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Young Scientists Forum

Abstracts

Young Scientists Forum YSF-1 The role of TGF-b1 on chondrocyte focal adhesion and regulation of extracellular matrix synthesis

YSF-3 Role of WT1 in the regulation of expression of pro- and anti-angiogenic isoforms of VEGF

S. Ab Rahim1, L. Selvaratnam2, I. Othman2 and T. Kamarul1 1 Department of Orthopaedic Surgery, Faculty of Medicine, University of Malaya, Kuala Lumpur, MALAYSIA 2School of Medicine and Health Sciences, Monash University, Sunway Campus, Selangor, MALAYSIA

E. Amin1,2, D. Nowak2, M. Saleem3, S. Harper2, D. Bates2 and M. Ladomery1 1 Centre for Research in Biomedicine, Faculty of Health and Life Sciences, University of the West of England, Bristol, UK, 2Microvascular Research Laboratories, Department Physiology, University of Bristol, Bristol, UK, 3 Academic & Children’s Renal Unit, University of Bristol, Bristol, UK

Introduction: Focal adhesion plaques are vital prerequisites for articular cartilage cells (chondrocytes) to be responsive to growth factors such as IGF and FGF. Such cell adhesions via integrin ligation are responsible for regulating the activation of growth factor receptors. This phenomenon is important to demonstrate the dependence between chondrocyte adhesion to the extracellular matrix (ECM) and the regulation of chondrocyte behaviour by growth factors to create a harmonious optimal environment for the chondrocytes. Methods: In proving our hypothesis, the synthesis of ECM by chondrocytes in two different culture systems and the effect of TGF-b1 on these systems were investigated. Chondrocytes were cultured in monolayer sheets and as chondrocyte-alginate constructs. Results: It was demonstrated that alginate, an anionic polysaccharide biomaterial, did appear to be an ideal scaffold for chondrocyte support and differentiation, primarily by increasing synthesis of glycosaminoglycan and collagen type II as compared to monolayer constructs. In addition, supplementation of TGF-b1 further increased glycosaminoglycan and collagen type II elaboration in chondrocyte-alginate constructs. The observations made are most likely related to matrix regulation involving the aggregation (condensation) of chondrocytes which enhances cell–cell interactions, leading to propagation of signal transduction events. Furthermore, aggregation of chondrocytes into colonies within such constructs leads to extensive formation of adhesion plaques by integrin receptor signalling, which in turn synergistically up regulates the amount of cellular protein being expressed. Conclusions: Thus, in this study, chondrocytes have been shown to be more responsive to TGF-b1 when cultured in alginate constructs due to an increased adhesive interaction cascade.

YSF-2 Proteomic analysis of age-related changes and oxidative modified proteins in mice kidney H. Amelina and S. Cristobal Department of Biochemistry and Biophysics, Stockholm University, Stockholm, SWEDEN Proteomics is a powerful tool to screen cellular or tissular changes in protein expression. Two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight mass-spectrometry (MALDI-TOF/ MS) are key technologies used in proteomic analysis to gain information about protein expression profiles and post-translational modifications. Analysis of age-related changes in protein expression could lead to revolutionary new insights into age-related functional changes and improve our understanding of the molecular mechanisms of aging. To contribute this goal, we applied proteomic techniques based on liquid chromatography coupled with 2-DE (LC/2-DE). The kidney, that consists of mainly postmitotic cells and undergoes age-related deleterious changes, was selected as a model for this proteomic analysis. The elution fractions from 28-, 52- and 76-weeks mice kidney (CD1-Swiss strain, male and female specimens) were analysed by 2-DE. We detected more than 300 spots on the 2-DE images, and 54 spots showed up to 3-fold significant alterations in expression levels (p < 0.05) between young and old specimens. Further, these proteins were identified by MALDI-TOF MS; the results showed increases in oxidative and proteolytic proteins and decreases in cytoskeletal proteins and antioxidant enzymes. Moreover, using 2,4-dinitrophenyl hydrazine (DNP) immunoblotting approach, we evaluated specific carbonylation level of proteins, that were targets to oxidation. Our results provide insight into how differences in protein expression may be associated with age-related decline of cellular function. Thus, we also demonstrate that proteomic analysis can be effectively applied to the assessment of the age status of protein expression, and thereby provide a valuable information on age-related proteomic changes.

The Wilms tumour suppressor gene WT1 is mutated in 10–15% of Wilms tumours and mutations in WT1 are also associated with Denys Drash Syndrome (DDS). DDS is characterised by childhood nephrotic syndrome, glomerulosclerosis, end-stage renal failure and Wilms tumours. WT1 encodes a zinc finger transcription factor that also regulates gene expression at the post-transcriptional level. The search for WT1’s DNA and mRNA targets is ongoing. A recent study shows that WT1 regulates the expression of a key growth factor, VEGF (Vascular Endothelial Growth Factor). There are two families of VEGF isoforms; an angiogenic family (VEGFxxx where xxx denotes the number of amino-acids); and a newly discovered sister family of anti-angiogenic isoforms (VEGFxxxb) that result from a distal alternative 3¢ splice site in exon 8. VEGFxxxb is downregulated in all cancer cell lines tested to date. Although WT1 regulates VEGF expression transcriptionally, the extent to which WT1 affects VEGF splice isoform levels is not known but splicing is altered in DDS patients (Schumacher, et al., 2007). We measured VEGF isoform expression in differentiated wild-type and DDS patient-derived podocytes with a mutation in WT1 (R366C). The differentiated podocytes represents the closest in vivo model of podocytes in the kidney within the constraints of a cell culture model. Total VEGF and VEGFxxxb levels were determined by ELISA and RT-PCR. In the differentiated DDS podocytes there was a 8.73 ± 0.003 fold reduction in VEGFxxxb compared to wildtype. DDS podocytes stably transfected with a plasmid expressing wildtype WT1 restored the expression of VEGFxxxb to 0.73 ± 0.08. In the differentiated DDS podocytes there was a 2.32 ±0.48 fold increase in the total amount of VEGF compared to wildtype differentiated podocytes. Transfection of a plasmid encoding wildtype WT1 into differentiated DDS podocytes rescued the DDS phenotype by decreasing the amount of total VEGF to 0.62 ±0.11 fold compare to wildtype podocytes. These findings suggest that WT1 plays a role in the regulation of VEGF splice isoform expression; either by direct binding to VEGF pre-mRNA or indirectly by modulating splice factor activities.

YSF-4 Evidence for the presence of n-acylethanolamines and acyglycerols in Tetrahymena D. Anagnostopoulos1, C. Freni2, J. Wood2, L. Pandarinathan2, A. Makriyannis2 and A. Siafaka Kapadai1 1 Department of Chemistry, University of Athens, Panepistimioupolis, Athens, GREECE, 2Center for Drug Discovery, Northeastern University, Boston, MA, USA The endocannabinoid system is a ubiquitous lipid signaling system of great biological significance in vertebrates. Many of the actions of the most studied endocannabinoids, N-arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerols (2-AG) are mediated by binding and activation of the cannabinoid receptors CB1 and CB2. The action of these molecules is down regulated by specific degrading enzymes, the fatty acid amide hydrolase (FAAH) and the monoacylglycerol lipase (MAGL). We have already provided strong evidences that the endocannabinoid system is present in the unicellular eukaryote Tetrahymena pyriformis being able to metabolize both [3H]AEA and [3H]2-AG, through the action of a corresponding FAAH-like and MAGL-like enzyme. In the present study we report the identification and quantification of N-acylethanolamines (NAEs) and acylglycerols (AcGs) of Tetrahymena pyriformis and Tetrahymena thermophila by LC-atmospheric pressure chemical ionization-MS. The method has been validated to analyze 24 analytes, including the corresponding free fatty acids, in a single chromatographic run. N-alpha-linolenoyl, N-eicosenoyl, N-linoleoyl, N-palmitoyl, N-steroyl and N-oleoyl ethanolamines where identified as long as the glycerol analogs. Anandamide and 2-AG seem to be present in much lower concentration that could not be quantified. NAEs and AcGs were found for the first time in Tetrahymena but their biological role has not been found yet. Preliminary experiment thought, show that NAEs and AcGs affect the growth of Tetrahymena cultures. The evolutionary origin of the endocannabinoid system and its presence is lower organisms may provide important knowledge about its role in life and cell harmony.

FEBS Journal 275 (Suppl. 1) 439–470 (2008) ª 2008 The Authors Journal compilation ª 2008 FEBS

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YSF-5 Mutations and complex genomic rearrangements in the BRCT domain of the tumor suppresor gene BRCA1 S. Armaou1, M. Pertesi1, I. Konstantopoulou1, T. Anagnostopoulos1 and D. Yannoukakos1,2 1 National Center for Scientific Research ‘‘Demokritos’’, Athens, GREECE, 2 BioGenomica, Center for Genetic Recearch & Analysis, Athens, GREECE Introduction: The BRCA1 protein contains a tandem of two BRCT domains at its C-terminal region. Several proteins interact with this region and collaborate with BRCA1 in important functional pathways, such as DNA repair and transcriptional regulation. These interactions are disrupted by BRCA1 mutations that affect the BRCT repeats. The majority of cancerassociated germline BRCA1 mutations result in truncated products lacking one or two of the BRCT domains. Such mutations confer severe susceptibility to breast and/or ovarian cancer. Methods: We have previously shown, after screening a cohort of highly affected Greek families, that mutations in the BRCT domain are dominant in the BRCA1 mutational spectrum of breast and/or ovarian cancer patients. Here, we aimed to determine the frequency of each of the five recurrent BRCT-domain mutations in consecutive cases of breast cancer patients. Mutation screening methods employed were quantitative multiplex PCR of short fluorescent fragments (QMPSF), multiplex ligation-dependent probe amplification (MLPA), and DNA sequencing as a final confirmation step. Results: In a total of 785 random breast cancer patients, 27 (3.44%) carried one BRCA1 BRCT domain germline mutation. 26% of those were complex genomic rearrangements not detectable by routine screening methods. The break points of the three genomic rearrangements (0.9% frequency) were mapped and one of them was proven to produce no transcript. A combination of biochemical and genetic data was used to evaluate the pathogenicity of some missense variants. Conclusions: Germline mutations in the BRCT domain of BRCA1 are the main cause of breast cancer predisposition with hereditary etiology in the Greek population.

YSF-6 Interaction of aminoacid molecules with phospholipid bilayer. Molecular dynamics simulation S. A. Aramyan, A. H. Poghosyan and A. A. Shahinyan The International Scientific-Educational Center of the National Academy of Sciences of Armenia, Yerevan, REPUBLIC OF ARMENIA The aim of this work was to determine the ways of interaction of aminoacids with phospholipids bilayers using computer simulation methods. Molecular dynamics simulations make it possible to get insight of the system and answer questions in structural biology that are difficult to find experimentally. There were used the molecules of alanine, and valine and phospholipids bilayer consisting of 128 Dipalmitoilphosphatidilcholine (dppc) molecules and 3655 water molecules, which corresponds to fully hydrated state of dppc bilayer. For the computer experiment the GROMACS simulation package and GROMOS force fields were used. After energy minimizations molecular dynamics simulations were done with duration of 2 nanoseconds each. All simulations were made at same conditions with same parameters. Temperature was 303 K in an NPT ensemble. The Coulomb interactions were cut off to 2.5 nm and the van der Waals interactions were cut off to 1.4 nm. The isotropic pressure coupling type was used. Several parameters, describing the structure of bilayer, such as area per lipid on the surface of bilayer, thickness of bilayer, arrangement of aminoacid molecules in the bilayer were investigated. We have got that area per lipid reduces after aminoacid addition from 0.628 nm2 to 0.613 nm2. Thickness also reduces from 6.77 nm to 6.68 nm.

Results: Intracellular ROS and the level of superoxide anions were increased after 24 hours of dRib treatment. Moreover, intracellular antioxidant status including antioxidant enzymes activities such as catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glucose-6-phosphate dehydrogenase (G6PD) as well as non-enzymatic antioxidant glutathione (GSH) were diminished. U937 cells exposed to dRib (30 mM) exhibited biochemical features of apoptosis including annexin staining and sub-G1 populations. The use of N-acetylcysteine (NAC), a thiol antioxidant and GSH precursor, prevented the extent of oxidative damages in U937 cells. The NAC completely prevented superoxide production along with suppression of DCF fluorescence. In addition, treatment of U937 cells with NAC restored the intracellular GSH level and enhanced antioxidant enzyme activities. High dRib-induced DNA fragmentation and annexin staining were suppressed in the presence of NAC. Conclusions: Our results clearly demonstrated that dRib induces ROS formation and apoptosis as well as suppression of antioxidant defense system in U937 cells.

YSF-8 HPV type 16 and 18 E7 protein disrupt direct interaction of pluripotent protein CTCF with Y box binding protein 1 (YB-1) and up-regulates c-myc gene V. Balakrishnan1, N. H. Othman1 and S. Shamsuddin2 School of Medical Sciences, University Sains Malaysia, Kelantan, MALAYSIA, 2School of Health Sciences, University Sains Malaysia, Kelantan, MALAYSIA 1

Introduction: Human Papillomaviruses (HPVs) type HPV-16 and HPV-18 are known as high risk HPV, which are cause 95% of cervix cancer. E7 protein of HPV is able to interact with cellular protein pRB which inactivate pRB thus allowing progression of the cell cycle thus trigger cancer development. CTCF is a multivalent transcription factor which has been found to interact directly with YB-1. Both binds synergistically on c-myc gene promoter and down regulates the gene. Materials and Methods: In-vivo – Cell lysate were prepared from HeLa and Caski cell lines using RIPA buffer, which were then used for co-immunoprecipitation assay using CTCF and YB-1 antibody, respectively. In-vitro – Full domain of HPV 18 and 16 E7 proteins, and truncated CTCF and YB-1 proteins were produced in bacterial expression system and purification. These proteins were coupled to sepharose beads and preceded with pull-down assay. EMSA assay was performed to confirm the pull-down assay. Results: In-vivo – CTCF was found to bind with HPV 16 E7 but did not have interaction with HPV 18 E7. Meanwhile, YB-1 interacts with E7 protein vice-versa to CTCF. In-vitro – HPV 16 E7 bind with Zn-domain of CTCF specifically and HPV-18 E7 bind with CSD-domain of YB-1 specifically. EMSA reveals negative shift in the promoter region of c-myc gene, which indicates that HPV 16 & 18 E7 disrupt the interaction of YB-1 and CTCF.

YSF-7 Levels of reactive oxygen species, antioxidant defense system and apoptosis in U937 cells following 2-deoxy-D-ribose treatment A. Ardestani and R. Yazdanparast Institute of Biochemistry and Biophysics, University of Tehran, Tehran, IRAN Introduction: Reducing sugars are known to produce reactive oxygen species (ROS) mainly through the glycation reaction. 2-deoxy-D-ribose (dRib), a sugar with a large reducing capacity, has been known to induce oxidative stress and cell death among different cell lines. Methods: In the present study, the effect of dRib on ROS production, antioxidant defense system and induction of apoptosis in human monocytic cells (U937) was investigated. Intracellular ROS was detected using the fluorescence probe dichlorofluorescein diacetate (DCFH-DA). Furthermore, the level of superoxide was measured using intracellular reduction of nitroblue tetrazolium (NBT) to formazan.

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Conclusion: HPV 16 E7 bind to CTCF at Zn-domain and HPV 18 E7 bind to YB-1 at CSD domain. However CTCF and YB-1 have no binding with HPV 18 E7 and HPV 16 E7, respectively. It is known that CSD-domain of YB-1 binds to Zn-domain of CTCF and produce synergistic effect towards c-myc promoter by down-regulation the c-myc gene expression. EMSA has shown that interaction of HPV E7 protein towards CTCF/YB-1 complex have release CTCF/YB-1 complex from c-myc gene promoter site. Therefore it is postulate that the interaction of E7 protein with CTCF and YB-1 significantly reduces the interaction of YB-1 and CTFC. Thus, enhances the up-regulation of c-myc promoter and promotes tumourogenesis of cervix.


Young Scientists Forum YSF-9 Hydrogen peroxide in the endoplasmic reticulum E. Balazs Department of Physiology, Semmelweis University, HUNGARY Formation of disulfide bonds and proper folding of newly synthesized proteins in the endoplasmic reticulum (ER) requires an oxidative environment. Protein oxidation is driven by an enzymatic relay which involves protein disulfide isomerase (PDI) and endoplasmic reticulum oxidoreductase (Ero1). In yeast Ero1 is known to produce H2O2, but in mammalian cells the precise source and regulation of ROS formation in the ER has not yet been elucidated. To study these questions, we targeted a previously described H2O2-sensitive fluorescent protein, Hyper to various organelles of HeLa cells. After expressing these probes at different intracellular locations we measured the highest fluorescent signal in the ER. To determine whether the activity of PDI-Ero1 system is related to these findings, we manipulated the level of Ero1 by different means. Modifying the expression of Ero1 affected the oxidative folding capacity of the ER as expected, While these alterations had no influence on the measured H2O2 level. In further experiments we found that the H2O2 level of the ER decreased parallelly with the calcium level of the ER during histamine or thapsigargin induced calcium mobilization. These treatments at the same time had no effect on the protein folding. Our results suggest that the H2O2 level of the ER is regulated by the luminal calcium concentration. We also propose that an alternative source of H2O2 exists, which is independent of the PDI-Ero1 system.

YSF-10 Targeting neutrophil serine proteases in inflammation with a new recombinant protease inhibitor K. Baranger, M.-L. Zani, S. Dallet-Choisy and T. Moreau Inserm U618 ‘Proteáses et Vectorisation Pulmonaire’, IFR 315 Imagerie Fonctionnelle, Faculte´ de Medecine, Universite´ François Rabelais, Tours, FRANCE Introduction: In inflammatory lung diseases, the large influx of neutrophils is associated with the subsequent extracellular release of huge amounts of active serine proteases including neutrophil elastase (NE), proteinase 3 (Pr3) and cathepsin G (CatG). The consequence is a strong imbalance between these proteases and their endogenous inhibitors including elafin, its active precursor trappin-2 and secretory leukocyte protease inhibitor (SLPI). Methods: In order to evaluate a therapeutic strategy for lung diseases based on the aerosol administration of protease inhibitors to limit the deleterious effects of proteases, we have designed a new recombinant inhibitor called CemSLPI2. This inhibitor is composed of the cementoin domain of trappin-2 followed by the inhibitory domain of SLPI. The cementoin domain contains transglutamination sequences that would allow a covalent anchoring of the inhibitor to extracellular matrix proteins and thus would improve the bioavailibility of the inhibitor at inflammatory sites. The inhibitor was produced in the yeast Pichia pastoris and purified by cation exchange chromatography. Results: We found that CemSLPI2 is a potent inhibitor of free NE and CatG with Ki values similar to those determined for wild SLPI. NE and CatG bound to neutrophil membrane were also inhibited by CemSLPI2 in a dose-dependent manner. We have also shown that it can be covalently linked to fibronectin by a tissue transglutaminase and retains its inhibitory capacity. Conclusion: CemSLPI2 is a new, potent inhibitor of elastase and cathepsin G with anchoring properties that make it attractive in the context of an inhibitor-based therapeutic strategy for lung diseases.

Abstracts YSF-11 Sic1 can regulate the fundamental events in the budding yeast cell cycle M. Barberis1 and E. Klipp1,2 1 Computational Systems Biology, Max Planck Institute for Molecular Genetics, Berlin, GERMANY, 2Theoretical Biophysics, Humboldt University Berlin, Institute for Biology, Berlin, GERMANY Introduction: The modeling of the G1/S transition in budding yeast showed that a systems biology analysis highlighted the implications of the cell size determination that impinge the events driving DNA replication (1). Activation of the replication machinery has still to be highlighted in many regulatory events. However, the balance between the Cdk1-Clb5,6 kinase that induces firing of the DNA replication origins (2, 3) and the Sic1 cyclin-dependent inhibitor is crucial to regulate DNA replication events. In budding yeast, passage through the cell cycle employs multiple Cdk1-Clb complexes that are activated and inactivated in a fixed sequence by different cyclins. This originates the oscillatory behavior widely known as ‘waves of cyclins’. Here we address the intriguing question of how this coordinated fashion is achieved, and if the Sic1 inhibitor plays a regulative role in this process. Methods: Dynamics of the Cdk1-Clb kinases concentrations in time are described by ordinary differential equations, as reported (1), and we explicitly consider one single compartment. The mathematical model has been modeled using the computational software Mathematica Version 5.1, Wolfram Research. Results: We provided a rational explanation of the activation of DNA replication initiation due to the Sic1 localization, and its role as promoter of Cdk1-Clb5 to start DNA replication (4), as experimentally reported (5). Comparing mathematical models that differ for the regulatory interactions in which Sic1 is involved, we proposed a specific sequence of events that reproduces the successive activation of the phase-specific Cdk1-Clb complexes (6). This suggested that Sic1 can offer a feedback loop regulation to trigger the ‘waves of cyclins’, and the timing of their appearance. Conclusions: The understanding of biological processes is strongly improved by the construction of mathematical models able to predict the dynamics of the process in a variety of conditions. Here we show that the order of cell cycle critical events is determined by the specificity of regulatory interactions. References: 1. Barberis, et al., PLoS Comput. Biol. 2007; 3: 649–666. 2. Bell, et al., Annu. Rev. Biochem. 2002; 71: 333–374. 3. Takeda, et al., Oncogene 2005; 24: 2827–2843. 4. Barberis, et al., Genome Inform. 2007; 18: 85–99. 5. Rossi, et al., Cell Cycle 2005; 4: 1798–1807. 6. Barberis, et al., Submitted 2008.

YSF-12 Molecular understanding on curcumin-glucoside, a novel curcumin derivative inhibits a-synuclein oligomer formation: relevance to Parkinson’s disease D. Bharathi and K. S. J. Rao Department of Biochemistry and Nutrition, Central Food Technological Research Institute, Mysore, INDIA a-Synuclein aggregation or fibrillation is centrally implicated in Parkinson’s disease. It involves multi-step nucleated polymerization process via the formation of dimers, soluble toxic oligomers and insoluble fibrils. In the present study, we synthesized a novel compound viz., Curcumin-glucoside (Curc-gluc), a modified form of curcumin and studied its anti-aggregating potential with a-synuclein. Under aggregating conditions in vitro, Curc-gluc prevents oligomer formation as well as inhibits fibril formation indicating favorable stoichiometry for inhibition. The binding efficacies of Curc-gluc to both a-synuclein monomeric and oligomeric forms were characterized by Micro-Calorimetry. It was observed that titration of Curc-gluc with a-synuclein monomer yielded very low heat values with low binding. However, interestingly in case of oligomers it showed significant binding with a KB of 4.5 · 104, DH is )1414 kcal/mol and the entropy DS is 25.18. Addition of Curc-gluc inhibited aggregation in a dose–dependent manner and enhanced a-synuclein solubility, which proposes that Curc-gluc stabilizes the monomer and prevents oligomerization. The data is supported by confocal microscopy, transmission electron microscopy, circular dichroism spectroscopy, immunoblot, 1D and 2D NMR-NOESY, COSY etc. Overall, the data suggests that Curc-gluc binds to a-synuclein and prevents further oligomerization of a-synuclein monomers into fibrils. Thus the present investigation might aid in the development of disease modifying agents as therapeutic models for PD.


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YSF-13 Breaking up amyloid fibrils: methionine oxidation dissociates preformed amyloid fibrils K. J. Binger1,2, M. D. Griffin1 and G. J. Howlett1 1 Department of Biochemistry and Molecular Biology, BIO21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Victoria, AUSTRALIA, 2CSIRO Molecular and Health Technologies, Parkville, Victoria, AUSTRALIA Methionine is one of the most readily oxidized amino acids and its oxidative state is proposed to reflect the balance of oxidant and anti-oxidant reactions within the cell, where accumulation of oxidized proteins is an indicator of oxidative damage. Methionine residues are also linked to the pathogenicity of several amyloid diseases however the mechanism of action is unknown. These diseases are characterized by the accumulation of insoluble amyloid plaques of which the major constituents are amyloid fibrils. More than 25 different proteins are shown to assemble into amyloid fibrils in vivo and in several systems methionine oxidation has been shown to completely inhibit fibril assembly. Human apolipoprotein (apo) C-II readily self-assembles in vitro to form homogeneous amyloid fibrils and thus provides a convenient model to examine the effect of methionine oxidation on amyloid fibril formation and stability. ApoC-II contains two methionine residues (Met-9 and Met-60), and upon oxidation of these residues fibril formation was inhibited. Oxidized apoC-II molecules did not inhibit native apoC-II assembly, indicating that the oxidized molecules were unable to interact with the growing fibrils. The treatment of pre-formed fibrils with hydrogen peroxide caused dissociation of the fibrils. However, mutants in which the Met-60 was substituted with a valine were protected from this peroxide-induced dissociation, indicating that this is a specific effect of oxidation of this core methionine. This work highlights an important role for methionine in the formation of amyloid fibril structure and gives new insight into how oxidation affects the stability of mature fibrils.

YSF-15 Thrombospondin 1. A novel functional affector of the Reelin pathway with a role in postnatal neuronal migration S. Blake, V. Strasser, R. Hofbauer, W. J. Schneider and J. Nimpf Max F. Perutz Laboratory, Vienna, AUSTRIA Introduction: Postnatal neurogenesis and neuronal migration are key processes in establishing functionality and complexity of the brain. However, we are far from understanding the molecular mechanisms underlying the complex interplay of neuronal adhesion, neurogenesis, and migration. Reelin, a known key player in embryonic neocortical development has recently been shown to also be involved in postnatal neuronal migration. The contribution of Reelin to postnatal neurogenesis and migration is, however, less well understood than in embryonic development and is clearly not the only factor contributing to these events. Here we introduce a new role for the extracellular matrix protein Thrombospondin 1 in postnatal neuronal migration as a novel functional affector of the Reelin pathway. Materials and Methods: Solid phase binding assay, primary neuronal cultures, Dab1 phosphorylation assay, ApoER2 fragmentation assay, subventricular zone ex vivo migration assay, immunohistochemistry, immunofluorescence. Results: In this study TSP1 was identified as a novel functional ligand for ApoER2 and VLDLR. Like Reelin, it induces the signaling cascade including receptor clustering, Dab1 phosphorylation and ApoER2 fragmentation. Interestingly, TSP1 increases neuroblast adhesion in ex vivo assays which stands in clear contrast to the action of Reelin which is known to induce disassembly of neuroblast chains. We found a similar effect on postnatal neuronal migration in vivo in TSP1-/- mice that display a changed morphology of the Rostral Migratory Stream. Conclusions: Here we describe TSP1 as a novel functional affector of the Reelin signaling cascade and as a new player in neural migration with a specific role in postnatal development.

YSF-14 Control of the synthesis and the fate of gene transcripts by transcriptional coregulators D. Bittencourt, M. Dutertre, L. Gratadou and D. Auboeuf INSERM U685/AVENIR, Hoˆspital Saint-Luis, Paris, FRANCE Gene expression is a multi-step process starting in the nucleus and including transcription, pre-mRNA processing and export of the mRNAs to the cytosol where they are translated. During processing mRNAs associate with many proteins (assembly into ribonucleoprotein particles) and there is evidence that processing reactions (such as splicing) are necessary for the interaction of the RNA with the export machinery. We have previously shown that steroid hormones simultaneously affect transcription and splicing processes through the recruitment at target-promotor level of transcriptional coregulators involved in both transcript synthesis and maturation (Auboeuf et al. Science 2002 and PNAS 2004). Here we report that these coregulators interact not only with their target-genes and target-gene pre-mRNAs but also with the mature spliced mRNAs in both the nucleus and cytosol. We also observed that some of these coregulators shuttle between the nucleus and the cytoplasm and their depletion from cultured MCF7 breast cancer cell lines results in a robust accumulation of poly(A)+ RNAs within the nucleus suggesting an essential role for these coregulators in mRNA export. Therefore, our working model is that a subset of transcriptional coregulators are involved in mRNA maturation and participate in the export of their target gene products allowing to control in a coordinated manner the synthesis, the maturation, and the fate (export) of their target gene products. Thus, our work extends the unexpected large spectrum of action of transcriptional coregulators that appear to play a key role in the coordination of the different steps of the gene expression process.

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YSF-16 Expression of HIF-1a, HIF-2a mRNA and HIF-dependent genes in colon, gastric and pancreatic cancer cells: effect of hypoxia A. Bobarykina1, K. Tsuchihara2, H. Esumi2 and O. Minchenko1 1 Molecular Biology, Palladin Institute of Biochemistry, Kiev, UKRAINE, 2 Research Center for Innovative Oncology, National Cancer Center Hospital East, Kashiwa, JAPAN Introduction: The expression of VEGF, Glut1, 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase-3 (PFKFB-3) and PFKFB-4 genes is HIFdependent. We studied effect of hypoxia on the expression of HIF-1a, HIF2a mRNA and mRNA HIF-dependent genes in colon and gastric cancer cells. Methods: mRNA expression was measured by ribonuclease protection method. Results: We have identified several unique alternative splice variants of rat PFKFB-4 mRNA. One variant of PFKFB-4 mRNA has short and modified carboxyl-terminal part as a result of 22 bp deletion in fructose2,6-bisphosphatase region which changes reading frame and create stop codon after J-domain. It has all 6-phosphofructo-2-kinase domains but does not have one fructose-2,6-bisphosphatase domain. Moreover, Jdomain in this variant is modified. Second variant of PFKFB-4 mRNA has also shorter and modified carboxyl-terminal part as a result of long (193 bp; exon 11 and 12) deletion in fructose-2,6-bisphosphatase region. This variant of PFKFB-4 mRNA has all 6-phosphofructo-2-kinase domains and five, instead six, fructose-2,6-bisphosphatase domains. Third alternative splice variant of PFKFB-4 has insert (94 bp, intron 11) which contains stop codon. This unique alternative splice variant of PFKFB-4 has all 6-phosphofructo-2-kinase and fructose-2,6-bisphosphatase domains and short (only 12 amino acids) C-terminus encoded by intron 11. We have shown that all alternative splice variants of PFKFB-4 mRNA are expressed in the lung and that expression of second and third variants significantly induced by methyl tretbuthyl ether which possibly affect some steps of alternative splicing. Conclusion: Our results indicate that rat PFKFB-4 mRNA has several unique alternative splice variants for isozymes with reduced fructose-2,6-bisphosphatase and its expression induced by methyl tretbuthyl ether.



Abstracts

YSF-17 Expression of OsCam1-1 gene under salt stress in rice Oryza sativa L. is mediated by abscisic acid

YSF-19 Chromosome aberrations in response to combined mycotoxins

B. Boonburapong1, S. Phean-o-pas1, S. Chadchawan2 and T. Buaboocha1 1 Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok, THAILAND, 2Department of Botany, Faculty of Science, Chulalongkorn University, Bangkok, THAILAND

A. Bouslimi, I. Ayed-Boussema, C. Bouaziz and H. Bacha Laboratory of Research on Biologically Compatible Compounds, Faculty of Dentistry, Monastir, TUNISIA

2+

Introduction: Calmodulin (CaM) is a small Ca sensor, which transduces increased [Ca2+]cyt by binding to and altering the activities of a variety of target proteins that affect physiological responses to the vast array of specific stimuli received by plant cells. Methods: OsCam1-1 gene was isolated by RT-PCR from Oryza sativa L. ‘KDML105’ and its sequence was determined. The copy number and expression of OsCam1-1 were determined by gel blot analysis and real-time PCR. Result: From the OsCam1-1 sequence, it is highly conserved compared with other plant Cam genes. Southern blot analysis suggests that it is a singlecopy gene. Expression of OsCam1-1 in rice cultivars KDML105 and LPT123 in comparison with their salt-tolerant varieties FL530 and LPT123C171, respectively, was examined. Salt stress (80 mM NaCl) was shown to increase OsCam1-1 mRNA level in leaf but not in root of KDML105 and FL530. Exogenous ABA application two hours before salt stress had no effect in KDML105 but increased OsCam1-1 induction in leaf and increased mRNA level in root of FL530 under salt stress. For LPT varieties, salt stress increased OsCam1-1 mRNA level in LPT123C171 but not in LPT123. When exogenous ABA was applied before salt stress, OsCam1-1 mRNA level was increased in LPT123 while the induction in LPT123C171 was observed earlier than that of plants experiencing salt stress alone. In addition, ABA inhibitor application was shown to inhibit expression of OsCam1-1 induced by salt stress. Conclusion: OsCam1-1 plays an important role in salt stress response and its induction under salt stress is probably mediated by ABA.

YSF-18 Implication of mitochondria in the signalling cascade of fusarial toxin induced apotosis C. Bouaziz-Taleb1, E. El Golli1, O. S. El Dein2, S. Abid-Essefi1, C. Lemaire2, C. Brenner2 and H. Bacha1 1 Laboratoire de Recherche sur les Substances Biologiquement Compatibles (LRSBC), Faculte de Medecine Dentaire, Monastir, TUNISIA, 2Laboratoire de Geńe´tique et Biologie Cellulaire (LGBC) University of Versailles Saint Quentin en Yvelines, FRANCE Introduction: There is now compelling evidence implicating mycotoxins produced by the Fusarium molds in livestock disorders and human diseases in different parts of the world. Fusaria produce predominantly two types of mycotoxins: The non-estrogenic trichotecenes including T-2 toxin and the mycoestrogens, including Zearalenone (ZEN). Methods: Using human cervix carcinoma cells (hela), we tried to better understand the role of mitochondria in fusarotoxins-induced apoptosis and to elucidate the similarities and the differences between them. Several flow cytometry and immunofluorescence analyses were performed. We measured the cells viability by fluorescein diacetate, the oxidative stress induction by hydroethidine, the loss of mitochondrial transmembrane potential by DiOC(6) (3,3¢-dihexyloxacarbocyanine), the relocalisation of mitochondrial bax and the release cytochrome C. Results: Our results clearly showed that hela cells respond to ZEN and t-2 toxin by loss of cell viability. besides, both fusarotoxins produced an increase in O2-production. A concomitant time-dependent decrease of mitochondrial transmembrane potential (DYm) was observed. Subsequently, increase of caspase-3 activity, relocalisation of mitochondrial bax and cytochrome C release were detected. Moreover, cell pre-treatment with a broad-specificity caspase inhibitor (Z-VAD) abolished apoptosis. We also demonstrated that this death signalling pathway and the mitochondrial membrane permeability induction can be regulated by some of the Bcl-2 family members, such as Bax, Bak, BClxl and Bcl-2. Conclusion: Taken together, all this data suggests that apoptosis in both ZEN and t-2 toxin-treated cells is correlated with the changes of intracellular ROS levels, is mediated by a mitochondrial caspase-dependent pathway regulated by BCl-2 family members at different levels.

For many years it has been known that several food items, which are derived from plants infected by fungi in the field when growing the plants or during the harvest and storage of the food item, can contain concomitantly different mycotoxins. A lot of mycotoxins may be produced by one mould; many Aspergillus and Penicillium species, in particular, can produce several mycotoxins simultaneously, depending on the environmental and substrative conditions. This is particularly true for ochratoxin A (OTA) and citrinin (CTN). Considering the concomitant production of OTA and CTN, the aim of the present study is to investigate whether genotoxicity is a key determinant of renal diseases, and to show how it is enhanced by a combination of both mycotoxins as compared to their effect separately. The combined effects of OTA and CTN observed in vitro is clearly of synergistic nature. To further assess OTA, CTN and OTA with CTN genotoxicity induced damages to Vero cells, we choose to monitor the effect of both mycotoxins either individually or concomitantly on the induction of chromosome aberrations in vivo. The different types of structural chromosome aberrations induced by each of the OTA, CTN and the combination of OTA and CTN treatments are gaps, rings, breaks and centric fusions. While breaks and rings represent the majority of chromosome abnormalities, gaps are the least frequent lesions. Our results clearly show that the combination of the two toxins leads to an important increase of chromosome aberrations as compared to each toxin taken alone.

YSF-20 A novel approach to identifying calpain substrates in vivo Z. Bozoky1, A. Alexa1, J. Dancsok1, G. Gogl1, E. Klement2, K. F. Medzihradszky2 and P. Friedrich1 1 Institute of Enzymology, BRC, Hungarian Academy of Sciences, Budapest, HUNGARY, 2Proteomics Research Group, BRC, Hungarian Academy of Sciences, Szeged, HUNGARY Introduction: Calpains the ubiquitous, cytoplasmic SH-proteases, are held to regulate intracellular pathways by limited proteolysis of their substrate proteins. Many so-called calpain substrates have been identified. However, these assignments have been made in vitro, and only a small part of them are confirmed in vivo. To gain deeper understanding of calpain action and function, one must apply a no-bias, proteomic approach. This paper is an attempt in this direction. Methods: Drosophila Schneider S2 cells were used in our substrate finding method, based on analysing the protein concentration differenties in three types of sample by classical 2D gel electrophoresis followed by densitometric evaluation. Calpain substrate candidates are the spots with the density relation: control >> Ca2+ ionophore added < Ca2+ ionophore and calpain inhibitor added. These spots are identified by LC-MS/MS. Results: We found 11 candidate spots and the proteins were identified, from which only one had been described in the literature as calpain substrate. Four other candidates were selected to be tested in in vitro experiments. We found that all of them were cleaved by calpains. Conclusions: We developed a calpain substrate finding method with which 11, mostly new, candidates were found. Four proteins were randomly selected to confirm the reliability of the technique. Acknowledgements: OTKA60723.


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Abstracts


YSF-21 IFNb induces the expression of secreted IL-1 receptor antagonist in human monocytes through a MEK2 dependent, ERK1/2 independent pathway

YSF-23 Proline residues affect the recognition of transmembrane segments by the TIM23 complex in the mitochondrial inner membrane

K. J. Brandt, N. Molnarfi, L. Gruaz and D. Burger Division of Immunology and Allergy, Clinical Immunology Unit, Department of Internal Medicine, University Hospital, Geneva, SWITZERLAND

S. Calado Botelho, H. Kim and G. Von Heijne Department of Biochemistry and Biophysics, Center of Biomembrane Research, Stockholm University, Stockholm, SWEDEN

Interferon-b (IFNb) is currently used to treat multiple sclerosis patients. However, its mechanisms of action are not fully understood. We previously demonstrated that IFNb induces the production of the anti-inflammatory cytokine sIL-1Ra (the secreted form of interleukin-1 receptor antagonist) in human monocytes. Here we show that the induction of both sIL-1Ra protein and mRNA expression were inhibited in the presence of UO126 (MEK1/2 inhibitor) but not of PD98059 (MEK1 inhibitor). This was confirmed by silencing experiments in which only MEK2 silencing inhibited the expression of sIL-1Ra. However both UO126 and PD98059 diminished the phosphorylation of the MEK1/2 canonical substrates, ERK1/2. This suggests that MEK2 used another downstream pathway to control sIL-1Ra gene transcription upon IFNb activation. The latter did not involve the transcription factors NFjB and PU.1. In conclusion, the production of sIL1Ra induced by IFNb in human monocytes is regulated by MEK2 through a pathway which is different from ERK1/2 and remains to be identified.

Introduction: The TIM23 complex is one of the translocases responsible for the insertion of proteins into the mitochondrial inner membrane (IM). Very little is known about how the TIM23 complex recognizes and differentiates between proteins that should be imported to the matrix from proteins that should be integrated into the mitochondrial IM. An early report by Meier et al. (1) demonstrated that the presence of proline residues in transmembrane domains (TMDs) strongly disfavour their insertion into the IM. Methods: We have recently developed a new approach to measure the membrane-insertion efficiency of TMDs in the mitochondrial IM using the S. cereveisiae mitochondrial protein, Mgm1p as a model protein. To investigate the effects of proline residues on TMD insertion by the TIM23 complex, the first hydrophobic domain of Mgm1p was substituted with a set of 19 amino-acid segments carrying a single or a pair of prolines in varying positions. The membrane-insertion efficiency was determined by a simple gel-based assay. Results: Our results show a strong positional dependence in the effect of proline residues on the membrane insertion of TMDs into the mitochondrial IM. Prolines placed near the middle of the TMD reduce insertion efficiency, whereas prolines placed near the N- and C-terminal ends of the TMD have little effect. Conclusions: These results show a similar trend to the data obtain by Hessa et al. (2) for the endoplasmic reticulum (ER) insertion machinery, suggesting that protein-lipid interactions may provide the basis for the recognition of TMDs by the TIM23 complex. References: 1. JCB; 170: 881–888 2. Nature 435: 377–381

YSF-22 CD44 is involved in TIMP-1-induced signalling pathway leading to UT-7 cell survival L. Bridoux, E. Lambert, J. Devy, M.-L. Sowa, L. Martiny and E. Petitfrere-Charpentier Laboratoire de Biochimie UMR CNRS MeDyC 6237, URCA, Reims, FRANCE The Tissue inhibitors of metalloproteinases (TIMPs) regulate several cell functions such as cell proliferation and survival independently of matrix metalloproteinases (MMPs) inhibition. Among the four TIMPs identified in the mammals, only TIMP-1 and TIMP-2 display an anti-apoptotic activity. Previous studies in our laboratory demonstrated that (i) TIMP-1 induces UT-7 erythroid cell survival via the JAK2/PI 3-kinase/Akt pathway and (ii) proMMP-9, localized at the UT-7 plasma membrane, is crucial for this effect. In several normal and cancerous cell lines, pro-MMP-9 is anchored at the plasma membrane by the transmembrane glycoprotein, CD44. Although it is obvious that TIMP-1 activates intracellular signalling pathways, the receptor of TIMP-1 is not yet identified. In this work, we studied the role of CD44 in TIMP-1-induced signalling pathway leading to UT-7 cell survival. We demonstrated (i) by immunoprecipitation that CD44 was constitutively associated with pro-MMP-9 and (ii) by immunofluorescence that these two proteins were localized at the plasma membrane of UT-7 cells. To confirm the role of CD44, we transfected UT-7 with short interferring RNA (siRNA) targeting CD44 and showed that CD44 expression silencing inhibited TIMP-1 anti-apoptotic effect (mesured by flow cytometry and JAK2 tyrosine kinase phosphorylation). Moreover, we demonstrated that CD44 was constitutively associated with JAK2 and CD44 expression silencing abolished TIMP-1-induced JAK2 phosphorylation. The set of our data highlights the involvement of a pro-MMP-9/CD44/JAK2 complex in the TIMP-1-activated signalling pathway leading to UT-7 erythroleukemic cell survival.

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YSF-24 Invadopodia: specialized membrane microdomains G. Caldieri, G. Giacchetti, M. Baldassarre, F. Attanasio and R. Buccione Consorzio Mario Negri Sud, S. Maria Imbaro, Chieti, ITALY Introduction: When invasive cells are grown on a flat substratum they extend proteolytically active protrusions, termed invadopodia, into the surrounding matrix where focalized degradation of the Extra-Cellular-Matrix (ECM) takes place. Invadopodia feature a very intense local signalling activity, and the concentration of a number of key components among which membrane-type-1-matrix-metalloprotease and certain integrins, previously shown to be sorted to cholesterol-enriched Detergent-Resistant-Membrane (DRM) fractions. This suggests that invadopodia are specialized membrane domains where polarized traffic might occur. Methods: We performed cholesterol depletion experiments in A375 MM melanoma cells with different approaches: treatment with beta-methyl-cyclodextrin, caveolin 1 siRNA and transfection with a caveolin mutant known to block arrival of cholesterol to the plasma membrane. Further, we analyzed the floatation profile of typical invadopodia components by equilibrium ultracentrifugation from cells with or without active invadopodia. The role of traffic at invadopodia was investigated treating cells with Brefeldin A and Nocodazole, able to inhibit anterograde and apical polarized transport of proteins respectively. Results: Invadopodia biogenesis and function was impaired by direct and indirect cholesterol depletion and efficiently recovered upon cholesterol re-addition. Moreover, invadopodia components shifted to lighter (i.e. cholesterol-rich) fractions in actively degrading cells. Finally, inhibition of traffic blocked the progression of ECM degradation in an acute fashion. Conclusions: We propose that invadopodia are DRM-enriched domains, acting as platforms for signaling and specialized transport of newly synthesized proteins required for focal degradation.


Young Scientists Forum YSF-25 Natural chaperone ligands as new therapies for the treatment of genetic diseases related to catecholamine synthesis

Abstracts YSF-27 The role of RhoB in lovastatin-induced reversion of cisplatin resistance in human laryngeal carcinoma cells

A. C. Calvo1, A. L. Pey1, B. Thony2 and A. Martinez1 1 University of Bergen, Bergen, NORWAY, 2University of Zurich, Zurich, SWITZERLAND

T. Cˇimbora-Zovko1, G. Fritz2 and M. Osmak1 1 Ruder Bosˇkovic Institute, Zagreb, CROATIA, 2University of Mainz, Mainz, GERMANY

Introduction: Tyrosine hydroxylase (TH) is the rate limiting enzyme in the synthesis pathway of catecholamine neurotransmitters. TH activity is highly regulated at the protein level by phosphorylation and reversible feed-back inhibition by its end-products catecholamines. In humans, inherited defects in TH have been associated to L-DOPA responsive dystonia and infantile parkinsonism. Mutations in the homologue enzyme phenylalanine hydroxylase (PAH) cause phenylketonuria (PKU). Some PKU patients are nowadays treated with tetrahydrobiopterin (BH4) supplementation, the natural cofactor of TH and PAH. BH4 is thought to stabilize PAH in vivo and thus increase PAH activity in patients. However, little is known about the effect of BH4 in TH. Materials and Methods: The effect of BH4 and dopamine on the activity and stability of wild type and mutant TH was measured by a combination of enzyme kinetic analysis and circular dichroism (CD) spectroscopy using recombinant TH. Treatment with BH4 in wild type mice was carried out to determine the effect on TH mRNA, protein levels and activity in brain. Results: BH4 and dopamine stabilize TH in a concentration dependent manner as seen by CD spectroscopy. TH activity and protein in brain of BH4 treated mice show a slight increase compared with non treated controls. Conclusions: We conclude that BH4 and the catecholamine end products of the TH reaction may have a role as natural chaperone ligands on TH. This open the possibility of using BH4 as a new therapy in the treatment of TH related diseases.

Introduction: Acquired resistance to cisplatin represents a major obstacle to successful chemotherapy. Our cisplatin-resistant CA3ST and CK2 cells display cytoskeleton alterations comparing to parental human laryngeal carcinoma HEp-2 cells. Since lipid modification of Rho GTPases is essential for biological function, we analyzed their expression as well as the cellular response to HMG-CoA reductase inhibitor lovastatin. Methods: We used semiquantitative RT-PCR and Western blot analysis, transient transfection, flow cytometry and cytotoxicity assays. Results: Among several Rho GTPases analyzed, cisplatin-resistant cells displayed strong decrease of RhoB on both mRNA and protein level. Lovastatin treatment induced concentration-dependent increase in RhoB in all cell lines tested and cell cycle arrest in G1 phase, which was more pronounced in cisplatin-resistant than parental cells, whereas at later time points cisplatin-resistant cells were highly susceptible to apoptosis. This effect was suppressed by the addition of geranylgeranyl pyrophosphate, and to less extent farnesyl pyrophosphate. Furthermore, lovastatin pretreatment restored sensitivity of resistant cells to cisplatin to the level found in parental cells. In line with this, transient transfection of EGFP-RhoB in CA3ST cells restored their sensitivity to cisplatin. Conclusions: We conclude that in cisplatin-resistant and lovastatin-sensitive laryngeal carcinoma cells RhoB is involved in resistance to cisplatin. Moreover, since lovastatin has already entered clinical trials for several types of cancer, these data could lead to new clinical strategies aimed to overcome cisplatin resistance and improve efficacy of cancer treatment.

YSF-26 Partial proteasome inhibition triggers p53-dependent premature senescence

YSF-28 Novel bioactive peptides from the venom and mucous from the Brazilian stingrays Potamotrygon gr.

N. Chondrogianni1, I. P. Trougakos1, D. Kletsas2 and E. S. Gonos1 1 National Hellenic Research Foundation, Institute of Biological Research and Biotechnology, Athens, GREECE, 2NCSR ‘Demokritos’, Institute of Biology, Athens, GREECE Proteasome-dependent degradation has been extensively investigated and has been shown to play a vital role in the maintenance of cellular homeostasis. Proteasome activity and expression is reduced during aging and replicative senescence. Proteasome activation has been shown to confer lifespan extension in human diploid fibroblasts (HDFs) whereas partial proteasome inhibition triggers an irreversible premature senescent phenotype in young HDFs. As p53 and Rb tumor suppressors regulate both replicative and premature senescence, in this study we investigated their implication in proteasome inhibition-mediated premature senescence. By taking advantage of a variety of HDFs with defective p53 or/and Rb pathways, we revealed that partial proteasome inhibition results in an immediate growth arrest and/or a moderate increase of apoptotic death which is independent of the cellular genetic context. However, in the long term, proteasome inhibition-mediated premature senescence can only be established in the presence of functional p53. In total, we demonstrate that following partial proteasome inhibition, senescence is dominant in HDFs with functional p53 and Rb molecules, crisis/death is induced in cells with high p53 levels and defective Rb pathway, whereas stress recovery and restoration of normal cycling occurs in cells that lack functional p53. These data reveal the strong interplay between the integrity of proteasome function, senescence and cell survival.

K. Conceiçaõ1, R. Lopes Melo1, E. E. Marques2, M. H. Borges3, F. M. Bruni1 and M. Lopes-Ferreira1 1 Laboratorio Especial de Toxinologia Aplicada (LETA), Center for Applied Toxinology (CAT/CEPID) Instituto Butantan, BRAZIL, 2Nucleus of Environmental Studies, Federal Univesity of Tocantins, Tocantins, BRAZIL, 3 FUNED, Fundaçaõ Ezequiel Dias, Belo Horizonte MG, BRAZIL Stingrays of the family Potamotrygonidae are widespread throughout river systems of South America. Some species are endemic to the most extreme freshwater environment of the Brazil and cause frequent accidents to humans. Due to their complexity and diversity, these venoms represent an extensive source of bioactive compounds such as peptides and proteins. So, the characterization of the content of venoms has a growing number of potential benefits for basic research, clinical diagnosis, development of new therapeutic agents, and production of antiserum. In order to analyze in detail the peptides and small proteins of Potamotrygon venoms and mucous, proteomic techniques (high-performance liquid chromatography (HPLC) separation, matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS), and quadrupole time-of-flight tandem mass spectrometry (Q-TOF-MS/MS) have been employed. Taking advantage of these methodologies the present study describes the isolation, biochemical characterization, and sequence determination of novel bioactive peptides. These peptides were shown to be effective in microcirculatory environment causing effects like vasoconstriction, increasing the number of leucocytes and inhibiting thrombin. These peptides were fully sequenced by de novo amino acid sequencing with mass spectrometry and identified as novel peptides. Also, we describe the activity of new analogues from the vasoactive peptide ‘Orpotrin’. Taken together, the results indicate the usefulness of this approach for low molecular mass compounds in complex samples such as venoms. Financial support: FAPESP.


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Abstracts


YSF-29 Human 1-8D gene, a new pro-apoptotic gene, is overexpressed in colon cancer: characterization of an absurd situation V. Daniel Carmi, E. Makovizky Avraham, E. Reuven, E. Vadai, E. Tzehoval and L. Eisenbach Department of Immunology, Weizmann Institute of Science, Rehovot, ISRAEL Introduction: Screening of peptides derived from overexpressed genes in colon carcinoma was resulted in many HLA-A2 binding peptides. Seven peptides were shown to be immunogenic. Three of them were derived from the 1-8D gene, which belongs to the 1-8 interferon-inducible gene family. Three members of the family 1-8D, 1-8U and 9-27 are linked on chromosome 11 and are highly homologous. Methods and Results: The human 1-8D gene was confirmed to be overexpressed in colon tumor tissues compared to paired normal tissue using Real Time PCR. Sequence comparison of the human 1-8D in pairs of tumor/normal colon tissues revealed six different alleles. Transient expression of the human 1-8D gene in several mammalian cell lines showed accumulation of cells in G1 phase followed by elevation in the subG1 phase. This increase was dependent on caspase activity but not on p53 expression. To further confirm this induction of apoptosis, Annexin V binding assays in combination with propidium iodiode staining and TUNEL assays were performed. These experiments showed that 1-8D expression induces apoptosis in a timedependent manner. Furthermore, transformation assays revealed a possible role of 1-8D gene as a cell growth inhibitor. Using the human 1-8D-GFP fusion protein, we found that the protein localized to a compartment identified as the Golgi body. Conclusions: Our data suggest, for the first time, a novel role for the 1-8D as a pro-apoptotic gene. The 1-8D gene induces apoptosis in a caspase but not p53 dependent manner.

YSF-30 A single amino acid change converts mortalin from a gene associated with longevity into aging C. Deocaris1, K. Yamasaki2, S. Kaul3 and R. Wadhwa3 1 Institute of Health and Sports Sciences, University of Tsukuba, Tsukuba, JAPAN, 2Age Dimension Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, JAPAN, 3Research Institute for Cell Engineering, AIST, Higashi, Tsukuba, JAPAN Introduction: Chaperone functions of the heat shock protein (HSP) family are essential factors that govern lifespan of organisms. Paradoxically, the mitochondrial heat 70 exists with two isoforms that have opposing phenotypes: mortalin 1 (which induces accelerated aging or senescence) and mortalin 2 (which leads to life-span extension and immortalization). Methods: Using expression plasmids of single point mutants of mortalin and recombinant proteins, in vivo and vitro chaperone assays were performed, respectively. Senescence studies were done on mouse embryonic fibroblasts and on tumor cells transfected with the mortalin constructs and. Finally, a homology model of mortalin was developed to provide a biophysical perspective of its aging functions. Results: A G624R replacement in mortalin’s substrate lid reduces its ability to chaperone misfolded protein aggregates leading to the induction of senescence in tumor cells. Embryonic fibroblasts from in-bred mice carrying the natural allelic form of mortalin-R624 (or mot-1) readily acquires aged cell morphologies with life-span reduced by 10–12 population doublings compared to fibroblasts homozygous for mortalin-G624 (or mot-2). Conclusions: Here we show that a single amino acid change in the longevity-associated mitochondrial HSP70 mortalin is able to alter chaperone activity and sub-cellular distribution, and remarkably, reverse its ageing phenotype. These results have implications for the role of the mitochondria and its chaperones in longevity, the development of therapies for switching ageing phenotypes for ageing-prone normal cells and ‘immortal’ tumors, and for the uncanny century-old evolution of the mitochondrial Hsp70 challenging the suitability of the laboratory mouse as a model for ageing research.

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YSF-31 Effects of new Pd(II) complexes on the allergenicity and proteolysis of bovine b-lactoglobulin A. Divsalar and A. A. Saboury Institute of Biochemistry & Biophysics, Tehran University, Tehran, IRAN b-lactoglobulin (BLG), the major protein in whey, is a globular protein with a monomer molecular weight of 18 kDa. Since native BLG is resistant to peptic and chymotryptic hydrolysis, then it has suggested that BLG is among the major causes of intolerance and/or allergenic response to cow’s milk in humans. Reports represent that the susceptibility of BLG to enzymatic hydrolysis often increases after protein denaturation by temperature, high pressure and different solvent conditions, because denaturation leads to removal of conformational epitops. In this study, a new class of four structurally related anticancer Pd(II) complexes including 2,2¢-bipyridin n-butyl dithiocarbamato Pd(II) nitrate, 2,2¢-bipyridin n-hexyl dithiocarbamato Pd(II) nitrate, 2,2¢(-bipyridin octylglycinato Pd(II) nitrate and 2,2¢(-bipyridinglycinato Pd(II) nitrate was designed. The aim of this study is to investigate the effects of these new synthesized complexes on the chymotryptic hydrolysis of BLG type A and B. After incubation of BLG(s) with different Pd(II) complexes for 20 hours at 4C, digestibility and susceptibility of the proteins were tested at relatively high chymotrypsin/BLG(s) ratio (1:10) in the presence and absence of different Pd(II) complexes. Electrophoresis and spectroscopic results revealed that in the presence of Pd(II) complexes, the protease-resistant of BLG(s) significantly decreased. Results suggested that some regions of BLG that were transiently exposed during the denaturation or incubation with Pd(II) complexes effectively hydrolyzed by chymotrypsin and that were not accessible in the native proteins. Hence, Pd(II) complexes can lead to decreasing in protease-resistant activity or allergenicity of BLG.

YSF-32 Host susceptibility to schistosomes: effect of portal sera on cell proliferation of S. mansoni schistosomules H. Draz1, Y. Shaker1, E. Ashour1 and G. Wu2 1 Biochemistry Department, National Research Centre, Dokki, Cairo, EGYPT, 2 Division of Gastroenterology-Hepatology, University of Connecticut Health Center, Farmington, CT, USA Introduction: It was hypothesized that the site preference of S. mansoni could be dependent on a constituent of portal blood that is not present in the periphery. Methods: Schistosomules of Schistosoma mansoni (20 days old) were incubated in medium containing hamster portal venous or peripheral venous serum (highly susceptible host) or rat portal venous or rat peripheral venous serum (poorly susceptible host) in presence of bromodeoxyuridine (BrdU) in order to determine effects of these substances on cell proliferation. Results: Compared to schistosomules cultured in presence of control medium, BLIs were increased by 41% in the presence of portal, but not in peripheral serum of the highly susceptible (hamster) host. In contrast, no significant differences were observed in the BLIs in portal sera from rat (poorly susceptible host) and control media, or peripheral sera. In vivo results revealed that there was no detectable cell proliferation in S. mansoni schistosomules (6 days old) in the lungs. However, cell proliferation was detected in schistosomules beginning at 17 days. The results indicated that portal venous serum from a highly susceptible host, but not from a poorly susceptible host stimulates schistosomule cell proliferation in vitro. The timing of the increase in cell proliferation during schistosomal development corresponds to liver portal mesenteric localization of schitosomules. Conclusion: Taken together, the data support the conclusion that in susceptible hosts, portal serum signals schistosomal cell proliferation, which bring to an end schistosomal migration. This may explain the co-localization of adults, and the known organ selectivity of disease.


Young Scientists Forum YSF-33 Structural and functional analysis of BRCA1-BRCT protein mutants implicated in breast cancer I. Drikos1, A. Thanassoulas2, G. Nounesis2 and C. E. Vorgias1 1 Department of Biochemistry and Molecular Biology, Faculty of Biology, National and Kapodistrian University of Athens, Athens, GREECE, 2National Center of Scientific Research ‘Demokritos’, IRRP, Athens, GREECE Breast cancer is the most common malignancy among women in western world. One third of the familial breast cancer cases can be attributed to mutations of certain genes as BRCA1 and BRCA2. The breast cancer tumor suppressor BRCA1 is involved in DNA repair and cell cycle control through interactions with other partner molecules such as BACH1 and CtIP. Mutations at the two C-terminal tandem (BRCT) repeats of BRCA1, disrupting BRCA1 complexes with BACH1 and CtIP, were identified in breast tumor patients. The aim of this study is to analyze how structural modifications of the mutated BRCT domain changes the proper function of BRCA1 protein. For this purpose, the cDNAs encoding the wild type (wt) and the mutant carboxyl-terminal regions (BRCT-domain) of BRCA1 were independently cloned in suitable prokaryotic expression vectors. The corresponding proteins were obtained in highly soluble and stable form. Biophysical analysis of the secondary structure and the thermodynamic stability of the wild type and selected mutant BRCT domains were performed by Circular Dichroism (CD) and Differential Scanning Microcalorimetry (DSC), respectively. Modification in binding affinity of the wt and mutant BRCT domains with BACH1 and CtIP, were studied by Isothermal Titration Calorimetry (ITC). These experiments demonstrated that the studied mutations of the BRCA1-BRCT (i) change the thermodynamic stability of the domain and (ii) influence the binding affinities with synthetic phosphopeptides, corresponding to BACH1 and CtIP binding sites. This loss of interactions may explain the abolition of the cell cycle control, mutation accumulation and breast cancer development.

Abstracts YSF-35 Betulinine JS8 induces apoptosis in tumor cells by mitochondrial disruption via specific non-covalent interactions with cytochrome c P. Dzubak1, J. Sarek2, P. Anzenbacher1, V. Masek1, P. Novak3, V. Havlicek3, M. Otyepka4, D. Vydra1 and M. Hajduch1 1 Laboratory of Experimental Medicine, Departments of Pediatrics and Department of Pharmacology, Faculty of Medicine and Dentistry, Palacky University, Olomouc, CZECH REPUBLIC, 2Department of Organic and Nuclear Chemistry, Charles University, Prague, CZECH REPULIC, 3Laboratory of Molecular Structure Characterization, Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, CZECH REPUBLIC, 4 Department of Physical Chemistry, Palacky University, Olomouc, CZECH REPUBLIC Synthetic betulinine, 3b,28-Diacetoxy-18-oxo-19,20,21,29,30-pentanorlupan22-oic acid (JS8) induces rapid apoptotic responses at micromolar concentrations in tumor cells. Several lines of evidence reported here indicate that JS8-induced apoptosis is mediated by its effects on mitochondria and induces cytochrome c release with consequent activation of caspases. The release of cytochrome c and apoptosis can be prevented by applications of the antioxidant N-acetyl-L-cysteine and KCN, an inhibitor of complex IV, but not other tested inhibitors of oxidative phosphorylation. The data suggest that the target of JS8 lies between complexes III and IV where cytochrome c is located. Incubation of JS8 with purified cytochrome c in vitro resulted in the formation of specific non-covalent complexes that were characterized using UV/VIS, Raman, and mass spectrometry. Molecular modeling corroborates the suggested location of the putative JS8 binding sites on the surface of cytochrome c. We propose that cytochrome c is both the effector molecule and primary target for triterpenoid anticancer compounds such as JS8.

YSF-36 Podoplanin as a malignancy promoter: a new variant with a diferent role? B. Fernandez-Munõz, E. Marfin Villar, M. M. Yurrita, J. Renart and M. Quintanilla Instituto de Investigaciones Biome´dicas ‘‘Alberto Sols’’ (CSIC-UAM), Madrid, SPAIN

YSF-34 Modulation of topoisomerase I activity: pharmacological approach to prevent CFTR exon 9 aberrant splicing G. Dujardin1, A. Mbopda1, E. Buratti2, F. Pagani2, A. Kornblihtt3, C. Ferec1 and L. Corcos1 1 INSERM U613, Brest, FRANCE, 2ICGEB, Trieste, ITALY, 3Laboratorio de Fisiologia y Biologia Molecular, Buenos Aires, ARGENTINA Introduction: CF is the most common autosomal recessive disease in Caucasians, and it is caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. Less-severe forms depend on an allelic variation upstream of CFTR exon 9 and responsible for its skipping, which results in a non functional protein. This region is composed of 9–13 UG repeats followed by 5–9 U. The aim of this work was to try to design a pharmacological approach to improve the level of exon 9 inclusion. Methods: We used transfections of CF exon 9-centered minigenes and RNA polymerase II vectors and pharmacological treatments modify the levels of exon 9 skipping. Results: We show that camptothecin (CPT), a DNA topoisomerase I (TopoI) inhibitor, known to modify pre-mRNA splicing, or a slow RNA pol II, lead to a decrease in CFTR exon 9 inclusion. As CPT blocks TopoI on DNA, we used an expression vector of RNA polymerase II lacking the Carboxy Terminal Domain (CTD) and we demonstrate that this domain is absolutely necessary for the CPT effect. We propose that TopoI kinase domain could phosphorylate a component of the transcription elongation complex, leading to the alteration of splicing. Strikingly, a specific inhibitor of TopoI kinase activity restores exon 9 inclusion. Conclusions: Our results suggest that the specific ability of TopoI to modulate pre-mRNA splicing depends on its kinase domain. Pharmacological targeted interventions on TopoI kinase activity could provide new means to restore sufficient levels of functional CF mRNA.

Podoplanin (PDPN) is a small transmembrane glycoprotein linked to the actin cytoskeleton whose expression is strongly related to tumour progression. PDPN is induced in a wide variety of cancers, specially those derived from germ cells and stratified epithelia and is frequently located in invasive areas of the tumours. We reported that PDPN is induced during mouse skin chemical carcinogenesis and wound healing. PDPN expression is significantly increased during carcinogenesis, particularly on later stages associated with development of highly malignant poorly differentiated carcinomas. Ectopic expression of PDPN in epithelial cells results in an epithelial-mesenchymal transition (EMT) and the acquisition of a more migratory and metastatic phenotype. These changes seem to require PDPN association with ERM proteins and RhoA activation. We have found that PDPN expresses two isoforms by alternative splicing: the standard isoform (PDPNs) and a variant, PDPNv(DYS), differing only in two cytoplasmic aminoacids. PDPNs and PDPNv(DYS) appear to perform distinct cellular roles as in contrast to the standard isoform, PDPNv(DYS) was unable to promote EMT. In order to understand the function of PDPN on cell migration, invasion and signal transduction, we are analyzing PDPN-mediated protein–protein interactions and the role of PDPN phosphorylation on these interactions.


447

Abstracts


YSF-37 Doxorubicin inhibits TGFb-signaling via blocking translocation of Smad-proteins into nucleus of tumor cells Y. Filyak1,2, O. Filyak1,3, S. Souchelnytskyi2 and R. Stoika1 Institute of Cell Biology, NASU, Lviv, UKRAINE, 2Karolinska Institutet Biomics Center, Stockholm, SWEDEN, 3Lviv State University of Vital Activity Safety, Lviv, UKRAINE 1

Introduction: Doxorubicin (Dx, adriamycin) is a DNA-damaging anticancer drug, possessing both cytotoxic and anti-metastasis activity. However, the anti-metastasis activity of Dx cannot be explained by earlier described mechanisms of its action. TGFb1 is a cytokine that is often up-regulated in human tumors and can decrease cytotoxic action of anti-cancer drugs and induce metastasis-formation. Methods: Western-Blotting, RT-PCT, Luc-reporter assay, immunohistochemistry and nuclei-isolation, etc. were used for investigation of geneexpression, protein-phosphorylation and protein-translocation. Radioactive 35 S and 3H were used for DNA and protein labeling. Results: Dx inhibited TGFb-signaling in human lung adenocarcinoma A549 cells, namely, it blocked TGFb1-induced activation of Smad3-responsive CAGA12-Luc reporter, as well as five other Smad-dependent reporters. However, it didn’t affect functioning of TGFb-independent c-Myc-Luc reporter. That was observed as early as after 1–3 hours of treating these cells with Dx, while such DNA-damaging drugs (cisplatin or methotrexate) didn’t alter activation of CAGA12-Luc reporter under the same conditions. Besides, after 1 hour action, Dx abrogated TGFb-induced translocation of Smad3-protein from cytoplasm to the nucleus. Down-regulation of expression of Smad2-, Smad3-, Smad4-proteins, and TGFb-receptor type-II mRNA, and up-regulation of inhibitory Smad7-protein upon Dx treatment, were found after 12– 24 hours of Dx treatment. Phosphorylation of Smad2- and Smad3-proteins was also affected by Dx. Conclusions: Tumor cell treatment with Dx is resulted in inhibition of TGFb-signaling at both early (1 hour) and later (12 hours) stages of the drug action. Such inhibition can be a potential molecular explanation of the antineoplastic action of Dx towards tumor cells.

YSF-38 Candida species trigger oxidative stress response in innate immune cells I. E. Frohner, C. Bourgeois, O. Majer, W. Glaser and K. Kuchler Max F. Perutz Laboratories, Medical University Vienna, AUSTRIA The clinical spectrum Candida spp. ranges from mucocutaneous infections to systemic, life-threatening diseases in immunocompromised patients. One of the immediate early responses of host phagocytes facing pathogens is the production of reactive oxygen species (ROS), which aim at destroying invading microbial pathogens. We therefore asked whether Candida spp. can induce ROS in innate immune cells, and which genes are implicated in counteracting the oxidative burst mounted by host cells. Hence, we constructed homozygous C.a deletion strains, each lacking one of the six superoxide dismutase genes (SOD1-6). Using an in vitro interaction system with primary immune cells (primary bone-marrow-derived macrophages, BMDMs or dendritic cells, mDCs), we investigated the role of C.a deletion strains in the ROS response. Real-time in vitro ROS assays based on chemiluminescence and dihydrofluoresceine staining of host and fungal pathogens, we showed that C.a, as well as C. dubliensis (C.d) and C. glabrata (C.g) strongly trigger ROS production in BMDMs, although to a different extent. The marked differences in ROS production by BMDMs infected by C.a, C.d and C.g may relate to different expression of cell surface proteins, and may perhaps contribute to distinct virulence properties observed for these pathogens in vivo. Most strikingly, a dramatic increase in extra-cellular ROS accumulation was observed when BMDMs were co-cultured with C.a deletion strains lacking a single fungal dismutase gene. Thus, our data identify a novel C.a. defence gene possibly playing a role in extra-cellular ROS detoxification during host invasion, since this gene may destroy host-derived ROS. Acknowledgements: Supported by the ERA-Net Pathogenomics FunPath project funded through the Austrian Science Foundation (FWF-I125-B09), and by a Fellowship of the Vienna Biocenter PhD Programme to IF.

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YSF-39 Matrix chondroitin sulphate chains participate in the major intracellular signalling pathways which affect the proliferation, adhesion and migration of fibrosarcoma cells E. Fthenou1, A. Zafiropoulos1, N. K. Karamanos2 and G. N. Tzanakakis1 1 Department of Histology, School of Medicine, University of Crete, Heraklion, GREECE, 2Laboratory of Biochemistry, Department of Chemistry, University of Patras, Patras, GREECE Intoduction: Fibrosarcoma is an uncommon soft tissue tumor whose cell microenvironment is rich in ECM components and particularly in glycosaminoglycans/proteoglycans (GAGs/PGs). Platelet derived growth factor (PDGF) has been shown to physically interact with GAGs which are abundant in the fibrosarcoma cell microenvironment. PDGF is involved in the autocrine growth stimulation of malignant cells, the stimulation of angiogenesis and the recruitment and regulation of tumor fibroblasts. In this study the role of chondroitin sulfate (CS) on fibrosarcoma cell proliferation, adhesion, motility, migration and PDGF-BB signaling pathway was investigated. Methods: Exogenously added GAGs, regulators of endogenous GAG synthesis (sodium chlorate as selective inhibitor and b-D-xyloside as a stimulator) and specific glycosidases, to cleave cell-associated GAGs, were utilized. Results: Cleavage of cell-associated CS as well as specific inhibition of endogenous CS production severely impaired these fibrosarcoma cell functions. Treatment with free CS chains enhanced cell motility and chemotaxis, whereas adhesion was inhibited. A significant co-stimulatory effect of CS in combination with PDGF-BB on the growth of HT1080 was found. The stimulatory effect of CS was not due to transcriptional up regulation of PDGF receptor genes, but rather to more efficient signalling of PDGF-Rb through tyrosine 1021 phoshorylation. When inhibitors of the main cellular signaling pathways regulating actin cytoskeleton rearrangements were applied, CS chains were found to upregulate cell motility through the MAPK pathway, specifically through JNK, whereas CS-induced chemotaxis was found to require tyrosine kinase dependent pathways. Conclusion: This study introduces a new crucial role of CS chains on tumor cell proliferation, adhesion, motility and chemotaxis.

YSF-40 Human histone chaperone nucleophosmin (NPM1) alters the higher order chromatin structure by interacting with a chromatin associated protein S. Gadad1, K. Hizume2, S. Venkatesh1, J. Shandilya1, C. Das1, K. Takeyasu2 and T. K. Kundu1 1 Transcription and Disease Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, INDIA, 2Laboratory of Plasma Membrane and Nuclear Signaling, Graduate School of Biostudies, Kyoto University, Sakyo-Ku, Kyoto, JAPAN Introduction: Reversible posttranslational modifications of core histones and chromatin associated proteins and ATP dependent chromatin remodeling complexes are not enough to maintain the dynamicity of chromatin, which is an essential step for the transcriptional regulation. Recent evidences suggest that the replication independent histone chaperone activity could be one of the key players of this phenomenon. Recently, we have shown that the multifunctional human histone chaperone, NPM1 enhances chromatinmediated transcription in an acetylation dependent manner. Methods: By employing co-immunoprecipitation assay, we have found that NPM1 interacts with one of the most important chromatin associated protein (CAP). Different C-terminal and internal deletion mutants of NPM1 were used in vitro pull down studies to identify the domain of NPM1 interacts with CAP. To elucidate the functional role of this interaction, apart from biochemical analysis, two approaches viz., circular dichroism (CD) spectroscopy and atomic force microscopy (AFM) were used taking in vivo as well as in vitro reconstituted chromatin. Results and Conclusion: In vitro pull down studies suggest that acidic stretch 1 domain of NPM1 is responsible for interaction. Biochemical analysis along with CD experiments lead us to conclude that NPM1 decondenses chromatin by removing CAP and visualization through AFM shows that NPM1 alleviates CAP mediated compaction. Thus, NPM1 not only activates transcription in an acetylation dependent manner but also disrupts higher order chromatin structure by laying path for various DNA dependent physiological processes.



Abstracts

YSF-41 YSF-43 Structure-function analysis of PAR1, an atypical bHLH Interaction between heavy metals and sarcoplasmic protein, regulating shade avoidance responses in reticulum Ca2+-ATPase E. Gramigni1, G. Santini1 and M. K. Moncelli2 plants 1

A. Galstyan1, I. Roig Villanova1, J. Bou-Torrent1 and J. F. Martinez Garcia1,2 1 Laboratori de Genetica Molecular Vegetal, Consorsi CSIC-IRTA, Barcelona, SPAIN, 2Institucio Catalana de Recerca I Estudis Avançats, Barcelona, SPAIN

Light is arguably the most important abiotic factor controlling plant growth and development throughout their life cycle: from germination to flowering induction. Therefore to sense multiple parameters of ambient light signals, plants have evolved at least four different photoreceptor systems: phytochromes, cryptochromes, phototropins, and UV-receptors. The red/far-red light (R/FR) sensing phytochromes (phys) are the best characterized of these photoreceptors. One of the processes regulated by phys, the shade avoidance syndrome (SAS) refers to a group of responses induced when fully de-etiolated plants grow in close proximity of neighbouring vegetation. The perception of this single environmental signal (low R: FR) by phytochromes initiates a complex transduction network that triggers sophisticated SAS responses. In the laboratory, we have identified PHY RAPIDLY REGULATED1 (PAR1), a gene, whose expression is rapidly affected by low R: FR ratio light. PAR1 encodes a novel and atypical bHLH protein. We show data about structure-function relationship of this novel factor. By analyzing biological activity and subcellular localization in transgenic lines overexpressing different deletion series of PAR1 fused to the reporter green fluorescence protein (GFP), we have identified the functional domains of the protein that confer biological activity and nuclear localization. Moreover, yeast two hybrid analyses allowed us to demonstrate that PAR1 dimerization ability is crucial for having biological function in plants. In this communication the latest results will be shown.

YSF-42 Plant cellular self/non-self discrimination is orchestrated by bacterial pathogens V. Goëhre, T. Spallek, H. Ha¨weker and S. Robatzek Max-Planck Institute for Plant Breeding Research, Department of Plant-Microbe Interactions, Cologne, GERMANY Introduction: Self/non-self discrimination is the underlying principle by which higher organisms sense microorganisms including potential pathogens. Presence of microbes is monitored by pattern recognition receptors (PRRs) by recognizing so-called microbe-associated molecular patterns (MAMPs). Upon perception of MAMPs, plants initiate a suite of defense responses that lead to plant immunity. To overcome this level of perception, pathogens deliver effector molecules into the plant cell that interfere with immune responses. Results: We are studying the Arabidopsis thaliana pattern recognition receptor FLS2, which recognizes bacterial flagellin, flg22, and triggers defense reactions such as stomata closure, ROS production, cell wall fortification, and expression of defense genes. Due to recognition at first contact, FLS2 restricts bacteria from entry into plant tissues and limits bacterial proliferation. Therefore, pathogenic bacteria target FLS2 function. AvrPtoB, an effector from Pseudomonas syringae pv. tomato, suppresses immune responses triggered by FLS2. Expression of AvrPtoB in planta leads to degradation of flg22 activated FLS2. AvrPtoB is a modular protein, which carries an E3 ligase activity in its C-terminal part. Interaction of AvrPtoB with FLS2, which does not require the C-terminal domain, was shown in pull-down assays. Furthermore, FLS2 is ubiquitinated by AvrPtoB in vitro. Conclusions: We propose a model, in which AvrPtoB binds to FLS2 independently of the E3 ligase function and prevents acute signaling. Ubiquitination of FLS2 is a subsequent step, which allows degradation of this receptor and thereby permanent down-regulation of defense signaling. Our studies provide mechanistic insights about how plants and microbes interact and how host immune response are subverted by pathogens.

Dipartimento di Biologia Animale e Genetica ‘L. Pardi’, Universita degli Studi di Firenze, Firenze, ITALY, 2Dipartimento di Chimica, Universita degli Studi di Firenze, Firenze, ITALY

Introduction: Sarcoplasmic Reticulum (SR) Ca2+-ATPase is an integral membrane protein responsible for transport of Ca2+. This protein plays an essential role in cellular calcium homeostasis. SR Ca2+-ATPase couples the hydrolysis of ATP to the active transport of Ca2+ ions from the cytoplasm into the SR lumen, thus inducing muscle relaxation. Our study aims at investigating the effect of heavy metals of environmental impact (Cd2+, Cu2+ and Pb2+) on the molecular mechanism of SR Ca2+-ATPase. Methods: We are making use of an electrical technique recently developed (B. Kelety et al., Assay Drug Dev. Technol. 2006; 4: 575–582). The protein is first adsorbed on a solid supported membrane (SSM) and then activated with Ca2+ and ATP concentration jumps through a rapid solution exchange. If at least one electrogenic step is involved in the reaction following such activation, a current transient can be measured along the external circuit. Numerical integration of each transient is related to a net charge movement, which depends upon the particular electrogenic event, i.e. cytoplasmic Ca2+ binding and lumenal Ca2+ release. Results: We observed a decrease of the current signal in the presence of a specific heavy metal concentration. Therefore, we determined an inhibitory effect of xenobiotics (Cd2+, Cu2+ and Pb2+) on the SR Ca2+-ATPase activity in the concentration range 1–100 lM. Conclusions: We can conclude that heavy metals of environmental impact have an inhibitory effect on SR Ca2+-ATPase.

YSF-44 Positive regulation of osteoclast - mediated bone resorption and podosome organization by protein tyrosine phosphatase epsilon S. Granot-Attas1, C. Luxenburg2 and A. Elson1 Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot, ISRAEL, 2Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot, ISRAEL 1

We present genetic and biochemical evidence that protein tyrosine phosphatase epsilon (PTPe) is required for osteoclast activity in vivo and in vitro. Young female mice lacking PTPe (EKO mice) exhibit increased bone mass due to reduced osteoclast-mediated bone resorption. EKO osteoclasts adhere to bone poorly in vivo, and serum concentrations of products of osteoclastmediated bone resorption are reduced in EKO mice. EKO osteoclasts function poorly in in vitro assays of bone resorption, indicating that they are inherently defective. Organization of podosomes, the actin-rich structures by which osteoclasts adhere to bone, is severely disrupted in EKO osteoclasts. Disorganization is evident in the internal structure of individual podosomes that are part of sealing zone-like structures as well as in their reduced ability to participate in these structures in freely-growing cells and after replating. Furthermore, EKO podosomes located at the peripheral belt exhibit defective actin-core dynamics and are longer-lived than WT belt podosomes. At the molecular level quantitative immunofluorescence microscopy revealed increased phosphotyrosine content in podosomes of EKO osteoclasts. Studies also suggest that PTPe is phosphorylated at its C-terminal Y638 following activation of integrin, and that this phosphorylation of PTPe is required for activation of Src following adhesion to matrix. We hypothesize that lack of PTPe reduces Src activation following activation of integrins in osteoclasts, leading to reduced cellular response following contact with matrix and to reduced osteoclast function in EKO mice. In all, we conclude that PTPe-mediated dephosphorylation of Src and other podosomal proteins is required for regulating podosome dynamics in osteoclasts, and for proper maturation and function of these cells.


449

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YSF-45 Oxidative stress markers in the cerebellum during hypokinesia in rats A. S. Grigoryan, G. A. Navasardyan, M. A. Simonyan and A. A. Sanosyan Department of Pathophysiology, Yerevan State Medical University, Yerevan, ARMENIA Hypokinesia (HK) is considered as a risk factor in a variety of diseases, including nervous system pathology. Oxidative stress is involved in the pathogenesis of HK-induced disorders, affecting movement and motor coordination functions, in which cerebellum plays significant role. The aim of this study was to evaluate the role of oxidative stress in cerebellum during HK. Experimental HK was achieved by placing rats in narrow individual cages for 2, 7, 15, 45 and 70 days with 22 hours exposure to HK each day. The activity of antioxidative enzymes: superoxide dismutase (SOD) and catalase, and malondialdehyde (MDA) level were measured in cerebellar tissue homogenates. It was found that at early stages of HK (2 and 7 days) MDA level in the cerebellar tissue slightly increased by 9.7% (p < 0.05). It was accompanied by decrease in antioxidant enzyme activities. Then, on the 15th day SOD activity increased reaching control values; the level of catalase activity reduced by 35.3% (p < 0.05), which however was not accompanied by MDA increasing. Behavioral studies showed improved motor coordination functions (rotorod test) in animals kept 15 days at HK. We suggested that at this stage of HK the level of H2O2 mildly enhanced due to decrease in antioxidant enzyme activity, particularly catalase activity and molecules of H2O2 may act as signal molecules to activate molecular adaptation system in cerebellar cells. Increased SOD and catalase activities and reduced level of MDA were observed at late stages of HK. These observations were distinguished as allostatic adaptation in chronic HK conditions.

YSF-46 TRPV1 C-terminus multiple binding sites studies using fluorescence spectroscopy and molecular modeling L. Grycova, Z. Lansky, E. Friedlova and J. Teisinger Institute of Physiology, Academy of Sciences of the Czech Republic, Progue CZECH REPUBLIC Introduction: TRPV1 is a member of a TRP ion channel family. This ion channel is predicted to be composed of six transmembrane domains and two intracellular termini. TRPV1 plays an important role in pain sensation and its activity is regulated by various stimuli and molecules. The main point of our interests was isolated C-terminus (CT) and its interaction with ligands like ATP and calmodulin, which play a role in TRPV1 activity regulation. Methods: C-terminus TRPV1 was subcloned into expression vectors pGEX4t and pET32b. Protein was expressed in bacteria E. coli and purified in two steps purification protocol. Based on computer model of TRPV1-CT, several amino acid residues were selected and using PCR site directed mutagenesis were replaced by alanine. Molecular models predictions were verified by steady–state fluorescent spectroscopy experiments with FITC, TNP-ATP, AlexaFluor 488. Results: We have localised ATP binding site, pivotal role in this interaction plays lysine 735. Our molecular model predicted single calmodulin binding domain and fluorescence anisotropy measurements confirmed its position. Isolated TRPV1-CT interacts with calmodulin in a Ca2+ dependent manner. Basic residues R771, R785, R778, and R797 interacted with negatively charged edges of calmodulin binding sites. Dissociation constants of all these constructs were estimated. Conclusions: Protein modelling combined with spectroscopic techniques served as a suitable tool in mapping binding domains of isolated TRPV1CT. TRPV1-CT interacts with ATP as well as calmodulin and the crucial amino acid were pointed out. Acknowledgements: This work was supported by GAAVIAA600110701, GACR303/07/0915, project H148, Centre of Neurosciences LC554 MSMT CR.

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YSF-47 Natural inhibitor of plasmepsin II from the gorgonian Plexaura homomalla: partial purification and characterization Y. Guerra1, A. Ramı´ rez1, M. L. Reytor1, R. Robles1, C. Berry2, J. Mendiola3, A. Hernandez-Zanui4 and M. Cha´vez1 1 Center for Protein Studies, Faculty of Biology, University of Havana, Havana, CUBA, 2Cardiff School of Biosciences, Cardiff University, Cardiff, UK, 3Tropical Medicine Institute ‘Pedro Kouri’, Havana, CUBA, 4Cuban Institute of Oceanology, CITMA, Havana, CUBA Introduction: The increasing resistance of malaria parasites to the current therapies determines the necessity of new antimalarial drugs. Growth inhibition of Plasmodium falciparum by aspartic proteinase inhibitors demonstrated their potentialities as antimalarials drugs. Plasmepsin II is the best characterized of these enzymes in Plasmodium falciparum. The aim of this work was the partial purification and characterization of a Plm II inhibitor detected in the gorgonian Plexaura homomalla. Methods: The inhibitor from Plexaura homomalla (PhPI) was partially purified combining affinity chromatography and gel filtration chromatography. The inhibitory assays were performed using the chromogenic peptide Leu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe as substrate and a recombinant form of Plm II. A preliminary PhPI specifity profile against other proteinases was evaluated as part of its functional characterization. Results: Kinetics parameters of the recombinant Plm II using the substrate Leu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe were KM: 27.4 ± 2.0 (M, kcat: 10.4 ± 1.5/s and kcat/KM: 378 ± 56/mM/s. During the inhibitor purification procedure gel filtration chromatography yields a main peak containing 98.3% respect to the total units of Plm II inhibitory activity initially applied to the column. PhPI Ki value against Plm II was 2.2 nM and the inhibition curve showed a concave behaviour. The inhibitor was able to inhibit Pepsin and Plm IV, both aspartic proteinases. PhPI is not able to inhibit the serine, metallo and cysteine proteinases tested. Conclusions: The PhPI Ki value and the specificity profile suggest that it is an aspartic proteinase thight binding inhibitor. Interactions studies of this molecule with Plasmepsins would be useful for the development of new antimalarials drugs. Acknowledgements: International Foundation for Science, and Organization for the Prohibition of Chemical Weapons (IFS grant F/3772-1), UNDP/World Bank/WHO Special Program for Research and Training in Tropical Disease (TDR, ID 990948) and British Council.

YSF-48 A novel RET kinase-b-catenin signaling pathway contributes to tumorigenesis in thyroid carcinoma T. S. Gujral1, W. Van Veelen2, D. S. Richardson1, S. M. Myers1, J. A. Meens1, D. S. Acton2, M. Dunach3, B. E. Elliott1, J. W. M. Hoeppener2 and L. M. Mulligan1 1 Division of Cancer Biology and Genetics, Cancer Research Institute, Queen’s University, Kingston, ON, CANADA, 2Department of Metabolic and Endocrine Diseases, University Medical Center Utrecht, Utrecht, THE NETHERLANDS, 3Unitat de Biofı´sica, Departament de Bioquı´mica i Biologia Molecular, Facultat de Medicina, Universitat Autonoma de Barcelona, Barcelona, SPAIN Introduction: The RET receptor tyrosine kinase has essential roles in cell survival, differentiation, and proliferation. Oncogenic activation of RET causes the cancer syndrome multiple endocrine neoplasia type 2 (MEN 2), and is a frequent event in sporadic thyroid carcinomas. However, the molecular mechanisms underlying RET’s potent transforming and mitogenic signals are still not clear. Results: Here, we demonstrate that nuclear localization of b-catenin is frequent in both thyroid tumours and their metastases from MEN two patients, suggesting a novel mechanism of RET-mediated function, through the b-catenin signalling pathway. We show that RET binds to, and tyrosine phosphorylates, b-catenin and demonstrate that the interaction between RET and b-catenin can be direct and independent of cytoplasmic kinases, such as SRC. As a result of RET-mediated tyrosine phosphorylation, b-catenin escapes cytosolic downregulation by the APC/Axin/GSK3 complex and accumulates in the nucleus, where it can stimulate b-cateninspecific transcriptional programs in a RET-dependent fashion. We show that downregulation of b-catenin activity decreases RET-mediated cell proliferation, colony formation, and tumour growth in nude mice. Conclusions: Together, our data show that a b-catenin-RET kinase pathway is a critical contributor to the development and metastasis of human thyroid carcinoma.



Abstracts

YSF-49 Tpl2 transduces GPCR signals that promote actin cytoskeleton reorganization and cell migration

YSF-51 Functional selection of randomized library of MC4 receptor in yeast Saccharomyces cerevisiae

M. Hatziapostolou, C. Polytarchou, D. Panutsopulos, L. Covic and P. N. Tsichlis Molecular Oncology Research Institute, Tufts-New England Medical Center, Boston, MA, USA

V. Ignatovica and J. Klovins Latvian Biomedical Research and Study Centre, Riga, LATVIA

Introduction: Tpl2 (tumor progression locus 2) is a mitogen activated protein kinase kinase kinase (MAP3K) that is activated by provirus insertion in retrovirus-induced rodent lymphomas and mammary adenocarcinomas. Physiologically Tpl2 is required for the transduction of TLR, IL-1, TNF-a and CD40 signals and plays a very important role in inflammation. Methods: Mouse embryonic fibroblasts (MEFs) from Tpl2+/+ and Tpl2-/C57BL6 embryos were used. Immortalized Tpl2-/- MEFs were also used to establish cell lines reconstituted either with the wild type Tpl2 or the kinase dead mutant. Phalloidin staining was used to survey the actin cytoskeleton, wound healing and transwell filter assays were applied to study cell migration. The activity of Tpl2 and MMPs was assessed by in vitro kinase and zymography assay, respectively. Gene expression was evaluated by real time RT-PCR. Intracellular calcium concentration was measured by using a fluorescent Ca++ indicator. Results: Here we show that Tpl2 is required for the transduction of cell migration signals originating in the G-protein coupled receptor (GPCR) PAR1. Proteinase-activated receptor 1 (PAR1) signals transduced by Tpl2 activate Rac1 and Focal adhesion kinase (FAK), and they are required for reorganization of the actin cytoskeleton and cell migration. In addition, we demonstrate that Tpl2 is required for Ca++ release from the endoplasmic reticulum in response to GPCR signals. Conclusions: Our data suggest that signals that regulate cell migration and gene expression flow between stromal and tumor cells in both directions and that Tpl2 plays a pivotal role in this process.

YSF-50 Marginally hydrophobic transmembrane helices: how do they stay in the membrane and what do they imply for protein structure and function? L. Hedin, K. Geiger, K. Enqvist, M. Lerch-Bacher, I.-M. Nilsson, A. Elofsson and G. Von Heijne Center for Biomembrane Research, Stockholm University, Stockholm, SWEDEN Membrane proteins are crucial for cellular communication and transport. Due to their hydrophobicity, they often follow different rules regarding folding and function than soluble proteins. It is usually assumed that a transmembrane helix overall must be hydrophobic to be inserted into and remain in a cellular membrane bilayer. However, predicting the hydrophobicity of transmembrane helices from all known 3D-structures, we have found that there are a number of transmembrane helices that are not sufficiently hydrophobic to insert in isolated form. One logical deduction is that these helices are stabilized in the membrane by interactions with other membrane helices. We are now investigating these marginally hydrophobic helices by comparing their insertion efficiency with or without neighbouring helices. The interesting sequence is inserted between two engineered glycosylation sites in the LepB-protein. When expressing the construct in vivo in the presence of microsomes, the extent of single and double glycosylation depends on the insert’s insertion efficiency. Quantification is performed by comparing band intensities of glycosylated proteins separated by SDS-PAGE. So far we have confirmed the low insertion efficiency for more than 20 helices. By analysing a large number of potential helix-helix interactions we will be able, for the first time ever, to get an overview of the general importance of helix-helix interactions for the insertion of marginally stable helices. We believe that this information will be of great importance for the overall understanding of membrane protein folding and structure.

Introduction: G protein coupled receptors (GPCR) are the largest transmembrane recpetor family. Members of GPCR family regulate wide range of very important physiological functions, being therapeutic target for most of recently developed drugs. Melanocortin 4 receptor (MC4R) controls feeding and energy homeostasis and MC4R ligands may serve as potential drugs for adiposity treatment. In development of new therapeutic ligands it is crucial to study regions of receptor that are involved in ligand binding. These regions can be identified by receptor mutagenesis and analysis of functional activity of mutant receptor. Methods: In this study Asp at position 126 of MC4R gene was randomized in PCR reaction using random primers. Obtained library of randomized MC4R variants was cloned into expression vector and transformed in corresponding yeast Saccharomyces cerevisiae cells. Clones from library containing different amino acid codons were isolated and screened for functional activity. Additionally the yeast cells were grown under conditions of functional selection (presence of active ligand) after transformation with randomized MC4R library. These cells were passed through subsequent number of growth cycles followed by sequence estimation at each cycle. In this experiment only the cells that contained functionally active variants of MC4R were able to grow and multiply. Results: From all randomized variants only aspartic acid (GAC) and tyrosine (TAC) codon variants showed functional activity during activation with MSH, but MC4R containing aspargine (ATT), cysteine (TGC), glutamic acid (GAA), glycine (GGG and GGT), isoleucine (ATC and ATA), leucine (CTA and CTT), phenylalanine (TTT and TTC) and valine (GTT and GTC) codon variants were functionally inactive. During functional selection GAC codon coding for aspartic acid was selected. Conclusions: This functional study shows that amino acid in position 126 of MC4R has overall functional importance in ligand recognition and must be considered as significant binding site determinant in development of novel therapeutics on MC4R.

YSF-52 Regulation of human ribosomal protein biosynthesis at the pre-mRNA splicing step A. Ivanov, N. Parakchnevitch, A. Malygin and G. Karpova Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Science, Novosibirsk, RUSSIA Introduction: Accurate regulation of ribosomal protein gene expression is indispensable for eukaryotic cell life function. However, there is a gap in knowledge on regulation of splicing of human ribosomal protein pre-mRNAs. The present study was devoted to the investigation of the role of human ribosomal proteins (rp) S13 and S26 in regulation of their own pre-mRNAs splicing. Methods: Standard molecular biological methods (PCR, cloning, immunoprecipitation, footprinting etc) were used in the work. Results: Comparison of the sequences of rpS13 and rpS26 genes among eukaryotes revealed that the 1st intron is more conserved than other introns. It was shown that rpS13 binds specifically to its own pre-mRNA near the 5¢and 3¢-splicing sites of the 1st intron and rpS26 near the 3¢- splicing site of the 1st intron. The 1st intron excision from these ribosomal protein pre-mRNAs was inhibited by addition of the respective ribosomal protein into the in vitro splicing reaction. Besides, transfection of HEK 393 cells with mini-gene expressing rpS13 showed that the presence of the 1st intron reduced expression by a factor of four. Cloning, expression and purification of rpS26 mutants were performed. Binding of these mutants to rpS26 pre-mRNA revealed that the central and C- domains of rpS26 are necessary for binding of this protein to its pre-mRNA. Conclusion: Overproduction of human rpS13 and rpS26 interferes with splicing of their pre-mRNAs by a feedback mechanism. Acknowledgement: This work was supported by grant from RFBR (# 0804-00593-a).


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Abstracts


YSF-53 Human interferon-gamma derivates to be used as potential drugs for treatment of multiple sclerosis E. Ivanova1, S. Petrov1, D. Dangova1, V. Posheva1, A. Berzal-Herranz2, G. Nacheva1 and I. Ivanov1 1 Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia, BULGARIA, 2Instituto de Parasitologia y Biomedicina ‘Lopez-Neyra’, CSIC, Granada, SPAIN

YSF-55 Oligopeptides of human ribosomal proteins involved in formation of the mRNA binding centre of the ribosome Y. Khairulina, K. Bulygin, A. Ven’yaminova, D. Graifer and G. Karpova Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, RUSSIA

Multiple sclerosis (MS) is an autoimmune disease leading to demyelination of the central nervous system, which is accompanied by an abnormal production of interferon gamma (hIFNc). The latter itself suppresses myelin biosyntheses and deteriorates the MS patients’ health. Besides the current methods for suppressing the overexpression of hIFNc (application of hIFNb, anti-hIFNc antibodies, etc.), a new approach for counteracting hIFNc could be based on the competitive inhibition by hIFNc analogues incapable to trigger the hIFNc signal transduction pathway but active towards the hIFNc receptor. Our data show that the deletion of the entire C-terminus (21 aminoacids) of hIFNc lead to 10-fold decrease in its biological activity. Using PCR and appropriate primers we have constructed two new 3¢-end derivative genes coding for hIFNc proteins truncated by 24 (hIFNc-24Ct) and 27 aminoacids (hIFNc-27Ct). The last alpha-helix (F) in these proteins is shortened by three and six aminoacids respectively. The two genes were expressed in E. coli LE392. Both proteins in clear cell lisates were investigated for antiviral and antiproliferative activities and proved biologically inactive. When co-incubated in equimolar concentrations with the wild type hIFNc (standard) in the antiproliferative assay, hIFNc-24Ct competed for the cell receptors that led to 40% (±10%) inhibition of the standard activity whereas hIFNc-27Ct showed decreased affinity. Based on these and other results we conclude that hIFNc-24Ct has preserved better its affinity to the hIFNc receptor and therefore it is a potential candidate for anti-hIFNc therapy of MS and/or other autoimmune diseases. Acknowledgements: Supported by NSF, grant K-1405.

Introduction: The understanding of molecular mechanisms of translation requires detailed information on the structure–function relationship of the ribosome. The information on eukaryotic ribosomes that to date could not be studied by X-ray analysis is much more limited than on prokaryotic ones. Methods: Structural and functional topography of eukaryotic ribosomes was studied with the use of an affinity cross-linking approach. Results: We determined protein S3 and S15 fragments neighboring mRNA nucleotides in positions +4 to +12 with respect to the first nucleotide of the P site codon on the human ribosome. The proteins were cross-linked to mRNA analogues containing perfluorophenyl azide-modified uridine in ribosomal complexes where it was located in these positions. To identify oligopeptides cross-linked to 32P-labeled mRNA analogues, the modified proteins were cleaved with CNBr at the methionine residues and modified oligopeptides were identified by PAGE. We found that cross-linker at the uridines in positions +4 to +12 modified C-terminal fragment 111-145 of protein S15 (yield of cross-linking strongly decreased when modified uridine moved from position +4 to +12). Locations of cross-linking sites in protein S3 depended on the positions of modified uridines and were found in the N-terminal half (fragment 2-217) and/or in the C-terminal fragment 190-236. Conclusions: We found that S15 protein fragment 111-145 is involved in formation of the eukaryotic ribosome decoding site and a new feature of arrangement of mRNA in the ribosome, neighborhood of mRNA with S3 protein fragment 190-236. Acknowledgement: The work was supported by grant from RFBR (0804-00508).

YSF-54 CREB signalling in the maturation of newborn neurons in the adult dentate gyrus

YSF-56 Life cycle-dependent remodelling and limited environmental responsiveness in trypanosome membrane transport

R. Jagasia and C. Lie Helmholtz Zentrum Munchen, IDG, Institute of Developmental Genetics, Neuherberg, GERMANY Neural stem cells continuously generate new functional neurons in the hippocampal dentate gyrus throughout adulthood. Hippocampal network activity regulates the maturation and integration of new hippocampal neurons. The intracellular signalling pathways involved in these processes are largely unknown. Here we investigate the role of CREB in the development of hippocampal neurons in the adult mouse. We demonstrate that cyclic AMP response element binding protein (CREB)-dependant is strongly active in new hippocampal neuron. To manipulate CREB signalling, in vivo, we have generated retroviruses expressing constitutively active and dominant negative forms of CREB. Modulating CREB activity has profound affects on hippocampal adult neurogenesis in vivo. Loss of CREB-signalling leads to loss of expression of immature neuronal markers, aberrant dendritic maturation and subsequent loss of newborn neurons in the adult hippocampus. Interestingly, increased CREB activation leads to increase maturation and then subsequent loss of new born neurons compared to wild-type cells. We conclude that CREB signalling is tightly regulated and involved in the maturation and integration of adult born dentate granule neurons. Using our retroviral strategy we are now investigating on a single cell level what upstream pathways are modulating CREB signalling in hippocampal neurogenesis, including both GABA- and NMDA-mediated neuronal activity. Acknowledgement: Supported by the Alexander von Humboldt Foundation (to R. J.).

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V. L. Koumandou, S. K. A. Natesan, T. Sergeenko and M. C. Field Department of Pathology, University of Cambridge, Cambridge, UK Mechanisms controlling and integrating membrane traffic remain incompletely understood. Trypanosoma brucei exhibits strong developmental alteration in endocytic flux between the principal life stages and hence offers a convenient system to assess transcriptional and other changes. We constructed a targeted oligonucleotide microarray to assay differential expression of trafficking genes. Approximately 6% of this cohort are developmentally expressed, including several small GTPases and SNAREs upregulated in mammalian stage parasites, consistent with increased activity versus insect stages. Differential expression is also evident amongst vesicle coat factors and protein kinases. We used the microarray to assess the effects of specific gene overexpression, knockdown, exposure to altered culture conditions and stress, on transcription. While trypanosomes overexpressing Rab5 have significantly enhanced endocytosis and levels of clathrin protein, no significant alterations to steady-state mRNA levels are detectable. Knockdown of clathrin or the major surface glycoprotein failed to perturb transcription, and exposure to DTT or tunicamycin revealed no evidence for a classical unfolded protein response. Finally, altered serum levels invoked little transcriptional alteration. Therefore, while trypanosomes regulate mRNA abundance to effect the major changes accompanying differentiation, a given differentiated state appears transcriptionally inflexible, revealing fundamental differences between control of membrane transport in trypanosomes and higher eukaryotes.



Abstracts

YSF-57 Interaction of nucleotide excision repair factors XPC-HR23B, XPA and RPA with damaged DNA

YSF-59 Molecular beacon with dual FRET for detecting heterodimeric DNA binding proteins

Y. S. Krasikova1, N. I. Rechkunova2 and O. I. Lavrik1,2 1 Novosibirsk State University, Novosibirsk, RUSSIA, 2Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, RUSSIA

T. Krusin´ski, A. Ozyhar and P. Dobryszycki Wroclaw University of Technology, Faculty of Chemistry, Division of Biochemistry, Wroclaw, POLAND

In eukaryotes, nucleotide excision repair (NER) is a versatile and highly conserved repair system capable of removing a wide range of DNA lesions. The interaction of nucleotide excision repair factors, Xeroderma pigmentosum complementation group C protein in complex with human homolog of RAD23B (XPC-HR23B), replication protein A (RPA) and Xeroderma pigmentosum complementation group A factor (XPA) with 48-mer DNA duplexes imitating damaged DNA structures were investigated. Research methods: photoaffinity labeling technique, electrophoretic mobility shift assay. It was demonstrated that proteins under investigation exhibited low specificity of binding to damaged DNA compared to undamaged DNAduplexes. RPA stimulates XPC-HR23B-DNA complex formation and when XPA and XPC-HR23B were added simultaneously to DNA, synergistic effect in binding of these proteins with DNA was observed. RPA crosslinkes to DNA containing photoreactive nucleotide residue, 5I-dUMP, in one of the strands and fluoresceine substituted analogues of dUMP as a bulky lesion in the opposite strand of DNA-duplex and stimulates crosslinking of XPC-HR23B. Therefore, RPA can be one of the main regulation factors on different stages of nucleotide excision repair process. The obtained data are in agreement with the model of the random order assembly that proposes cooperative damage recognition by NER factors forming preincision complex.

Introduction: A molecular beacon for detecting DNA-binding proteins activity has been previously described (1). We present for the first time a double molecular beacon (DMB) system allowing to investigate two proteins interacting simultaneously with DNA at two nearby binding sites. To describe spectroscopic features of DMB we used the heterodimeric ecdysteroid receptor proteins, ultraspiracle (Usp) and ecdysone receptor (EcR) from D. melanogaster and a response element from the promoter of hsp27 gene. Methods: Steady-state fluorescent measurements were performed for sample solutions of Left (dabcyl-labeled) and Right (BHQ-3-) ds-oligonucleotides with 9-base overhangs and Middle 18-base ss-oligonucleotide (5’-fluorescein- and 3’-Cy5.5-) sequences, containing 5’ (hsp275’) and 3’ (hsp273’) protein binding half-sites in absence and presence of DNA-binding domains (DBD) of Usp and/or EcR. Results: The dissociation constants, Kd, UspDBD-hsp275’ and UspDBDhsp273’ complexes were determined to be 0.57 and 6.01 nM respectively. The Kd values for EcRDBD-hsp275’ and EcRDBD-hsp273’ were 1.99 and 9.49 nM. Obtained results are consistent with those obtained by conventional fluorescence titrations and by FRET measurements with molecular beacons (2). Conclusions: Double molecular beacon was successfully applied for detecting and quantitative analysis of the sequence-specific interaction of a natural hsp27 response element with UspDBD and EcRDBD. Both proteins belong to the group of nuclear receptor family and Usp is a homolog of human RXR protein. This new approach allows for sensitive detection of heterodimeric proteins such as dimeric transcription factors in a way that could be readily applied to real-time imaging. References: 1. Heyduk & Heyduk, Nature Biot. 2002; 20: 171–176 2. Krusin´ski et al. J. Fluoresc 2008; 18: 1–10

YSF-58 Influence of Dpb2p, a non-catalytic subunit of DNA polymerase epsilon, on the fidelity of DNA replication in Saccharomyces cerevisiae J. Kraszewska1, M. Jaszczur1, J. Rudzka1, K. Flis1, M. E. Budd2, P. Polaczek2, J. L. Campbell2, P. Jonczyk1 and I. J. Fijalkowska1 1 Laboratory of Mutagenesis and DNA Repair, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, POLAND, 2Braun Laboratories, California Institute of Technology, Pasadena, CA, USA Understanding of the mechanisms that control the generation of mutations on normal and damaged DNA templates is crucial in studies of carcinogenesis and mutagenesis. One likely source of spontaneous mutations are errors occurring during DNA replication. DNA polymerases are central to replication fidelity. Most replicases are multisubunit complexes. With regard to fidelity, the DNA polymerase catalytic subunits have received most of the attention but still little is known about the role of the non-catalytic subunits in replication fidelity. DNA polymerase epsilon, one of the major replicative DNA polymerase in Saccharomyces cerevisiae, comprises of four subunits: catalytic Pol2p and three accessory subunits- Dpb2p, Dpb3p, and Dpb4p. Pol2p and Dpb2p are essential in yeast. In order to investigate a possible role for the Dpb2p subunit in maintaining the fidelity of DNA replication, we isolated temperature-sensitive mutants in the DPB2 gene. Several of the newly isolated dpb2 alleles increase level of the replication errors and dpb2 strains present mutator phenotype. We further demonstrated that Dpb2p influences the fidelity of polymerase e holoenzyme. Results of the two hybrid assay reveal that the mutator variants of Dpb2p have an impaired interaction with the polymerase e catalytic subunit-Pol2p what provides a possible explanation of the loss of high fidelity of replication. Our results uncover that DNA polymerase subunits other than those housing the DNA polymerase and 3’ fi 5’ exonuclease activities are essential in controlling the level of spontaneous mutagenesis and genetic stability in yeast cells.

YSF-60 Mechanisms of vitamin K3-induced ROS generation in glioma cells T. Kulahava1, G. Semenkova1, Z. Kvacheva2, N. Krylova1 and S. Koran2 1 Department of Biophysics, Physics Faculty, Belarusian State University, Minsk, BELARUS, 2State Scientific Research Institute of Epidemiology and Microbiology, Minsk, BELARUS Reactive oxygen species (ROS) formation in brain leads to the oxidative stress, which has been considered to be involved in the pathogenesis of neurodegenerative diseases. The purpose of this work was to establish the mechanism of menadione- and vikasol-induced ROS generation in rat C6 glioma cell and human U251 glioma cell using lucigenin-enhanced chemiluminescence (CL), 2’7’-dichlorodihydrofluorescein diacetate fluorescence as well as inhibitory analysis. It was shown that the addition of enadione and vikasol to suspension or monolayers of glioma cells leads to the generation of superoxide outside C6 cells and hydrogen peroxide inside cells of both cell lines. At that intensity of menadione-induced CL has maximal value immediately after menadione addition, whereas maximal intensity of CL after vikasol addition is observed through 7–8 minutes. Therefore diverse solubility of vitamin K3 analogs in lipidic phase could influence their interaction with donors of electrons and the rate of ROS generation. It was established that menadione concentration for maximal hydrogen peroxide generation in C6 glioma cells is 10-fold higher than for superoxide production and vikasol concentration is the same. The received data permit to conclude that menadione in dose-dependent manner could be used as an inducer of superoxide or hydrogen peroxide formation in cells. It was also established that neuronal NO-synthase, ND: ubiquinone oxidoreductase of mitochondria and arachidonic acid metabolism enzymes on lipoxygenase pathway take part in superoxide generation under menadione and vikasol action on glioma cells. Acknowledgement: This work was supported by the Belarusian Republican Foundation for Fundamental Research (projects B07MC-064, B07M081).


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Abstracts


YSF-61 PI3K/Akt signaling is a target of cyclosporin A affecting invasiveness in human glioblastoma LN229 cells A. Kwiatkowska and B. Kaminska Laboratory of Transcription Regulation, Nencki Institute, Warsaw, POLAND Introduction: Invasiveness is a complex process regulated by common intracellular pathways and several studies implicated PI-3K/Akt pathway in those processes. PTEN, a negative Akt regulator, is mutated in many malignant gliomas. We demonstrated anti-invasive effects of cyclosporin A (CsA) in gliomas. In the present study, we sought whether CsA impairs tumor invasiveness by interfering with Akt signaling in human glioblastoma cells differing in PTEN status. Methods: Effects of CsA on invasiveness of LN229 (wt PTEN) and T98G (mutated PTEN) cells were evaluated in invasion assay. Akt phosphorylation was determined by Western blotting in cells exposed to CsA. Luciferase reporter assay was employed to study CsA effects on NFjB and AP-1 activities. Changes in expression of genes correlated with invasiveness were determined by QRT-PCR. Results: We demonstrated that CsA decreases the level of phosphorylated Akt in LN229, but not in T98G cells, concomitantly with the down-regulation of LN229 invasiveness. Furthermore, CsA treatment strongly reduced NFjB and AP-1 activities and decreased mRNA levels for metalloproteinases (MMPs) involved in extracellular matrix degradation. Conclusion: Our results demonstrate that CsA down-regulates Akt signaling in LN229 cells impairing invasion and migration. CsA did not affect phosphorylated Akt level or invasiveness of T98G cells bearing PTEN mutation. It is likely that down-regulation of Akt activity results in suppression of NFjB and AP-1–driven transcription, and changes in MMP gene expression.

YSF-62 Involvement of CIP2A, a novel human oncoprotein inhibiting protein phosphatase 2A (PP2A), in breast cancer A. Laine1, C. Come1,2, M. R. Junttila1, P. Puustinen1, M. Niemela1, M. Boulfroy3, S. The´zenas3, J.-M. Darbon3, J. Isola2, O.-P. Kallioniemi4 and J. Westermarck1,2 1 Centre for Biotechnology, University of Turku and Ebo A˚kademi, Turku, FINLAND, 2Institute of Medical Technology, University of Tampere, Tampere, FINLAND, 3Research Center of Cancerology, Montpellier, FRANCE, 4VTT Biotechnnology, Turku, FINLAND Introduction: Inhibition of protein phosphatase 2A (PP2A) activity is one of the prerequisites for human cell transformation. Our laboratory has recently identified a novel protein, designated as Cancerous Inhibitor of PP2A (CIP2A), that inhibits PP2A in human malignancies. We have demonstrated that CIP2A is required for the malignant cellular growth, for in vivo tumor formation and that it is overexpressed in common human malignancies. Thus, our results demonstrate that CIP2A is a novel human oncoprotein that inhibits PP2A in human malignancies (Junttila et al., Cell 2007; 130: 51–62). Methods and results: In breast cancers, we show a significant overexpression of CIP2A at the mRNA level in invasive human mammary tumors as compared to non-invasive tumors or to normal breast. We observe also an increased cytoplasmic expression of CIP2A protein in breast cancer tissues. Microarray data from a study based on 251 breast tumors indicate that CIP2A is overexpressed in these tumors and this overexpression is correlated with a high expression of Ki67 and PCNA, two proliferative markers. Functionally, we have confirmed this role for CIP2A as depletion of CIP2A by siRNA treatment decreases the viability of breast cancer cells. Conclusions: Taken together, our current results indicate that CIP2A is involved in human breast cancer progression and strongly suggest that CIP2A expression is linked to agressiveness of the mammary tumors. It is expected that our future results will reveal fundamental novel information about general mechanisms of cellular transformation and about the specific role of CIP2A in breast cancer.

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YSF-63 Characterization of Ets-1 p27, a novel isoform of Ets-1 oncoprotein C. Laitem, G. Leprivier and M. Aumercier Institut de Biologie de Lille, Lille, FRANCE Introduction: The transcription factor Ets-1 is implicated in physiological processes (development, haematopoiesis, angiogenesis…) as well as invasive pathologies (rheumatoid arthritis, cancers…). It is a transcriptional activator with a DNA-binding activity downregulated by an intramolecular mechanism, named auto-inhibition. To counteract it, Ets-1 interacts with its partners to bind to target promoters. Among them are matrix metalloproteinases, stromelysin-1 and collagenase-1. Two isoforms of Ets-1 are described in human: Ets-1 p51, the majority full-length protein and Ets1 p42, a non auto-inhibited isoform with different transcriptional properties. In this study, we characterized, Ets-1 p27, a novel isoform with dominant negative functions towards Ets-1 p51. Methods: We analyzed the variant expression by RT-PCR experiments and the cellular localization of isoforms by immunofluorescence assays. Then, we investigated binding properties by gel shift and Biacore experiments and transcriptional properties by transient transfections of luciferase reporter vectors. Results: The novel identified splicing variant is co-expressed in human with full-length ets-1 in a tissue specific manner and in mammary adenocarcinoma. It encodes Ets-1 p27 which is co-localized in the nucleus with Ets-1 p51. Ets-1 p27 is auto-inhibited and binds cooperatively to the stromelysin-1 promoter from which it chases away Ets-1 p51. This binding competition correlates with Ets-1 p27 abilities to block in a dose-dependent manner the transactivation of the stromelysin-1 and collagenase-1 promoters mediated by Ets-1 p51. Conclusions: Ets-1 p27 is a natural dominant negative isoform of Ets-1 p51 oncoprotein and the ratio Ets-1 p51/Ets-1 p27 constitutes a transcriptional rheostat adjusting the activity of Ets-1 p51.

YSF-64 LRP1: a versatile endocytic receptor that promotes malignant cell invasion by regulating actin cytoskeleton and focal adhesion dynamics B. Langlois, J. Devy, B. Sid, H. Emonard, L. Martiny and S. Dedieu Universite´ de Reims Champagne-Ardenne (URCA), Laboratoire de Biochimie, Riems, FRANCE Introduction: Low-density receptor-related protein-1 (LRP-1) mediates the endocytic clearance of numerous biological molecules from the pericellular environment, including matrix proteinases involved in extracellular matrix degradation during cancer progression. However, its accurate functions remain highly controversial. Methods: In order to specify the role of LRP-1 in tumor progression, we developed a long-term vector-based short hairpin RNA strategy against LRP-1. Results: We show that LRP-1 silencing results in a drastic inhibition of cell invasion despite a strong stimulation of pericellular MMP-2 and uPA proteolytic activities. Cell migration in both two- and three-dimensions is decreased by LRP-1 silencing. LRP-1-silenced carcinoma cells display major cytoskeleton rearrangements and atypical overspread morphology with a lack of membrane extensions. Furthermore, LRP-1 silencing accelerates cell attachment, inhibits cell-substrate deadhesion and induces the accumulation at the cell periphery of abundant talin-contaning focal adhesion complexes deprived of FAK and paxillin. Conclusions: We conclude that, in addition to its ligand-uptake activity, LRP-1 regulates the cytoskeletal organization, focal complexes composition and dynamics, thus leading to the most favourable adhesive state to promote invasion. Altogether, our data provide new insights about the function of LRP-1 and support the concept that LRP-1-silencing would be of interest to develop new therapeutic anti-cancer strategies.



Abstracts

YSF-65 Characterization of mycobacterial lipidic antigens and of the molecular bases of their presentation by CD1B to human T cells

YSF-67 EGFR and death receptor pathways leading to caspase-independent apoptosis: the role of c-tocotrienol and lovastatin

E. Layre1, G. De Libero2, H. De La Salle3, G. Puzo1 and M. Gilleron1 1 IPBS-CNRS, Toulouse, FRANCE, 2University of Basel, Basel, SWITZERLAND, 3EFS-INSERM, Toulouse FRANCE

L. Loizou1,2, A. Evdokiou3 and A. Odysseos1,2 1 University of Cyprus, Nicosia, CYPRUS, 2EPOS-Iasis, R&D, Nicosia, CYPRUS, 3Adelaide Royal Hospital, Adelaide, AUSTRALIA

Mycobacterium tuberculosis is the causative agent of tuberculosis which remains today a major health problem in the world. The intracellular location of the bacilli shields it from antibodies detection. Therefore protective response requires activation of T lymphocytes. Lipids can activate these cells thanks to new non polymorphic antigen-presenting molecules: CD1 proteins. The majority of CD1-restricted antigens are mycobacterial cell wall components. All these antigens are amphipatic molecules composed of acyl chains and a polar headgroup. Hydrophobic channels of CD1 groove allow binding antigen acyl chains and positioning polar headgroup for T cells recognition. This loading takes place in lysosome and the antigenic complex is addressed to plasma membrane for T cells activation. If the processing of proteic antigens is well described, little is known about processing mechanisms of lipidic antigens. In this context, we work on a better characterization of the lipidic antigens repertory. Indeed a lot of others mycobacterial cell wall compounds have structural properties to be presented by CD1 proteins. So, several T-cell clones have been generated against global mycobacterial lipidic fractions. This allowed us to isolate and describe a new mycobacterial lipidic antigen using different structural biochemistry approaches. In addition, we study glycolipids processing mechanisms. Our model study is a mycobacterial glycolipid containing one to four acyl chains and two or six mannose units. After different degradations of polar/apolar parts, activity of resulting compounds on specific T cell permits to determine the minimal active structure and then to highlight enzymes implied in processing of lipidic antigens.

The mevalonate (MVA) pathway is often dysregulated in tumour cells. Lovastatin and the Vitamin E isoform c-Tocotrienol work synergistically to inhibit this pathway, representing a potential drug combination for cancer therapy. The sensitivity of prostate carcinoma cell-lines with different androgen-responsiveness phenotype to c-Tocotrienol was analyzed following treatment(s) with (a) Lovastatin, (b) Z-VADfmk, a pan-caspase inhibitor, (c) Geranylgeraniol (GGO), a mevalonate-pathway modulator and (d) TNFrelated apoptosis-inducing ligand (TRAIL). Sensitization of both cell-lines to c-Tocotrienol after modulation of the mevalonate pathway by Lovastatin was not rescued but further enhanced by pan-caspase inhibition and mevalonate modulation by GGO. Immunoblot analysis revealed augmentation of cleaved effector caspases and concurrent modulation of the mitochondrial apoptotic cascades with upregulation of BAX in a c-Tocotrienol–dose dependent manner. Lovastatin sensitized both cell-lines to c-Tocotrienol– induced inhibition of G-protein prenylation and activation of the TRAIL/ Apo2 apoptotic cascade as shown by RAP1A and Death Receptor levels, respectively. This effect was enhanced by pan-caspase inhibition while a low-level ERK-phosphorylation induced by c-Tocotrienol in the androgen unresponsive cells was reversed by Lovastatin. It is suggested that the antiproliferative effects of the combined Lovastatin/c-Tocotrienol treatment involve cross-talks between the TRAIL/Apo2 cascade and the MAP kinase cascade. These cascades also form links with the EGFR pathway, a major target for cancer therapy since many tumours are dependent on aberrant EGFR signaling. Notably, c-Tocotrienol also inhibits early events in the EGFR signaling cascade. On-going studies using our fluorescent EGFR inhibitors focus on the dependency of the Lovastatin/c-Tocotrienol effects on the EGFR pathway.

YSF-66 Regulation of the membrane fissioning activity of CtBP1/BARS during the mitotic Golgi checkpoint G. Li, C. Valente and D. Corda Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro, ITALY Introduction: As a member of the multifunctional C-terminal-binding protein family, CtBP1/BARS is involved in membrane fission and gene transcription. Its nuclear activity regulates numerous cellular functions, including epithelial differentiation, tumorigenesis and apoptosis. In cytosol, CtBP1/BARS controls a membrane-fissioning machinery involved in several membrane trafficking steps and in mitotic Golgi partitioning. This partitioning results from Golgi fragmentation during mitosis, and is required for entrance into mitosis; its block causes G2 cell-cycle arrest, defining the ‘Golgi mitotic checkpoint’, a process dependent on CtBP1/ BARS. Methods: Cytosol extracts have been prepared from interphase and mitotic cells. CtBP1/BARS interactors were isolated from interphase cytosol by coimmunoprecipitation. With mitotic cytosol, similarly precipitated proteins were compared to a mock column (coupled to pre-immune IgGs) and to CtBP1/BARS precipitation from interphase cytosol. The differentially precipitated bands have been cut from gels and isolated for sequencing by MALDI-TOF and nano-electrospray mass spectrometry. Results: With interphase cytosol, lipid kinases and proteins involved in microtubule assembly and cytoskeleton regulation were revealed as part of the CtBP1/BARS protein complex. Following its validation, this approach has now been applied to the CtBP1/BARS-driven molecular machinery during mitosis. This has revealed different specific elements that intervene during mitotic progression. Conclusions: This approach successfully led to identification of CtBP1/ BARS interactors from interphase cytosol. The subsequent identification of CtBP1/BARS interactors that are specific to mitotic cytosol allows their functional analysis, to provide us with important information regarding the composition and regulation of the CtBP1/BARS membrane-fissioning machinery that defines the Golgi mitotic checkpoint.

YSF-68 Uptake of cell penetrating peptides triggers the membrane repair response A. Lorents1, C. Palm2, M. Ha¨llbrink2 and M. Pooga1,3 1 Institute of Molecular and Cell Biology, University of Tartu, Tartu, ESTONIA, 2Deparment of Neurochemistry, Arrhenius Laboratories, Stockholm University, Stockholm, SWEDEN, 3Estonian Biocentre, Tartu, ESTONIA Introduction: Cell penetrating peptides (CPP) are capable of translocating into cells and delivering different cargoes. However, the internalization mechanism of CPPs is still debated and it’s not known how cells compensate the disturbances induced by peptides in plasma membrane. The proteins known to cause cell lysis are taken up in a similar manner with CPPs. Internalization of perforin leads to damage of plasma membrane, which is repaired by using cellular vesicles donating their membranes. In this study we examined whether uptake of CPPs into cells triggers the membrane repair response. Methods: Uptake of different cell penetrating peptides by eukaryotic cells was assessed by flow cytometry and distribution of peptides was mapped at ultra-structural level by transmission electron microscopy. The membrane repair response was studied by detecting translocation of lysosomal marker LAMP-2 to the plasma membrane by fluorescence microscopy. Results: All studied peptides were taken up by HeLa cells. CPPs accumulated in/on the cellular plasma membrane preferentially at protrusions and translocation into cells was observed. The peptides seemed to slightly disorder the packing of membranous lipids. In addition, the used CPPs (for example penetratin and MAP) induced accumulation of LAMP-2 to the plasma membrane. The induced repair was dependent on the character and concentration of used peptide. Conclusions: The disturbances in the plasma membrane and exposure of LAMP-2 at the cell surface corroborates that CPPs induce the membrane repair response, which helps cells to reseal the damaged regions and to overcome the stress caused by the uptake of peptides.


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Abstracts


YSF-69 Methyltransferase-directed transfer of activated groups (mTAG) for targeted covalent labeling of DNA

YSF-71 Single-molecule DNA biosensors for transcriptionfactor detection

G. Lukinavicius1, V. Lapiene1, A. Lapinaite1, E. Weinhold2 and S. Klimasauskas1 1 Laboratory of Biological DNA Modification, Institute of Biotechnology, Vilnius, LITHUANIA, 2Institute of Organic Chemistry, RWTH Aachen University, Aachen, GERMANY

K. Lymperopoulos, M. Heilemann, L. C. Hwang, R. Crawford and A. N. Kapanidis Department of Physics and IRC in Bionanotechnology, Oxford University, Oxford, UK

Methyltransferases catalyze highly specific transfers of methyl groups from the ubiquitous cofactor S-adenosyl-L-methionine (AdoMet) to various biopolymers like DNA, RNA and proteins. Being the second most ubiquitous cofactor after ATP, AdoMet is involved in numerous essential biochemical processes in all living organisms. Despite the high importance of transmethylation reactions in biology, the naturally transferred methyl group has a very limited utility for practical applications. We have demonstrated that allylic and propargylic side chains can be efficiently transferred by DNA MTases with high sequence- and base-specificity (Dalhoff et al., Nat. Chem. Biol., 2(1) 31–32). Recently we have synthesized the first AdoMet analog with an extended propargylic side chain carrying a primary amino group (Lukinavicˇius et al., Journal of American Chemical Society, 129(10) 2758– 2759). Using model C5-cytosine and N6-adenosine DNA MTases, we demonstrate that: (i) methyltransferase-directed transfer of the extended side chain from such cofactors leads to efficient sequence-specific modification of DNA; (ii) the efficiency of transalkylation is further enhanced by rational engineering of the cofactor binding center in a DNA cytosine-5 methyltransferase; (iii) covalent attachement of reporter groups (biotin, fluorophores) at modification sites could be achieved via amino-specific chemoligations with corresponding NHS esters; (iv) labeled plasmid DNA can be used to efficiently transform E. coli cells even in the presence of an endogenous methylcytosine-specific endonuclease, McrBC. These findings envision numerous applications of the mTAG labeling technique in Cell and molecular biology, DNA-based nanotechnologies and medical diagnostics (Klimasˇ auskas et al., Trends Biotechnol., 25(3) 99–104).

YSF-70 Restriction of alphaviral replication by viral self-downregulation mechanisms V. Lulla and A. Lulla Institute of Technology, University of Tartu, Tartu, ESTONIA Semliki Forest virus (SFV) is among the best known models for the studies of the replication strategies of positive-strand RNA viruses. SFV replication relies on the production of replicase proteins in the form of non-structural polyprotein precursor P1234, which is then processed by viral non-structural protease (nsP2) in a highly regulated manner. Non-structural polyprotein cleavage strategy in its turn defines regulation of the viral RNA replication by causing rearrangements of the viral replication complex, so that its RNA template preference is changed in favour of minus-strands over plus-strands during the course of viral infection. Our results of the real-time monitoring of the viral protease activity using intracellular FRET substrates provided evidence that restriction of the minus-strand synthesis at 4–5 hours after infection is associated with greatly enhanced specific trans-activity of the SFV nsP2 indicating the release of the protease from the transformed replication complexes. In view of our recent findings that alphaviral nsP2 proteases support cross-processing of the homologous non-structural polyproteins specifically at the 2/3 sites by unique mechanism despite the significant dissimilarities in their sequences, our studies provided existing theoretical model with important experimental results. Newly discovered molecular details can now be translated into a model in which alphaviral non-structural protease not only plays crucial role in the regulation of the viral macromolecular synthesis during infection but is also a major causative factor of the homologous interference described for alphaviruses, suggesting that superinfection exclusion and establishment of the persistent infection are rather the two facets of the same process.

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We have developed a single-molecule fluorescence assay that detects transcription factors in solution with high sensitivity and specificity. The single-molecule assay is based on protein-detected coincidence of two DNA fragments (Heyduk et al., Nat Biotech 2002; 20: 171), each containing one half-site for transcription-factor binding; this coincidence is detectable using two-colour alternating laser excitation (ALEX) spectroscopy (Kapanidis et al., PNAS 2004; 101: 8936). In the absence of a transcription factor that can bind to the fully assembled site, the two half-site DNA fragments (which carry short and complementary single-stranded DNA tails) diffuse independently in solution. In the presence of a transcription factor specific to the fully assembled DNA site, the two DNA fragments diffuse as a single molecular complex; such species can be distinguished from the free DNA half-sites and can be counted, reporting on the presence and concentration of transcription factors. Using this assay, we demonstrated protein-dependent DNA coincidence to detect transcription factors in dilute (sub-nM) protein solutions; multiplexing capability by detecting two transcription factors simultaneously in the same solution; compatibility with complex biological samples such as nuclear extracts; and sensing of changes in gene expression in bacterial cells. Also, this assay can be used for sensing small molecules like cAMP and IPTG and creating logical gates. Finally, we have shown that the assay can be implemented on surfaces using a single immobilized DNA half-site and a complimentary half-site in solution; in this case the protein presence is monitored by fluorescence coincidence analysis using total internal reflection fluorescence microscopy.

YSF-72 New genes affecting alginate biosynthesis in Azotobacter vinelandii M. Maerk, H. Ertesvaag and S. Valla Norwegian University of Science and Technology, Trondheim, NORWAY Alginates are industrially important polysaccharides with a wide range of applications in medical, pharmaceutical and food technology. They consist of variable amounts of mannuronic (M) and guluronic (G) acid. Polymer chain length and the distribution and ratio of M and G units greatly affect the physiochemical properties, thus making different alginates ideal for different areas of use. Alginate is produced by brown algae and by bacteria in the genera Pseudomonas and Azotobacter. In A. vinelandii, alginate is produced during both vegetative growth and encystment. Today, commercial alginate production is based on harvest from algae, which gives varied mixtures of different alginates. There is great interest in developing bacterial bioproduction strains, in order to be able to produce high yields of tailored alginates with desired properties. It is therefore of interest to study which genetic factors affect alginate biosynthesis. In addition, microbial alginate synthesis is currently used as a biological model system in a European systems biology project coordinated by our research group. The structural genes and the main regulatory genes directly involved in bacterial alginate biosynthesis are well known. However, there is reason to believe that other factors also have an effect on the channeling of sugar precursors towards and through the alginate biosynthetic pathway. Phenotypic characteristics (e.g. oxygen consumption, tendency to encyst) could also influence the overall production process. In the search for new genes affecting alginate synthesis, we have constructed an A. vinelandii transposon mutant library and identified a range of interesting mutant candidates through high-throughput screenings. Verification of some selected candidates has been completed, and more are under way. The A. vinelandii genome sequence is available online, which makes it possible to identify the inactivated gene in each mutant by sequencing. This work has revealed several genes not previously known to affect alginate production, and further investigations of the most interesting mutants have begun. This project is expected to result in an increased knowledge of the global factors affecting alginate biosynthesis, and thus contribute to the groundwork for future development of alginate bioproduction strains. In addition, this work could lead to the elucidation of novel gene functions, and new insights with regards to other cellular processes in this organism.


Young Scientists Forum YSF-73 MicroRNAs involved in cerebral cortex development N. A. Maiorano and A. Mallamaci Neurobiology Sector, International School for Advanced Study, Trieste, ITALY MicroRNAs are a newly recognized class of small, non coding RNAs that post-transcriptionally regulate the expression of target mRNA transcripts (1). Several microRNA databases allow systematic in silico searching for microRNA putative targets (2,3). We selected a set of genes relevant to the main morphogenetic subroutines active during cerebral cortex development: Lhx2; Tbr2; Fez1; Otx1. We used the miRBase search engine (2) to select microRNAs that match conserved putative regulatory elements within their 3’UTRs. MicroRNAs selected by in silico screening were analyzed via in situ hybridization of coronal brain slices from mouse embryos of different gestational ages, by Exiqon LNAbased technology (4). Those of them displaying interesting spatial and temporal expression patterns have been tested by neuroblast lipofection, in order to assay their capability to target specific sequences within the 3’UTR of the selected genes. At the moment, we are setting up in vivo functional analysis of some of these microRNAs, by in utero electroporation (5). References: 1. Bartel DP. Cell 2004; 116: 281–297. 2. Griffiths-Jones. Methods Mol. Biol. 2006; 342: 129–138. 3. Marks DS, John B, et al., PLoS Biol. 2004; 2, No. 11, e363. 4. Kloosterman WP, Wienholds E, de Bruijn E, Kauppinen S, Plasterk RH. Nat. Methods 2006; 3(1): 27–29. 5. Saito T. Nat. Protoc. 2006; 1(3): 1552–1558.

YSF-74 The short isoform of TRF2 protein is produced from full-length TRF2 mRNA on owing to internal initiation of translation O. Maksimenko, M. Yerokhin and P. Georgiev Institute of Gene Biology, Russian Academy of Sciences, Moscow, RUSSIA TBP is a transcription factor participated in the transcription initiation. There is family of proteins named TRF with high homology to TBP. Recently we showed that p175 and p75 TRF2 isoforms are generated from a single mRNA. The C-end of p175 contains the open read-frame of p75. We hypothesized that p75 is produced by internal initiation of translation from IRES at an in-frame AUG codon. Interestingly that in this case the potential IRES is located in the coding region of mRNA. In work we used bicistronic system: the promoter drives transcription of mRNA containing lacZ and eGFP ORFs. The lacZ is translated in a cap-dependent manner. However since the eGFP coding region is downstream of lacZ stop codon, it is expressed if a sequence at the spacer region can mediate internal initiation of translation. We made and transfected into S2 cells bicistronic constructs containing trf2 fragment (upstream of p75 AUG start), reaper IRES (positive control), bicistronic (negative control) and monocistronic (control of cap-dependent translation) vectors. A comparison revealed that the trf2 fragment and the rpr IRES increased eGFP expression. We monitored protein expression by western blotting showed that fusion lacZ-eGFP protein was not detected. Thus, we report that translation of p75 TRF2 is initiated at internal inframe AUG and is mediated by IRES present in the coding region of trf2 mRNA.

YSF-75 Comparison of bead-based bioassay and enzyme-linked immunoassay for the determination of osteocalsin in biological samples C. J. Malavaki, I. Kanakis and N. K. Karamanos Laboratory of Biochemistry, Department of Chemistry, University of Patras, Patras, GREECE Introduction: Bioanalytical microarrays are continuously been developed in order to achieve faster, more reliable and automated typing analyses in clinical and research laboratories. ELISA is currently the most commonly used technique due to the high specificity and sensitivity. Although this method is well established for single-molecule analysis, it is more desirable to simultaneously quantitate multiple analytes from a relatively small sample size, which is a critical factor, especially when monitoring disease or treatment states. The Luminex xMAP system, a combination of ELISA-based technology with flow cytometry with the utilization of fluorescent-labeled microspheres has opened new horizons in analytical bioassays. Methods: The aim of this experimental work was to compare ELISA and Luminex, concerning their analytical characteristics. For this purpose osteocalcin (OCN), a small protein which is produced by osteoblasts and is considered as a reliable biomarker of bone formation, was determined in serum of 17 patients with bone metastases from breast cancer. Results: Linear regression showed excellent correlation of the obtained results. However, the intra- and inter-assay CVs obtained by Elisa were

Abstracts much higher (10.6% and 13.7%, respectively) than those by Luminex (2.6% and 3.7%, respectively). Additionally, the lower limit of detection was 39.3 pg/ml for Luminex and 540 pg/ml for ELISA. Conclusions: In conclusion, bead-based bioassay has lower cost, is less time consumable, as compared to ELISA and furthermore is much more sensitive, reproducible and accurate assay with a wider dynamic range of concentrations.

YSF-76 Phytoecdysteroids from Silene viridiflora and their actoprotective, adaptogenic and antimicrobial activity N. Z. Mamadalieva1, V. N. Syrov1, D. Egamberdieva2 and V. Edwards Jones3 1 S.Yu.Yunusov Institute of the Chemistry of Plant Substances AS RUZ, Tashkent, UZBEKISTAN, 2National University of Uzbekistan, Department of Biotechnology and Microbiology, Tashkent, UZBEKISTAN, 3Manchester Metropolitan University, Manchester, UK Many medicinal plants contain ecdysteroids, among them S. viridiflora is unique for the great variety of such compounds produced. It has been used as folk medicinal plant for treatment several disease such liver, kidney disorders, rheumatic arthritis, healing of wounds and antimicrobial agents. In this paper, we describe isolation, structural elucidation of phytoecdysteroids from plant Coronaria flos-cuculi and their antibacterial activity. Using column chromatography and HPLC the six ecdysteroids were found in plant namely 2-Deoxyecdysone (1), 2-deoxy-20-hydroxyecdysone (2), 20-hydroxyecdysone (3), polypodine B (4), integristerone A (5), 26-hydroxypolypodine B (6), 2,22-diacetate- (7) and 3,22- diacetate-20,26-dihydroxyecdysone (8), sileneoside A (9), sileneoside D (10). Table Phytoecdysteroids of the plant Silene viridiflora

No

R1

R2

R3

R4

R5

R6

R7

1 2 3 4 5 6 7 8 9 10

H H H H OH H H H H H

H H OH OH OH OH OAc OH OH OH

OH OH OH OH OH OH OH OAc OH OGal

H H H OH H OH H H H H

H OH OH OH OH OH OH OH OH OH

OH OH OH OH OH OH OAc OAc OGal OH

CH3 CH3 CH3 CH3 CH3 CH2OH CH2OH CH2OH CH3 CH3

Some of those ecdysteroids showed in vivo experiment adaptogenic and actoprotective activities (5–10 mg/kg per oral) in rats. These compounds were tested for antimicrobial activity using the agar diffusion technique against twenty pathogenic bacteria strains E. faecalis NCTC775, M. luteus T33, S. aureus MRSA16, S. epidermis NCTC7944, K. aerogenes NCTC8172, B. cereus T80, S. saprophyticus T415, K. oxitoca 6653, K. pneumoniae 40602, C. freundii 82073, A. haumanii 60649, E.coli NCTC9001, P. aureginosa NCTC6749, P. rettgerri NCIMB9570, E. hormaechei T2, A. faecalis T3, E. hormaechei T10, Acinetobacter sp. T16, P. agglomerans T26, P. aureginosa T145. 2-Deoxyecdysone (1) showed antibacterial activity against fifteen bacteria, whereas 20,26-dihydroxyecdysone (8) against only six bacterial strains.


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YSF-77 Up-regulation of type II collagen gene expression by 17-b-estradiol in articular chondrocytes

YSF-79 An editable histone modification pattern that marks and configures mitotic chromatin

L. Maneix1, A. Servent1, B. Poreé1, N. Boujrad2, G. Flouriot2, K. Boumediene1, S. Moslemi1 and P. Gale´ra1 1 Laboratory of Connective Tissue Biochemistry, Faculty of Medicine, University of Caen-Lower Normandy, FRANCE, 2Laboratory of Molecular Endocrinology of Reproduction, University of Rennes I, Rennes, FRANCE

Y. Markaki1, A. Christogianni1,3, G. Papamokos2, A. S. Politou2,3 and S. D. Georgatos1,3 1 Foundation for Research and Technology-Hellas, Biomedical Research Institute, Ioannina, GREECE, 2Laboratory of Biological Chemistry, School of Medicine, University of Ioannina, Ioannina, GREECE, 3Laboratory of Biology, School of Medicine, University of Ioannina, Ioannina, GREECE

Introduction: Type II collagen, encoded by COL2A1 gene, is a phenotypic marker of articular cartilage whose expression is strongly altered during osteoarthritis (OA) process. As reflected by the elevated risk of OA in postmenopausal women, 17b-estradiol (17b-E2) is a steroid involved in the regulation of cartilaginous matrix homeostasis. The aim of present study was to investigate the molecular mechanisms regulating type II collagen expression under the effect of 17b-E2, and to characterize the genomic pathway via estrogen receptors (ER). Methods: Differentiated articular chondrocytes were assayed for real time PCR, western blot and collagen neosynthesis experiments. Transient cotransfections were performed with human deleted COL2A1 constructs, in combination with wild type or mutated ERa. In addition, interactions between transcription factors were determined by immunoprecipitation and validated in vivo by ChIP experiments on COL2A1 promoter. Results: 17b-E2 could stimulate the expression of its own receptor and also type II collagen neosynthesis and mRNA steady-state levels. Indeed, ERa induces an activating effect on COL2A1 gene transcription, mediated by the proximal promoter covering the –266/+121bp region. This sequence contains eight GC boxes, known to be binding sites for Sp1/Sp3 transcription factors. Protein interactions between ERa and Sp1, and also of these transcription factors with COL2A1 promoter, allowed us to confirm the involvement of GC cis sequences in the transactivation of COL2A1 by AF-1 domain of ERa. Conclusions: Understanding the molecular basis for 17b-E2 induction of COL2A1 expression provides new insights into molecular mechanisms of OA and may facilitate identification of novel approaches for its treatment.

YSF-78 Modulation of PARP-1 activity by hydrocortisone in rat liver nuclei A. V. Margaryan, M. A. Melnikova-Sharova and T. R. Torgomyan Yerevan State University, Yerevan, ARMENIA Poly(ADP-ribose)polymerase (PARP-1) is chromatin-associated enzyme which is involved in broad range of nuclear processes. PARP-1 activation is implicated in a various pathophysiological conditions including tumorogenesis. Nowadays, PARP inhibitors are widely used in anticancer therapy. Often, PARP-1 inhibitors are administrated in combination with dexamethasone (synthetic analogue of hydrocortisone) to prevent side-effects of anticancer drugs. In present study we were interested to examine whether hydrocortisone can modulate PARP-1 activity in rat liver nuclei, arising a possibility to interfere with effects of PARP inhibitors. Hydrocortisone was administrated peritoneal (5 mg/1000 g) to white outbred 3- to 4-week-old rats. Animals were decapitated after light ether anesthesia. PARP activity in isolated nuclei was determined by (1). Our data showed that in early phase of hydrocortisone action (4 hours after administration) PARP-1 activity in rat liver nuclei is not altered. In 24 hours of hormone action we detected dramatic suppression of PARP-1 activity. Induction of olygonucleosomal DNA fragmentation via activation of nuclear Ca/Mg-dependent endonuclease unexpectedly didn’t cause PARP-1 activation at least under our experimental conditions. This knowledge will help to improve more effective practical outcomes in anticancer chemotherapy which involves PARP-1 inhibitors. Reference: 1. Putt et al. Anal. Biochem. 2004; 326: 78–86.

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Introduction: Previously we identified nuclear envelope-bound kinases recriuted by heterochromatin protein 1 that phosphorylate histone H3 at threonine-3 in vitro. By producing affinity purified antibodies and performing indirect immunoflurescence we showed that histone H3, in vivo, is specifically phosphorylated at threonine-3 during mitosis (1). Methods: Exploring this process further, we have immunoisolated mononucleosomal particles from synchronized mitotic cells and analyzed the modification status of the H3 tail by western blotting and mass spectrometry. Results: As it turns out, threonine-3 phosphorylation is part of a complex epigenetic ‘signature’, which includes trimethylation of lysine-4 and asymmetric dimethylation of arginine-8 (PMM mark). Molecular dynamics simulations reveal that PMM-containing H3 tails have a propensity to associate with one another, forming higher order assemblies. Consistent with in silico data, morphological dissection of naturally occurring mitotic cells shows that PMM-marked chromatin accumulates in compact, spatially segregated ‘domains’. The spatial arrangement of these domains changes in a programmed fashion in the course of mitosis. Furthermore, when metaphase chromosomes are stretched or unraveled, PMM domains are axially displaced and occasionally resolve into a train of smaller patches that are repeated quasi-regularly along the length of extended chromatin fibers. Conclusions: These observations strongly suggest that PMM represents an intrinsic chromatin folding determinant that supports the formation of transient ‘stem-and- loop’ structures and confers topological specificity to the different regions of mitotic chromosomes. Acknowledgements: This work is co-funded by the European Union-ESF & National Sources in the framework of ‘Pythagoras II’. Reference: 1. Polioudaki et al., FEBS Lett. 2004; 560: 39–44.

YSF-80 Regulated synthesis of prostaglandin D2 by cultured adipocytes Md. A. Mazid1, Md. A. Rashid1, M. Jisaka2, T. Nagaya2 and K. Yokota2 1 Faculty of Pharmacy, University of Dhaka, Dhaka, BANGLADESH, 2 Department of Life Science and Biotechnology, Shimane University, Shimane, JAPAN PGD2, a major prostanoid of arachidonate cyclooxygenase (COX) pathway, undergoes non-enzymatic dehydration to form PGJ2 derivatives including 15-deoxy-D12,14-PGJ2 (15d-PGJ2) and D12-PGJ2. These PGJ2 derivatives are well-known natural activators of peroxisome proliferator-activated receptor (PPAR) c. Recently, we found specific gene expression of COX isoforms and the increased expression level of lipocalin-type PGD synthase during the maturation phase of 3T3-L1 adipocytes. However, the regulation and roles COX isoforms in the formation of PGs are yet to be clarified. To investigate the relative contributions of COX isoforms in the endogenous production of PGD2 and 15d-PGJ2, we performed immunological assay for them. Treatment of the cultured adipocytes with aspirin or indomethacin significantly inhibited the production with appreciable attenuation of adipogenesis. These data indicated that endogenous 15d-PGJ2 can serve as an autocrine signaling molecule. Moreover, exposure to phorbol 12-myristate 13-acetate (PMA) in presence of calcium ionophore, A23187 showed increase level of PGD2 and 15d-PGJ2 in 3T3-L1 adipocytes. PMA were effective in the induced gene expression of COX-2. However, stimulated synthesis of those prostanoids was blocked partially by the treatment with either COX-1 inhibitor, SC-560 or COX-2 inhibitor, NS-398. These finding imply the contribution of both COX isoforms to the stimulated synthesis of PGD2 and PGJ2 derivatives in adipocytes through the coupling with L-PGDS.


Young Scientists Forum YSF-81 Antimicrobial peptides: membrane affinity as the basis for selectivity and high local concentrations as the basis for activity M. Melo1, R. Ferre2, A. D. Correia1, L. Feliu2, M. Planas2, E. Bardajm2 and M. A. R. B. Castanho1 1 Institute of Biochemistry – Faculty of Medicine, University of Lisbon, Lisbon, PORTUGAL, 2Laboratori D’ Innovacio en Processos i Productes de Sintesi Organica – University of Girona, Girona, SPAIN Introduction: Antimicrobial peptides (AMPs) represent a wide variety of short, cationic, gene-encoded peptide antibiotics, found in virtually every organism. BP100 (HKKLFKKILKYLNH2) and omiganan (HILRWPWWPWRRKNH2) were designed from naturally occurring AMPs, and their detailed mechanism of action is still unknown. Methods: Fluorescence and light scattering spectroscopies were used to characterize the peptide interactions with model lipid bilayers, as well as their functional properties (induced leakage and translocation). Preliminary cytotoxicity assessments using eukaryotic models were carried out to further understand the overall low haemolytic activity of BP100. Results: A strong preference of both peptides towards anionic membrane model systems – which mimetize bacterial membranes – was observed. BP100 was observed to be cytotoxic, but for concentrations higher than the minimum inhibitory concentration (MIC) against bacteria. There were marked differences in the interaction and functional patterns at high peptide:lipid (P:L) ratios; these differences, despite also occurring for omiganan, were most evident for BP100. Relatively welldefined P:L ratios were determined (as high as one peptide per 6 phospholipid molecules) above which the peptides exhibited the behavior changes. Conclusions: The selectivity of these peptides seems to be correlated to their partition behaviour. In addition, the events at high membrane coverage could represent the molecular scale equivalent of the conditions at which the activity of the peptides is triggered: although the P:L ratios at these transitions seem very high, it can be shown that this is precisely the membranebound concentration to expect in a bacterial membrane under physiological conditions.

YSF-82 Tmg1 facilitates efficient telomere elongation via recombination F. L. Meng, Y. Hu and J.-Q. Zhou Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (CAS), Shanghai, CHINA Introduction: Telomeres are highly ordered DNA-protein structures at the ends of linear eukaryotic chromosomes. They not only help to distinguish chromosome ends from DNA double-strands breaks, but also provide a way to replicate chromosomes completely. Telomeric DNA can be efficiently replicated by telomerase or recombination in Saccharomyces cerevisiae (budding yeast). Genome-wide screening of telomere length in non-essential gene deletion yeast strain set had identified more than 280 genes which are involved in telomere maintenance. Methods: As a complementarity, we screening the telomere lengths in additional 66 non-essential genes which previous were taken for essential genes in large-scale deletion project. Results: We found that deletion of TMG1 (Telomere Maintenance Gene 1) displays a progressively-shorter-telomere phenotype at the early passages. This phenotype is also observed in different strain background. Single telomere sequences reveal that telomerase (or recombinational telomere maintenance mechanism) cannot efficiently replicate telomeric DNA at the early passages of tmg1D. Either over-expression telomerase or increasing the telomerase recruitment at telomere cannot restore the telomere length in tmg1Dstrain. Epitasis analysis also suggested that Tmg1 function independently with the well characterized telomere maintenance genes. Surprisingly, we found that tmg1D accelerates senescence in telomerase deficient cells by inhibiting recombinational telomere replication. Further study suggested that TMG1 is essential for type II survivor production in telomerase deficient cells. The recombinational role of Tmg1p is also supported by the observation that tmg1D is hyper-sensitive to Methyl Methanesulfonate (MMS). Purified Tmg1p can specifically binds telomeric DNA in vitro, and its DNA binding activity is essential for the Tmg1p function in vivo. Conclusions: Our results indicate that Tmg1p facilitates telomere elongation through recombination regardless of telomerase, and these findings may help to understand the relationship between telomerase-dependent and recombination-mediated telomere replication.

Abstracts YSF-83 Theoretical study of the lectin-carbohydrate interactions N. K. Mishra1, Martin Petek1, M. Wimmerova1, A. Imberty2 and J. Koa1 1 National Center for Biomolecular Research, Faculty of Science, Masaryk University, Brno, CZECH REPUBLIC, 2CERMAV-CNRS, BP53, Grenoble, FRANCE Understanding the intimate details of lectin saccharide interactions are getting importance due to its capability in deciphering the saccharide code. The specificity of the lectin for the saccharide capitalizes them as a target of specifically tailored drugs, and inhibitors for viruses. It opens an avenue for the lectin-carbohydrate interaction research, which is also a big challenge for theoretical modeling due to the polar flexible saccharide moiety often interacting via ions. PA-IIL is one of the lectins produced by Pseudomonas aeruginosa which shows high specificity towards L-fucose. The pathogen adhesion to the airways is influenced by the lectin. The pathogen is especially dangerous for individuals with cystic fibrosis (Govan et al., Microbiol. Rev. 1996; 60: 539–574). The binding studies on the lectin with a set of carbohydrates disclosed the synchronized arrangement of electrostatic and hydrophobic interactions in the recognition of carbohydrate. We used Molecular Mechanics Poisson Boltzmann model in combination of normal mode analysis to elucidate the free energy of the binding and conversely it’s consequences on the complex conformations. Solvent analysis in the apo form lectin exhibited that the water cluster exactly fits to the key hydroxyl groups of the carbohydrate in the holo form (Mishra et al. Proteins: Structure, Function, and Bioinformatics, Accepted). Dissection of the binding energy into its contributing terms revealed that the desolvation costs associated with the saccharide binding compensated by the coulombic interaction energy driven by calcium ions. However, in case of mutated lectin the local ordering of water around the calcium ions (formed due to absence of terminal glycine) disfavors the binding due to more desolvation cost, which is not compensated by coulombic interactions. The recognition of carbohydrate is mainly depends on the increasing non-polar nature of the carbohydrate, which mainly derived by two factors: positive cooperative intramolecular hydrogen bonding, and presence of non-polar group.

YSF-84 The oxidative stress induced by deoxynivalenol increases the mRNA expression of some Bcl2-family genes in Caco2 cell line M. C. Munteanu, L. Postolache, M. Costache and A. Dinischiotu University of Bucharest, Faculty of Biology, Bucharest, ROMANIA Introduction: Fusarium toxins such as, deoxynivalenol (DON), a carcinogenic mycotoxin, cause diverse toxic effects in animals and are suspected for disease induction in humans. DON carcinogenic effects might be related to oxidative cell damage and cell proliferation induction. Several studies suggested that DON binds to the ribosomal peptidyl-transferase inhibiting protein synthesis. In this study, DON mediated induction of oxidative stress, modulation of reduced glutathione and lipid peroxidation as well as modification of Bcl2 genes expression were investigated in Caco-2 cell line. Methods: The concentration of 80 lM DON for Caco2 cells treatment was established by the MTT test. The level of reduced glutathione and lipid peroxidation (LPO) were assessed after 24, 48 and 72 hours. The gene expression of Bcl2 was analyzed by quantitative real time PCR (qRT-PCR) after 4, 6, 12 and 24 hours of exposure. Results: The reduced glutathione level in Caco-2 cells was depleted starting with 24 hours (62%), and it remained higher than control until 24 hours of exposure to DON. Conclusions: According to our results, oxidative stress might represent an important factor in the chain of cellular events leading into intestinal carcinogenicity of DON.


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YSF-85 Bacterial binding and opsonizing effect of extracellular Hsp70

YSF-87 Cathepsin X is a crucial factor for maturation of dendritic cells

M. T. Nguyen, T. K. Fa´bian´, M. Singh, P. Csermely and C. So˜ti Department of Medical Chemistry, Semmelweis University, Budapest, HUNGARY

N. Obermajer1, U. Sˇvajger2, M. Jeras2 and J. Kos1,3 University of Ljubljana, Faculty of Pharmacy, Department of Pharmaceutical Biology, Ljubljana, SLOVENIA, 2Department for Blood Supply, Blood Transfusion Center of Slovenia, Ljubljana, SLOVENIA, 3Jozef Stefan Institute, Department of Biochemistry and Molecular Biology, Ljubljana, SLOVENIA

Introduction: Stress (or heat shock) proteins form an ancient defense system of our cells. Cells under stressful conditions can express stress proteins on their surface and release them to the extracellular space, where these proteins funtion as a ’danger signal’ and induce apoptosis of target cells as well as activate both humoral and cellular immune responses. One of the most important stress protein implicated in extracellular signalling is Hsp70, the inducible cytosolic isoform of the 70 kDa heat shock protein family. Methods: In the present experiments, we investigated the effect of extracellular Hsp70 on the interaction of bacteria with the immune system by using immunoblot, immunofluorescence and killing assay. Results: Our results demonstrated that extracellular Hsp70 was able to bind both to the surface of Gram-positive Streptococcus mutans and mitis and Gram-negative Escherichia coli bacteria. Acidic milieu (pH 5.5) and high temperature (42C) facilitated while ATP partially inhibited the formation of the Hsp70-bacterium complex. We found that extracellular Hsp70 exerted an opsonizing effect, it was able to activate the killing activity of polymorphonuclear neutrophil leukocytes. Discussion: Our results provide a novel mechanism by which extracellular Hsp70 may induce the activation of the innate immune response. These findings may lead to the deeper understanding of the relationship between stress and immunity, as well as may promote the development of immune stimulatory drugs.

YSF-86 Identification of novel MEK1-interacting protein complex from cancer cells M. Niemela1,2, M. R. Junttila1,2, S. Saarinen1, S. Tuomi1,3, J. Kast4, J. Ivaska1,3 and J. Westermarck1,5 1 Centre for Biotechnology, University of Turku and Abo Akademi University, Turku, FINLAND, 2Turku Graduate School of Biomedical Sciences, University of Turku, Turku, FINLAND, 3VTT Technical Research Centre of Finland, Medical Biotechnology, Turku, FINLAND, 4The Biomedical Research Centre, University of British Columbia, Vancouver, BC, CANADA, 5Institute of Medical Technology, University of Tampere, Tampere, FINLAND Introduction: Mitogen-activated protein kinase (MAPK) signaling mediated by Raf-MEK1,2-ERK1,2 kinase cascade has been shown to play an important role in cancer cell proliferation and survival. Therefore, understanding of the molecular mechanisms that maintain increased MEK1,2 activity in the cancer cells might provide novel opportunities for development of cancer treatments. Based on our previous results, HT-1080 human fibrosarcoma cells display constitutive MEK1,2 phosphorylation that is resistant to stress-mediated dephosphorylation. Methods: In order to understand how constitutive MEK1,2 activity is maintained, we have searched for proteins that associate with MEK1 in HT-1080 cells. By using single-step Strep-tag protein complex purification method combined with mass-spectrometric protein sequencing we have recently been able to identify a novel MEK1 interacting protein complex. Results and conclusions: Identified protein complex contains several proteins that have not previously been shown to be associated with MEK1. However, based on published data, at least some of the identified proteins might have potential role in the regulation of MEK1 activity. The overall aim of this project is to study the role of novel MEK1 interacting protein complex in the regulation MEK1 activity and cancer cell behaviour. In particular we are interested to study if identified protein complex would maintain constitutive activity of MEK1 in cancer cells.

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1

Introduction: DCs play a critical role in the initiation of adaptive immune response. Immature DCs capture and process antigens and migrate into lymphoid organs where they maturate with the ability to activate T cells. Although many different activating signals stimulating DC maturation have been identified and assessed, maturation as a process itself is less well characterized. DC maturation is accompanied by a range of morphology and cytoskeleton structure changes. Upon addition of maturation stimuli, DCs rapidly adhere (cell-substratum contact), develop polarity and assemble podosomes. The intracellular, cytoskeletal components of podosomes are linked to the extracellular matrix through Mac-1 integrin receptor, specifically recruited to the podosomes. Cathepsin X is a cysteine protease primarily expressed in antigen presenting cells, macrophages and DCs and is involved in Mac-1 activation. Methods: Phenotypic and functional characteristics of DCs were assesed with flow cytometry, confocal and time-lapse video microscopy, ELISAs, adhesion and migration assays and allogeneic T lymphocyte proliferation assay. Results: We show that cathepsin X is translocated to the plasma membrane in DCs upon maturation stimuli. Inhibition of cathepsin X during DC differentiation and maturation resulted in altered phenotype and function of DCs, with reduced surface expression of co-stimulatory molecules and increased expression of ILT3 and ILT4, almost completely abolished cytokine production, diminished migration and reduced capacity to stimulate T lymphocytes. Conclusions: Cathepsin X enables adhesion of DCs and formation of podosomes, providing an essential stimulus via Mac-1 integrin receptor. Its action results in mature DCs phenotype, capable of efficient migration and T cell stimulation.

YSF-88 Novel factor of nervous system development in Drosophila A. Orlova, N. Vorobyeva and Y. Shildovski Department of Regulation of Gene Expression, Institute of Gene Biology RAS, Moscow, RUSSIA Mammalian protein p34 initially was described as a ribosome-binding protein. Recently it was found that p34 interacts with mitogenic form of maltose-binding protein-aFGF (acidic Fibroblast Growth Factor) and it might be involved in the regulation or mediation of aFGF signaling. aFGF belongs to the large family of FGF(Fibroblast Growth Factors), and, particularly, it is involved in nervous system development. In our work, we discovered and investigated homologue of p34 in Drosophila. We have generated antibodies against different parts of this protein. Western analysis showed existence of several forms of this protein with different mobility on PAGE. We have found p34 both in nucleus and cytoplasm in cells. We determined sites of p34 binding on the polytene chromosomes. We have found several p34 transcripts by Northern blot analysis and determined structure of these transcripts by 5¢- and 3¢RACE. Chromatographical analysis showed that p34 proteins variants are present mainly in a large 2–3 MDa protein complex. We have also determined level of p34 gene expression on different stages of Drosophila development. We found that p34 is expressed in nervous system in developing embryo. Genetic crosses were done to check interaction of p34 mutations with mutation in genes participating in nervous system development and genes, encoding participants of aFGF pathway.


Young Scientists Forum YSF-89 Determination of novel nuclear localization signal elements within Drosophila EcR and Usp

M. Orlowski1, B. Greb-Markiewicz1, T. Gwo´z´d´z´1, J. Dutko-Gwo´z´d´z´1, M. Jakob1, S. Krzywda2, J. Dobrucki3 and A. Ozyhar1 1 Wroclaw University of Technology, Faculty of Chemistry, Department of Biochemistry, Wroclaw, POLAND, 2Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University, Poznan, POLAND, 3Division of Cell Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, POLAND Introduction: Ecdysteroids are an important class of steroid hormones that regulate essential biological processes in insects and other arthropods. The functional ecdysteroid receptor is a transcription factor comprised of two nuclear receptors, the 20-hydroxyecdysone (20E) receptor (EcR) and the Ultraspiracle protein (Usp). EcR and its ligands are attractive targets for development of gene switches in humans and in agricultural insect control. EcR and Usp from D. melanogaster exhibit differential distribution of molecular determinants directing subcellular trafficking. Our previous study suggested that N-terminal region (A/B) has the significant impact for distribution of all EcR isoforms. The C-terminal extension (CTE), a part of DNA-binding domain (DBD) is a characteristic structural element, which mediates for a particular receptor its dimerization on asymmetric DNA sequences. Methods: In order to determine the sequence of nuclear localization signals (NLSs), series of deletion and point mutants tagged to yellow fluorescent protein (EYFP-C1) were prepared and examined in mammalian cells: HeLa, COS-7 and CHO-K1. Results: Our data revealed the presence of the novel NLS in A/B region of EcR, close to the DBD-core. Moreover, the EcRDBD-CTE harbors probably a part of complex NLS spreading within the DBD and the N-terminal extension (NTE) of EcRDBD. The DNA-recognition helix from UspDBD is responsible for Usp distribution, in contrast to EcRDBD, where the second Zn-module possesses a part of NLS. Conclusions: EcR and Usp exhibit novel asymmetric localization of NLS sequences. Acknowledgements: This work was supported by the Ministry of Science and Higher Education grants: N302/035 32/2827[2827/P01/2007/32] and partially- 3/TO9A/040/28.

YSF-90 Comparative proteomic analysis of alcoholic fermentation employing an indigenous strain of S. cerevisiae from Zitsa, Epirus M. Parapouli1, L. Fragkos-Livanios2, M. Samiotaki2, A.-I. Koukkou1, A. Perisynakis1, E. Hatziloukas3, G. Panayotou2 and C. Drainas1 1 Biochemistry Lab, Department of Chemistry, University of Ioannina, Ioannina, GREECE, 2Biomedical Sciences Research Center’’ Alexander Fleming’’, Athens, GREECE, 3Laboratory of Molecular Biology, Department of Biological Applications and Technologies, University of Ioannina, Ioannina, GREECE Introduction: The aim of this study was to investigate the biochemical pathways induced during must fermentation of the S. cerevisiae, strain Z622, isolated from the traditional wine area of Zitsa (Epirus, Greece). Methods: Proteomic analysis (two-dimensional gel electrophoresis-2D-electrophoresis combined with LCMS/MS analyses) was used to assess the response of strain Z622 cultured in Debina grape must, Yeast extract-Malt extract (YM) and Minimal medium (MM) under fermentation conditions, at the beginning and at the end of fermentation. Proteomic data were verified by a reverse transcriptional-PCR analysis. Results: Through the comparative analyses of 2D gels, intracellular yeast proteins induced during must fermentation were involved in various metabolic pathways such as glycolysis, amino-acid biosynthesis/biodegradation, regulation of DNA replication, transcription and formation of antioxidants. Conclusions: The induction or repression of different metabolic pathways under various conditions of fermentation confirms the importance of employing indigenous yeast strains for the production of traditional wines with improved quality. Acknowledgement: This work was supported financially by PENED 2003-E.U/ G.S.R.T.

Abstracts YSF-91 Drosophila LRP1 modulates BMP signaling E. Parra-Peralbo and J. Culi Centro Andaluz de Biologia del Desarrollo (CABD), Universidad Pablo de Olavide, Sevilla, SPAIN The Low Density Lipoprotein Receptors (LDLRs) are members of an ancient and evolutionarily conserved family of proteins implicated in endocytosis of multiple ligands as well as in cell signaling. The Drosophila genome contains seven LDLRs. Here we present results about the LDLreceptor-related protein 1 (LRP1). In mammals, LRP1 acts as a scavenger receptor having a broad ligand-binding specificity. Its ligands include apolipoproteins, proteases, protease inhibitors, lipases and other functionally diverse macromolecules. Additionally, LRP1 modulates cell signaling pathways such as PDGF in vascular smooth muscle cells. We have generated null lrp1 mutations in Drosophila, which are homozygous viable and display extra veins in the wing. To identify the molecular basis of this phenotype, we tested if lrp1 mutations genetically interact with the signaling pathways known to be involved in wing vein formation. We detected an interaction solely with the integrin subunits myospheroid and multiple edematous wings. Integrins play an important role in vein formation by regulating Bmp signaling, which is essential for vein differentiation. It was shown that integrins regulate the diffusion of Short gastrulation (Sog), a Bmp antagonist. Accordingly, we detected a strong interaction between lrp1 mutations and Sog overexpression in the wing. Based on our results on Drosophila vein differentiation, we suggest a novel molecular model linking LRP1 activity and BMP signaling.

YSF-92 Inhibition of pyroglutamyl peptidase II (thyrotropin releasing hormone-degrading ectoenzyme) by HcPI, a natural inhibitor isolated from the marine annelid Hermodice carunculata enhances anterior pituitary secretions I. Pascual Alonso1, M. Cisneros2, M. A. Vargas2, I. Lazcano2, P. Joseph-Bravo2, M. de los Ange´les Chavez1 and J. L. Charli2 1 Center for Protein Studies, Faculty of Biology, University of Havana, Havana, CUBA, 2Department of Genetics and Molecular Physiology, Institute of Biotechnology, UNAM, MEXICO Introduction: Natural inhibitors of ectopeptidases are useful to elucidate their physiological role and as therapeutic agents. Pyroglutamyl peptidase II (PPII) is a highly specific metallo-ectopeptidase that hydrolyzes thyrotropin releasing hormone (TRH; pGlu-His-ProNH2), a neuropeptide with neural and endocrine functions. We searched for inhibitors of PPII to establish PPII and TRH roles in the central nervous system (CNS) and to obtain therapeutic agents in the treatment of various CNS disorders. Methods: A PPII inhibitor named HcPI was purified from the marine Annelide Hermodice carunculata. The Ki value was determined using tight binding inhibition strategy and its specificity tested against members of the M1 family. In vitro and in vivo effects of HcPI on PPII activity and anterior pituitary hormones were assayed in primary anterior pituitary cell cultures and BalbC mice and Wistars rats, respectively. Cells and animals were stimulated with TRH. Results: HcPI inhibited rat PPII with a Ki value of 71 nmol/l. The maximum inhibitory effect required at least 5 minutes of preincubation; inhibition was stable for at least 1 hour, and was reversible in the presence of increasing amount of substrate. HcPI was highly specificity for PPII, since nor other M1 family metallo peptidases were inhibited. PPII inhibition was also confirmed in cell cultures and in vivo with a dose-dependent behaviour. In vivo potency was higher in hypophysis and hypothalamus than in other brain regions. Inhibition of PPII activity produced an increase in prolactin secretion in vitro, and an increased secretion of thyrotropin in vivo, suggesting that PPII controls anterior pituitary secretions at multiple sites in vivo. Conclusions: HcPI is a highly efficient and specific PPII inhibitor. The data demonstrate that PPII controls some anterior pituitary secretions in vivo. The transport properties and strict selectivity of this biomolecule make it a very interesting candidate tool to explore the role of PPII and TRH in vivo and a drug candidate/leader compound. Acknowledgements: IBRO Larc program 2006, Wood Whelan Fellowship Program from IUBMB 2006, International Foundation for Sciences (IFS) grants 3276-1, 3276-2 and CONACYT Me´xico-Cuba 2003-2005.


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YSF-93 Identification of large conductance Ca2+-activated potassium channel in rat brain mitochondria – a novel neuroprotective target? M. Piwonska, G. Wilczynski and A. Szewczyk Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, POLAND A large amount of evidence has implicated mitochondria as the potential target for cytoprotective strategies. It has been shown that increased K+ uptake catalyzed by mitochondrial ATP—regulated potassium channel (mitoKATP channel) or large conductance Ca2+-activated potassium channel (mitoBKCa channel) may induce protection in different models of cell death. Our recent findings derived from the double-label immunofluorescence experiments, revealed that the distribution of immunoreactivity generated by BKCa b4 subunit antibody colocalizes with the mitochondria labeled with anti-COX antibody. We have shown that the punctate mitochondrial b4 immunoreactivity is preferentially expressed in neurons with much less dense staining in glial cells. Therefore, these results suggest the presence of novel K+ channel in rat brain mitochondria. These observations were additionally supported by functional studies with the use of isolated rat brain mitochondria. Hence, in the present study, in order to investigate the potential neuroprotective effects of BKCa channel openers, we used organotypic hippocampal slice cultures (OHC) exposed to glutamate. We demonstrated that preincubation of the slices with BKCa channel opener – NS1619 resulted in decreased neuronal cell death measured as propidium iodide (PI) uptake. This neuroprotective effect was reversed by the application of BKCa channel inhibitors – paxilline and iberiotoxin. Taken together, these findings seem to be relevant for the further studies concerning the role of mitochondrial potassium channels in neuroprotection.

YSF-94 Assesment of in vitro effects of atrazine on peripubertal rat Leydig cell steroidogenesis K. Pogrmic, S. Kaisarevic, R. Kovacevic and S. Fa Department of Biology and Ecology, University of Novi Sad, Faculty of Sciences, Novi Sad, SERBIA Worldwide used pesticide, atrazine, a chloro-s-triazine herbicide, that selectively inhibits photosynthesis in broadleaf and grassy weeds, has been reported to interfere with reproduction and development and may be associated with risk of cancer. Here we studied in vitro effects of atrazine on testicular steroidogenesis in purified Leydig cell (LC) from peripubertal rats. Obtained results revealed dose-dependent increase of basal and human chorion gonadotropin (hCG)-supported androgen production in LC after 24-hour exposure to atrazine (doses: 1 nM to 50 lM). Both basal and hCG/supported androgen production were accompanied by increased levels of cAMP in the incubation medium. At the same time, in the presence of subsaturated doses of hCG, progesterone production was stimulated, while in the presence of saturated hCG concentrations, progesterone production was inhibited. In addition, conversion of progesterone (P) to testosterone (T), which reflects the activity of CYP17 and 17b-hydroxysteroid dehydrogenase, was stimulated when Leydig cells challenged with atrazine for 24 h, and incubated in the presence of progesterone for additional 2 hours. Obtained results demonstrated that 24-hour incubation of peripubertal rat Leydig cells in the presence of atrazine increased steroidogenic capacity of that cells and indicated possible modulatory effect of atrazine in steroidogenic pathway, at points before, but also after progesterone production. Further studies should clarify by which mechanisms atrazine modulated testicular steroidogenesis.

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YSF-95 Bone marrow-derived stem cells contribute to regeneration and repair of the kidney E. Prodromidi1, R. Poulsom2, C. Pusey1 and T. Cook 1 Imperial College London, Hammersmith Campus, London, UK, 2Cancer Research UK, London Research Institute, London, UK Introduction: Following injury or disease, the kidney has a limited intrinsic ability to regenerate. Emerging data suggest that cells derived from adult bone marrow may reconstitute part of the damaged kidney by forming various renal cell types. This study explored the contribution of bone marrow-derived cells (BMDCs) in regeneration and repair of the kidney in murine models of renal disease. Methods: Sex-mismatched bone marrow transplantation allowed the detection of the origin of transplanted cells by virtue of the Y chromosome, and lineage-specific markers identified the phenotype of engrafted cells. Following transplantation, acute tubular injury in chimaeric mice was induced by ischemia-reperfusion or administration of a nephrotoxic dose of mercuric chloride (HgCl2). Mice deficient in the a3 chain of collagen IV, a model of the human Alport syndrome that leads to progressive glomerular disease, were also transplanted with healthy bone marrow. Results: BMDCs repopulated the proximal tubular epithelium of chimaeric mice, and this was significantly enhanced following acute tubular injury. For the first time, intracellular specific markers (abnormal lysosomal beige granules) confirmed acquisition of tubular phenotypic features by donorderived engrafting cells in damaged host kidneys. Evidence for infrequent cell fusion between indigenous renal tubular cells and BMDCs was also found. In addition, whole bone marrow transplantation significantly improved renal function and glomerular histology in Alport mice by replacing podocytes and partially restoring the gene defect. Conclusion: These results show that BMDCs contribute to renal regeneration, and highlight the potential of these cells in the treatment of acute tubular injury and genetic renal disease.

YSF-96 Dual targeted tRNA-dependent amidotransferase ensures both mitochondrial and chloroplastic Gln-tRNAGln synthesis in plants C. Pujol1, M. Bailly2, D. Kern2, L. Marechal-Drouard1, H. Becker2 and A.-M. Duchene1 1 Institut de Biologie Moleculaire des Plantes, Strasbourg, FRANCE, 2Institut de Biologie Moleculaire et Cellulaire, Strasbourg, FRANCE Aminoacyl-tRNA formation is an essential process in protein biosynthesis, generally achieved by direct attachment of an amino acid to tRNA by the aminoacyl-tRNA synthetases. An exception is Gln-tRNAGln synthesis, which in eukaryotic cytosol is catalyzed by glutaminyl-tRNA synthetase (GlnRS), while most bacteria an all archae employ the transamidation pathway. In this pathway tRNAGln is first mischarged with glutamate (Glu) by a non-discriminating glutamyl-tRNA synthetase (ND-GluRS). The glutamate attached to tRNAGln is then amidated into glutamine (Gln) by a tRNA-dependent amidotransferase (AdT) generating Gln-tRNAGln. My PhD work aims to explore the mitochondrial Gln-tRNAGln formation in plant. Mitochondria and chloroplasts having their own translation machinery, a complete set of aa-tRNAs is expected in these organelles. The pathway of Gln-tRNAGln formation has been explored in few eucaryote and in each case, mitochondrial Gln-tRNAGln is formed directly. In Arabidopsis thaliana, we did not detect mitochondrial GlnRS activity. By contrast the dual-targeted mitochondrial-chloroplastic GluRS can attach Glu to both tRNAGlu and tRNAGln and thus appears to be non-discriminating. Moreover, we find the three genes, GatA, GatB and GatC which encode a tRNA-dependent amidotransferase (AdT), active in the mitochondrial and in the chloroplastic extracts. In conclusion we demonstrate that in plants GluRS and GatCAB, via the indirect transamidation pathway, serves to form Gln-tRNAGln in both mitochondria and chloroplasts. We therefore find the first example of multimeric protein GatCAB, with three subunits dual-imported in the two organelles.


Young Scientists Forum YSF-97 Crystallographic and functional analysis of thermoactive nitrilase from Pyrococcus abyssi J. Raczynska1, C. Vorgias2 and W. Rypniewski1 1 Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, POLAND, 2National and Kapodistrian University of Athens, Faculty of Biology, Department of Biochemistry-Molecular Biology, Athens, GREECE Nitrilase superfamily consists of enzymes that catalyse the cleavage of various types of carbon–nitrogen bonds such as those present in nitriles, acid amides, secondary amides or N-carbamoyl amides. They utilize a novel catalytic triad: glutamic acid, lysine and cysteine. To gain more insight into the catalytic mechanism we have obtained a structure of a putative nitrilase from a hyperthermophilic archeon Pyrococcus abyssi which was shown recently to hydrolyse small aliphatic nitriles [1]. Methods: Crystallographic data for the nitrilase crystals were collected at EMBL-Hamburg. The structure was solved by molecular replacement using 1j31.pdb as a model. Activity tests were performed using Nessler’s reagent to detect ammonia. Results: The protein is a dimer in solution as well as in the crystal and the whole dimer is present in the asymmetric unit. Each subunit is a four-layer abba-sandwich and a C-terminal part protruds from the core and interacts with the other subunit. We have found that it also catalyses the breakdown of b-ureido propionic acid and urea with the release of ammonia. We have obtained a structure of the enzyme with b-alanine, the product of the first reaction. The same mode of binding has been observed for acetate molecule in another crystal form that we have examined.

Abstracts YSF-99 Ostreolysin binding to cholesterol-rich lipid domains K. Rebolj1, M. Sentjurc2 and K. Sercic1 1 Department of Biology, Biotechnical Faculty, University of Ljubljana, Ljubljana, SLOVENIA, 2Jozef Stefan Institute, Ljubljana, SLOVENIA Introduction: Ostreolysin (Oly) is a 15-kDa cytolytic protein that requires a specific combination of cholesterol (Chol) and sphingomyelin for the effective binding to the lipid membrane. This interaction can be diminished (i) by the addition of unsaturated phosphatidylcholine (DOPC), (ii) by replacing Chol with other natural sterols or synthetic Chol derivatives, and (iii) by the addition of lysophospholipids. In this work, we investigated the determinants of the lipid structure dictating Oly membrane binding. Methods: Surface plasmon resonance (SPR) was applied to study the binding of Oly to lipid bilayers with different compositions. Electron paramagnetic resonance (EPR) was applied to evaluate fluidity characteristics of certain lipid mixtures. Results: Membranes containing domains with high order parameters are much more susceptible for Oly binding. Lysophosphatidylcholine and DOPC dramatically altered membrane domain structure and subsequent Oly binding. Conclusion: Combining SPR and EPR techniques enabled us to observe the correlation between membrane fluidity and Oly binding in detail. Our results indicate that fluorescently labelled Oly mutants deprived of toxic activity could be useful markers for studing the harmony of ordered membrane domain called lipid rafts.

YSF-100 SWAP-70 is required for proper stress-induced erythropoiesis T. Ripich and R. Jessberger Institute of Physiological Chemistry, Faculty of Medicine, Cal Gustav Carus Dresden University of Technology, MTZ, TU Dresden, GERMANY

Conclusions: The manner in which both ligands bind suggests specificity for the carboxylate group. Most likely, the enzyme from P. abyssi should be classified as b-ureidopropionase (b-alanine synthase).Fig. 1 Acetate molecule bound in the active site of nitrilase from P. abyssi Reference: 1. Mueller et al. Protein Expr. Purif. 2006; 2: 672–681.

YSF-98 Stability of K+-channels: differences between prokaryotic KcsA and eukaryotic ROMK1 channels M. Raja, E. Vales and P. Pohl Institute for Biophysics, Johannes Kepler University, Linz, AUSTRIA K+-channels are believed to be stabilized by K+-ions in the selectivity filter. However, it was shown that all ions may be rinsed out of the selectivity filter of bacterial K+-channel KcsA by osmotic flow and the channel was still active. On the other hand, the eukaryotic potassium channel ROMK1 was reported to be water-impermeable which could be due to its inactivation when the amount of K+ ions in the pore is decreased. We checked the hypothesis that both channels differ in their intrinsic stability. Mutations were performed in the selectivity filter of KcsA towards ROMK1 in order to identify respective residues. All mutants were active when reconstituted in a planar lipid bilayer, but in detergent they differed in the degree of oligomerization as determined by gel electrophoresis. We conclude that the selectivity filter contributes to the stability of tetramer. In addition, mutant channels were permeable to Na+ as well, resembling the physiological properties of NaK channels.

Introduction: SWAP-70, an unusual phosphatidylinositol-3-kinase (PI3K)dependent, F-actin binding protein that interacts with the RhoGTPase Rac is expressed in mast cells, B cells (1) and dendritic cells. In these cells, SWAP-70 acts as an important regulator of cell polarization, migration in vitro and in vivo, adhesion under static and flow conditions and lymphoid tissue homing and formation (2). We further showed that SWAP-70 plays significant roles also in cell effector functions such as mast cell degranulation (3) and production of specific immunoglobulins (4). Results: Recently we discovered expression of SWAP-70 in red blood cell precursors and set out to investigate its role in erythropoiesis. Erythropoiesis in the bone marrow appears normal, but splenic erythropoiesis does not: although in steady state conditions SWAP-70-/- mice show wild type-like basic blood parameters, the mice harbor much fewer erythroblasts in the spleen. This may indicate a possible impairment of stress-induced erythropiesis, which was confirmed by phenylhydrazine treatment to induce anemia, from which SWAP-70-/- mice recover poorly. Newly formed red blood cells from SWAP-70-/- mice show increased hemoglobin content, hematokrit and reticulocyte counts are altered. Currently we are investigating the mechanisms that cause these and additional phenotypes. Erythropoiesis, precursor populations as well as Epo and c-kit signaling are studied. Our central hypothesis, however, takes the known features of SWAP-70 in F-action dependent processes into account and thus proposes that migration and homing of erythrocyte precursors to the spleen, and cell–cell interactions within erythroblast islands, the specialized niches in which erythropoiesis happens, composed of erythroblasts surrounding a central macrophage, is impaired. This hypothesis is currently being tested in vitro and in vivo. Reference: 1. Shinohara M. et al., Nature 2002 Apr 18; 416(6882): 759–763 2. Pearce G. et al., Nat. Immunol. 2006 Aug; 7(8): 827–834, Quemeneur et al., Blood 2007; Dec 19; [Epub ahead of print] 3. Gross B. et al., Eur. J. Immunol. 2002 Apr; 32(4): 1121–1128 4. Borggrefe T. et al., Eur. J. Immunol. 2001 Aug; 31(8): 2467–2475.


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Abstracts


YSF-101 Modulation of type I collagen in stromal fibroblasts by breast tumour cells B. A. Rose1, V. D. Leaner1 and M. I. Parker2 1 Division of Medical Biochemistry, Faculty of Health Sciences, University of Cape Town, Cape Town, SOUTH AFRICA, 2International Centre for Genetic Engineering and Biotechnology, Faculty of Health Sciences, University of Cape Town, Cape Town, SOUTH AFRICA Introduction: In previous studies in our laboratory type I collagen mRNA levels were found to be decreased in stage II and III breast tumour tissue compared to adjacent normal tissue. In vitro studies suggested that direct contact between cultured fibroblasts and tumour cells was necessary for type I collagen down-regulation. The aim of this study is to determine how breast tumour cells modulate type I collagen levels in co-cultured fibroblasts. Methods: CCD-1068SK breast fibroblasts were co-cultured with either MCF12A non-tumorigenic epithelial cells (control) or MDA-MB-231 breast tumour cells for 48h in serum-free medium. Co-culture methods included (1) direct contact between cells, (2) indirect (cell types separated by a 0.2 lm transwell membrane), or (3) fixed (tumour cells fixed before co-culture with fibroblasts). Cell types were differentially labelled and fluorescent microscopy was used to determine if co-cultured cells were in direct contact. Type I collagen mRNA expression in fibroblasts was quantified by means of realtime RT-PCR. Results: Real-time RT-PCR results showed a down-regulation of both a1(I) and a2(I) procollagen mRNA levels in fibroblasts directly co-cultured with tumour cells. However, no change in mRNA expression was observed when cells were co-cultured under indirect or fixed conditions. Conclusion: Results suggest that tumour cells are producing a factor that down-regulates type I collagen mRNA expression only when in direct contact with fibroblasts. This factor does not appear to be secreted into the medium. More conclusive evidence needs to be gathered to determine the role of tumour membrane factor(s) in type I collagen down-regulation.

YSF-103 Expression enhancement of bubaline somatotropin in E. coli through gene modifications in the 5¢-end coding region S. Sadaf1,2, M. A. Khan2 and M. W. Akhtar2 1 Institute of Biochemistry and Biotechnology, University of the Punjab, Lahore, PAKISTAN, 2School of Biological Sciences, University of the Punjab, Lahore, PAKISTAN Introduction: Heterologous proteins, especially of eukaryotic origin, are often difficult to express efficiently in E. coli due primarily to the differences in codon usage, unique and subtle structural features of foreign gene sequence, non-availability of machinery for posttranslational modifications, potential toxicity of certain proteins to the host-cell and/or degradation of the foreign protein by host cell proteases. This study addresses the problem of poor expression of somatotropin (ST) gene in E. coli and describes expression enhancement through silent and non-silent gene modifications. Methods: A series of constructs with codon optimization, substitution, deletion or addition in the 5¢-region of the sequence encoding bubaline ST (BbST) were prepared. Results: In the native form, the BbST expression was barely discernible on SDS-gel of the total E. coli cellular proteins (TCP). Introduction of silent and non-silent mutations in +2 to +8 codons, however, raised the expression levels to varying extents. In some constructs, a single base variation, i.e., G fi A or G fi C led to a remarkable increase in BbST expression (up to 28 % of the TCP) whereas in the case of G fi T substitution the expression dropped to undetectable levels. Deletion of native GCC codon and addition of CAUCAC repeat thrice at +2 position enhanced the expression up to 48 %, while insertion of NGG codons at the same position caused just a modest increase in expression. Conclusion: Differences in expression appeared as if related to the nature of early downstream codons (especially +2) and the stability of mRNA secondary structure although the levels of intracellular mRNA pools, as analyzed by real-time RT-PCR were quite similar. Overall, the study highlights the importance of 5¢-end codon adaptations in solving the problems encountered in expressing the eukaryotic genes in E. coli.

YSF-104 YSF-102 Development of a novel strategy for obtaining GPCRs Destabilization of messenger RNA with long 3¢UTR in plants in crystallizable form A. Roy1,2, A. K. Shukla1 and H. Michel1 1 Department of Molecular Membrane Biology, Frankfurt am Main, GERMANY, 2Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Frankfurt Am Main, GERMANY Introduction: G protein-coupled receptors (GPCRs) constitute an important class of integral membrane proteins, that are involved in several signaling pathways. About 50% of the currently available drugs are targeted against these receptors hence, high-resolution structures of these receptors will be of immense importance from the perspective of designing specific and potent drugs. Crystallization of GPCRs is difficult owing to their flexible nature and limited hydrophilic surface area available for crystal contacts. Methods: Here we present a novel approach in which, R. sphaeroides was used as an expression system to generate a fusion protein complex of the photosynthetic reaction center (RC) with a GPCR (human Angiotensin 1a receptor), expecting that such a complex would be easier to crystallize than the receptor alone. The notion behind this approach is that the RC will act as a scaffold in providing surface area to create crystal contacts and at the same time, it will also reduce the flexibility, hopefully without perturbing the functionality of the receptor. Results: Out of several fusion constructs generated, one was found to be functional. It was possible to functionally solubilize, purify and crystallize the fusion complex in the ligand bound state. The functional expression level of the fusion complex was also higher than that of the receptor expressed alone. Conclusion: This approach could in principle be extended to other membrane proteins that fail to crystallize on its own.

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A. Schwartz, T. Komarova and M. Skulachev Moscow State University, Moscow, RUSSIA Our group has been investigating so-called ‘quality control’ mechanism in plants that was named as nonsense mediated decay (NMD). This machinery can destroy aberrant messenger RNA (mRNA), in particular mRNAs that have premature termination codon (PTC). At the moment most of NMD features in mammals and yeast are well-known, but in plants the details of NMD mechanism remains obscure. Identification of PTC is imperative stage of NMD. We performed experiments, which allowed us to establish some principles of PTC recognition in plants. Firstly, it was shown that efficient PTC recognition requires synthesis of mRNA in the nucleus. In addition, we proved that stop codon, which is placed more than 200 nucleotides upstream of polyA signal will be identified as PTC in plant cells. We also provide evidence concerning poly-A-binding protein, which is involved in RNA quality control in plants as well. It supports the hypothesis that accessibility of the poly(A)-tail for the terminating ribosome complex is crucial for NMD triggering. Finally we can conclude – the distance between poly(A) tail and open reading frame is quite important for stability and fate of plant messenger RNAs.



Abstracts

YSF-105 Comparative proteomic studies of ‘Rhodospirillum rubrum’ grown under different nitrogen conditions

YSF-107 New screening system demonstrates persephin potential in Parkinson’s disease treatment

T. Selao, S. Nordlund and A. Noreń Department of Biochemistry and Biophysics, Stockholm University, Stockholm, SWEDEN

Y. A. Sidorova, M. M. Bespalov, M. Lindahl, M. Paveliev and M. Saarma Institute of Biotechnology, University of Helsinki, Helsinki, FINLAND

Introduction: The diazotrophic alpha-proteobacterium Rhodospirillum rubrum has the capacity to grow under a wide range of metabolic conditions. This versatility creates a demand for regulation both at a transcriptional as well as at the metabolic level for the cell to be able to adapt to different environmental conditions. Methods: Three independent cultures were grown in nitrogen fixing or nitrogen rich medium. Cells were lysed and the soluble fraction used to prepare 2D-PAGE samples. 2D-PAGE gels were analysed according to differential expression. The differentially expressed protein spots were excised, in-gel digested and identified using MALDI-TOF mass spectrometry. Results: Forty-four differentially expressed proteins have been identified in R. rubrum grown anaerobic and photoheterotrophically, with different nitrogen sources, using 2D-PAGE and MALDI-TOF. A higher level of expression was found under nitrogen rich growth, for proteins involved in carbon (reductive tricarboxylic acid cycle, CO2 fixation and poly-hydroxybutyrate metabolism) and amino acid metabolism. Ntr and Nif regulated proteins, such as glutamine synthetase and nitrogenase were, as expected, induced under nitrogen fixing conditions and glutamate dehydrogenase was down regulated. In addition to differential expression, two of the identified proteins revealed variable pI values in response to the nitrogen source used. Conclusions: This is the first time a differential study of the proteome of this organism has been performed, using 2D-PAGE and MALDI-TOF mass spectrometry. This study allows for a broader view of the regulatory networks responding to nitrogen availability. We have shown the ‘cross talk’ between nitrogen and carbon signaling for protein expression, since a major effect of the different nitrogen sources is a response in the expression pattern of many enzymes related to carbon metabolism.

YSF-106 Novel export complex is responsible for efficient transcription and mRNA transport through the nuclear pore Y. Shidlovskii1, M. Kurshakova1, A. Krasnov1,2, D. Kopytova1, J. Nikolenko1, E. Nabirochkina1,2, D. Spehner3, P. Schultz3, L. Tora3 and S. Georgieva1,2 1 Department of Regulation of Gene Expression, Institute of Gene Biology RAS, Moscow, RUSSIA, 2Centre for Medical Studies in Russia, University of Oslo, Moscow, RUSSIA, 3Institut de Genetique et de Biologie Moleculaire et Cellulaire (IGBMC), CNRS UMR, Illkirch Cedex, FRANCE SAGA/TFTC-type multiprotein complexes play important roles in the regulation of transcription. We have investigated the importance of the nuclear positioning of a gene, its transcription and the consequent export of the nascent mRNA. We show that E(y)2 is a subunit of the SAGA/TFTC-type complex in Drosophila and that E(y)2 concentrates at the nuclear periphery. We demonstrate an interaction between E(y)2 and the nuclear pore complex (NPC) and show that SAGA/TFTC also contacts the NPC at the nuclear periphery. We further show that E(y)2 acts together with X-linked male sterile 2 (Xmas-2) to regulate mRNA transport. Importantly, E(y)2 and Xmas-2 knock down decreases the contact between the heat-shock (hsp70) gene loci and the nuclear envelope and interferes with transcription. Thus, E(y)2 and Xmas-2 function together with SAGA/TFTC in the anchoring of a subset of transcription sites to the NPC basket to achieve efficient transcription and mRNA export.

Glial-derived neurotrophic factor (GDNF) family ligands (GFLs) play an important role in the development and maintenance of the nervous system. GDNF and NRTN signalling via GFRa1-2/RET complexes support dopaminergic and motor neurons. Thus, both neurotrophic factors are considered for the treatment of Parkinson’s disease and amiotrophic lateral sclerosis (ALS). ARTN signalling via GFRa3/RET can be beneficial for the patients suffering from chronic pain. The fourth member of GDNF family – PSPN does not have functional receptor in the nervous system. However, several studies demonstrated that PSPN supports survival of several neuronal populations by unknown mechanism. Therefore, RET activators hold promises for the treatment of several neurological disorders. Bad pharmacokinetics properties, pronounced side-effects, extreme stickiness, inconvenient delivery and costly production make GDNF themselves unsuitable for therapeutical purposes. Development of small molecular weight GFLs mimetics could help to overcome these difficulties. We established new sensitive, cell-based system for the screening of the small molecules RET agonists and antagonists. Developed cell line expresses RET, GFRa1 and genetically engineered system for the monitoring of RET downstream target mitogene-activated protein kinase (MAPK) induction using luciferase for read-outs. We characterized and validated our system with known RET activator and inhibitor. Using this cell line we showed the ability of PSPN to signal through GFRa1. Our findings were proven by other biochemical methods. From all GFLs PSPN has the best therapeutical profile: it is less sticky than other GFLs and has the lowest number of negative side-effects. Our results support PSPN potential as anti-Parkinson’s and anti-ALS therapeutical.

YSF-108 Significance of microRNA-21 in colorectal cancer pathogenesis O. Slaby1,4, R. Hrstka1, L. Dubska3, M. Svoboda2,4, E. Ruckova1, J. Ovesna5 and R. Vyzula2,4 1 Masaryk Memorial Cancer Institute, Department of Clinical and Experimental Pathology, Brno, CZECH REPUBLIC, 2Masaryk Memorial Cancer Institute, Department of Comprehensive Cancer Care, Brno, CZECH REPUBLIC, 3Department of Laboratory Medicine, Masaryk Memorial Cancer Institute, Brno, CZECH REPUBLIC, 4Faculty of Medicine, Masaryk University, Brno, CZECH REPUBLIC, 5Crop Research Institute, Praha Ryzyneˇ, CZECH REPUBLIC MicroRNAs (miRNAs) are endogenously expressed short non-coding RNAs, that repress protein translation through binding to target mRNAs. Although the number of verified human miRNA is still expanding, only few have been functionally described. However, emerging evidences suggest the involvement of altered regulation of miRNA in pathogenesis of cancers and these genes are thought to function as both tumours suppressor and oncogenes. Previous studies, mainly based on microarrays technology applied on colorectal cancer cell lines, showed altered expression levels of several miRNAs in colorectal cancer (CRC). In our study, we examined by Real-Time PCR expression levels of microRNA-21 (miR-21) in tumors of 29 colorectal cancer patients. For six cases of CRC samples also adjacent non-tumor tissues were analyzed. For data normalization we tried different approaches (18S rRNA, GAPDH, let-7a-1). Finally, variability of let-7a-1 expression was shown to be the lowest. Expression levels of miR-21 were significantly higher in tumors comparing to normal mucosa (p = 0.0001). High expression of miR-21 was also associated with lymph node positivity (p = 0.025) and the development of distant metastases (p = 0.009) in CRC patients. To elucidate function of miR-21 in colon cancer cells (DLD1, SW837, HCT116) we tested effects of synthetic 2ÓMe-anti-sense-miR-21 (anti-miR-21) transfection (2ÓMe-EGFP as control) on their proliferation, cell cycle regulation, sensitization to chemotherapeutic agents (5-fluorouracil, irinotecan, oxaliplatin) and invasive properties. We observed 15–30% decrease of cells proliferation by MTT test after transfection of anti-miR-21 in comparison with control cells. Moreover, cytostatic effect of treatments was enhanced about 20– 30% in transfected colon cancer cells. Interestingly, these actions of antimiR-21 were not associated with significant changes of cell cycle. We hypothesize, that miR-21 affects directly proliferation and now we are testing common markers of proliferation (Ki-67) and those which are possibly under miR-21 post-transcriptional control. Simultaneously, we evaluate changes in invasive properties of anti-miR-21 transfected cancer cells by matrigel invasion assay. Our results suggest possible role of miR21 in colorectal cancer pathogenesis. Acknowledgements This work was supported by IGA MZ CR NR/9076 – 4 and project MZ0MOU2005.


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Abstracts


YSF-109 Novel mechanism of coupling of chromatin remodeling and transcription initiation

YSF-111 Insight into the molecular details of domain movements of 3-phosphoglycerate kinase

N. Soshnikova1, N. Vorobyeva1, Y. Shidlovskii1, S. Georgieva1,2 and Y. Ilyin1 1 Department of Regulation of Gene Expression, Institute of Gene Biology RAS, Moscow, RUSSIA, 2Centre for Medical Studies in Russia, University of Oslo, Oslo, Norway

J. Szabo´1, A. Varga1, B. Flachner1, P. Konarev2,3, D. Svergun2,3, P. Zavodszky1 and M. Vas1 1 Institute of Enzymology, BRC, HAS, Budapest, HUNGARY, 2EMBL Outstation, Hamburg, GERMANY, 3Institute of Crystallography, RAS, Moscow, RUSSIA

Coupling of different nuclear processes on a certain gene is one of the most important issues of modern molecular biology. Novel transcription factor of RNA Polymerase II SAYP participates in activation of transcription of a plenty of genes. ChIP experiments showed that SAYP is located predominantly on promoters of genes under its control. Chromatographical analysis of SAYP showed that it is a subunit of 2 MDa DNA-binding protein complex. Preparative purification from drosophila embryos showed that SAYP organizes complex which contains all subunits of PBAP and TFIID complexes, which are principal factors of chromatin remodeling and transcription initiation in higher eukaryotes, respectively. Immunoprecipitation experiments showed that SAYP is a subunit of both TFIID and PBAP complexes and these complexes are directly linked via SAYP. Presence of TFIID, PBAP and SAYP on promoters of SAYP-controlled genes are interconnected, what was confirmed by ChIP combined with RNAi of indicated complexes. Using RNAi of separate subunits of PBAP and TFIID complexes and in vitro binding assays we found direct partners of SAYP in PBAP and TFIID complexes. Thus SAYP is potent activator of transcription which serves as an organizer of interplay of different activities in nucleus.

Introduction: Molecular movements, such as domain closure by operation of hinges, are essential for functioning of multidomain enzymes. 3-Phosphoglycerate kinase (PGK) is a typical hinge-bending enzyme with two domains: each of them binds one of the two substrates, 3-PG and MgATP, respectively. Location of the main molecular hinge of PGK has been identified at b-strand L in the interdomain region, but the mechanism of domain closure has not been explored at atomic level. Methods: Here the role of individual side-chains (located at the hinge and in substrate binding sites) in domain closure have been tested by site-directed mutational, kinetic, binding and biophysical (DSC, SAXS) studies. Results and conclusions: We have found that one catalytic residue (R38), that interacts with the substrate 3-phosphoglycerate, as well as of several residues at the nucleotide site (K219, N336 and E343) are essential in inducing domain closure. The other catalytic residue (K215), that moves about 10 A˚ distance upon interaction with the transferring c-phosphate of MgATP, however, has no direct role in domain closure. We also present experimental evidence for the importance of the shape and length of bL in operation of the hinge by deleting T393. The way of transmission of the substrate effects towards the main hinge is described at atomic details.

YSF-110 PKA and StAR are involved in Leydig cells adaptive response to stress

YSF-112 Antibiotics as translation inhibitors: new methods – new insights

S.N. Stojkov, S.A. Andric, M.M. Janjic and T.S. Kostic Faculty of Sciences, University of Novi Sad, Novi Sad, SERBIA

W. Szaflarski1,2 and K. H. Nierhaus1 1 Max-Planck-Institut fu¨r Molekulare Genetik, AG Ribosome, Berlin, GERMANY, 2Department of Histology and Embryology, Poznan University of Medical Sciences, Poznan, POLAND

It was previously observed that the immobilization stress (IMO) decreased Leydig cell steroidogenesis, but the possible mechanisms involved in the adaptation to repeated stress have not been identified. Here we investigated the effects of acute (15 min, 30 min or 2h) and repeated (2 or 10 days, 2h daily) IMO on Leydig cells steroidogenesis and proteins involved in regulation of steroidogenesis. The results showed that basal and hCG-stimulated cAMP production by Leydig cells isolated from rats exposed to both acute and repeated IMO was significantly reduced. Treatment with cholesterol, the steroid substrate transported into mitochondria by steroidogenic acute regulatory (StAR) protein, significantly increased androgen and progesterone production by Leydig cells isolated from rats exposed to repeated IMO. In contrast, Leydig cell steroidogenesis was still reduced by IMO when other steroid supstrates (22(R)-OH-cholesterol, pregnenolone, progesterone, D4androstendione) were present in the culture media. In Leydig cells obtained from rats exposed to repeated IMO immunoblot analysis revealed increased immunoreactivity for both protein kinase A (PKA) and StAR protein. Also, phosphorylation and production of mature StAR protein was evident during the exposure of rats to repeated IMO. This findings reveal that PKAmediated phosphorylation of StAR protein is an important mechanism in the adaptive response of Leydig cells to repeated IMO.

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The increasing resistance against antibiotics is a major threat for infectionmedicine. A detailed knowledge of both the antibiotic mechanisms and their binding sites is an important tool for the development of new classes of drugs and for avoiding functional overlaps. Since most of the antibiotics target the ribosome – about 70% – we optimized biochemical conditions for the analysis of ribosomal functions in poly(U)-dependent poly(Phe) synthesis and in cell-free coupled transcription-translation system with Green Fluorescence Protein as reporter, both systems yielding almost in vivo perfection in respect to the translational errors, which then could be applied for the analysis of drug inhibition mechanisms. Number of antibiotics and new features of their inhibition mechanisms were revealed and three examples will be discussed:Edeine and pactamycin: The effects of these two drugs were studied and compared, since the binding sites of these drugs overlapped. In fact, we demonstrated that the drugs showed the strongest antagonism in their inhibition activities known up to now. Further new features were: (i) Edeine disturbed the accuracy of amino acid selection as strong as streptomycin, well known for its strong increase of errors in protein synthesis. This finding was a surprise, since edeine does not bind to the decoding center as streptomycin, but rather far away at the E site of the ribosome. (ii) Pactamycin turned out to be a specific blocker of the translocation reaction for some tRNAs such as tRNALys, but not for others, e.g. tRNAPhe, again a surprise.Kasugamycin: We demonstrated that kasugamycin inhibits exclusively the small ribosomal subunit 30S, specifically the P-site tRNA binding through perturbation of the mRNA-tRNA codon-anticodon interaction near the E-site. Our data explain, why this drug does not inhibit the 70S-type initiation on leaderless mRNA.



Abstracts

YSF-113 The PII signal transduction proteins as sensors of cellular carbon, nitrogen and energy status in photosynthetic bacteria

YSF-115 Apoptotic inductors specific influence on the composition and activities of 26S proteasomes in K562 cells

P. F. Teixeira and S. Nordlund Department of Biochemistry and Biophysics, Stockholm University, Stockholm, SWEDEN

A. Tsimokha, J. Vatazhok, I. Kozhukharova and V. Kulichkova Institute of Cytology RAS, St. Petersburg, RUSSIA

Introduction: The PII family of signal transduction proteins is widespread amongst the three domains of life and its members have a fundamental role in the general control of nitrogen metabolism [1] . These proteins exert their regulatory role by direct protein-protein interaction with a multitude of cellular targets, modulated by the binding of metabolites like ATP, ADP, 2oxoglutarate and also by reversible post-translational modification. In the photosynthetic nitrogen fixing bacterium Rhodospirillum rubrum three PII paralogs were identified and termed GlnB, GlnJ and GlnK. In this work we have analysed the interaction of GlnJ with several cellular targets such as the ammonium transporter AmtB1, the adenylyltransferase GlnE and the uridylyltransferase GlnD. Methods: Interaction of GlnJ with AmtB1 was analysed using isolated wt R. rubrum membranes and purified GlnJ in the presence of different effectors (ATP, ADP, 2-oxoglutarate, Mg2+, Mn2). Enzyme activity assays and protein purification strategies were performed as described [2,3]. Results and conclusions: Our results show that the interaction of GlnJ with cellular targets is regulated by the concentration of manganese, 2-oxoglutarate and the ADP/ATP ratio. 2-oxoglutarate abolishes interaction of GlnJ with AmtB1 and GlnE while favouring interaction with GlnD. Conversely, ADP acts by relieving the 2-oxoglutarate signal indicating an additional mechanism of regulation. This study provides further evidence for the proposal that PII proteins are sensitive to the carbon, nitrogen and energy status in the cell. References: 1. Ninfa and Jiang, Curr. Opin. Microbiol. 2005; 8: 168–163. 2. Jonsson et al., FEBS J. 2007; 274: 2449–2460. 3. Jonsson and Nordlund, J. Bacteriol. 2007; 189: 3471–3478.

YSF-114 Immune complexes of glucose-regulated protein94 (Grp94) with IgG, present in the plasma of type 1 diabetic subjects, show strong angiogenictransforming capacity on HUVECs E. Tramentozzi1, A. Pagetta1, M. Frasson2, M. Montopoli1 and P. Finotti1 1 Department of Pharmacology and Anesthesiology, University of Padova, Padova, ITALY, 2Department of Biochemistry, University of Padova, Padova, ITALY Introduction: We previously showed that plasma concentration of the heat shock protein (HSP) Grp94 was increased in type 1 diabetes, and that Grp94 circulated in complexes with IgG displaying serine-like proteolytic activity. The findings raised the question of whether Grp94-IgG complexes were linked to diabetes, representing a marker of the development and progression of vascular complications frequently associated with disease. Methods: Complexes of Grp94 with IgG were purified from plasma by chromatographic procedures on mono-Q and Protein G columns, followed by immunoblotting and immunoprecipitation with anti-Grp94 and anti-Fabspecific antibodies. Immune complexes were tested on HUVECs, and both cell growth proliferation and differentiation were evaluated by counting cells and by means of optical and confocal microscopic analyses. Conditioned media and cell lysates were also analyzed for the expression and activity of specific proteins. Results: Grp94 was found irreversibly linked with both fragmented and integer IgG in complexes that caused a significant stimulation of the cell growth and strong angiogenic transformation of HUVECs. Cell proliferation was accompanied by an intense activation of the P-ERK1/2 pathway, whereas angiogenic differentiation was mediated by an increased expression of both matrix metalloprotease-9 and HSP70 that appeared to be structurally and functionally coupled. Neither proteolytic activity of metalloprotease-9 nor VEGF were responsible for effects of Grp94-IgG complexes on HUVECs. Conclusions: Plasma Grp94-IgG complexes may represent a marker of vascular damage in vivo, in any inflammatory/autoimmune disease in which steady increases in extra-cellular Grp94 trigger the mechanisms leading to the onset of disease and its complications.

The 26S proteasome is a multisubunit protease complex which catalyzes the non-lysosomal proteolytic pathway in eukaryotic cells. The proteasomes realize programmed proteolysis and processing of various regulatory proteins. Thus, they play an important role in major cellular processes, including transcription, cell cycle progress, signal transduction, immune response, oncogenesis and apoptosis. Apoptosis is one of the major protective cellular processes providing the control over quality and quantity of cells in multicellular organism. Studies using the proteasome selective inhibitors have provided direct evidence for the ubiquitin–proteasome system functions in regulation of apoptosis. The data of the present paper show for the first time that apoptosis inductors (doxorubicin or diethylmaleate) regulates subunit composition, enzymatic (proteolytic and endoribonuclease) activities, and phosphorylation and glycosilation states of 26S proteasomes in neoplastic (proerythroleukemic K562) cells. Thus, the specificity of proteasomal enzymatic activities is changed in K562 cells undergoing apoptosis. Furthermore, apoptosis inductors treatment of K562 cells lead to modification of 20S proteasomal subunits including catalytic subunits a1, a5 and b2 associated with proteasomal RNase and trypsine-like proteolytic activities respectively. Moreover, we showed the selective effect of dephosphorylation of proteasomal subunits on their enzymatic activities. Our results for the first time demonstrate changes in phosphorylation of proteasomes in apoptotic cells and present evidence for the role of these changes in the control of enzymatic activities of these particles in apoptotic cells. Acknowledgements: The work was supported in part by RFBR (project 08-04-00834) and by the Scientific Cooperation Center ‘Material science and characterization in high technology’.

YSF-116 14-3-3c regulates CtBP1/BARS-mediated fission of post-Golgi carriers C. Valente, G. Turacchio, A. Colanzi, A. Luini and D. Corda Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Chieti, ITALY Introduction: Membrane fission is an essential process in cell functions such as membrane trafficking, cell division and neurotransmission. While several fissioning steps in membrane trafficking require the GTPase dynamin, we have previously shown that other dynamin-independent events require C-terminal-binding-protein-1/ brefeldinA-ADP ribosylated substrate (CtBP1/BARS). In particular, CtBP1/BARS has a crucial role at the Golgi complex for formation of post-Golgi carriers, which transport protein cargoes from the trans-Golgi network to the plasma membrane. Methods: I have used a proteomic approach to define this CtBP1/ BARS-driven fissioning machinery further (affinity chromatography, mass spectrometry, Western blotting), with RNA interference and the temperature-sensitive mutant of a viral protein (VSVG; as a cargo reporter) used to investigate its individual components. Results: Among the BARS-interactors that I have identified, I am focussing on 14-3-3c, a member of the 14-3-3 protein family involved in various physiological processes: membrane trafficking, apoptosis, cell-cycle control and signal transduction. BARS and 14-3-3c knock-down demonstrate that 14-33c controls CtBP1/BARS-mediated fission/ formation of plasma-membranedirected, post-Golgi carriers. 14-3-3c also interacts with phosphatidylinositol 4-kinase IIIb (PI4KIIIb), a Golgi-resident kinase that is another regulator of this transport step. Indeed, 14-3-3c binds both CtBP1/BARS and PI4KIIIb, while CtBP1/BARS does not bind directly with PI4KIIIb. Conclusions: Thus, 14-3-3c appears to function as an adaptor protein by binding to PI4KIIIb and CtBP1/BARS, thus bringing together PI4KIIIb, which has a role in formation/extrusion of post-Golgi carriers, and CtBP1/ BARS, which controls their fission. This study provides us with the first definition of components of the CtBP1/BARS-driven fissioning machinery in the secretory pathway.


467

Abstracts


YSF-117 The role of laforin and malin in Lafora disease and the regulation of glycogen metabolism in neurons J. Valles Ortega, D. Vilchez, S. Ros, D. Cifuentes, M. Garcia-Rocha and J. Guinovart IRB-Institute for Research in Biomedicine, Barcelona, SPAIN Introduction: Lafora progressive myoclonus epilepsy (LD, MIM 254780) is an autosomal recessive neurodegenerative disorder characterized by the presence of polyglucosan intracellular inclusion bodies (Lafora bodies), structurally defined as poorly branched glycogen. Mutations have been identified in two genes, EPM2A (6q24) and EPM2B (6p22.3). EPM2A encodes laforin, a dual-specificity phosphatase with a functional carbohydrate-binding domain. EPM2B encodes malin, an E3 ubiquitin ligase that interacts with laforin and promotes its degradation. The physiological roles of laforin and malin are unknown and the cellular processes altered by mutations in these proteins that give rise to the devastating disorder of LD remain to be elucidated. Results: We have published recently that laforin and malin control the glycogen synthase (GS) activity impeding glycogen accumulation. We show a new mechanism of regulation of glycogen synthesis in which malin stimulates the degradation of GS and PTG (a PP1 regulatory subunit that leads to the activation of GS) by the ubiquitin-proteasome system in a laforindependent manner (Fig. 1). Furthermore, we report that the accumulation of glycogen induced by PTG overexpression causes apoptosis in neurons. Fig 1. New mechanism of regulation of glycogen synthesis

Perspectives: However, the phosphatase activity of laforin is not required for this process. It has been reported a laforin phosphatase activity against phosphorylated polyglucosans. Nevertheless, the involvement of this activity on LD still remains unclear. We are investigating the putative role of the laforin dual-specific phosphatase domain in the development of Lafora Disease by looking for new substrates. In addition, the role of the laforin-malin mechanism in the regulation of the glycogen metabolism of neurons and the other tissues and its possible link to other neurological alterations or diseases are being studied.

YSF-118 A critical role for the karyopherin proteins, Crm1 and Kpnb1, in cancer cell survival and proliferation P. J. Van Der Watt1, C. P. Maske2, D. T. Hendricks1, M. I. Parker3, L. Denny4, D. Govender2, M. J. Birrer5 and V. D. Leaner1 1 Division of Medical Biochemistry, Faculty of Health Sciences, University of Cape Town, Cape Town, SOUTH AFRICA, 2Division of Anatomical Pathology, Faculty of Health Sciences, University of Cape Town, Cape Town, SOUTH AFRICA, 3International Centre for Genetic Engineering and Biotechnology, Faculty of Health Sciences, University of Cape Town, Cape Town, SOUTH AFRICA, 4Department of Obstetrics and Gynaecology, Faculty of Health Sciences, University of Cape Town, Cape Town, SOUTH AFRICA, 5 Department of Cell and Cancer Biology, National Cancer Institute, National Institute of Health, Bethesda, MD, USA Introduction: The Karyopherin superfamily comprises nuclear transport proteins that are involved in nucleocytoplasmic protein trafficking and are thus critical for correct protein localisation. Misregulation of the nuclear transport machinery is likely to contribute to cancer development. In this study, we examined expression of the nuclear transport proteins, Crm1, Karyopherin b1 (Kpnb1), and Karyopherin a2 (Kpna2), in cervical cancer and investigated their functional relevance to the disease. Methods: Real-time RT-PCR and immunofluorescence were used to analyse expression of these genes in normal and cervical cancer tissues, and Western Blot analysis to detect their expression in cultured cell lines. siRNA technologies were used for inhibition of Crm1, Kpnb1 and Kpna2, and assays performed to analyse biological effects of inhibition.

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Results: Crm1, Kpnb1 and Kpna2 were significantly overexpressed in cervical cancer tissue compared to normal cervical tissue, and in cervical cancer cell lines compared to normal cervical epithelial cells. Oesophageal cancer cell lines similarly showed increased expression compared to normal oesophageal cells, thus the upregulation is not specific to cervical cancer. Inhibition of Crm1 and Kpnb1 in cervical, oesophageal and breast cancer cells resulted in significantly reduced cell proliferation, while inhibition of Kpna2 had no effect. Cell cycle analysis indicated that the reduced proliferation associated with an increase in the subG1 population, and Caspase-3/7 assays indicated that apoptosis was occurring. Crm1 siRNA-induced cell death was accompanied by nuclear accumulation of p53. Conclusions: Our data suggests that nuclear transport proteins may serve as markers of cancer, and that their targeted disruption may be of benefit in cancer therapy.

YSF-119 The antifungal activity of a plant defensin requires membrane permeabilization and internalization N. Van Der Weerden1, F. Lay1, R. Hancock2 and M. Anderson1 1 Department of Biochemistry, La Trobe University, Melbourne, AUSTRALIA, 2Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC, CANADA Plant defensins are a family of small (45-54 amino acids), basic, cysteine-rich peptides. Members of this family have been characterized in several plant species and in various tissues, where they are believed to constitute a vital component in the plant’s innate immune system. Many plant defensins show inhibitory activity against various fungal pathogens although the molecular basis of this activity remains unknown. Permeabilization of membranes is a common activity for many antimicrobial peptides and while this was originally thought to be their sole mechanism of action, recent findings indicate that intracellular targets may also be involved. We are interested in a defensin (NaD1) that is abundantly produced in the flowers of the ornamental tobacco, Nicotiana alata. We have previously shown that NaD1 is inhibitory towards a number of agronomically important fungal pathogens. We are using fluorescence techniques and electron microscopy to examine the interaction of NaD1 with fungal hyphae. NaD1 appears to bind to the cell wall before permeabilizing the hyphal membrane. The kinetics of permeabilization differ from those seen for typical membrane-active peptides and the peptide is able to enter fungal hyphae. Permeabilization may represent a way for NaD1 to cross the plasma membrane and interact with intracellular targets rather than the sole mechanism of action.

YSF-120 Structure-based distinction in antiviral drug activation: 3-phosphoglycerate kinase (PGK) and nucleoside diphosphate kinase (NDPK) A. Varga1, J. Szabo´1, C. Lionne2, S. Arold3, B. Roy4, P. Za´vodszky1, T. Barman2 and M. Vas1 1 Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, HUNGARY, 2UMR5236 CNRS, Institute de Biologie, University of Montpellier I, Montellier, FRANCE, 3UMR5048 CNRS, Centre de Biochimie Structurale, University of Montpellier I, Monpellier, FRANCE, 4 UMR5247 CNRS, Institute de Biologie, University of Montpellier I, Montpellier, FRANCE Introduction: Nucleoside analogues are privileged leads in antiviral drug design. In the last years L-nucleoside analogues, mirror images of the natural D-nucleosides emerged as a new class of antiviral agents. They are converted intracellularly to their corresponding triphosphate. This active form can be built into the viral DNA and thereby prevent chain elongation as well as protein synthesis. The enzymes responsible for the formation of nucleoside-triphosphate in the cell remains largely unexplored. This phosphorylation step involve kinases of low specificity and/or enantioselectivity e.g. nucleoside diphosphate kinase (NDPK) and 3-posphoglycerate kinase (PGK). Methods: Here the phosphorylation reaction have been tested by steadystate experiments, ligand binding and biophysical (DSC) studies. The results have been explained in the light of the known high resolution X-ray structural data. Results and conclusions: Naturally existing nucleotides e.g. ADP, GDP, CDP and UDP and the chemically syntetised L-ADP and L-CDP were investigated as model compounds in order to understand the structural basis of phosphorylation process of these nucleotides. We have found that D-purine nucleotides can be efficiently phosphorylated by both PGK and NDPK. D-pyrimidine analogues are better substrates for NDPK, but the presence of the ribose 3¢-OH group is essential for its activity, independently from the base type. L-nucleotides are activated faster by PGK and the presence of the ribose OH-groups are not required for the enzyme activity. These results show that NDPK and PGK have complementary structure-based distinction in the cell. Testing of these rules for the drug molecules is in progress.



Abstracts

YSF-121 The RNA component of ribonuclease P from Dictyostelium discoideum

YSF-123 Cloning and sequence analysis of the human CA9 promoter

A. Vourekas1, V. Stamatopoulou1, C. Stathopoulos2 and D. Drainas1 1 Department of Biochemistry, School of Medicine, University of Patras, Patras, GREECE, 2Department of Biochemistry and Biotechnology, University of Thessaly, Larissa, GREECE

H. Yildirim and F. Ko¨çkar Balikesir University Faculty of Science and Literature, Department of Biology, Balikesir, TURKEY

Introduction: Ribonuclease P is an essential enzyme that endonucleolytically cleaves all precursor tRNA transcripts to produce their mature 5¢ ends. The biological importance of RNase P is underscored by its presence in all organisms. All forms contain a similar in size RNA subunit which is absolutely required for catalysis. However, the size and number of protein subunits of the holoenzyme varies significantly, from one small subunit in bacteria to ten subunits in human RNase P. The putative gene of the RNA subunit of D. discoideum RNase P was recently identified through phylogenetic comparison (1), but provided no clues for its possible catalytic attributes reported by our lab previously (2). Methods: D. discoideum RNase P RNA sequence identified by Marquez et al. (1) was cloned by PCR. The gene sequence was certified by primer extension and 3¢ RACE of RNA extracted from active RNase P fractions. Northern blot analysis was used for size estimation of various fragments of RNA subunit present in RNase P preparations. Active RNase P fractions were treated with micrococcal nuclease for the destruction of the native RNA subunit. Various in vitro transcripts of the RNA gene were assayed for ribozyme activity and used in reconstitution assays for the rescue of the catalytic activity of the MN treated enzymatic complex. Results and discussion: D. discoideum RNase P has distinctive characteristics compared to other eukaryotic RNase P enzymes. It harbors fewer protein subunits, which are larger than their homologues in other eukaryotes. Our biochemical analysis certified the RNase P RNA gene. Two transcripts of this gene are present in active fractions, the full length and a smaller transcript lacking 70 nts at the 5¢ end. Both fragments were cloned and were used in reconstitution assays where they substituted the native RNA subunits. Both fragments were capable of rescuing RNase P enzymatic activity, which was previously eliminated by nuclease treatment. These results provide novel insight on RNase P RNA structure and function. References: 1. Stathopoulos C, Tekos A, Zarkadis IK and Drainas D. Eur. J. Biochem. 2001; 268: 2134–2140. 2. Marquez SM, Harris JK, Kelley ST, Brown JW, Dawson SC, Roberts EC and Pace NR. RNA 2005; 11: 739–751.

YSF-122 Putative posttranslational modifications of Paramecium RAB7 isotypes may result in distinct electrophoretic migration pattern E. Wypych, M. Osinska, J. Wiejak and E. Wyroba Nencki Institute of Experimental Biology, Warsaw, POLAND Introduction Traffic along endocytic pathway is regulated by the family of Rab proteins directing internalized cargo to different destinations. Two paralogous genes encoding Rab7 were cloned by us in evolutionary ancient Paramecium. The overall predicted protein sequences of Paramecium Rab7 isotypes exhibit higher similarity to human counterpart than to Rab7 deriving from parasitic protozoa. The deduced sequences of 206 amino acids are 97.6% identical. Interestingly, specific antipeptide antibodies detected single cross-reacting polypeptides of ~21 kDa for Rab7a and ~23 kDa for Rab7b. Methods Mass spectrometry and detection of putative phosphorylation/glycosylation modifications were performed to clarify this pattern. Cell homogenate prepared with phosphatase inhibitors was subjected to SDS-PAGE: one half of the gel was silver-stained whereas the identical second half was electrotransferred onto nitrocellulose and immunoanalyzed with specific antibodies. Results Mass spectral data revealed that silver-stained protein bands corresponding to those observed in Western blot contained the peptides matching both the Rab7 isotypes. Phosphorylation pattern was analyzed with Pro-Q Diamond that detects all phosphorylated residues and with antibodies specific for phospho-serine, -threonine and –tyrosine, respectively. In both cases the subsequent immunodetection of the same blot was performed. No significant difference was observed in phosphorylation pattern for both isotypes. Pro-Q Emerald staining revealed the glycosylated band corresponding to Rab7b isotype. Conclusions These results indicate that distinct pattern of Paramecium Rab7 isotypes migration in SDS-PAGE could be the result of posttranslational glycosylation of Rab7b. Acknowledgments: This work was supported by grant 2 P04A 020 29 of the Ministry of Science and Higher Education.

Carbonic anhydrases (CAs) are a group of zinc-containing metalloenzymes that catalyse the reversible reaction from H2O and CO2 to HCO3- ions. There are fifteen mammalian CA isozymes with different tissue distributions and locations. Of the fifteen isozymes, CA IX has been linked to the tumors and neoplastic invasion. CA IX is ectopically expressed at relatively high levels and with a high prevalence in some tumor tissues whose normal counterparts do not contain this protein, e.g. carcinomas of the cervix uteri, esophagus, kidney, lung and breast. On the other hand, tumors originating from tissues with high natural CA IX expression, such as the stomach and gallbladder, often lose some or all of their CA IX upon conversion to carcinomas. The aim of the study is to cloning, sequence and functional analysis of human CA9 promoter. Intially, 1284 bp of human CA9 promoter was amplified using PCR-based strategy using the human blood genomic DNA as a template and cloned into the luciferace vector, pGL2 basic. Then, the sequence analysis of human CA9 promoter was performed. Comparison of the sequence of human CA9 promoter to other promoter of mammalian homologues have also been carried out. The putative transcription factor binding sites was found by using TRANSFAC database. For functional analysis of hCA9 promoter, the full length promoter in the pGL2 basic vector was transfected into Hep3B cell lines by Ca phosphate transfection method. The basal promoter activity of human CA9 promoter was determined as time and concentration dependent manner.

YSF-124 Intermediate filament misregulation, GLAST downregulation and MAPK activation in developing brain of rats with congenital hypothyroidism A. Zamoner, L. Heimfarth and R. Pessoa-Pureur Departamento de Bioquimica, Instituto de Ciencias Basicas da Saude, Porto Alegre, BRAZIL Developmental thyroid hormone (TH) deficiency leads to mental retardation and neurological deficits in humans. In this study congenital hypothyroidism was induced in rats by adding 0.05 % 6-propyl-2-thiouracil in the drinking water during gestation and suckling period. This treatment induced hyperphosphorylation of neuronal intermediate filament (IF) proteins, neurofilaments of high, medium and low molecular weight (NF-H, NF-M and NF-L, respectively) without altering the phosphorylation level of astrocyte IF proteins, glial fibrillary acidic protein (GFAP) and vimentin in cerebral cortex of rats. Furthermore, the immunocontent of GFAP and NF subunits was down-regulated, while vimentin was unaltered in tissue homogenate of hypothyroid animals. Nevertheless, the immunocontent of the Triton-insoluble IF proteins showed that NF subunits and GFAP were also decreased, while vimentin was unaltered, suggesting that hyperphosphorylation partially interfered with NF polymerization/aggregation ability. Moreover, we verified the immunocontent of astrocyte glutamate/aspartate transporter (GLAST), as well as activation of mitogen-activated protein kinases (MAPK) in hypothyroid rats. Results showed that hypothyroidism is associated with decreased GLAST immunocontent. Otherwise, we demonstrated increased extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation without altering Jun N-terminal kinase (JNK) and p38MAPK phosphorylation. However, total JNK levels were down-regulated. Taken together, these results suggest that the thyroid status could modulate the integrity of neuronal cytoskeleton acting on the endogenous NF-associated phosphorylating system and that such effect could be related to glutamate-induced excitotoxicity, as well as ERK1/2 and JNK modulation. These events could be somehow related to the neurological dysfunction described in hypothyroidism.


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YSF-125 The tumor-targeted efficiency of shRNA vector harboring hTERT/U6 promoter with silicon nanoparticles P. Zhang1, Y. Chen2, X. Jiang2, Z. Tu2 and L. Zou1 Children’s Hospital, Chongqing Medical University, Chongqing, CHINA, 2 Key Laboratory of Laboratory Medical Diagnostics of Ministry of Education, Chongqing Medical University, Chongqing, CHINA 1

Introduction: RNA interference (RNAi) has been widely used in the research of functional genomics, gene therapy and signal transduction. However, there are limitations for the application in the clinical research because of siRNA’s interference uncertainty, the safety and the low transfection efficiency. hTERT promoter was applied to overcome the above-mentioned shortages in targeted tumor therapy. Methods: We firstly constructed shRNA-EGFP-U6 and shRNA-EGFPhTERT/U6 expression vector respectively to suppress the GFP expression in telomerase negative and positive cells. Then we further constructed the shRNA-hTERT-U6 and shRNA-hTERT-hTERT/U6 to affect hTERT expression and the malignant phenotypes in telomerase negative and positive cells.

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Results: The mosaic hTERT/U6 promoter was identified by sequencing. shRNA-EGFP-U6, shRNA-EGFP-hTERT/U6, shRNA-hTERT-U6 and shRNA-hTERT-hTERT/U6 were constructed successfully. Telomerase negative human fibroblast HELF cells and the telomerase positive human hepartocarcinoma SMMC-7721 cells stably expressed EGFP are screened by G418. Further results showed that GFP expression was inhibited in shRNA-EGFP-hTERT/U6 cells other than shRNA-EGFP-U6 in SMMC7721 cells, which was measured by fluorescence microscopy, real-time PCR and Western blot assays. hTERT expression and tumor malignancies were suppressed more in SMMC-7721 cells expressed shRNA-hTERT-hTERT/ U6 than cells expressed shRNA-hTERT-U6. Compared to the traditional transfection medium cation liposome, silicon nanoparticles were better transfection efficient for these vectors. Similar results were obtained from the mice liver cancer model. Conclusions: shRNA-hTERT-hTERT/U6 could inhibit hTERT expression and tumor malignant phenotypes, which was stronger tumor-oriented capacity in telomerase positive cells. In addition, silicon nanoparticles could help the inhibition as the transfection medium. This will benefit for the tumor target therapy.
