Journal compilation ª 2007 FEBS C5-5 Peroxisome degradation in mammalian cells: development of a study model by using dominant-negative peroxin variants S J Huybrechts, P P Van Veldhoven and M Fransen Catholic University of Leuven, Leuven, BELGIUM During the past decade, considerable progress has been made in the elucidation of the function of proteins involved in peroxisome biogenesis In recent years, there has also been a growing interest in how peroxisomes are degraded To date, methylotrophic yeasts are the most popular model organisms to study selective peroxisome degradation – a process called pexophagy – and little is known about the molecular mechanisms underlying peroxisome degradation in mammalian cells In order to get more insight into this process, we are currently generating a mammalian cell model in which peroxisome membrane biogenesis can be switched off in a controlled manner Pex3p, Pex16p, and Pex19p – three peroxins that play an essential role in peroxisomal membrane biogenesis – were selected as a target protein If the expression level (or function) of one of these peroxins is down-regulated (or suppressed), no new peroxisomes are formed anymore, and the degradation process of pre-existing peroxisomes can be studied Here we report the initial results of our work 217 Abstracts Signal Transduction C5-6 Analysis of E2 sumoylation in Saccharomyces cerevisiae C5-8 Regulation of the ARC105-mediated transcription by TRIM11 H Klug1, P Knipscheer2, J V Olsen3, N Nigam1, K Maderbock1, M Mann3, T K Sixma2 and A Pichler1 ă MFPL, Wien, AUSTRIA, 2The Netherlands Cancer Institute, Amsterdam, THE NETHERLANDS, 3Max-Planck Institute for Biochemistry, Martinsried, GERMANY H Ishikawa1,2, H Tachikawa3, Y Miura1 and N Takahashi1,2 United Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Tokyo, JAPAN, 2Japan Science and Technology Agency, CREST, Saitama, JAPAN, 3Department of Applied Biological Chemistry, Faculty of Agricultural and Life Science, University of Tokyo, Tokyo, JAPAN Posttranslational modification of proteins with ubiquitin and SUMO (small ubiquitin related modifier) plays an important role in many cellular processes Both modifiers are conjugated to their target proteins via an ATP-dependent enzyme cascade involving an E1 activating enzyme, an E2 conjugating enzyme and mostly an E3 ligating enzyme Recently, we have shown that both the mammalian ubiquitin E2 conjugating enzyme E2–25K and the SUMO E2 Ubc9 are modified with SUMO Whereas sumoylation of the ubiquitin E2 at its N-terminal a-helix interferes with E1 interaction in vitro, modification of the mammalian Ubc9 at a structural similar site has only minor effects on E1 interaction Via SUMO conjugation mammalian Ubc9 can interact and modify a substrate in dependence of its non-covalent SUMO interaction motif To gain insights into the biological function of E2 sumoylation we investigated Ubc9 modification in budding yeast Here we can show that also yeast Ubc9 is sumoylated in vitro and in vivo In contrast to mammalian Ubc9, which is modified at Lys14 in vitro, we mapped the modification site in yeast Ubc9 to Lys153 in vitro and in vivo by mass spectrometry analysis Although mammalian and yeast Ubc9 involve different sites for sumoylation one could imagine a similar function because both sites are positioned at similar distances from the catalytic cysteine To test this hypothesis we currently investigate sumoylated yUbc9 for thioester formation ability and aim to identify putative targets specifically interacting with sumoylated Ubc9 In addition we examine in vivo phenotypes of Ubc9 sumoylation C5-7 The transcriptional repression activity of KyoT2 on the Notch/RBP-J pathway is regulated by PIAS1-catalyzed Sumoylation J Wang, H Qin, J Liang, Y Zhu, L Liang, M Zheng and H Han Fourth Military Medical University, Xian, CHINA The LIM domain protein KyoT2 negatively regulates the Notch signaling pathway through interaction with RBP-J, the core element of the Notch signaling pathway in nucleus Here we show that PIAS1 (the protein inhibitor of activated STAT1) interacts with KyoT2 directly and attenuates KyoT2-mediated transcriptional repression We found that KyoT2 is modified by SUMOylation at two lysine residues, K144 and K171 The SUMOylation of KyoT2 is catalyzed by PIAS1 but not hPc2, another KyoT2-interacting protein with SUMO E3 ligase activity Using mutants disrupting either or both of the SUMO sites, we showed that SUMOylation of KyoT2 did not influence its expression, intracellular localization, or interaction with known partners However, disruption of the K171 SUMOylation site did reinforce the transcriptional repression activity of KyoT2, suggesting that SUMOylation of this site counters the repression activity of KyoT2 Finally, we show that PIAS1 failed to attenuate the repression activity of the K171R mutant of KyoT2, suggesting that PIAS1 antagonizes the transcriptional repression activity of KyoT2 through catalyzing its SUMOylation at K171 These results demonstrated that KyoT2 is a substrate of SUMO modification catalyzed by PIAS1, and that SUMOylation may modulate the transcriptional repression effect of KyoT2 on the Notch/RBP-J signaling pathway 218 TRIM11 belongs to tripartite motif containing protein family and contains RING Finger domain that is one of the consensus domains of many ubiquitin ligases (E3) TRIM11 destabilizes humanin, an inhibitor of Alzheimer-like neuronal insults; however, its physiological role remains to be explored In this study, we show that TRIM11 interacts with activator-recruited cofactor 105-kDacomponent (ARC105); it is a mediator of chromatin-directed transcription activation and is a key regulatory factor for TGFb signaling and lipid metabolism Co-expression of TRIM11 increased ARC105 degradation but a proteasome inhibitor suppressed this Co-expression of TRIM11 and ARC105 also increased ubiquitination of ARC105 In addition, TRIM11 suppressed ARC105-mediated transcriptional activation induced with TGFb in a reporter assay, suggesting that TRIM11, with the ubiquitin-proteasome pathway, regulates ARC105 function in TGFb signaling Because TRIM11 has multimeric activity and was localized in the nucleus by the ARC105 co-expression; it is very intriguing to postulate that the ARC105 degradation may be regulated by TRIM11’s multimerizing activity and occur at the site of on-going transcription We are trying to clarify these possibilities and will discuss the possible mechanism by which TRIM11 regulates ARC105-dependent transcription C5-9 Ubiquitin over-expression promotes E6AP autodegradation and reactivation of the p53/mdm-2 pathway R Crinelli, M Bianchi, M Menotta and M Magnani ` Universita degli Studi di Urbino ‘Carlo Bo’, Urbino, ITALY Ubiquitin is induced by several types of stress and in response to external stimuli Moreover, ubiquitin content varies among mammalian tissues, suggesting that its relative abundance may have a functional relevance It has been established that intracellular ubiquitin pools are subject to regulatory constrains Less certain is the mechanism by which the pool of conjugated ubiquitin shifts in parallel with total ubiquitin, and how this regulation affects the flux of the substrates through the pathway In this study we demonstrate that ubiquitin over-expression in HeLa cells promotes the destabilization of the ubiquitin protein ligase E6AP by a mechanism involving self-ubiquitination, leading to the stabilization of a transcriptionally active form of the tumor suppressor p53 Under the same experimental conditions, the levels of other well known ubiquitin proteasome substrates remained unchanged, suggesting that ubiquitin over-expression affects only a subset of ubiquitinprotein species These results represent the very first evidence that the activity of a ubiquitin ligase can be regulated by ubiquitin levels, supporting the idea that a strict interrelationship between pathway component activities and ubiquitin pool size exists These findings may have important implications in developing more specific anti-cancer therapies targeting the ubiquitin proteasome pathway ª 2007 The Authors Journal compilation ª 2007 FEBS Signal Transduction Abstracts C5-10 The role of proteasome manipulation on cellular senescence, stress response and longevity C5-12 Mechanisms of TNF-a-induced matrix metalloproteinase-9 expression in MC3T3-E1 osteoblastic cells N Chondrogianni, I P Trougakos and E S Gonos National Hellenic Research Foundation, Athens, GREECE C L Tsai1,2 and C M Yang1 Chang Gung University, Tao-Yuan, TAIWAN, 2Chang Gung Institute of Technology, Tao-Yuan, TAIWAN The proteasome is the major cellular proteolytic machinery responsible for cellular homeostasis Alterations of proteasome function have been recorded in various biological phenomena including cellular response to oxidative stress, ageing, replicative senescence and longevity Here we report that proteasome can be manipulated (up- or down-regulated), resulting in different functional effects on cellular ageing, survival and longevity Once it is activated through stable overexpression of beta catalytic subunit or hUMP1/POMP accessory factor, proteasome activities are stimulated, due to elevated amount of assembled proteasome These increased levels of assembled proteasome result to enhanced cellular capacity to cope better with oxidative stress, mainly through an up-regulated rate of proteolysis Moreover, stable transfectants exhibit an extended lifespan and a delay of senescence In contrast, upon replicative senescence of primary fibroblasts, the proteasome is down-regulated resulting to lower amount of assembled and thus, functional proteasome Additionally, when proteasome is partially inhibited in young primary human fibroblasts (remaining proteasome activity in levels similar to the ones found in replicative senescent fibroblasts), the cells exhibit an irreversible senescence-like phenotype within weeks following the treatment By taking advantage of cell lines with absent p53 and/or Rb pathways, we have identified which of these pathways is mainly responsible for this accelerated appearance of senescence, rendering cells capable of escaping from this phenotype In conclusion, these data demonstrate the central role of the proteasome during cellular senescence, response to stress and longevity C5-11 Carboxyl terminus-truncated alpha1D-adrenoceptor is functional and susceptible of desensitization ´ ´ C E Rodrı´ guez Perez1, M T Romero Avila1, G Reyes Cruz2 and ´ J A Garcı´ a Sainz1 ´noma de Me´xico, Me´xico, D.F., Universidad Nacional Auto MEXICO, 2CINVESTAV-Instituto Polite´cnico Nacional, Me´xico, D.F., MEXICO Alpha1D adrenoceptors play a key role in the regulation of blood pressure The function of these adrenoceptors is regulated by desensitization/resensitization processes We generated two truncated forms of human a1D-AR: (i) one deleted at the N-terminus, to increase the membrane expression and (ii) another mutant truncated both at the C- and N-terminus, to achieve a good expression and explore the role of the carboxyl tail in their function and regulation The mutant receptors were expressed in Rat-1 fibroblasts and were identified by photoaffinity labelling and radioligand binding techniques Both truncated receptors had high affinity (KD 0.3 nM) for [3H] tamsulosin and were expressed at high density (Bmax G1.5 pmol/mg of protein) Truncated receptors were fully functional and exhibit intrinsic activity as evidenced in quantitative studies of the inositol phosphates production and the intracellular [Ca2+] Interestingly, the activation of protein kinase C with phorbol esters blockades the receptor intrinsic activities and also the ability of noradrenaline to activate them These results demonstrate that the carboxyl terminus of human a1D-ARs is not essential for signalling or desensitization Acknowledgement: This research was supported by Grants from DGAPA (IN200206) and CONACyT (45837-Q) ª 2007 The Authors Journal compilation ª 2007 FEBS Mature bone mass is maintained by bone remodeling cycle Matrix metalloproteinases (MMPs) such as MMP-9 is the major factor involving in remodeling process MMP-9 is induced by several proinflammatory cytokines including tumor necrosis factor-a (TNF-a) in various cell types However, the mechanisms underlying TNF-ainduced MMP-9 expression in MC3T3-E1 osteoblastic cells remained unknown In this study, we showed that TNF-a-induced MMP-9 expression in a time- and concentration-dependent manner, was attenuated by the inhibitors of Ca2+ (BAPTA, Ni2+), calmodulin (calmidazolium chloride), CaM kinase II (KN-62), and NF-jB (helenalin) The involvement of NF-jB in MMP-9 expression was further confirmed by transfection with the dominant negative mutants of IKKa, IKKb, and NIK Furthermore, TNF-a-stimulated translocation of NF-jB into the nucleus was revealed by immnofluorescence staining and was attenuated by the inhibitors of MEK1/2 (U0126) and NF-jB (helenalin) These results suggested that TNF-a induced MMP-9 expression mediated through Ca2+, calmodulin, CaM kinase II, and NF-jB pathways in MC3T3-E1 osteoblastic cells C5-13 Molecular mechanisms for the control of the function of transcriptional coactivator p300 Q Li, J J R St-Germain and J Chen University of Ottawa, Ottawa, ON, CANADA Transcriptional coactivator p300 is required for a broad array of cellular activities including embryonic development and cell proliferation High levels of p300 are found in prostate cancer and correlated with aggressiveness of the cancer Yet, p300 appear to also function as a tumor suppressor Mutations in p300 genes have been found in many epithelial cancers In general, cells are very sensitive to gene dosage of p300 There is abundant information on how p300 participates different cellular pathways through chromatin remodeling or coordination of gene regulation, but not much is known on how p300 activity per se is regulated, except that p300 can be phosphorylated, acetylated, sumolated, ubiquitinated and is a substrate of the 26S proteasome We have reported that Akt/protein kinase B, plays an important role in maintaining p300 activity (Chen J et al Cell Mol Life Sci.2004; 61: 1675–1683) We have also established that deacetylase inhibitors such as valproic acid and butyrate induce p300 degradation through the 26S proteasome activity In addition, the negative effects of valproic acid and butyrate on p300 activity are mediated through augmentation of the B56c3 regulatory subunit of PP2A (Chen J et al Mol Cell Biol 2005; 25: 525–532) Overexpression of the B56c3 subunit enhances p300 degradation and suppresses p300-dependent transcription Conversely, knockdown of the B56 subunit by RNAi elevates both the level of p300 protein and the p300-dependent transcription Our study provides the first ever evidence for a functional interaction between PP2A and p300, and the sheds of new light into potential mechanisms that control p300 function The role of cellular distribution in the regulation of the function of transcriptional coactivator p300 will also be discussed 219 Abstracts Signal Transduction C5-14 Insulin-like growth factor-I induces alpha-1Badrenoceptor desensitization, phosphorylation and internalization T Molina, T Romero and A Garcia Instituto de Fisiologia Celular, Universidad Nacional Autonoma de Mexico, MEXICO Insulin-like growth factor-I (IGF-I) induces alpha-1B-adrenoceptor phosphorylation associated to receptor desensitization The effect of IGF-I was markedly decreased by pertussis toxin suggesting a role of pertussis toxin-sensitive G proteins Transfection of the carboxyl terminus of the b-adrenergic receptor kinase or the Dp85 mutant of phosphoinositide 3-kinase markedly decreased the a1Badrenoceptor phosphorylation induced by IGF-I without decreasing the receptor phosphorylation induced by noradrenaline Inhibitors of PI3K and PKC also blocked this effect In addition, it was observed that AG1478, an inhibitor of the EGF receptor kinase and BB-94, a metalloproteinase inhibitor, also diminished IGF-Iinduced adrenoceptor phosphorylation The IGF-I-induced alpha1B-adrenoceptor desensitization is associated to adrenoceptor internalization The following sequence of events are suggested: (i) stimulation of IGF-I receptors activate pertussis toxin-sensitive G proteins; (ii) this activates metalloproteinases, which catalyze heparin bindingEGF shedding, and transactivation of EGF receptors and (iii) dissociated Gbc subunits and phosphotyrosine residues stimulate PI3K activity, which leads to activation of PKC, resulting in a1Badrenoceptor phosphorylation, desensitization and internalization Acknowledgement: This research was supported by Grants from DGAPA (IN200206) and CONACyT (45837-Q) 220 ª 2007 The Authors Journal compilation ª 2007 FEBS ... regulatory proteins Involvment of particular signal transduction pathway was also studied ª 2007 The Authors Journal compilation ª 2007 FEBS Signal Transduction Abstracts C2-48 Sterols and Multi... both cholinergic signal transduction pathways and apoptotic cell death and presenilin mutations are responsible for both sensitizing neurons to death and altering neuronal signal transduction C2-75... cytotoxicity was related to MAPK signaling, which was involved oxidative stress, and Fas signaling, detected growth factors inhibit AAP cytotoxicity through MAPK signaling Thus, MAPK is involved