Báo cáo khoa học: POSTER PRESENTATIONS P1 Functional Genomics, Proteomics and Bioinformatics docx

POSTER PRESENTATIONS P1 Functional Genomics, Proteomics and Bioinformatics P1–1 The influence of the HERV-K LTR on KIAA1245 subfamily gene expression N. Abrarova, E. Stoukacheva, T. Vinogradova and E. Sverdlov Laboratory of structure and function of human genes, Institute of Bioorganic Chemistry, Moscow, RUSSIA Long terminal repeats (LTR) of endogenous retroviruses com- prise about 8% of human genome. Typical LTR contains a set of regulatory elements: promoters, enhancers, polyadenilation sites, which can take part in neighbouring genes expression regu- lation. Earlier, we have described a subfamily of closely related genes highly similar to the KIAA1245 mRNA, part of which contains a HERV-K LTR in their structure, whereas the other lacks it. Using this subfamily as a model for studying regulation of gene transcription, we showed that LTR serves as an alterna- tive promoter and possess high enhancer activity. Genes that contain LTR differ from genes lacking it in cell type specificity of transcription. We generated a series of successive 5¢- and 3¢-dele- tion mutants with a 200 bp increment, and also two middle vari- ants, 200 and 400 bp in length (Mid200 and Mid400, respectively). To determine promoter activity of LTR and its fragments they were cloned into pGL3-BV vector, containing luciferase reporter gene. The full-sized LTR demonstrated high promoter activity in Tera1 and NT2/D1 cell lines. To determine the enhancer activity LTR and subfragments were cloned in pGL3-PV vector, which has SV40 promoter in its structure besides luciferase gene. Transient transfections demonstrated that Mid200 fragment of the LTR exhibits high cell type specific enhancer activity. In Tera1 cell line it was comparable to the activity of universal SV40 enhancer. This fact allows to suggest that enhancer is localized in this region of the LTR. The analysis of transcription of KIAA1245 subfamily genes have shown that LTR-lacking gene Al592309 is not transcribed in Tera1 and NT2/D1 cell lines, whereas LTR-containing genes are transcribed in these cell lines. Thus, we may suggest that insertion of the LTR in second intron of KIAA1245 genes may lead to change in cell type specificity of their transcription. This fact allows to sug- gest the role of the LTR in evolution of KIAA1245 subfamily genes. P1–2 Study of DNA interactions with cerium (III), lanthanum (III) and gadolinium (III) ions by using of Raman spectroscopy V. Kohoutkova 1 , P. Babula 1 , R. Opatrilova 1 , O. Vrana 2 , V. Adam 3 , J. Zehnalek 3 and R. Kizek 3 1 Department of Natural Drugs, University of Veterinary and Pharmaceutical Sciences, Brno, CZECH REPUBLIC, 2 Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, CZECH REPUBLIC, 3 Department of Chemistry and Biochemis- try, Mendel University of Agriculture and Forestry, Brno, CZECH REPUBLIC The biological properties of the lanthanides, based on their simi- larity to calcium, have stimulated research into their therapeutic application. Up-to-date we have been successfully using at least two pharmaceuticals cerium nitrate as a topical cream with silver sulfadiazene for the treatment of burn wounds and lanthanum carbonate as a phosphate binder for the treatment of hyperphos- phatemia. Lanthanides3+ compounds have also been investi- gated for their anti-cancer potential. Clinical reports suggested that several compounds such as cerium (III) iodide posed cyto- toxic effect, however the biochemical mechanism of their action is still unclear (1–3). Therefore, we aim our attention on study of interactions DNA with inorganic salts of cerium (III), lanthanum (III) and gadolinium (III). Raman spectroscopy was employed to characterize the perturbations to DNA conformation induced in DNA by three inorganic salts of the above mentioned lantha- nides (2). All studied lanthanides coordinated to N7 of two neighbouring guanine bases, as this nitrogen does not form H bonds with other bases, in the same or in opposite DNA strands. The results of the present work demonstrate that Raman spec- troscopy represents a suitable tool to provide insights into struc- tural factors involved in the mechanisms underlying antitumor effects of drugs. Acknowledgement: This work was supported by GA CR 522/ 07/0692. References: 1. Fricker SP. Chem. Soc. Rev. 2006; 35(6): 524–533. 2. Vrana O et al., J. Struct. Biol. 2007; 159(1): 1–8. 3. Babula P et al., Curr. Pharm. Anal. 2009; 5(1): 47–68. P1–3 Real-time polymerase chain reaction with electrochemical detection for detection pandemic viruses V. Adam 1 , D. Huska 1 , S. Krizkova 1 , J. Hubalek 2 and R. Kizek 1 1 Department of Chemistry and Biochemistry, Mendel University of Agriculture and Forestry in Brno, Brno, CZECH REPUBLIC, 2 Department of Microelectronics, Brno University of Technology, Brno, CZECH REPUBLIC Viral infections pose a threat for man kind. Diagnostic systems focus on the direct cultural proof of identity. Almost all systems use polymerase chain reaction. Real-time polymerase chain reac- tion is based on the polymerase chain reaction and routinely used to amplify and simultaneously quantify a targeted DNA molecule. Fluorescent dyes intercalating with double-stranded DNA and modified DNA oligonucleotide probes fluorescing when hybridized with a complementary DNA are used for real time quantification of the amplicons. The process of DNA quantification has demands on robust instrument and on high cost chemicals. Electrochemical detection is low cost and sensitive alternative method to detect nucleic acids (1, 2). The main aim of this work is to propose novel electrochemical detector for monitoring PCR. Miniaturized poten- tiostat with nanotubes-based screen-printed electrodes was used for detection of specific sequence of severe acute respiratory syn- drome, avian influenza virus and Ebola virus amplified by poly- merase chain reaction. The amplicons were obtaining after 2, 4, 6, 8, 10, 15, 20, 25, 30 and 35 cycles, whereas the amount of amplified DNA was between 10 and 100 pg. The amplicons obtained even after two cycles were detectable by using the electrochemical instrument. Moreover we compared the results with agarose gel electrophoresis. The lowest detectable amount of DNA was obtained after 15 PCR cycles. Acknowledgement: This work was supported by GA AV KAN208130801. Poster Presentations Abstracts FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies 95 References: 1. Palecek E et al. Anal. Chim. Acta. 2002; 496(1): 73–83. 2. Huska D et al. Electrophoresis 2008; 29(24): 4964–4971. P1–4 Spatial distribution of heavy metals in healthy and melanoma animal tissues V. Adam 1 , O. Zitka 1 , D. Huska 1 , S. Krizkova 1 , J. Strnadel 2 , V. Horak 2 , T. Vaculovic 3 , K. Novotny 3 , V. Kanicky 3 , J. Kaiser 4 and R. Kizek 1 1 Department of Chemistry and Biochemistry, Mendel University of Agriculture and Forestry in Brno, Brno, CZECH REPUBLIC, 2 Academy of Sciences of the Czech Republic, Insti- tute of Animal Physiology and Genetics, Libechov, CZECH REPUBLIC, 3 Department of Chemistry, Masaryk University, Brno, CZECH REPUBLIC, 4 Institute of Physical Engineering, Brno University of Technology, Brno, CZECH REPUBLIC Levels of some of heavy metals are monitored and their altera- tions can be used as diagnostic markers. Nevertheless, spatial dis- tribution of the essential metals in animal tissues is still not clear. The aim of this work is detection of copper and zinc in healthy and tumour tissues of miniature pigs by using of the laser induced breakdown spectroscopy (LIBS). Concentration of essen- tial-heavy-metal-transporting protein metallothionein (MT) was determined by the Brdicka reaction. Tissue cryosections (thick- ness 40 lm) were obtained from the MeLiM strain of miniature pigs with hereditary melanoma, particularly from healthy skin, cutaneous nodular melanomas and metastases in the liver, spleen and lymph nodes. Using LIBS we measured maps of spatial dis- tribution of the essential heavy metals in cryosections and found that the maps of healthy and tumour cryosection markedly dif- fered. The highest content of MT was determined in the tumours localised on the back of animals and was nearly 500 lgofMT per gram of tissue. The MT levels determined in metastases in spleen, lungs and liver were within the range from 40 to 160 lg/g of the tissue, the average level was 110 ± 40 lg/g of the tissue. Acknowledgements: This work was supported by the GA AS CR (IAA401990701) and by the Ministry of Education, Youth and Sport CR (2B08063) and Liga proti rakovine Praha (2009). References: 1. Krizkova S et al. Sensors 2008; 8(5): 3106–3122. 2. Kaiser J et al. Spectrochim. Acta Part B 2009; 64(1): 67–73. P1–5 Chromatin accessibility of MHCII locus and enhanceosome stability determine transcriptional competence through mitosis P. Arampatzi, M. Gialitakis, T. Makatounakis and J. Papamatheakis Department of Biology, Institute of Molecular Biology and Biotechnology, Heraklion, GREECE During mitosis, transcription factors and RNA polymerase II are displaced from mitotic chromatin, which is condensed and tran- scription is interrupted. However specific gene regions are sug- gested to be bookmarked by TBP and histone modifications for rapid reactivation after cell division. The MHCII locus is trans- criptionally activated by the assembly of an enhanceosome (MCE) which recruits the master regulator, CIITA. We found that endogenous or GFP-fusions subunits of the MCE remain associated and dynamically exchanged on mitotic chromosomes. Chromatin immunoprecipitation assays show significant occu- pancy by the MCE, CIITA and general transcriptional machinery at various phases of the cell cycle including mitosis in B lym- phoid cells. Mitotic maintenance of the MCE does not depend on CIITA expression and correlates with a chromatin state that is fully or partly maintained in mitotic lymphoid or non lym- phoid cells, respectively. Monitoring of newly synthesized RNA shows reduced transcriptional activity of the DRa gene in mito- sis. Constitutive expression of exogenous CIITA is sufficient to fully support mitotic expression in lymphoid but not in epithelial cells. Using an inducible system we show that CIITA synthesised in cells arrested in mitosis rescues MHCII transcription to asyn- chronous levels in lymphoid cells demonstrating full expression competence of DRa gene. These results show there are two pat- terns of mitotic deficiency of gene expression: in B lymphoid cells low but significant MHCII transcription can be fully rescued, as opposed to epithelial cells that show minimal mitotic transcrip- tion may due to enhanced chromatin condensation. P1–6 Associations between PTGS1 gene mutations and aspirin resistance in patients with congenital heart diseases I. Coskun 1 , Y. Atay 1 , A. Berdeli 2 , M. Mecidov 1 , A. Atay 3 , M. F. Ayik 1 , T. Yagdi 1 and E. A. Alayunt 1 1 Cardiovascular surgery, Ege University, izmir, TURKEY, 2 Pediatric Molecular Medicine Laboratory, Ege University, izmir, TURKEY, 3 Department of Clinical Biochemistry, Ataturk Training Hospital, izmir, TURKEY Introduction: This study was aimed to evaluate that frequency and existance of PTGS1 gene mutation which coded cyclooxy- genase-1 (COX-1) enzyme and associations with aspirin resistance in patients with congenital heart diseases (CHD) in Turkish pop- ulation. Methods: It was included 90 patients with CHD treated by aspirin after their operations. 24 healthy participants were enrolled as control group. Direct DNA coding sequence method was used for analysing PTGS1 gene mutation. PCR amplification was made by using specific oligo primers (MWG) and AmpliTaq Gold PCR kits for four exons. After capillary gel electrophoresis, nucleotide sequences of PTGS1 gene was obtained and compared with cDNA Genbank (NM_000962) and protein (NP_000953) sequences to determine single- nucleotide polymorphisms and amino acid mutations. Results: W8R missense aminoacid mutation was the most fre- quent mutation (83.3%) in exon-2. P188P sinonym aminoacid polimorphism (34%) and D189E amino acid mutation (20%) were other mutations detected in exon-6 in patient group. In con- trol group, W8R missense aminoacid mutation (83.3%) in exon- 2, Q41Q sinonim aminoacid mutation (8.3%) in exon-3, D189E missense aminoacid mutation (12.5%) in exon-6 were found. Nine new missence amino acid mutations (L13P; L15V; P18R; C58T; I45M; R53S; D189E; F200L; R239L) and eight sinonym amino acid mutations (L23L; P39P; R53R; G44G; G62G; K185K; P18P) were detected. It was observed the association between aspirin resistance and missence amino acid mutations in patients. Conclusion: Genetic variations in PTGS-1 may affect aspirin resistance in CHD. Detecting these variations may help to predict drug responce and to select optimal therapy and dosage regi- mens. This is the first study in our country dealing with PTGS1 gene mutation and polymorphism which coded COX-1 enzyme and association with aspirin resistance in CHD. Abstracts Poster Presentations 96 FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies P1–7 Analysis of the ID1 gene expression level and promoter methylation in patients with benign and malignant thyroid nodules A. Banaszewska 1 , M. Piechota 1 , K. Drobnik 2 , K. Ziemnicka 2 , K. Ziemnicki 1 and R. Plewa 1 1 Department of Animal Physiology and Development, Adam Mickiewicz University, Poznan, POLAND, 2 Department of Endocrinology Metabolism and Internal Disease, University of Medical Science, Poznan, POLAND Inhibitor of DNA binding (ID1) has been proposed to serve a dual function as a regulator of cell proliferation and differentia- tion in both normal and pathological conditions associated with cancer development. ID is a transcription family of factors lack- ing basic DNA binding domain, which form inactive heterodi- mers with basic helix-loop-helix transcription factors and inhibits expression of tissue specific genes and cells differentiation. Recent research shows, that ID1 mRNA level correlates with malignant thyroid carcinoma phenotype. Thus, dual role of ID1 gene in benign and malignant thyroid nodules was studied. ID1 mRNA level of 33 patients was investigated in human papillary thyroid carcinoma (PTC), non-toxic nodular goiter, toxic nodular goiter and nodular goiter with Hashimoto disease using Real time PCR and Delta Delta Ct method to estimate relative amount of ID1 mRNA. Furthermore, genomic DNA was extracted and ID1 pro- moter methylation was examined in the majority of patients spec- imens. Six of seven PTC patients samples were found to have barely detectable ID1 mRNA level. Expression of ID1 was higher in non-toxic nodular goiter and in nodular goiter with Hashimoto disease in comparison to PTC and toxic nodular goiter (P < 0.05). No hypermethyleted ID1 gene promoter was found in the majority of analyzed cases. In our study no significant association was observed between ID1 gene expression and malignant thyroid nodules. This findings indicate that ID1 could not be a marker of aggressive phenotype in PTC. P1–8 TNF alpha -308 polymorphism and matrix metalloproteinase-9 level in children with juvenile idiopathic arthritis threated with etanercept J. Basic 1 , D. Pavlovic 1 , T. Jevtovic-Stoimenov 1 , J. Vojinovic 2 , M. Marinkovic 1 and A. Veljkovic 1 1 Medical Faculty, Institute of Biochemistry, Nis, SERBIA, 2 Clinic of Pediatrics, Clinical Centre Nis, Nis, SERBIA Juvenile idiopathic arthritis (JIA) is the most common form of persistent arthritis in children. Tumor necrosis factor-a (TNF- a) is likely to have a primary role in the pathogenesis of JIA, including matrix metalloproteinase-9 (MMP-9) production. The aim of this study was to investigate G-A -308 single nucleotide polymorphism (SNP) in the promoter region of TNF a gene, as well as to evaluate the eventual influence of etanercept (TNF receptor II-Fc fusion protein) on MMP-9 level in children with JIA. A total of 42 patients with JIA were screened for the poly- morphism using the PCR-RFLP method. Plasma MMP-9 level was determined before starting etanercept therapy and 12 months after, using Elisa kit. The genotype -308GG was present in 13 (31%) patients and the -308AA genotype was present in two (4.7%) patients. Twenty-seven (64.3%) patients were heterozy- gous. MMP-9 level in patients with the genotype -308GG was significantly decreased after 1 year of treatment with etanercept compared to the value before (P = 0.01). On the other hand, there were no significant decrease of MMP-9 levels after treat- ment in patients with the genotype -308GA and -308 AA (P = 0.147; P = 0.126). We discussed decrease of MMP-9 level in children with -308GG genotype as a possible consequence of better response to etanercept treatment compared to A allele car- riers. P1–9 Screening of Three Exons of the RET Proto- oncogene in Turkish Patients with Papillary Thyroid Carcinoma N. S. Bayramci 1 , L. Acik 2 , L. Y. Koc 3 , G. Vural 4 , M. Kilic 5 , M. Tez 5 , M. Koc 5 and N. Ercakmak 4 1 Faculty of Education, Department of Science Education, Bayburt University, Bayburt, TURKEY, 2 Faculty of Arts and Science, Department of Biology, Gazi University, Ankara, TURKEY, 3 Faculty of Science, Department of Biology, Ankara University, Ankara, TURKEY, 4 Nuclear Medicine Department, Dr. Abdurrah- man Yurtaslan Ankara Oncology Education and Research Hospi- tal, Ankara, TURKEY, 5 5th Surgery, Ankara Numune Education and Research Hospital, Ankara, TURKEY Different types of tumors can develop in thyroid gland cells. Thy- roid carcinoma represents the most frequent form of cancer of the thyroid glands, with prevalence in Europe of about 3 per 100 000. PTC is the most frequent histotype of differentiated thy- roid cancer. The RET proto-oncogene, localized to chromosome subband 10q11.2, comprises 21 exons, which encodes the protein RET, a receptor tyrosine kinase (RTK) expressed in derivatives and tumors of neural crest origin. The RET proto-oncogene is involved in the genesis of papillary thyroid carcinoma. To deter- mine the mutation frequency of RET proto-oncogene in Turkish papillary thyroid carcinoma patients, we conducted this retro- spective study. Eighty-two patients with PTC were screened for mutations in exon 10, 11 and 13 using PCR and sequence analy- sis. A negative control was included in each amplification analy- sis. Twelve of the patients exhibited mutation, 24 of them showed SNP. Twelve different mutations located in exon 11 and three different mutations in exon 13. 12.19% of the patients had mutation in exon 11. 3.65% of the patients had mutation in exon 13. Twenty-four of the patients exhibited SNP (29.26%). We found the SNP at codons 631 (1.21%), 769 (1.21%), 691 (3.65%) in exon 11 and at codons 769 (24.39%) and 763 (1.21%) in exon 13. The most frequent SNP that is found in patients with PTC (24.3%) involve codon 769 (exon 13) in the tyrosine kinase domain. The common mutation found in patients involves codon 630 (four patients) in the extracellular domain of RET encoded by exon 11, less frequently, mutation occurred codons 765, and 770 and 795 (exon 13) in the tyrosine kinase domain. The muta- tions were found in 41.46% of all thyroid carcinoma patients. The numbers of mutations were higher in exon 11 than exon 13. In our series, we detected that four patients in 82 of all PTC patients presented Cys630Ser (TGC fi AGC, 5.9%) is the most common mutation at codon 630 in exon 11. In addition to that, Cys630Ser mutation, is associated with medullary thyroid carci- noma, however in our genetic testing it was diagnosed on four patients with papillary thyroid carcinoma. Therefore, the rela- tionship between Cys630Ser variation and development of PTC should be further explored. All of the investigated polymor- phisms are silent mutations, leave out codon 691 polymorphism. We observed the codon 691 polymorphism in three patients (GGT/AGT) of all 82 PTC patients. In the 13rd exon region of the RET proto-oncogene, we found that these possible mutations, which expressed as an alteration in the form of TCC (serine) replacement with TGC (sistein) at codon 765, CGA (arginine) Poster Presentations Abstracts FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies 97 replacement with CAA (glutamic acid) at codon 770. The other mutation in exon 13 was at codon 770, Arg replacement with Glu. The large number of SNP at codon 769 in exon 13 with PTC patients was interesting and it should be further explored. P1–10 Metabolomic profiling of serum from obese adult females infected by Chlamydophila pneumoniae G. Bazylak 1 , C. Ludwig 2 , E. Fagadar-Cosma 3 and U. L. Gunther 2 1 Department of Pharmaco-Bromatology & Molecular Nutrition, Faculty of Pharmacy Collegium Medicum Nicolaus Copernicus University, Bydgoszcz, POLAND, 2 HWB NMR, CR UK Institute for Cancer Studies, University of Birmingham, Edbagston Birming- ham, UK, 3 Department of Organic Chemistry, Institute of Chemis- try Timisoara of Romanian Academy, Timisoara, ROMANIA Chlamydophila (Chlamydia) pneumoniae is a very common path- ogen causing an acute respiratory diseases and multidirectional inflammation progress during a past and persistent infections in humans of all age. Recently, significant associations between C. pneumoniane infection and obesity indices have been observed on results of epidemiological studies in various populations from several north Europe countries. However, until now the number of NMR- or MS-based metabolomic and proteomic studies on specific biomarkers related with variety of nutrition disorders, including obese subjects, is still scarce. Thus, the goal of our studies was to find by use of the 1D- and 2D-1H-NMR proce- dures the range of characteristic, specific and unique low weight metabolites (proteins) in serum samples from wide group of over- weight and obese adult females with increased hormonal activity of adipose tissue, and which indicating past (or persistent) infec- tion by C. pneumoniae as diagnosed by introductory serological immunosorbent assay. Proposed here 1H-NMR methodology should be effective for profiling changes in the serum metabolo- me (proteome) of subjects with obesity and its associated implica- tions with bacterial infection and disorders of immunological status, thus enabling future early diagnosis, risk assessment and advanced treatment of infection related obesity in humans. Acknowledgement: Supported by the grant from EU NMR project – Contract No. RII3-026145. P1–11 The study on Fnr-type transcription regulators functions in bacterium Paracoccus denitrificans using proteomics, transcriptomics and bioinformatics tools P. Bouchal 1 , I. Struharova 1 , E. Budinska 2 , O. Sedo 3 , T. Vyhlidalova 1 , Z. Zdrahal 3 , R. van Spanning 4 and I. Kucera 1 1 Department of Biochemistry, Faculty of Science, Masaryk Univer- sity, Brno, CZECH REPUBLIC, 2 Institute of Biostatistics and Analyses, Masaryk University, Brno, CZECH REPUBLIC, 3 Department of Experimental Biology, Masaryk University, Brno, CZECH REPUBLIC, 4 Department of Molecular Cell Physiology, Vrije Universiteit Faculty of Earth and Life Sciences, Amsterdam, THE NETHERLANDS Paracoccus denitrificans is a facultatively autotrophic soil bacte- rium which responds to decreasing level of oxygen in the environ- ment by a switch from aerobic to anaerobic (denitrifying) growth mode. In our work, we analyzed roles of fnr-type transcription regulators FnrP, NNR and NarR (with described key roles as sensors for oxygen, nitrate and nitrous oxide) using proteomics, transcriptomics and bioinformatics tools to consider their roles in the adaptation processes at global level. Protein compositions of four P. denitrificans strains (wild type, FnrP-, NNR- and NarR- mutants grown aerobically, semiaerobically and semiaerobically with nitrate) were analyzed using set of 36 large format 2-D PAGE gels, their statistical evaluation and MS/MS protein iden- tification of more than 500 proteins. Expression differences of key up-/down-regulated gene products were validated using qRT- PCR. Comparative proteomic analysis revealed, besides other results, differences between strains in the expression of three key denitrification enzymes (nitrate reductase, nitrite reductase, nitrous oxide reductase), OmpW and UspA proteins. These find- ings are in agreement with known mechanisms and/or confirmed the positions of proposed fnr binding sites in their promoters. Detection of additional group of up-/down- regulated proteins without fnr binding sites in their promoters (e.g. SDR dehydro- genases, TonB receptors) indicates involvement of additional reg- ulatory mechanisms into the adaptation processes studied. Evidences for potential roles of transcription regulators of other families (e.g. LysR, MarR and two-domponent regulators) are discussed. Acknowledgements: We are thankful to Czech Science Foun- dation (grant No. 203/07/P0471) and to Czech Ministry of Edu- cation (grant No. MSM0021622413) for financial support. P1–12 Analysis of proteome alteration in tomato roots after Meloidogyne hapla infection A. Obrepalska-Steplowska 1 , K. Nowaczyk 1 , M. Luczak 2 , M. Figlerowicz 2 , R. Dobosz 3 and M. Budziszewska 1 1 Interdepartmental Laboratory of Molecular Biology, Institute of Plant Protection, Poznan, POLAND, 2 Polish Academy of Sci- ences, Institute of Bioorganic Chemistry, Poznan, POLAND, 3 Department of Zoology, Institute of Plant Protection, Poznan, POLAND Root-knot nematodes from the genus Meloidogyne are parasitic to many economically important crop plants and cause great losses in the crop production. Out of fifteen European species, eight were found in Poland; among them the most common and widely distributed is M. hapla, parasiting on dicotyledonous plants. During the initial stage of infection characteristic root galls form on the roots of susceptible plant hosts. As a result of interaction between parasite and the host, in the vascular cylinder of the root permanent feeding sites are formed with specialized multinucleated feeding cells (giant cells). The redifferentiation of root cells and development of metabolically active giant cells is induced by the nematode. The mechanism of giant cells forma- tion and the role of plant proteins in this processs remain unclear. Our attempt was to analyse the changes in the plant pro- teome in the early stage of M. hapla infection. Tomato plants were grown in isolated conditions and infected with M. hapla. After isolation of proteins from roots of healthy and infected plants, as well as from nematodes, two-dimensional electrophore- sis was performed. The results of experiments were compared to ensure elimination of nematode proteins from further analysis. As some qualitative and quantitative changes were observed, cho- sen proteins were analysed using mass spectrometry. The decrease of cytosolic and mitochondrial dehydrogenases of cellular respi- ration and electron transport chain was observed as well as for proteins related to cytoskeleton (actins), relevant to changes in the cell’s shape and wound response (annexin). The qualitative changes are related with proteasome endopeptidase complex, absent in infected root cells. Nematode proteins secreted during Abstracts Poster Presentations 98 FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies infection had been the subject of many recent investigations. But, as the relationship between these parasites and the host is very close, determination of proteins engaged on the both sides of the interaction can give us the insight to the mechanism of plant sub- ordination to the parasite. P1–13 Determination of chromosomal regions affecting some production traits in F 2 intercross chickens Z. Bulut 1 , E. Kurar 2 , Y. Ozsensoy 2 , M. Nizamlioglu 1 , M. Garip 3 , A. Yilmaz 3 , T. Caglayan 3 , S. Dere 3 , V. Kurtoglu 4 and M. Dogan 1 1 Faculty of Veterinary Medicine, Department of Biochemistry, Sel- cuk University, Konya, TURKEY, 2 Faculty of Veterinary Medi- cine, Department of Genetics, Selcuk University, Konya, TURKEY, 3 Faculty of Veterinary Medicine, Department of Zoo- technics, Selcuk University, Konya, TURKEY, 4 Faculty of Veteri- nary Medicine, Department of Animal Nutrition, Selcuk University, Konya, TURKEY In this study, a F 2 level Denizli X White Leghorn population (n = 441) was used to dissect chromosomal regions, which have an affect on body weight and egg yield. DNA samples were iso- lated from all F 0 ,F 1 and F 2 animals using a standard phenol/ chloroform method. For chromosomal level screening, a total of 99 markers which were suitable for genome searching were ampli- fied using Polymerase Chain Reaction (PCR). The resulting PCR products were separated by capillary electrophoresis by using Beckman Coulter CEQ-8000 Genetic Analysis System and geno- types were identified at each marker loci. QTL Express Program was used for QTL data analysis. In study, Quantitative Trait Loci (QTL) were identified on chicken chromosome 1 (GGA1), GGA2, GGA4, GGA8 and sex chromosome (GGAZ) for hatch- ing weight, body weight at different ages and egg yield. There is a need for narrowing these QTL regions by typing new markers in these intervals and for identifying genes that have affect on these economically important traits. P1–14 Investigation of osmoprotection and cold adaptation of halomonas halophilia DSMZ 4770 by proteomics S. Ceylan 1 , B. Akbulut 1 , D. Kazan 1 and D. Kazan 2 1 Engineering Faculty, Bioengineering Department, Marmara University, Istanbul, TURKEY, 2 TUBITAK Marmara Research Center, Genetic Engineering and Biotechnology Institute, Kocaeli, TURKEY Halophiles are an important group of microorganisms that can adapt to extremely saline environments. Since, they have the abil- ity to function under extreme saline conditions, halophilic micro- organisms are used at different industrial processes such as in the production of protein/enzyme stabilizer metabolites (e.g. betain, ectoin), in the removal of pollution in salinity effluents and in the production of stable enzymes. Proteomics is the large-scale study of proteins, particularly their structures and functions. Proteins are vital parts of living organisms, as they are the main compo- nents of the physiological metabolic pathways of cells. By under- standing metabolic pathways of moderately halophilic microorganisms it will be possible to modulate their metabolism to enhance the production of their industrially important prod- ucts. In this study osmoprotection and cold adaptation metabo- lism of moderately halophilic Halomonas halophilia was investigated. Halomonas halophilia was grown on different salt concentrations (5% NaCL, -20% NaCl) and different tempera- tures (19–37°C). Growth at these conditions was investigated and microorganisms were harvested at the end of exponential phase. Afterwards, whole cell proteins of the DSMZ 4770 were extracted and analyzed by proteomic tools. Two DE maps are prepared according to tube gel systems with NEPHGE technique in the first dimension and normal SDS-PAGE in the second dimension. Protein profiles between normal and stress conditions were determined and compared. Protein expression differences were determined by MALDI-Tof Mass Spectrometer (Waters/Mi- cromass). Proteins that involved in the osmoprotection and cold adaptation were determined. P1–15 Molecular typing of Staphylococcus aureus strains from ovine mastitis by pulsed-field gel electrophoresis and polymerase chain reaction A. Ciftci 1 , E. E. Onuk 2 , A. Findik 1 , T. Yildirim 3 and M. Unlu Sogut 4 1 Veterinary Faculty, Microbiology, Ondokuz Mayis University, Samsun, TURKEY, 2 Veterinary Faculty, Diseases and Clinical Sciences, Ondokuz Mayis University, Samsun, TURKEY, 3 Science Faculty, Biology, Amasya University, Amasya, TURKEY, 4 Medicine Faculty, Microbiology, Ondokuz Mayis University, Samsun, TURKEY Staphylococcus aureus is one of the most important etiological agents of ovine mastitis. In order to develop effective control mea- sures for mastitis, it is important to type S. aureus strains that have considerable genetic heterogeneity. In the current study, 47 S. aureus strains isolated from ovine mastitis were typed by poly- merase chain reaction (PCR) based on coagulase (coa) and protein A (spa) polymorphisms and by pulsed-field gel electrophoresis (PFGE). Eight different coa-types and four spa-types were identi- fied by PCR. While the most prevalent coa-type was CG2 (42.56%), the spa-types, S4 and S1, were the most commonly observed (44.68% and 38.29%, respectively). Nineteen different pulsotypes were identified, and 12 of these were represented by a single isolate. Pulsotypes J and K were predominant, and each represented nine isolates (19.14%). All isolates belonging to J and K pulsotypes were CG2. While all nine isolates belonging to J pulsotype were S4, all isolates in K pulsotype were S1. Although PFGE was found to be the best discriminatory technique for dis- tinguishing strains, coa- and spa-types were found to be in correla- tion with PFGE types and can be used for quick, preliminary epidemiologic studies for detecting strains that may cause mastitis. P1–16 Development and optimization of multiplex polymerase chain reaction for identification of Flavobacterium psychrophilum, Yersinia ruckeri and Aeromonas salmonicida subsp. salmonicida E. E. Onuk 1 , A. Ciftci 2 , A. Findik 2 and Y. Durmaz 3 1 Veterinary Faculty, Fisheries and Fish Diseases, Ondokuz Mayis University, Samsun, TURKEY, 2 Veterinary Faculty, Microbiology, Ondokuz Mayis University, Samsun, TURKEY, 3 Veterinary Con- trol and Research Enstitute, Fisheries and Fish Diseases, Samsun, TURKEY Bacterial cold water, enteric red mouth and frunculosis are the common bacterial diseases of fish worldwide. The etiologic agents Poster Presentations Abstracts FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies 99 of these diseases are Flavobacterium psychrophilum, Yersinia ruckeri and Aeromonas salmonicida subsp. salmonicida, respectively. In this study, a Multiplex Polymerase Chain Reac- tion (M-PCR) method which can identify these fish pathogens, Aeromonas salmonicida subsp. salmonicida, Flavobacterium psy- chrophilum and Yersinia ruckeri, simultaneously was developed and optimized. It was observed that M-PCR developed in this study, with YER8/10-Fer3/4-FP1/3 primer pairs, occured neither false specific nor nonspecific amplification. The detection limits of M-PCR method using DNA extract from dilutions of pure cultures of bacteria were detected as 35 pg for Y. ruckeri and F. Psychrophilum and 70 pg for A. salmonicida subsp. salmonicida. It was determined that 15 CFU Y. ruckeri and F. psychrophilum and 30 CFU A. salmonicida subsp. salmonicida could be detected by M-PCR developed using genomic DNA extracted from dilu- tions of suspensions. The detection limits in the presence of tissue debris were detected as 125 CFU for Y. ruckeri ve F. psychrophi- lum and 250 CFU for A. salmonicida subsp. salmonicida. In con- clusion, we decided that M-PCR method developed and optimised in this study could be used for accurate and rapid identification of these bacteria. P1–17 Evaluation of protein profiles of Enterococcus faecalis strains in relation to antigenic glycoproteins G. Ciftci 1 and H. Uysal 2 1 Veterinary Faculty, Biochemistry, Ondokuz Mayis University, Samsun, TURKEY, 2 Veterinary Faculty, Biochemistry, Ankara University, Ankara, TURKEY The aim of this research is to determine the specific antigenic gly- coproteins and comparative analysis of antigenic protein profiles of Enterococcus faecalis (E. faecalis) strains distinguished with aggregation substance (AS), gelatinase and cytolysine. For the determination of the whole cell protein profiles of E. faecalis, the Sodium Dodecyl Polyacrylamide Gel Analysis (SDS-PAGE) was performed. All the E. faecalis strains showed protein bands between the sizes of 29 and 165 kDa. The protein bands of 44 and 83 kDa were determined as major band in E. faecalis OG1RF (gelatinase positive) and E. faecalis OG1X (pAM9058) (AS positive). On the other hand; E. faecalis OG1X (pAM944) (cytolisine positive) strain also showed a 43 kDa major band. For the antigenic characterization, the immunblot analysis was performed. All of the three strains of E. faecalis showed common antigenic protein bands which were at the size of 165, 35 ve 32 kDa. The other antigenic protein bands of E. faecalis OG1X (pAM944), E. faecalis OG1RF and E. faecalis OG1X (pAM9058) were determined as 85, 65, 55, 39 ve 29 kDa, 83, 75 ve 45 kDa and 111, 98, 83, 75, 55, 45, 29 kDa in sizes, respec- tively. The Glycan Detection Kit from Roche Diagnostics and Periodic Acid Schiff (PAS) stainings were used for the investiga- tion of glycoproteins of E. faecalis. After the PAS staining, no glycoprotein band was shown as the method has low sensitivity. When the Glycan Detection Kit used, E. faecalis OG1RF and E. faecalis OG1X (pAM9058) strains showed two glycoprotein bands which were at the size of 111 and 39 kDa. Moreover, E. faecalis OG1X (pAM944) strain was also showed some glyco- protein bands which were 111, 106 and 39 kDa in size. In conclu- sion; no difference was determined for the protein profiles among the strains of Enterococcus faecalis which have AS, gelatinase and cytolisine. The antigenic profiles of the glycoproteins and the other proteins of E. faecalis was found as varied among these strains. It was the first study in the literature that suggested E. faecalis strains include the glycoproteins. P1–18 RAPD-PCR analysis of the genus apodemus KAUP, 1829 (Mammalia:Rodentia) in Turkey R. Colak, G. Olgun, I. Kandemir, N. Yigit and E. Colak Faculty of Science, Biology, Ankara University, Ankara, TURKEY The aim of the present study is to survey genetic structure based on DNA markers and to make contribution to the taxonomic status, population genetics of the Genus Apodemus in Turkey. In order to analysis genetic variation in Apodemus species, a Ran- domly Amplified Polymorphic DNA (RAPD) marker system was used. A total of 82 specimens (22 A. iconicus,26A. flavicollis,7 A. sylvaticus,15A. uralensis,4A. agrarius and 8 A. mystacinus) collected from 16 locations in Turkey were used. The estimates of NEI’s (1972) standart genetic identity and standart genetic dis- tance were calculated to show the genetic relationships between studied populations. All estimations were calculated with the POPGENE software. The 60 RAPD markers were tested in Apodemus, 11 ones yielded 101 polymorphic DNA bands. Genetic differentiation values, H = 0.0721 (P = 16.83%), were the lowest in A. agrarius. A. uralensis has H = 0.2303 (P = 77.23%), being the highest value between Apodemus spe- cies. According to Nei (1972), A. agrarius and A. mystacinus are the least similar to each other (I = 0.6256), and A. iconicus and A. uralensis are the nearest to each other (I = 0.9406). Phyloge- netic relationships between Apodemus species were shown with UPGMA dendogram constructed based genetic distance values. P1–19 What does it take to make an activated cortical domain within a plant cell? F. Cvrckova 1 , R. Bezvoda 1 and V. Zarsky 2 1 Faculty of Science, Department of Plant Physiology, Charles University, Prague, CZECH REPUBLIC, 2 Faculty of Science, Department of Plant Physiology and Laboratory of Cell Biology, Institute of Experimental Botany ASCR, Charles University, Prague, CZECH REPUBLIC Exocytosis is central to plant cell morphogenesis. A single cell may possess multiple plasmalemma domains performing localized exocytosis-driven expansion (‘activated cortical domains’, ACDs). Several large families of paralogous regulatory proteins might be involved in generating the diversity of ACDs within a cell, such as e.g. small GTPases of the Rop family and their interactors, su- bunits of the exocyst complex, actin-nucleating FH2 proteins, receptor-like kinases, or enzymes locally modifying membrane composition (the ‘candidate set’ genes). Rhizodermis of Arabidop- sis thaliana consists of two cell types that differ by a single ACD – trichoblasts carrying root hairs, and atrichoblasts. We used publicly available cell-type specific transcriptome data to identify genes specifically induced in trichoblasts compared to atricho- blasts (i), and to examine their relationship to over 60 ‘candidate set’ genes (ii), as well as to known genes whose mutations exhibit root hair-specific phenotypes (iii). In addition, we have searched for homologues of all three gene classes in the published soybean Abstracts Poster Presentations 100 FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies root hair proteome (iv; Brechenmacher et al., 2009). We found only limited overlap among the gene classes (i–iv), with most ‘candidate set’ genes exhibiting <5· induction in trichoblasts compared to atrichoblasts and low protein abundance in root hairs; very few class iii genes were found in either the transcrip- tomic or proteomic set. Thus, unlike forward or reverse genetics, transcriptomics and proteomics alone are unlikely to identify genes involved in plant ACD specification. Acknowledgement: This work was supported by the MSM0021620858, LC06004 and LC06034 projects. P1–20 Drug resistant MCF-7 cells possessed epithelial-mesenchymal transition related gene expression pattern O. D. Iseri 1 , M. Demirel Kars 1 , F. Arpaci 2 , C. Atalay 3 , I. Pak 4 and U. Gu ¨ ndu ¨ z 1 1 Department of Biological Sciences, Middle East Technical University, Ankara, TURKEY, 2 Department of Oncology, Gu ¨ lhane Military Medical Academy, Ankara, TURKEY, 3 Department of General Surgery, Ankara Oncology Hospital, Ankara, TURKEY, 4 Department of Pathology, Ankara Oncology Hospital, Ankara, TURKEY Background: Epithelial-mesenchymal transition (EMT) is a developmental process by which cells of epithelial origin lose epi- thelial characteristics, and acquire a mesenchymal phenotype. EMT, an indicator of poor prognosis, may also occur during tumor progression. Altered expression levels of Snail family tran- scription factors (Snail, Slug and Twist) are the key regulatory elements of EMT. Slug gene is one of the downstream mediators of transforming growth factor beta1 (TGFB1) signaling. Objectives: In the present study evaluation of expression of genes related to EMT was aimed in docetaxel and doxorubicin resistant MCF-7 breast carcinoma cells in order to assess their involvement in drug resistance. Methods: Resistant sublines (MCF-7/DOC and MCF-7/DOX) were developed by docetaxel and doxorubicin applications in dose increments and development of resistance was confirmed by XTT proliferation assays. RNA was isolated from sensitive and resistant cells and cDNA microarray analysis was performed using Affymetrix Ò Human Genome U133 Plus 2.0 Arrays in duplicate experiments. GeneSpring GX 7.3.1 Software was used for data analysis. Immunocytochemistry was also performed to determine relevant protein expression. Results: XTT demonstrated that MCF-7/DOC and MCF-7/ DOX were resistant to docetaxel and doxorubicin, respectively. According to data analysis, estrogen receptor a was drastically downregulated in MCF-7/DOC and MCF-7/DOX. It is known as a transcriptional repressor of the Slug. Therefore, decreased ERa and increased Slug expression could cause transcriptional activation of key regulatory elements of EMT. In addition, increased expression levels of TGF beta receptor2 (TGFBR2) together with SMAD3 might have stimulated EMT in resistant cells. Furthermore, amplified epidermal growth factor signaling via epidermal growth factor receptor1 upregulation might have enhanced the pathways leading to EMT. Slug upregulation caused cadherin switch in resistant cells i.e. E-cadherin and occlu- din were downregulated, and N-cadherin was upregulated. N- cadherin associates with the fibroblast growth factor receptor1 (FGFR1) leading to increased FGFR1 expression at the cell sur- face which was also upregulated in resistant cells. Immunocyto- chemistry results confirmed microarray expression analysis. Conclusion: This report demonstrates that in docetaxel and doxorubicin resistant cells EMT process was induced indicating a possible relationship of this process and drug resistance. P1–21 Characterization and whole genome sequencing of Arthrobacter phenanthrenivorans, a new phenanthrene degrading bacterium C. Drainas 1 , A. Kallimanis 1 , K. Kavakiotis 1 , K. Mavromatis 2 , N. C. Kyrpides 2 and A. I. Koukkou 1 1 Chemistry, University of Ioannina, Ioannina, GREECE, 2 Genome Biology Program, DOE-Joint Genome Institute, Walnut Creek, CA, USA Several bacterial strains were isolated from a creosote polluted site at Perivleptos (Epirus, Greece), based on their ability to uti- lize various polycyclic aromatic hydrocarbons as a sole carbon and energy source. One of them, Sphe3, was proved to be effi- cient degrader of phenanthrene, a polycyclic aromatic hydrocar- bon (PAH). Biochemical tests and 16S rDNA analysis showed that Sphe3 belonged to the genus Arthrobacter comprising a novel species named Arthobacter phenanthrenivorans sp. nov. Whole genome sequencing of the isolated organism revealed that it had a single circular chromosome of 4.25 Mb and two circular plasmids of 190 and 94 kb, respectively. A total of 4288 genes were predicted in the Sphe3 genome. With BLAST searches using peptide fragment sequences from a purified 1-H-2-N dioxygenase activity determined by Mass Spectrometry, we identified two encoding genes with over 90% homology to each other at the nucleotide level, one (diox1) located on the 190 kb plasmid and the other (diox2) on the chromosome of the Sphe3 genome. Fur- thermore, BLAST analysis of the 190 kb plasmid revealed that it additionally contained several genes involved in phenanthrene degradation. These findings revealed novelties that confirm the extended biodiversity of PAH catabolic genes. P1–22 Stress response to aromatic compounds in Acinetobacter radioresistens S13 P. Fattori, M. Zapponi, C. Lamberti, A. Pessione, R. Mazzoli, C. Giunta and E. Pessione University of Turin, Human and Animal Biology, Turin, ITALY Acinetobacter radioresistens S13 is a strain selected for its ability in phenol degradation. It is also able to degrade other aromatic compounds (i.e., Benzoate) through the b-ketoadipate pathway (1). Comparative proteomics alkaline studies on bacterium grown either on phenol or benzoate as sole carbon source reveal a dif- ferent response to stress induced by these two aromatic com- pounds. Phenol is a solvent able to solubilize outer membrane lipoproteins, lipooligosaccharides (LOS) and phosphatidyletha- nolamine, damaging cell wall (2). This compound induces an over expression of two proteases (ClpX and Serine protease) and of two RNA polymerase modulator factors (NusA and Rho) suggesting a gene modulation based on rE regulation. Instead Poster Presentations Abstracts FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies 101 benzoate induces an alternative phosphorylation-dependent stress-sensing response, maintained by a two-component regula- tory system, which consist of a membrane-embedded sensory kinase and a response regulator (3). The kinase auto-phosphory- lates a conserved histidine on the receipt of a stress-signal before transferring the phosphoryl group to an invariant aspartate in its cognate response regulator and in doing so activates its latent biological function (4). References: 1. Mazzoli R, Pessione E, Giuffrida MG, Fattori P. et al. Degra- dation of aromatic compounds by Acinetobacter radioresistens S13: growth characteristics on single substrates and mixtures. Arch. Microbiol. 2007; 188(1): 55–68. 2. Mrozik A, Piotrowska-Seget Z & Labuzek S. Cytoplasmatic bacterial membrane responses to environmental perturbations. Polish J Environ. Studies 2004; 13(5): 487–494. 3. Bekker M, Teixeira de Mattos MJ & Hellingwerf KJ. The role of two-component regulation systems in the physiology of the bacterial cell. Sci. Prog. 2006; 89: 213–242. 4. Mizuno T. His-Asp phosphotransfer signal transduction. J. Biochem. (Tokyo) 1998; 123: 555–563. P1–23 Identification of a(1,6)fucosylated proteins as biomarkers for human colorectal carcinoma L. M. Romay, S. V. Portela, R. F. Poceiro, E. G. Martı ´ n and A. F. Briera Department of Biochemistry Genetics and Immunology, University of Vigo, Vigo, SPAIN a(1,6)-fucose residues within the N-glycan core structures are commonly observed in glycoproteins and are often altered under pathological conditions. This type of fucosylation is product of a(1,6)fucosyltransferase [a(1,6)FT] activity and is regarded as an important manner of posttranslational modification and func- tional regulation of glycoproteins. Our previous studies showed that a(1,6)fucosyltransferase activity and expression are altered in human colorectal cancer (CRC). Therefore, the a(1,6)fucosylation of some glycoproteins might be implicated in the development and progression of colorectal carcinomas, being potential bioindi- cators for the diagnosis and prognosis or targets for CRC ther- apy. In the present study we have employed a Lens culinaris agglutinin lectin (LCA) chromatography combined with 1-DE separation and followed by MALDI-MS/MS analysis to identify distinctive core fucosylation patterns in five healthy and tumour tissue samples from CRC patients. Indeed, a lectin and western blotting methodology was performed to validate our preliminary results. We demonstrated that colorectal tumour tissues have higher levels of a(1,6)fucosylation compared to healthy ones. Besides, among the several protein bands observed after the lectin chromatography and the electrophoretic separation, we selected the bands with a higher difference of intensity between healthy and tumour specimens. Two proteins were identified as possible biomarkers for CCR, the heat shock protein gp96 precursor and the Fc binding protein receptor. The western blot analysis con- firmed the enhanced expression of heat shock protein gp96 pre- cursor and the diminution of Fc binding protein receptor levels in tumour tissues compared to healthy ones. In conclusion, these glycoproteins could be considered as candidates for future studies focused on their function in colorectal tumourigenesis and their potential clinical usefulness. P1–24 Molecular typing and methicillin resistance profile of Staphylococcus aureus strains isolated from the noses of healthy dogs A. Findik 1 , N. Akan 2 , E. E. Onuk 3 , D. Cakiroglu 4 and A. Ciftci 1 1 Veterinary Faculty, Microbiology, Ondokuz Mayis University, Samsun, TURKEY, 2 Veterinary Faculty, Ondokuz Mayis University, Samsun, TURKEY, 3 Veterinary Faculty, Diseases and clinical sciences, Ondokuz Mayis University, Samsun, TURKEY, 4 Veterinary Faculty, Internal Medicine, Ondokuz Mayis University, Samsun, TURKEY It was aimed to detect the carriage of methicillin-resistant Staph- ylococcus aureus (MRSA) strains in the nasal flora of healthy dogs and to genotype these S. aureus isolates. The swabs were taken from the nasal region of 80 dogs examined in the veteri- nary clinics for the check-up in Samsun. The methicillin resis- tance profile of 80 isolates which were identified as S. aureus were analysed phenotypically and genotypically. Agar Disc Diffu- sion Test was performed to determine the methicillin resistance phenotype of these S. aureus strains using the oxacillin antibiotic discs (5 g) and all the 80 strains were found as sensitive to methi- cillin. In Polymerase Chain Reaction (PCR) targetting nuc, mecA and fem genes, three strains (3.75%) was found to possess mecA. In the same analysis none of these strains were positive for fem gene. The PCR targetting coa and spa genes was performed for molecular typing of the S. aureus strains. Nine different coa genes and three different spa genes were determined according to the polymorphism of these genes. In conclusion, the determining of the nasal carriage of different genotypes of S. aureus in healthy dogs was considered as a basic finding for both the char- acterization of nasal isolates of S. aureus and molecular epidemi- ologic researches. In these strains, the detection of mecA gene, accepted as ‘Gold Standart’ for methicillin resistance, was evalu- ated as an important finding for community health. P1–25 Detection of methicillin resistance and slime factor production of Staphylococcus aureus in bovine mastitis A. Ciftci 1 , A. Findik 1 , E. E. Onuk 2 and S. Savasan 3 1 Veterinary Faculty, Microbiology, Ondokuz Mayis University, Samsun, TURKEY, 2 Veterinary Faculty, Diseases and Clinical Sciences, Ondokuz Mayis University, Samsun, TURKEY, 3 Veterinary Faculty, Microbiology, Adnan Menderes University, Samsun, TURKEY Mastitis is the most prevalent and costly disease of dairy cattle. Among the pathogens that cause mastitis, S. aureus is the most important species and the hardest to eliminate with antibiotics because of its multiple antibiotic resistance. It was aimed to detect methicillin resistant and slime producing S. aureus in the case of bovine mastitis. The triplex PCR was optimized targetting 16S rRNA, nuc and mecA for detection Staphylococcus species, S. aureus and methicillin resistance, respectively. For detection of slime producing, it was performed a PCR assay targetting icaA and icaD genes. In this study, 59 strains were detected as S. aur- eus by both conventional tests and PCR. Among the 13 S. aureus strains of them were found as methicillin resistant phenotypically and 4 (30.7%) were positive for mecA gene. Although 22 of 59 (37.2%) S. aureus isolates were slime-producing in Congo Red Agar in PCR analysis, only 15 of 59 were positive for both icaA and icaD genes. 16 of 59 strains were positive for icaA and 38 of them were positive for icaD gene. We found only two of 59 strains were positive for both methicillin resistance and slime pro- ducing, phenotypically. Abstracts Poster Presentations 102 FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies P1–26 Immobilization of acid phosphatase to magnetic particles and their use for dephosphorylation of proteins J. Frydlova 1 , L. Kubosek 1 , L. Kubosek 2 , Z. Kucerova 1 , M. Ticha 1 and M. Ticha 3 1 1st Faculty of Medicine, Institute of Pathophysiology and CEH, Charles University in Prague, Praha 2, CZECH REPUBLIC, 2 Faculty of Chemical Technology, Department of Biological and Biochemical Sciences, University of Pardubice, Pardubice, CZECH REPUBLIC, 3 Faculty of Science, Department of Biochemistry, Charles University in Prague, Praha 2, CZECH REPUBLIC In proteomics studies, the presence of phosphate group in a pro- tein or in a peptides obtained by proteolytic digestion is often confirmed by the comparison of mass spectra of peptides/proteins before and after the phosphatase treatment. A loss of phosphate groups results in a shift of peptide in mass spectrum. Generally, the use of enzyme immobilized to magnetic carriers has several advantages as compared with an application of soluble enzyme forms: an increased stability of enzymes, a possibility of direct use of enzyme reaction products for MALDI-TOF MS and an easy manipulation. Contrary to proteolytic enzymes, only limited information is available on properties of immobilized phosphata- ses. In the present study, acid phosphatase from potato was immobilized to glyoxal 4% agarose magnetic particles (20– 75 lm) via its free amino groups. The following properties of immobilized acid phosphatase were compared with those of the soluble enzyme: the pH dependence of the enzyme activity, the effect of the presence of cofactor (Mg 2+ ), the effect of tempera- ture and further storage and operational stability. A reusability of the immobilized phosphatase was also examined. Both forms of acid phosphatase were used for dephosphorylation of porcine pepsin A, bovine a-casein and chicken ovalbumin. Acknowledgements: This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic (grant MSM 0021620806 and project CEH LC 06044) and by the Czech Science Foundation (grant 203/09/0857). P1–27 Deletions with the presence of two repetitive DNA sequences in CEBPA gene O. Fuchs 1 , A. Kostecka 1 , D. Provaznikova 1 and J. Filkukova 2 1 Department of Cell Physiology, Institute of Hematology and Blood Tranfusion, Prague 2, CZECH REPUBLIC, 2 Department of Molecular Genetics, Institute of Hematology and Blood Tranfusion, Prague 2, CZECH REPUBLIC C/EBPalpha (CCAAT/enhancer binding protein alpha) belongs to family of leucine zipper transcription factors and it is neces- sary for transcriptional control of granulocyte and adipocyte dif- ferentiation, glucose metabolism and lung development. C/ EBPalpha is encoded by an intronless gene that is 2783 bp long and maps to human chromosome 19q13.1. Up to now, CEBPA gene mutations were detected only in patients with acute myeloid leukemia (AML), in patients wih myelodysplastic syndrome (MDS), in multiple myeloma and non-Hodgkin ´ s lymphoma patients. We detected 14 various heterozygous CEBPA gene dele- tions with the presence of two repetitions in CEBPA gene in AML patients, MDS patients, non-Hodgkin ´ s lymphoma patient, patients with peripheral artery disease, ischemic heart disease, hyperlipidemia and type 2 diabetes. These mutations are charac- teristic by the loss of one from these two same repetitions on the ends of deleted sequence. Two most frequent repetitions included in these deletions in CEBPA gene are GCCAAGCAGC (508– 517_907–916, GenBank Accession No. NM_004364.2) and CGC- GAG (493–498_865–870, GenBank Accession No. NM_004364.2). We found both these most frequent deletions in CEBPA gene in one young man (38 years old) after coronary thrombosis (heart attack). The same deletions were detected in his son (15 years old). In many cases (13 patients with AML, MDS, peripheral artery disease, heart disease, hyperlipidemia and type 2 diabetes) more than one type of these deletions was observed. These findings suggest that observed CEBPA gene dele- tions of this type are likely the result of unequal mitotic cross- over. We observed also in several patients in addition to these deletions other mutation which was created probably by mecha- nism of replication slippage in the repetitive sequence (for exam- ple 68dupl in addition to 912_929del or 311_313del in addition to 44_694del, in both cases GenBank Accession No. NM_04364.2). Acknowledgements: This work was supported by the Internal Grant Agency of the Ministry of Health of the Czech Republic (VZ 00023736). P1–28 Study of thermal tolerance in Cronobacter strains J. Gajdosova 1 , K. Kunikova 1 , I. Turcovsky 2 , E. Kaclikova 2 and H. Drahovska 1 1 Department of Molecular Biology, Comenius University, Bratislava, SLOVAK REPUBLIC, 2 Department of Microbiology, Food Research Institute, Bratislava, SLOVAK REPUBLIC Cronobacter spp., formerly named as Enterobacter sakazakii,is an opportunistic pathogen associated with sporadic cases of severe infections in neonates causing meningitis, necrotizing enterocolitis and sepsis. This organism is widely distributed in environment and rehydrated infant formula is the most common source of infection. Cronobacter was shown to be particularly tol- erant to osmotic stress and desiccation and some strains of this genus are also tolerant to elevated temperatures. Considering infant formula is not a sterile product, thermotolerant strains of Cronobacter spp. represent increased risk to survive during infant formula reconstitution. In our study, the genetic variability of 76 food isolates and 23 Cronobacter collection strains was measured by AFLP and 16S rRNA sequencing. Within AFLP profiles, a great variability was observed, the similarity among strains reached values 50–100%. Strains were clustered into 46 different clusters at the similarity level of 90%. Six main groups (desig- nated A-F) were clearly distinguished at the 75% similarity level; strain grouping was in concordance with their species identifica- tion and biochemical properties. Test of survival at 58°C sepa- rated strains into two groups; D values (decreasing of bacterial counts in one order) of thermosensitive strains fell within the range 17–50 s; on the other hand eleven strains (nine Cr. sak- azakii and two to Cr. malonaticus) were assessed as thermotoler- ant because their D values reached from 100 to more than 300 s. Thermotolerant strains were also positive for PCR thermotoler- ance marker homologous to a hypothetical protein Mfla_1165 from thermotolerant bacterium Methylobacillus flagellatus KT (Williams et al. 2005). 5.5 kbp DNA fragment around thermotol- erance marker was sequenced in strain Cr. sakazakii ATCC 2954. The region possessed 94% similarity with M. flagellatus KT com- plete genome including genes Mfla_1162 – Mfla_1168. Cloning of 2950 bp long part of Cr. sakazakii thermotolerance region into pGEM plasmid resulted in 3–5 times increased survival of Escherichia coli laboratory strain at 58°C. Our results have Poster Presentations Abstracts FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies 103 shown that this genetic region can be important in response to several stress conditions and may contribute to increased envi- ronmental resistance of Cronobacter strains. P1–29 Mitochondrial proteome changes during prereplicative phase of liver regeneration A. Gnoni 1 , R. Mangiullo 1 , R. Schiavone 2 , S. Vilella 2 , S. Papa 1,3 and F. Zanotti 1,3 1 Department of Medical Biochemistry, Physics and Biology, University of Bari, ITALY, 2 Department of Biological and Envi- ronmental Sciences and Technologies, Laboratory of Comparative Physiology, University of Salento, Lecce, ITALY, 3 Institute of Biomembranes and Bioenergetics, CNR, Bari, ITALY Introduction: The liver is usually a quiescent organ, but it is able to regenerate itself and replace lost tissue, after transplanta- tion and upon the loss of cells, following chemical and viral injury, and partial hepatectomy (PH). Liver regeneration is mainly divided in two phases: the prereplicative phase, during which the liver’s energy demand increases, and the replicative phase, during which increased DNA synthesis and mitosis occurs. In the prereplicative phase (0–24 h after PH), mitochondria show, a decrease in oxidative phosphorylation capability and production of oxygen free radicals (1). We applied a proteomic approach to mitochondria isolated 6 h after PH, to characterize mitochondrial proteins that are involved in the prereplicative phase of liver regeneration. Methods: Rats were subjected to PH at 6 h and mitochondria were isolated. Mitochondria from sham-operated rats were used as controls. Mitochondrial protein expression pattern were stud- ied by 2-DE electrophoresis, and subsequent spots identification was performed by nanoLC-ESI MS/MS analysis. Results: Compared to the sham-operated control group, 1 pro- tein was up-regulated and 13 proteins were down-regulated at 6 h. We identified eight differentially expressed proteins that were associated with lipid metabolism, the OXPHOS system, biotrans- formation and other metabolic pathways. Among these, Mn- superoxide dismutase, was down-regulated 6 h after PH. SOD2 is a matrix mitochondrial enzyme that catalyzes superoxide radicals dismutation. Reference: 1. Guerrieri F et al., Free Radic. Biol. Med. 1999; 26: 34–41. P1–30 Evolutionary dynamics of the Arabidopsis formin family M. Grunt and F. Cvrckova Faculty of Science, Plant Physiology, Charles University, Prague, CZECH REPUBLIC Formins (FH2 proteins) are a family of actin-organizing proteins exhibiting interesting evolutionary dynamics, with variable domain composition in some lineages including plants. Seed plants, in particular angiosperms, exhibit an extraordinary diver- sity of formins, which can be divided into two distinct classes on the basis of sequence of the conserved actin-nucleating FH2 domain, as well as domain composition (1). The genome of the common laboratory model, Arabidopsis thaliana, encodes 11 Class I formins and up to 12 Class II formins, which appears to be especially prone to gene duplication. Since the draft genome sequence of a closely related Arabidopsis species, A. lyrata, became available recently, we have used this sequence to identify members of the A. lyrata formin family, and compared them with their A. thaliana homologues. While we could easily distin- guish orthologues of each of the Class I formins, the situation is somewhat less clear in case of Class II proteins, consistent with gene duplications taking place even after separation of the thali- ana and lyrata lineages. Nevertheless, even one of the loci sus- pected to be pseudogenes in A. thaliana has an orthologue A. lyrata, suggesting that it might be a functional gene. Acknowledgement: This work was supported by the MSM0021620858 and LC06004 projects. Reference: 1. Grunt et al., BMC Evol. Biol. 2008; 8:115. P1–31 Impaired function of mitochondrial ATP synthase depending upon 9205delTA mutation load in ATP6 gene of mtDNA K. Hejzlarova, P. Jesina, V. Kaplanova, Z. Drahota, M. Kalous and J. Houstek Department of Bioenergetics, Institute of Physiology AS CR v.v.i., Prague 4, CZECH REPUBLIC Maternally transmitted disorders of mitochondrial ATP synthase are typically caused by heteroplasmic missense point mutations in mtDNA encoded subunit 6. In contrast, unique ATP synthase disorder due to microdeletion 9205delTA in ATP6/COX3 gene is associated with altered splicing of ATP6-COX3 mRNA and diminished synthesis of subunit 6 (F o -a). Up to now, only two patients with 9205delTA mutation have been found with very dif- ferent phenotypes and mutation load. To investigate phenotypic expression of the mutation, we have prepared transmitochondrial cybrids with varying mutation load (50–100%) and studied the relationship between heteroplasmy, mitochondrial respiration, ATP production and F o -a subunit content. Heteroplasmy in cell lines was analyzed by PCR/RFLP using NsiI restriction endonu- clease and digitonin-permeabilised cells were used for measuring oligomycin-sensitive, ADP-stimulated oxygen consumption. In parallel, ATP production was determined in DMSO-quenched samples by a luciferin-luciferase reaction. To evaluate how the mutation load affects on biosynthesis of F o -a subunit, patient cy- brids were analyzed by SDS-PAGE and WB using specific anti- bodies to ATP synthase subunits F o -a and F 1 -alpha. We have found that the decrease in ATP production and in ADP-stimu- lated respiration as well as the loss of subunit F o -a content exert similar threshold dependce on increasing mutation load. Pro- nounced decrease in all parameters was observed when mutation load reached about 80%. In contrast, near-linear relationship was found between ATP production, respiration and loss of F o -a subunit. In conclusion, our results demonstrate, that similarly as ATP6 missense mutations, 9205delTA biochemical phenotype exhibits distinct threshold effect that originates from a gene-pro- tein level. P1–32 Localisation of Pcb antenna complexes in photosynthetic prokaryote Prochlorothrix hollandica M. Herbstova and F. Vacha Institute of Plant Molecular Biology, Photosynthesis, Ceske Bude- jovice, CZECH REPUBLIC A filamentous freshwater phytoplankton Prochlorothrix hollandic- a belongs to an unusual group of cyanobacteria called prochloro- phytes. Typical cyanobacteria organise photosynthetic light- harvesting complexes into so-called phycobilisomes where no chlorophyll is bound. Prochlorophyta lost the ability to create Abstracts Poster Presentations 104 FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies [...]... results showed that dandelion and goosefoot plant PPO have 11.74 and 11.41 mM Km values for catechol at pH 6.5 and 7.5 However, common mallow and cress have 23.5 and 20.99 mM Km values for catechol at pH 7.0 and 5.0 have 23.5 and 20.99 mM Km values for catechol at pH 7.0 and 5.0 Dandelion and goosefoot plant PPO are more active and efficient to catechol substrate at different pH’s and 35°C Thus, different... total of 22 bands were determined, ranging from 156 to 16 kDa protein Of these bands, five protein (66, 32, 25, 224 and 23 kDa) were detected to be glycolyzed In addition to these protein and glycoprotein pattern of the secretion, they showed seasonal 108 Poster Presentations changes Protein band numbers and their optic density were high in posthibernation period Glycoprotein band numbers and their density... each cluster; 1: Kirikkale (subgroup1), Beynam, Haymana, Afyon, Konya and Yunak (subgroup2), 2: Aksaray (subgroup1) and Ayas, Kirsehir and Beypazari (subgroup2) P1 67 Associations between serum linolenic acid/ alpha-linolenic acid ratio and blood gene expression profiles K S Olsen1, C G Fenton2, E Anderssen3, L Froyland4, R H Paulssen2 and E Lund1 1 Institute of Community Medicine, University of Tromso,... triacylglycerol and cholesterol in 20 lipoprotein fractions) and transcriptomic (Affymetrix Rat Exon 1.0 ST, liver) profiles between SHR and SHR-Lx under conditions of standard, HSD and HSD + RA administration We observed noticeable distinction in effect of RA between SHR and SHR-Lx strains SHR-Lx reacted with significant impairment of glucose tolerance and less favorable distribution of cholesterol and triaylglycrols... fishes, amphibians, reptiles, birds and mammals (2) Two mammalian orthologs of Bv8, prokineticin 1 and prokineticin 2 (PKs), are the natural ligands for two G-protein-coupled receptors, PKR1 and PK-R2 Bv8/prokineticins and their receptor expression is restricted to specific endothelial cells of steroidogenic glands, central nervous system, peripheral blood leukocytes and cells of the innate immune system... compilation ª 2009 Federation of European Biochemical Societies 113 Abstracts P1 62 Posterior polymorphous corneal dystrophy – copy number, gene expression and candidate gene analyses within the PPCD1 candidate region on chromosome 2 0p11 .2 L Noskova1, P Liskova2, V Stranecky1, H Hartmannova1, R Ivanek3, K Jirsova4, S Merjava4, M Filipec5 and S Kmoch1 1 First Faculty of Medicine, Institute of Inherited Metabolic... the phosphopeptide adsorption and following desorption were optimized Acknowledgements: This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic (grant MSM 0021620806 and project CEH LC 06044) and by the Czech Science Foundation (grant 203/09/0857) P1 64 Functional analyses of Lupinus luteus cyclophiline promoter activities K Nuc1, P Nuc2 and R Slomski1 1 Poznan University... protein content and the changes about protein and glycoprotein profiles might be related to seasonal reproductive activity This means that they can be dependent on androgenic hormones P1 45 Comparison of protein and mRNA expression levels of titin isoforms in cardiac muscles of hibernating ground squirrels E Karaduleva, I Vikhlyantsev, J Shumilina and Z Podlubnaya Laboratory of Structure and Function of... are increased, the understanding for diversity and evolution of the GST classes would also be advanced FEBS Journal 276 (Suppl 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies Poster Presentations P1 71 Human Accelerated Region 20 (HAR20) operates as an enhancer: functional study of a HAR20 SNP associated with diabetes and cardiovascular disease... the fetal development and neonatal morbidity The aim of our study was to characterize the changes in expression of genes involved 118 Poster Presentations in the regulation and maintenance of mtDNA content (PGC1, NFR1, TFAM, POLG) DNA and RNA were isolated from 26 pairs of liver and muscle tissue samples obtained at autopsy from miscarriages after informed consent, between 13th and 28th week of gestation . POSTER PRESENTATIONS P1 Functional Genomics, Proteomics and Bioinformatics P1 1 The influence of the HERV-K LTR on. bands between the sizes of 29 and 165 kDa. The protein bands of 44 and 83 kDa were determined as major band in E. faecalis OG1RF (gelatinase positive) and