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VOLUME 45, No. 1, January–June 2008

C O N T E N T S

ANNUAL INTERNATIONAL CONFERENCE OF THE ROMANIAN SOCIETY OF BIOCHEMISTRY AND MOLECULAR BIOLOGY

(May 29–31, 2008, Bucharest, Romania)

ABSTRACTS OF PLENARY LECTURES, ORAL PRESENTATIONS AND POSTERS

SCIENTIFIC PROGRAMME .................................................................................................... 5

PLENARY LECTURES ............................................................................................................. 9

ORAL PRESENTATIONS ......................................................................................................... 15

POSTERS

SESSION 1 – Intracellular Trafficking ...................................................................................... 43 SESSION 2 – Protein Structure and Function ........................................................................... 49 SESSION 3 – Clinical and Immunological Biochemistry ......................................................... 63 SESSION 4 – Advanced Methods in Biochemistry and Molecular Biology ............................. 93 SESSION 5 – Bio-Nano-Technologies ...................................................................................... 103 SESSION 6 – Environmental Biochemistry ............................................................................... 129

AUTHOR INDEX ...................................................................................................................... 133

ROM. J. BIOCHEM., 45, 1, 1–138 (2008)

ROMANIAN JOURNAL OF

BIOCHEMISTRY

THE ANNUAL INTERNATIONAL CONFERENCE

OF THE ROMANIAN SOCIETY OF BIOCHEMISTRY

AND MOLECULAR BIOLOGY (May 29–31, 2008, Bucharest, Romania)

Abstracts of plenary lectures, oral presentations and posters

Held at the Faculty of Veterinary Medicine, 105 Splaiul Independenţei, and organized by

THE ROMANIAN SOCIETY OF BIOCHEMISTRY AND MOLECULAR BIOLOGY

THE INSTITUTE OF BIOCHEMISTRY, The Romanian Academy, Bucharest

THE FACULTY OF VETERINARY MEDICINE, The University of Agronomical Sciences and Veterinary Medicine, Bucharest

SCIENTIFIC PROGRAMME

THURSDAY, MAY 29th

14.00–16.00 Registration and poster display 16.00–16.15 Opening ceremony

16.15–17.00 Plenary lecture: Professor Robert W. Evans, Brunel University, Department of Biosciences, Uxbridge, United Kingdom

17.00–18.00 Oral presentations: Intracellular Trafficking Section Co-chairmen: Ştefana Petrescu, Norica Nichita

18.00–18.30 Sponsor presentations: Teknoleb/Millipore, Amex, Dexter/Beckman

19.00 Welcome cocktail

FRIDAY, MAY 30th

9.00–9.45 Plenary lecture: Professor Raymond A. Dwek, University of Oxford, Department of Biochemistry, United Kingdom

9.45–10.00 Coffee break

10.00–12.00 Oral presentations: Protein Structure and Function Section Co-chairmen: Raymond A. Dwek, Andrei Petrescu

12.00–12.20 Sponsor presentations: BioRad, Diamedix-Diagnostica

12.20–13.00 Poster viewing

13.00–14.00 Lunch

14.00–14.45 Plenary lecture: Dr. Bastian Hülsmann, Max-Planck-Institute for Biophysical Chemistry, Department of Cellular Logistics, Göttingen, Germany

14.45–14.55 Sponsor presentation: Novaintermed

15.00–16.00 Oral presentations: Clinical and Immunological Biochemistry Section Co-chairmen: Elena Ganea, Robert W. Evans

16.00–16.30 Coffee break

16.30–17.50 Oral presentations: Bio-Nano-Technologies Section Co-chairmen: Anca Roşeanu, Gabriela Negroiu

17.50–18.30 Poster viewing

18.30–19.00 Best Poster award

19.00–21.00 Dinner

ROM. J. BIOCHEM., 45, 1, 5–7 (2008)

RSBMM International Conference 2008: Scientific programme 2

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SATURDAY, MAY 31st

9.00–11.00 Workshop: Perspectives in Melanoma Research Chairman: Ştefana Petrescu 9.00–11.00 Workshop: Tools of Molecular Biology in Help of Applied Enzymology Chairman: Ştefan Szedlacsek 9.00–11.00 Workshop: The Investigation of Pore Forming Proteins in Model Membranes Co-chairmen: M. Radu, T. Luchian 11.00 Closing ceremony

SCIENTIFIC COMMITTEE

Prof. Dr. Raymond A. Dwek, Glycobiology Institute, University of Oxford, UK Prof. Dr. Robert W. Evans, Brunel University, Uxbridge, Middlesex, UK Dr. Ştefana Petrescu, Institute of Biochemistry, Bucharest, Romania Dr. Anca Roşeanu, Institute of Biochemistry, Bucharest, Romania Dr. Elena Ganea, Institute of Biochemistry, Bucharest, Romania Dr. Norica Nichita, Institute of Biochemistry, Bucharest, Romania Dr. Andrei Petrescu, Institute of Biochemistry, Bucharest, Romania

ORGANIZING COMMITTEE

Dr. Ştefana Petrescu, Institute of Biochemistry, Bucharest, Romania Dr. Anca Roşeanu, Institute of Biochemistry, Bucharest, Romania Dr. Elena Ganea, Institute of Biochemistry, Bucharest, Romania Dr. Norica Nichita, Institute of Biochemistry, Bucharest, Romania Dr. Andrei Petrescu, Institute of Biochemistry, Bucharest, Romania Prof. Dr. Aneta Pop, University of Agronomical Sciences and Veterinary Medicine, Bucharest, Romania Drd. Livia Sima, Institute of Biochemistry, Bucharest, Romania Paula Florian, Institute of Biochemistry, Bucharest, Romania Drd. Cristina Laslo, Institute of Biochemistry, Bucharest, Romania Drd. Mădălina Icriverzi, Institute of Biochemistry, Bucharest, Romania

TECHNICAL STAFF

Emilia Ardelean, Institute of Biochemistry, Bucharest, Romania Gabriela Scârneci, Institute of Biochemistry, Bucharest, Romania Adina Monea, Institute of Biochemistry, Bucharest, Romania

SPONSORS

Main sponsor

Dexter Com, representative of Beckman-Coulter and Promega

3 RSBMM International Conference 2008: Scientific programme

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Sponsors

(in alphabetical order)

Amex Import Export S.R.L. Bio-Rad

Coca Cola Dacchim

Diamedix-Diagnostica Nitech S.R.L. Novaintermed Sapaco 2000

Teknoleb, representative of Millipore

RSBMM International Conference 2008: Scientific programme 4

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PLENARY LECTURES

ROM. J. BIOCHEM., 45, 1, 9–13 (2008)

[PL1] GLYCOBIOLOGY – LEADING TO NOVEL THERAPIES FOR HEPATITIS AND HIV

R.A. DWEK

Glycobiology Institute, Department of Biochemistry, University of Oxford, UK

The word ‘Glycobiology’ entered the Oxford English Dictionary in 1992, defined as the study of the biological role of sugars attached to proteins (glycoproteins) and lipids (glycolipids). Research in glycobiology has made major contributions to understanding concepts in protein folding, immunology and virology, laying the foundations for applying glycobiology to the development of novel strategies for antiviral therapeutics and vaccines.

Viruses are a major public health concern associated with considerable morbidity and mortality worldwide. Over two billion people, of which 350 million are chronically infected, have been or are infected with Hepatitis B virus (HBV); 200 million people are infected with Hepatitis C virus (HCV), and over 40 million are infected with HIV. Each of these viruses is dependent on their properly folded coat glycoproteins for their infectivity. Iminosugar drugs that disrupt the folding of these glycoproteins are being investigated as antiviral therapeutics in the Oxford Antiviral Drug Discovery Unit.

One such compound, the glucose analogue N-butyl-deoxynojirimycin (NB-DNJ), was pioneered by the Oxford Glycobiology Institute and has world-wide approval for use in treatment of glycolipid storage disorders. But NB-DNJ is also effective against HCV, HBV, and HIV and methods to improve the targeted delivery of this drug including liposome encapusulation are in development. Building on this research, other mechanisms of inhibiting the morphogenesis (the assembly and secretion of infectious virus) of HCV are being investigated.

Knowledge of glycobiology is also being exploited in the design of a novel, antibody-based HIV vaccine. The basis for this is a rare neutralising antibody, obtained from a patient, that recognises clusters of sugars on the “immunologically silent” face of gp120 on the HIV. Methods for breaking tolerance to the sugars on gp 120 will be outlined.

These examples demonstrate how glycobiology is providing novel therapeutic approaches to viruses.

RSBMM International Conference 2008: Plenary lectures 2

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[PL2] IRON ACQUISITION BY NEISSERIA MENINGITIDIS – A BATTLE BETWEEN MICROBE AND MAN

R.W. EVANS

Biosciences, Brunel University, Uxbridge, Middlesex, UB8 3PH, UK

In the vast majority of mammalian cell types, cellular acquisition of iron is from transferrin via a receptor-mediated endocytic pathway. Although iron is an essential element for most bacteria, under physiological conditions (i.e. at neutral pH and in the presence of oxygen) it is rapidly oxidised to Fe3+ with the formation of insoluble hydroxides. As the “free” concentration of Fe3+ in extracellular fluids such as plasma has been estimated to be as low as 10–18 M, bacteria have developed ways to obtain their essential iron, for example by synthesising and secreting high-affinity chelators (siderophores) or by expressing surface receptors capable of scavenging iron from the host's iron-containing proteins such as transferrin and lactoferrin. Under iron-limiting conditions Neisseria meningitidis, the causative agent of meningococcal meninigitis and septicaemia, expresses transferrin- and lactoferrin-binding proteins (Tbps and Lbps) in its outer membrane. These receptors, which display an absolute specificity for human transferrin and human lactoferrin, respectively, comprise an integral membrane protein (TbpA and LbpA) and a surface-exposed lipoprotein (TbpB and LbpB). Although a vaccine is now available for the serogroup C meningococcal disease, no vaccine is available to prevent the serogroup B disease, the most prevalent in the Western world. As TbpA is highly conserved among meningococcal strains, surface-exposed and vitally important for pathogenicity, it is under consideration for inclusion in a vaccine effective against all serogroups.

An understanding of both the nature of the interaction of transferrins with the meningococcal receptor complexes and the mechanism whereby the pathogen acquires its ligand-bound iron is essential to the development of rational strategies against meningococcal meningitis. Solid-phase overlapping peptide libraries for TbpA and LbpA were therefore probed with their respective ligand and ligand-binding regions mapped onto 3D-homology models thus providing an insight into the regions involved in receptor-ligand interactions. Potential immunogenic epitopes have also been identified using convalescent-phase antisera.

3 RSBMM International Conference 2008: Plenary lectures 13

[PL3] USING THE TNT QUICK COUPLED TRANSCRIPTION/TRANSLATION SYSTEM IN CELL

BIOLOGICAL RESEARCH

B. HÜLSMANN

Department of Cellular Logistics, Max-Planck-Institute for Biophysical Chemistry, D-37077 Göttingen, Germany

Cell-free translation systems have become increasingly important in biochemical and cell biological research. This is mainly because lysates of eukaryotic origin, like the rabbit reticulocyte lysate, contain chaperones that are needed for the proper folding of newly synthesized eukaryotic proteins. In addition these lysates allow posttranslational modifications that in some cases are essential for the function of the translated protein. Taken together, rabbit reticulocyte lysate often successfully translates proteins that would be insoluble, inactive or even toxic when expressed in E. coli.

Another important feature of cell-free translation systems is the possibility to supply the reaction with radioactively labelled amino acids. As a result the translated protein is specifically labelled without any change in its primary amino acid sequence. Traditionally, a labelled protein then serves as a “prey” for interaction studies that test if such a protein binds to an immobilized binding partner (the “bait”). In addition to such well established binding assays the in vitro translation system can supply soluble proteins for more complex cell biological experiments. To illustrate these two examples will be given. First, it will be presented how an in vitro translated protein is sorted between two compartments of a living cell. Second, I will show what an in vitro translation system can tell us about the topology of a transmembrane protein.

RSBMM International Conference 2008: Plenary lectures 4

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ORAL PRESENTATIONS

ROM. J. BIOCHEM., 45, 1, 15–39 (2008)

[O1] CYTOPLASMIC PROTEINS AS DIAGNOSTIC BIOMARKERS IN PIGMENTED AND AMELANOTIC

MALIGNANT MELANOMAS

C. ARDELEANU1,2, D. TERZEA1,2, G. BUTUR1, Ş.M. PETRESCU3, G. NEGROIU3, M. MIHAI1, F. VASILESCU1, A. GEORGESCU1,

S. ZURAC2,4, F. STĂNICEANU2,4 1“Victor Babeş” National Institute of Pathology, Bucharest, Romania

2“Carol Davila” University of Medicine and Pharmacy, Bucharest 3Institute of Biochemistry of the Romanian Academy, Bucharest, Romania

4“Colentina” Clinical Hospital, Bucharest, Romania

Melanocytic tumors usually contain melanin, responsible for their brown color. About 50% of melanocytic tumors are devoid of melanin. The amelanotic malignant melanomas are frequently poorly differentiated tumors, their melanosomes being characteristically abnormal with fragmentation or absence of melanosomal proteins (tyrosinase, tyrosin related protein, HMB45, MELAN A). The loss of these proteins causes diagnostic difficulties in malignant melanomas. Recently, novel proteins were introduced as diagnostic biomarkers for the malignant melanomas.They are lacking of specificity, as other markers usually used (vimentin and S-100 protein). Among these new markers there are nestin, CD117, Tau, proteins of MAGE family, E-Cadherin, etc. Aiming to verify the practical usefulness of these markers in amelanotic melanoma diagnosis, we assessed their presence in pigmented and amelanotic melanomas. Fourteen amelanotic and 26 pigmented skin melanomas selected from the files of “Victor Babeş” Institute and “Colentina” Hospital were assessed immunohistochemically. Monoclonal antibodies or polyclonal sera against Nestin, CD117, MAGE-1, Tau protein (Ab3,) and E-Cadherin were used. All cases were positive for Vimentin and S-100 protein; all pigmented melanomas were positive for HMB45; 2 out of 26 cases were negative for MELAN A. Two out of 14 amelanotic melanomas were negative for HMB45 and other two cases for MELAN A. A good expression was found for Nestin: 12 positive/15 tested cases in amelanotic melanomas and 23 positive/26 tested cases for pigmented ones. Relatively good results were obtained also for MAGE-1 (7/12 in amelanotic and 16/23 in pigmented melanomas) and CD117 (7/12 in amelanotic and 13/16 in pigmented melanomas). The similar expression of Nestin, MAGE-1 and CD117 in the amelanotic and pigmented melanomas can suggest including them in a panel of biomarkers for characterization and diagnosis of malignant amelanotic melanoma.

RSBMM International Conference 2008: Oral presentations 2 18

[O2] EFFECT OF LIPID MICRODOMAINS ON OmpF INSERTION IN HETEROGENEOUS LIPID BILAYER

STUDIED BY FLUORESCENCE TECHNIQUES

M. BACALUM1, M. RADU1, M. WINTERHALTER2 1“Horia Hulubei” National Institute for Physics and Nuclear Engineering, Group of Biophysics and

Radiobiology, Atomistilor 407, Măgurele, Romania 2International University Bremen, Bremen, Germany

The interaction of transmembrane proteins with the lipid environment into the biological membrane is a very hot topic in the membrane biophysics considering the new paradigm of lipid rafts. The presence of microdomains in the lipid model membrane offers a good tool to investigate the influence of the heterogeneity of lipid composition on the protein functionality. In this approach the insertion of porine OmpF (outer membrane porine F) from the Gram-negative bacteria, into lipid bilayer of liposomes containing saturated and unsaturated lipids and cholesterol was investigated by fluorescence techniques.

To study the insertion of protein into the lipid bilayer small unilamellar vesicles were used as model membranes. Saturated, unsaturated lipids and cholesterol (Avanti Lipids) were used to prepare vesicles where lipid microdomains occur. The OmpF was extracted and purified by the group of Prof. Winterhalter. The insertion of OmpF into lipid bilayer was followed by FRET (Förster Resonance Energy Transfer), the donor being the tryptophan residues in OmpF and the acceptor being the DPH (diphenyl hexatriene) inserted in the lipid bilayer. The lipid bilayer fluidity was studied by DPH fluorescence depolarisation in the range of temperature 5–60ºC.

The insertion of OmpF in membranes made from mixtures of lipids with different molar ratios such as DOPC:DPPC (1:1) and DOPC:DPPC:cholesterol (1:1:1) was studied. The FRET efficiency was computed and Förster radius derived using an appropriate model. The presence of cholesterol, as a molecule that promotes the lipid rafts formation, does not appear to have a significant influence on OmpF insertion. The fluidity of membrane slightly decreases after the insertion of OmpF, as expected, without a change in the transition temperature of lipid bilayer (in the investigated temperature range).

The results suggest that OmpF insertion into lipid bilayer is not significantly influenced by the heterogeneity of bilayer composition.

3 RSBMM International Conference 2008: Oral presentations 19

[O3] DIRECTED THREE-DIMENSIONAL PATTERNING OF SELF-ASSEMBLED PEPTIDE FIBRILS

V. DINCĂ1,2, E. KASOTAKIS3, A. RANELLA2, M. FARSARI1, A. MITRAKI2,3, A. OVSIANIKOV5, B.N. CHICHKOV1,5, M. DINESCU1, C. FOTAKIS2,4

1National Institute for Lasers, Plasma and Radiation Physics, Romania

2Institute of Electronic Structure and Laser, Foundation for Research and Technology Hellas, 711 10 Heraklion, Crete, Greece

3Dept. Materials Science and Technology, University of Crete, Greece 4Department of Physics, University of Crete, Greece

5Laser Zentrum Hannover e.V., Hollerithallee 8, Hannover, Germany

Molecular self-assembly is emerging as a viable “bottom-up” approach for fabricating nanostructures. Self-assembled biomolecular structures are particularly attractive, due to their versatile chemistry, molecular recognition properties and biocompatibility. Amongst them, amyloid protein and peptide fibrils are self-assembled nanostructures with unique physical and chemical stability. The precise positioning of peptide-based nanostructures is an essential part of their use in technological applications and their controlled assembly, positioning and integration into microsystems is a problem of considerable current interest. Here, we propose a new method for the precise, three-dimensional patterning of amyloid fibrils. The technique is based on the selective attachment of photosensitive biotin (photobiotin) on surfaces and 3D structures and exploits thiol chemistry and self assembly of peptide fibrils. These structures consist of ORMOCER, a photostructurable organic-inorganic hybrid, on which photobiotin can be irreversibly attached when exposed to UV light from a laser or a lamp. The 3D structures are immersed in an aqueous solution of peptides that contain a cysteine residue. The self-assembly of the peptide fibrils into bridges on the structures is initiated through the controlled evaporation of water. The ability of the peptides to form fibrils in solution was confirmed using all the structural criteria that apply to amyloid fibril structural characterization. The length and the diameter of the peptide fibrils depend on the design of the 3D structures. When the peptide used does not contain cysteine, there is no formation of fibrils thin layer and subsequently no bridges.

We have demonstrated a new method for the controlled self-assembly of peptides on 3D structures.


[O4] INVESTIGATION OF THE INFLUENCE OF DIFFERENT PHYSICO-CHEMICAL PARAMETERS ON E. COLI

SUSCEPTIBILITY TO ANTIBIOTICS

O. DRACEA1,2, C. IORDACHE1,2, M. BUCUR2, G. NECULA2, C. UNGUREANU2, C. BALOTESCU1,2, D. CRISTEA1,

M.S. LIXANDRU1, I. RĂUŢ1, V. LAZĂR2 1“I. Cantacuzino” National Institute for Research and Development in Microbiology

and Immunology, Bucharest, Romania 2University of Bucharest, Faculty of Biology, Bucharest, Romania

Our aim was to evaluate the influence of different physico-chemical parameters on E. coli susceptibility to beta-lactam antibiotics. The influence of chemical composition of the culture medium was evaluated for: cephtriaxone (CRO), cefotaxime (CTX), imipenem (IMP), nalidixic acid (NA) and cefoxitine (FOX), by the comparative assessment of inhibition growth diameters on different solid media: Mueller Hinton (MH), Plate Count Agar (PCA), MacConkey (MC) and Eosin Methylen Blue (EMB). In order to evaluate the differences in antibiotic susceptibility of biofilm embedded cells comparatively to the planktonic one, an experimental model simulating the biofilm matrix was used. The influence of temperature was investigated by comparing the minimal inhibitory concentration (MIC) assessed by the classical serial broth dilutions method, performed at different temperatures: 4, 28 and 37°C, using different liquid media MH, MC, Nutrient Broth (CN), Tryptic Soy Broth (TSB). The inhibition diameter zone was much larger on PCA, EMB and MC as compared to MH media. When bacterial cells were included in agar matrix, the growth inhibition diameters obtained for different beta-lactams were different as compared to planktonic cells: for CTX and NA, a reduced zone diameter was obtained, in spite of CRO and IMP, which proved the same efficiency against planktonic bacteria or biofilm embedded bacteria. Our results have demonstrated that liquid media composition also influences the sensitivity of E. coli to CTX, as well as the incubation temperature, the MIC values obtained at 40°C being reduced in comparison with those obtained at 28°C and 37°C. This study demonstrates that culture media composition and incubation temperature strongly influence the antibiotic susceptibility. Our results underline the need for standard methods and specific parameters for the assessment of antibiotic resistance for bacterial cells isolated from biofilm associated infections.


[O5] IRON OVERLOAD AND NON-TRANSFERRIN-BOUND IRON

P. EVANS

Department of Haematology, UCL Cancer Institute, Paul O’Gorman Building, Huntley Street, London WC1E 6BT, UK

Several human diseases, particularly those requiring chronic blood transfusions, can result in iron overload. This has several deleterious consequences as excess iron is toxic to the body and may accumulate in certain organs resulting in dysfunction and even death.

During iron overload, as iron progressively accumulates, storage mechanisms are gradually overwhelmed and transferrin becomes more saturated with iron. Eventually a non-transferrin-bound fraction of iron (NTBI) appears in the circulation which is believed to distribute excess iron to vulnerable organs. We have been interested in characterising NTBI as understanding its nature will lead to more effective ways of removing it from blood plasma, thereby preventing its accumulation in vulnerable organs.

Initially NTBI was believed to be a low molecular mass fraction, possibly a single species such as iron citrate or acetate. However, selective filtration experiments show that it is both heterogeneous and of relatively high molecular mass, possibly through binding to plasma proteins. The heterogeneous nature of NTBI is borne out by its response to chelators both in vitro and in vivo. Only a small proportion of NTBI is redox active, and this form is believed to represent the most reactive and toxic form, reacting relatively rapidly with chelating agents which are used to remove iron during chelation therapy. However, the relatively large proportion of NTBI remaining after chelation therapy can also be taken up into cells causing cell and organ iron overload and eventual organ damage. We need to continue these studies to completely characterise NTBI which will enable us to either seek therapies to completely remove it or to be sure that fractions not removed by chelators are unable to cause cell and organ damage.


[O6] PROTEIN STRUCTURE AND FUNCTION CHARACTERISATION IN PROTEOME BIOMARKER

DISCOVERY

S.J. GADHER

Beckman Coulter International S.A., Nyon 1, Switzerland

Biomarker discovery is like panning for gold where finding the nuggets gets easier when there is less sand. Within bio-fluid proteomes, such as plasma and serum, that “sand” is comprised of well-characterized proteins. Identifying the potentially golden biomarkers in the presence of these highly abundant proteins has proven to be a serious challenge to current analytical techniques. Various novel approaches will be discussed. ProteomeLabTM IgY partitioning system initially plays a major role by reversibly capturing 12 of the more abundant proteins from human fluids (plasma, serum) yielding an enriched pool of low abundance proteins for further study. The application of multidimensional chromatography using ProteomeLabTM PF2D takes what could be a complicated process requiring specialist skills and makes it usable on a routine basis by everyday researchers. The system consists of automated hardware, pre-optimized chemistry and enhanced visualisation software that generate a proteomic map. The principle of Chromatofocusing is used for the first dimension, resulting in fractionation according to the pI of the proteins. The fractions from the first dimension are automatically injected into a reversed phase system where proteins are fractionated according to their hydrophobicity. The use of solid microparticles in this dimension results in highly efficient and fast separation. The end result is a series of liquid fractions containing proteins, making subsequent analysis using various other technologies such as mass spectrometry relatively easier. Additionally, the two-dimensional liquid chromatography protein separation system ProteomeLab PF2D when compared to classical 2D gel electrophoresis provides complementary data thus allowing comprehensive proteomic analysis of protein targets and pathways that are relevant to Biomarker discovery. Above technologies together with the ProteomeLab™ PA 800 Protein Characterisation System may facilitate protein analysis within research and pharmaceutical industry. Utilising an automated platform specifically for development and quality control processes, optimised characterisation methods allow for accurate protein analysis.


[O7] IMINOSUGARS WITH BIOLOGICAL ACTIVITY: SYNTHESIS AND STRUCTURE DETERMINATION BY NMR

A. HÎRTOPEANU1, R.M. MORIARTY2, A. NICOLESCU3, C. DELEANU1, A. POPESCU4, D. ISTRATI4, M. BĂLAŞU4

1“Costin D. Nenitzescu” Institute of Organic Chemistry, Romanian Academy, Bucharest, Romania 2University of Illinois at Chicago, Department of Chemistry, Chicago, USA

3“P. Poni” Institute of Macromolecular Chemistry, Iaşi, Romania 4Polytechnica University of Bucharest, Department of Organic Chemistry, Bucharest, Romania

Iminosugars, sugar analogs with the endocyclic oxygen atom replaced by nitrogen, form one of the most interesting classes of glycomimetics acting as inhibitors of glycoconjugate processing enzymes. Deoxynojirimycin (DNJ) and its N-alkylated derivatives proved to be promising lead compounds for the treatment of diabetes, viral infections (hepatitis B and C, HIV) and other metabolic disorders, due mainly to their action of inhibiting glycosidases. However, their role as inhibitors of glucosylceramide synthase, a key enzyme involved in the synthesis of glycosphingolipids linked to tumor formation, was less studied and only recently their potential in melanoma treatment began to be explored.

Synthesis and characterization of the DNJ core and of its N-alkylated derivatives of deoxynojirimycin with various substituents at nitrogen are presented. There is a close relationship between structure/stereochemistry and biological activity and, in a sufficiently broad series, analytical data may be used for predicting therapeutic leads. The structure and stereochemistry of iminosugars and their derivatives can be fully determined by NMR using 1D and 2D experiments, as will be exemplified in this presentation on the synthesized compounds.


[O8] INTERNALIZATION AND INTRACELLULAR TRAFFICKING OF THE PESTIVIRUS BVDV – A MODEL

FOR HCV

A. MACOVEI, C. LAZĂR, N. NICHITA

Institute of Biochemistry, Department of Viral Glycoproteins, Bucharest, Romania

Bovine viral diarrhea virus (BVDV) is a Pestivirus member of the Flaviviridae family of enveloped, RNA genome viruses. Its similarity with Hepatitis C virus (HCV) promoted BVDV as a good model to study HCV life cycle, a virus with a poor growth rate in tissue culture. Using pH inhibitors such as chloroquine, bafilomycin A1, or ammonium chloride, we showed that BVDV entry into target cells is massively inhibited, suggesting a dependence on low pH to initiate productive BVDV infection. The post-entry events, intracellular localization, virus morphogenesis as well as secretion pathways were investigated using brefeldin A (BFA). This drug blocks protein export from the endoplasmic reticulum (ER) and causes disruption of the Golgi complex with subsequent fusion of its cis and medial cisternae with the ER. BFA treatment of infected cells resulted in complete inhibition of BVDV secretion and increased co-localization of the envelope glycoproteins with the cis-Golgi marker GM130. Processing of the N-linked glycans was affected by BFA, however, virus assembly was not perturbed and the ER-cis Golgi blocked intracellular virions were fully infectious.These suggest that BVDV is using the classical secretory pathways to exit the infected cell; however, trafficking beyond the cis-Golgi compartment is not a prerequisite for pestivirus infectivity.


[O9] ROLE OF EDEM IN THE ER-ASSOCIATED DEGRADATION OF HUMAN TYROSINASE LACKING

THE TRANSMEMBRANE DOMAIN

A. MAREŞ, L.N. SPIRIDON, S. GHENEA, A.J. PETRESCU, Ş.M. PETRESCU

Institute of Biochemistry, Splaiul Independenţei 296, Bucharest, Romania

The endoplasmic reticulum (ER) represents the site of complex processes as folding and first maturation steps of newly synthesized proteins, including glycosylation and disulphide bond formation that take place under stringent quality-control monitoring mechanisms.

Proteins that acquire the correct conformation are transported to the Golgi apparatus. By contrast, misfolded proteins are retained in the ER by the calnexin cycle, and then targeted for proteasomal degradation after repeated trials of refolding.

A major pathway for misfolded protein disposal is the so-called ER-associated degradation (ERAD). The molecular sensors that discriminate between native and non-native proteins are under intense scrutiny. Experimental studies showed that one of the signals that mark a protein for degradation is the formation of isomer B of Man8GlcNAc2. Furthermore, it has been proposed that EDEM (ER Degradation Enhancing α-Mannosidase I-like protein) is involved in the selection of aberrant proteins for ERAD through its lectinic activity.

In this study, we are investigating the role of EDEM in the recognition of both protein and glycan determinants exhibited by misfolded glycoproteins such as mutant tyrosinases lacking the transmembrane domain which was previously shown to be an ERAD substrate. In parallel, we have generated EDEM point mutations predicted by molecular computational modeling to impair the lectin function, or lacking a 21 amino acids stretch within the N terminal region, in an attempt to elucidate the role of these domains on substrate recognition.


[O10] EFFECT OF CEFTAZIDIME ON THE ELECTROPHYSIOLOGICAL PROPERTIES OF OmpF

A. MARIN1, B.M. MACRI1, M. RADU2, M. WINTERHALTER3

1University of Bucharest, Faculty of Biology, Center of Neurobiology and Molecular Physiology, Bucharest, Romania

2National Institute for Physics and Nuclear Engineering “Horia Hulubei”, Group of Biophysics and Radiobiology, Măgurele, Romania

3International University Bremen, Bremen, Germany

The growing number of pathogenic bacteria that display resistance to multiple antibiotics is becoming an increasingly worrying clinical problem. Compounds of the cephalosporin family belong to the most important and most widely used group of antibacterial agents. They interfere with the biosynthesis of the peptidoglycan, a complex polymer of sugars and amino acids constituting the major component of the bacterial cell wall. Before reaching the target, the antibiotics have to cross the outer membrane. The general diffusion porin OmpF (outer membrane protein F) is considered to be the primary gateway for these antibiotics. To study translocation of these antibiotics of different structure across the outer bacterial membrane, we use the analysis of ion currents through single trimeric outer membrane protein F (OmpF) porins reconstituted in planar lipid bilayers, using the dip-tip technique. OmpF was extracted and purified by the group of Prof. Winterhalter. Ceftazidime was used without further purification.

In our experiments the OmpF has inserted into the lipid bilayers preferentially in saline conditions (3 M KCl). In these conditions, traces similar with the published data were recorded. The conductance was estimated at approximately 80.46±4.72 pS (n=8) and mean open time was calculated as 14.85±2.23 ms (n=8). OmpF was accessed from the pipette side, where ceftazidime (56.8 µg/ml) was added from the beginning of our experiments. Our observations indicate an increased conductance of OmpF in 3 M KCl compared to the values from the literature recorded at 0.1 M KCl or 1 M KCl. In addition our study suggests that ceftazidime increases the mean open time of the OmpF trimer. A possible mechanism of ceftazidime translocation through OmpF is discussed. These results are very important for the improvement of the pharmacological dosage of ceftazidime.


[O11] INFLUENCE OF MEMBRANE ELECTROSTATICS UPON REVERSIBLE PROTONATION REACTIONS TAKING PLACE ON THE CONSTRICTION REGION OF THE OmpF

PORIN

L. MEREUŢĂ, A. ASANDEI, T. LUCHIAN

“Alexandru I. Cuza” University, Faculty of Physics, Laboratory of Biophysics and Medical Physics, Blvd. Carol I nr. 11, Iaşi, Romania

In this work we employed single-molecule electric recording techniques to investigate effects of the transmembrane and dipole potential on the reversible protonation of acidic residues from the constriction zone of the OmpF porin. Our results support the paradigm according to which the protonation state of aspartate 113 and glutamate 117 residues from the constriction region of OmpF is influenced by the electric potential profile, via an augmentation of the local concentration of protons near these residues mediated by increasing negative transmembrane potentials. We propose that at constant bulk pH, pKa values for protons binding at these residues increase as the applied transmembrane potential increases in its negative values. Our data demonstrate that the apparent pKa for proton binding of the acidic aminoacids from the constriction region of OmpF is ionic strength-dependent. We present evidence suggesting that lower values of the membrane dipole potential lead to an increase in the values of the ‘on’ rate of the eyelet acidic residues protonation, caused by an elevation of the local concentration of hydrogen ions.


[O12] BEYOND THE TUMOR: FEAR AND HOPE

N. MIRANCEA, D. MIRANCEA

Institute of Biology, Splaiul Independenţei 296, Bucharest, Romania

In a healthy body, the rate of cell proliferation and the temporal-vectorial cell location in a multicellular organism are very carefully regulated processes. Sometimes, a cell achieves un-programmed genetic changes and the mechanisms of control that regulate cell multiplication break down so the cell starts to grow and divide, although the body has no need for further cells of its type. The consequence of such unwanted proliferating cells is the formation of a mass cell termed as cancerous tumor. Tumors resemble wounded tissues that do not heal. The major human cancers are derived from epithelial tissues. A tumor of epithelial origin is represented by per se tumor cells and stroma which consists in a multitude of cell populations and an extracellular matrix (ECM). The cross-talk between neoplastic cells and stroma determines the carcinoma architecture and invasive behavior of malignant cells. The step by step evolution of malignant cell phenotype to invasive tumorigenic behavior involved a cascade of gradually cellular and molecular events. Cell-cell and cell-ECM junctions as well as cytoskeleton are severely altered which leads to the loss of epithelial cell polarization and increase cell migration. The key to answering the major question of what determines tumor cells remains in one place retaining their associations with their neighbors and basement membrane (desmosomal and hemidesmosomal junctions) or dissociate and move elsewhere to arrest at the ectopic sites of metastatic lesions arises from the complex investigation at the ultrastructural and molecular level of changes in cell adhesion properties. Tumor cells are bad neighbors. There is a struggle between tumor cells and peritumoral stroma. Tumor cells made stromal cells their accomplice and grow invasively. Tumor cells induce tumor angiogenesis, but peritumoral blood vessels are immature: they lose basement membrane and pericytes, are fenestrated and exhibit inter- and transendothelial gaps, which favor tumor cell penetration into the blood circulation and multiple secondary tumor formation (the main cause of death). By modulating peritumoral stroma, targeting and halting tumor angiogenesis (using an anti-VEGF Receptor-2 antibody), reprogramming of nuclear genome by nuclear transplantation, despite the presence of irreversible genetic alterations, malignant phenotype can be reverted to the normal phenotype.


[O13] CHARMM FORCE FIELD PARAMETERIZATION OF BACTERIAL LIPOPOLYSACCHARIDES

A. MORARU1, I. ŞVAB1,2, D.F. MIHĂILESCU1

1University of Bucharest, Department of Anatomy, Animal Physiology and Biophysics, Splaiul Independenţei 91–95, Bucharest, Romania

2“N. Simionescu” Institute of Cellular Biology and Pathology, Str. B.P. Haşdeu 8, Bucharest, Romania

Bacterial lipopolysaccharides (LPS) are major components of the outer membrane of Gram-negative bacteria and are known to trigger a variety of inflammatory reactions in macrophages and other cells having a CD14 receptor. LPS consist of a hydrophobic domain known as lipid A (or endotoxin), a nonrepeating “core” oligosaccharide, and a distal polysaccharide (or O-antigen). Only few theoretical models of these structures have been created so far, due to the high complexity of the LPS. The purpose of this study was to obtain the parameters of the LPS molecule for the CHARMM molecular mechanics force field, to be used in Molecular Modeling simulations. It was used the structure of an integral membrane protein (FhuA) from E. coli, with the PDB ID: 1FI1. The parameters for the lipid A region were determined de novo. All the quantum chemical calculations for this molecule were completed using the Gaussian03 software at the Restricted Hartree Fock (RHF) level of theory using the Pople 6–31G* basis set. The optimized geometry delivered the equilibrium values for the bond length, bond angles and dihedrals, and the force constants were derived from the IR calculation. Partial atomic charges and van Der Waals parameters were assigned by finding equivalent parameters in CHARMM force field parameters and topology files version 27. The “core” oligosaccharide contains unusual sugars with seven or eight carbon atoms. This structure was built using a patching method for which a new topology file was determined using VMD extension Molefacture. The parameters obtained for the LPS molecule were tested by creating a hydrated bilayer consisting of LPS, dipalmitoylphosphoethanolamine and TIP3 water model molecules.


[O14] INTERNALIZED LIPID VESICLES TRAFFIC TO THE ENDOPLASMIC RETICULUM, USING

A pH-DEPENDENT ENDOCYTIC PATHWAY

R. MUSTAŢĂ, A. GRIGORESCU, R.A. DWEK, Ş.M. PETRESCU

Institute of Biochemistry, Department of Molecular Cell Biology, Splaiul Independenţei 296, 060031 Bucharest 17, Romania

Few endocytosed ligands, including bacterial toxins and Simian Virus 40 (SV40) have been shown to reach the endoplasmic reticulum (ER) in mammalian cells. Using calcein and fluorescently labelled lactoferrin encapsulated in liposomes we found that the cargo uses a microtubule based pathway with ER delivery. Endocytic uptake of the lipid vesicles was cholesterol dependent in all cell lines tested, including the caveolin-1-deficient human hepatoma 7 cell line. The ligand was transported in non-caveosome organelles requiring acidic pH for maturation, but able to escape the lysosomal route. These organelles were not recycling endosomes either, as shown by the lack of co-localization with recycling transferrin. Co-localization with the ER-Tracker and cholera toxin in live microscopy showed that the fluorescent ligand eventually reached the lumen of the ER with a half life of 1.5 h. Brefeldin A, which prevents Golgi dependent retrograde trafficking, does not disrupt the cargo delivery to the ER. This new endocytic pathway making use of acidic endosome-like organelles is an alternative to the SV40 caveolae pathways that exploits a cellular route linking the cell surface to the ER.


[O15] LUTEIN AND ZEAXANTHIN PROTECT AGAINST INDUCED OXIDATION IN CULTURED HUMAN RPE CELLS

A. PINTEA, D. RUGINĂ, C. MOMEU

University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, Romania

Lutein (L) and zeaxanthin (Z) are the only dietary carotenoids accumulating in the anatomic structures of human retina, including the retinal pigmented epithelium (RPE). They act as screening pigments by absorbing the damaging blue light. RPE plays a crucial role in the functioning of retina and is subject to continuos and high level of oxidative stress. The aim of this study is to investigate the effect of lutein and zeaxanthin on the oxidative status of RPE cultured cells in induced oxidative stress.

D407 RPE cells were cultivated in DMEM medium with 10% FCS. The cells viability was estimated by the MTT assay and the effect on the cell morphology by microscopy. The generation of intracellular ROS was determined by using a fluorescent probe (DCF-DA), reduced glutathione by DTNB assay, and antioxidant enzymes glutathione peroxidase (GPx) and superoxidedismutase (SOD) by using commercial kits. The oxidative stress was induced by adding a non-letal concentration of hydrogen peroxide.

Lutein and zeaxanthin were delivered to the cells in the physiological concentration of 10 mM. The incorporated amount/yield was 1.2 nmols/106 (6%) cells for lutein and 1.7 nmols/106 cells (8.5%) for zeaxanthin at 24 h. The cells viability was similar for control cells and cells treated with carotenoids. For cells treated with hydrogen peroxide the viability was significantly lower for both control (C) and carotenoids, but higher in carotenoids treated cells (59% for Z+H2O2, 49% for C+H2O2). The intensity of DCF fluorescence was lower in cells treated with L and Z comparing with C, explained by a quenching of oxygen reactive species. L did not significantly influence the GSH content but Z determined an increase of GSH concentration in control cells and cells with H2O2. Z also induced an increase of GPx and SOD activities in cells lysate.


[O16] INNOVATIVE TECHNOLOGIES FOR HYBRID ORGANIC-INORGANIC NANOBIOMATERIALS SYNTHESIS

R.M. PITICESCU1, G.C. CHITANU2, G. NEGROIU3, I. MIHĂILESCU4, L.E. SIMA3

1National Institute for Nonferrous and Rare Metals, Pantelimon, Romania 2“Petru Poni” Institute of Macromolecular Chemistry, Iaşi, Romania

3Biochemistry Institute of the Romanian Academy, Bucharest, Romania 4NationaI Institute for Lasers Physics, Plasma and Radiation, Măgurele, Romania

Nanotechnology allows for the improvement of nonresorbable biomaterials and effective manipulation of biological interactions at the nanometer level, which will dramatically increase the functionality and longevity of implanted materials. By applying bioactive nanoparticle coatings on the surface of implants, it will be possible to bond the implant more naturally to the adjoining tissue and significantly prolong the implant lifetime. Composite systems combining the advantages of polymers and ceramics seem to be a promising alternative, in particular for tissue engineering. We propose two innovative techniques to synthesise nanostructured organic-inorganic hybrids thin films on titanium alloys substrate, namely hydrothermal-electrochemical procedure and MAPLE technique. Hydrothermal-electrochemical procedure implies the in situ deposition of nanostructured hybrids under high pressures and low temperatures on titanium alloy substrate. MAPLE technique implies two steps: in the first step nanostructured organic-inorganic hybrids with strong bonding between components are synthesised by hydrothermal procedure under high pressures and low temperatures. In the second step stabilised suspensions of the as synthesised powders are deposited. Nanostructured hybrids powders were characterised by chemical quantitative analysis (inductively coupled plasma-ICP; atomic absorption spectrometry-AAS), XRD analysis to reveal crystallite sizes and microstructure, scanning electron microscopy to reveal morphology and Fourier transform infrared spectroscopy to reveal the strong bonding between components. Thin films were characterised by TEM to reveal the crystallite sizes and nanostructure. The analysis of cytoskeleton, nuclei and cell viability tests demonstrated that cells adhered and proliferated on hybrid active layers and that polymer enhances the biocompatibility properties.


[O17] THE EMERGENCE OF MULTICELLULARITY VIA GLYCAN SELF-ASSEMBLY

O. POPESCU1, G.N. MISEVIC2 1“Babeş-Bolyai” University, Institute for Interdisciplinary Experimental Research, Faculty of Biology and Geology, Molecular Biology Center, 42 August Treboniu-Laurian Street, 400271 Cluj-Napoca,

Romania 2Laboratoire “Assemblages Moléculaires: Modélisation Imagerie et SIMS”, FRE CNRS 2829,

Faculté des Sciences de l’Université de Rouen, 76821 Mont Saint Aignan cedex, France

Research done in the past century on marine sponges (Porifera) has provided insights into the molecular mechanism of the biological processes of cell adhesion, innate immunity, and self-recognition. Evidence that this mechanism is based on glyconectin self-assembly is shown by the structure-function relationships inferred from studies of isolated glycans from three different marine sponge species. The structural studies were performed on purified glyconectin glycans from Microciona prolifera, Halichondria panicea and Cliona celata using nuclear magnetic resonance and mass spectrometry. Seventeen novel, species-specific carbohydrate sequences that belong to the Porifera glyconectin family were revealed. The functional, cell recognition analyses of carbohydrate self-association were performed by measuring binding forces between individual glycan molecules under physiological conditions. The results show that the binding strength between homotypic pairs of glycans (400 pN) is higher than those between heterotypic pairs (20 pN). This difference is sufficient to explain the species-specific separation of glycan-coated beads in vitro and the sorting of sponge cells in vivo. We suggest that the glyconectin glycans, which are the constituents on the cell surface that are the most exposed to the environment, were responsible for the molecular recognition processes that underpinned the emergence of multicellularity.


[O18] PRIMARY HUMAN OSTEOBLASTS ISOLATION AND IN VITRO INTERACTION WITH IMPLANT

NANOMATERIALS

L.E. SIMA, Ş.M. PETRESCU

Institute of Biochemistry of the Romanian Academy, Department of Molecular Biology of the Cell, Spl. Independenţei 296, Bucharest, Romania

Osteoblasts are widely used to test the interaction of implant nanomaterials with bone cells. We have chosen two methods of obtaining primary human osteoblasts: i) isolation of mesenchymal stem cells (MSCs) from human bone marrow aspirates followed by in vitro differentiation to osteoblasts and ii) generation of osteoprogenitor cells by in vitro culture of trabecular bone pieces. Our goal was to characterise the interaction of bone cells with biomaterials.

Bone marrow MSCs were isolated by separation on ficoll gradient and adherent culture. Cells were seeded and expanded in DMEM until they reached 80% confluence. At second passage they were harvested and analysed by flow cytometry to confirm the MSC phenotype. Adult stem cells were differentiated for 21–28 days in osteogenic media. Osteoprogenitor cells were also obtained by bone explant. Both osteoblasts lines were analysed in terms of specific markers expression (alkaline phosphatase, osteocalcin, BMP-2 and BSP) using epifluorescence microscopy. Mineralization was tested by Alizarin Red staining. Differentiated cells were grown onto both biomaterial and standard surfaces. Biomaterials were obtained by pulsed laser deposition (PLD) or magnetron sputtering of thin films of hydroxyapatite on titanium. Biocompatibility tests were performed which consisted in morphology, viability, proliferation, and adhesion assays. Cell morphology was visualized by scanning electron microscopy. Osteoblasts viability was quantitated by MTS assay. Proliferative cells were assessed using Ki67 nuclear marker. We analysed cell adhesion on biomaterials by actin/vinculin fluorescent labeling. Specific markers for MSCs and osteoblasts have been determined in order to characterize the isolated cells. Viability, adhesion and proliferation of human primary osteoblasts on both PLD and plasma-sprayed HA as well as on standard material were determined. Biocompatibility studies proved that HA improves in vitro osteoprogenitor cell adhesion and function and is therefore a promising coating material for implantology.


[O19] PLANT AND FOOD METABOLOMICS: ADVANCED ANALYTICS AND STATISTICS

C. SOCACIU, F. RANGA, F. FETEA, A. BUNEA, M. TRIF, F. DULF, C. BELE

Department of Biochemistry, University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, Romania

“Metabolomics” aims the characterization of a specific metabolic profile (fingerprint) characterizing a living organism or tissues (including food matrices). Advanced methods are applied nowadays to characterize the most relevant metabolites (i.e., sugars, organic acids, pigments, hormones) in a sample such as plant tissues, fruits, blood, urine, using chromatographic (HPLC, LC-MS, GC-MS) or spectrometry (FTIR, NMR, isotopic analysis) techniques.

We used different plant varieties cultivated in Romania or other geographical areas (especially medicinal plants like Echinaceea sp. and Melissa off., but also edible oils and fruit juices) to identify their biochemical markers and to prove their authenticity. Generally, the techniques we used were HPLC-PDA, LC-MS and FTIR, coupled also with UV-Vis spectrometry and GC-FID. The target molecules used to fingerprint the metabolome were phenolic derivatives (including flavonoids and catechins, anthocyanins), carotenoids, fatty acids, phytosterols. We were able to identify by HPLC the specific molecules which characterize Echinaceea or Melissa varieties (e.g., rosmarinic acid, cichoric acid) as well to identify in oils and in juices the authenticity markers (phytosterols, fatty acids, and phenolics or pigments, respectively). Chemometric advanced statistics using Principal Component Analysis, Partial Least Square for FTIR were useful techniques to find a distinctive pattern of absorption, characterizing the fingerprint regions. These methods have a good perspective for the biological and geographical origin determination, as well as to find specific metabolic pathways related to taxonomic markers in plants or plant-derived food products.


[O20] BUILDING A DATABASE OF LRRs STRUCTURAL INFORMATION

L.N. SPIRIDON1, A. GOVERSE2, A.J. PETRESCU1

1Institute of Biochemistry, Department of Structural Biochemistry, Bucharest, Romania 2Wageningen University, Department of Nematology, Wageningen, Netherlands

Resistance gene proteins (R) are part of essential defense mechanisms in plants. R-proteins consist in a LRR (Leucine Rich Repeat) C-ter domain, a NB-ARC effector domain and a N-ter signal transducer domain. Modelling R proteins is very useful in engineering of breeding plants and by far the most challenging effort in this feild is to develop reliable representations of the LRR region.

LRR is an old structural motif found in proteins involved in very diverse cellular pathways. LRRs are found throughout the life kingdom, e.g., bacteria, algae, yeast, vertebrate and plant, and as it is known so far, they are almost entirely involved in recognition processes. LRR domains have a specific right-handed beta-alpha superhelix topology (CATH 3.80.10) of a horseshoe shape and their surface can be divided into a concave part, a convex hyper-variable part and two lateral parts.

Despite the similar topology, LRRs are very diverse both in terms of sequence and local 3D structure making critical a thorough investigation of sequence to local structure mapping for probabilistic modelling purposes. To this end we have developed a software to scan protein databases for structures containing LRR. The structures thus retrieved were clustered down to 28 non-redundant structural architectures. Using primary, secondary and tertiary structure information on this set a relational database was built. The structural characterization consisted in defining properties (parameters) such as the overall curvature, out-of-plane skew or twist. We correlated these properties with the information that can be extracted from sequence alone, in order to extrapolate it to eventual target sequences. This database and its analysis proved especially effective in designing a method for modelling LRRs of variable lengths between the repetitive motifs named the Optimized Joint Fragments Remote Homology Modelling procedure.


[O21] STRUCTURAL STUDIES ON KIM-CONTAINING PROTEIN TYROSINE PHOSPHATASES

Ş.E. SZEDLACSEK, M. BĂLAŞU, L.N. SPIRIDON, A.J. PETRESCU

Institute of Biochemistry, Bucharest, Romania

KIM-containing protein tyrosine phosphatases (PTP) represent a family of PTPs that are distinguished by the presence of a Kinase Interaction Motif (KIM) located N-terminal to the catalytic PTP domain. KIM can be subdivided into a highly conserved motif responsible for interacting mitogen activated protein kinases (MAPK) and a sequence playing a role in determining specificity for certain members of the MAPK family. These PTPs are highly specific in their substrate recognition, displaying preference to ERK1/2 (extracellular-signal-regulated kinase 1/2) and p38, over JNK (c-Jun N-terminal kinase). The activity of ERK2, an essential component of the MAPK pathway, is under the strict control of various effector proteins, including kinases and phosphatases. Protein tyrosine phosphatase SL (PTP-SL) is one of its major regulators. We succeeded to determine the 3D structure of PTP-SL, that representing the first structure of a KIM-containing PTP. However, we could not obtain crystals for its complex with ERK2. In general, despite numerous efforts, no crystal structure of ERK2 complexed with any of its protein partners has been obtained so far.

Using a combined procedure based on cross-linking of ERK2-PTP-SL complex, size exclusion chromatography, isothermal titration calorimetry and MALDI-TOF analysis of the non-digested or trypsin digested cross-linked complex, we obtained data regarding the interacting regions, stoichiometry and thermodynamics of the complex formation. The experimental data thus obtained, together with the crystal structures of ERK2 and catalytic domain of PTP-SL, were used to elaborate a structural model of the ERK2-PTP-SL complex. This was generated by docking, homology and ab-initio modeling, followed by structure refinement with molecular dynamics simulated annealing. The model provides insights into the structural basis of experimental findings reported in literature such as the dissociating effect of ATP on the complex, or the PTP-SL blocking effect on ERK2 export to the nucleus. In addition, new interacting residues at the complex interface are suggested, providing thus new elements for further basic and applied studies towards understanding the regulation of ERK2 activity in different signaling processes.


[O22] MOLECULAR SIMULATION OF GRAM-NEGATIVE BACTERIA OUTER MEMBRANE AND ITS INTEGRATED

PORINES

R. TĂCUTU, L.N. SPIRIDON, M. MICLUŢĂ, A.J. PETRESCU


Understanding the molecular mechanisms of antibiotic resistance is very important due to the high frequency of this phenomenon and helps to improve the efficiency of the therapies against bacteria. In the case of Gram-negative bacteria, beta-lactamic antibiotics and fluoroquinolones penetrate the outer membrane through special transport proteins named porines. One of the methods that bacteria are using for resistance is impermeability by using modified porines.

In studying Gram-negative resistance mechanisms the in silico approach might prove useful not only for the understanding of the system behavior at atomic level, but also to assist the experimental studies. The in silico description includes both building the structural model of the system and the simulation of its dynamics, in order to assess its evolution in time.

In our case the model is composed of a porine (OmpF) included in a bilayer composed of lipopolysaccharide (LPS) molecules on the one side and phosphatidyl ethanol amine on the other.

In order to build this model we first set the molecular dynamics parameters for a single LPS molecule using Glycam04 and Amber parameter DB force fields. The outer membrane was built by optimizing the geometry of a small bilayer (6 LPSs) and then multiplying it so that it can incorporate the porine protein in it. We used the in-house developed software Glyco-Pack to incorporate the porine with its channel perpendicular to the membrane plane. MD simulation was performed with Amber. This revealed sharply distinct dynamic regimes of the glucidic part of the LPS as compared to the lipid moiety. This is due to both the steric compactness of the glucidic part of LPS and the highly dense charge network formed by the LPS and its mobile counterions. The reference antibiotics were ceftazidime and ciprofloxacine.


[O23] RATIONAL DESIGN OF ANTIMICROBIAL PEPTIDES: STRATEGY FOR DRUG DISCOVERY

S. AVRAM, B.M. MACRI

University of Bucharest, Faculty of Biology, Department of Physiology & Biophysics, Splaiul Independenţei 91–95, Bucharest, Romania

Over the past decade, antimicrobial resistance has emerged as a major public health crisis. Antimicrobial polypeptides such as Melittin, Magainin and Dermaseptin are clinically important for the treatment of many infections. Here, structural and energetic information over 20 antimicrobial peptides were obtained by in silico methods.

3D structures of antimicrobial peptides: Melittin, Magainin-1, Magainin-2, Lactoferricin B , Gramidicin A , Gramicidin S, Dermaseptin, Dermaseptin derivative, Nisin, Indolicidin, Cecropin A, Cecropin B, Cecropin P1, Amphotericin B Bactenecin, Cinnamycin, Mastoparan-7 (wasp venom), Mastoparan (Polistes jadwagae), Mastoparan (vespula), Vancomycin and Alamethicin were performed in Sybyl 7.3 (BIOPOLYMERS) using (i) available crystal structures (PDB file) or (ii) computational mutagenesis tools. The minimum potential energy of antimicrobial peptides was calculated by conjugate–gradient method, convergence of 0.05, Tripos force field. Kollman partial charges were loaded. The peptides homology was used as tool to predict the antimicrobial mechanism.

The predicted secondary structures of Magainin-1, Dermaseptin, Cecropin A, Cecropin B, Cecropin P1, Mastoparan, performed by Garnier_Osguthorpe_Robson, are presented. For few antimicrobial peptides (Gramicidins, rms=0.22; Magainins, rms=0.8 and Mastoparans, rms=0.25) similarity of antimicrobial mechanism was evaluated by amino acid multiple alignment. Energetic terms (H-bond, van der Waals, torsion and electrostatic) were collected.

The structural information on antimicrobial peptides gives us the possibility to design new more potent antimicrobial peptides.

POSTERS

Session 1: Intracellular Trafficking

ROM. J. BIOCHEM., 45, 1, 41–47 (2008)

[P1] PROCESSING OF TYROSINASE MUTANTS IN HUMAN MELANOMA CELLS

G. CHIRIŢOIU, Ş.M. PETRESCU

Institute of Biochemistry, Molecular Cell Biology Department, Splaiul Independenţei 296, 060031, Bucharest 17, Romania

Tyrosinase (monophenol dihydroxyphenylalanine:oxygen oxidoreductase) is a type Ι membrane protein regulating the pigmentation process in humans. Tyrosinase is responsible for catalyzing the first two steps of the melanin synthesis pathway: hydroxylation of tyrosine to dihydroxyphenylalanine (DOPA) and its subsequent transformation to DOPA quinone. The absence of tyrosinase activity is associated with oculocutaneous albinism. Tyrosinase folds in the ER and is transported to the trans Golgi network where two copper ions are incorporated. From here it continues its journey to melanosomes where it initiates the melanin synthesis. This protein is a melanoma antigen and an important target for anti-melanoma vaccine therapies.

An important but yet unresolved problem is the dissection of the specific mechanism involved in tumor antigen down-regulation in some melanoma tumors.

In this project we aimed to characterise the processing of tyrosinase and tyrosinase mutants expressed in A375, an amelanotic human melanoma cell line which lacks tyrosinase. As tyrosinase mutants we used two soluble proteins, ST-474 and ST-405, which lack the transmembrane domain, and are retained in the ER and degraded through the proteasomal pathway.

In order to obtain transiently transfected cells, we have tested the following transfection reagents: lipofectamine 2000, FuGene, PEI and jet-PEI. In addition, several conditions of the electroporation methods were used. Transfected cells were characterized in terms of tyrosinase expression, DOPA-oxidase activity, degradation in proteasomes and glycosylation.

RSBMM International Conference 2008: Poster session 1 2

44

[P2] ROLE OF HYPOXIA IN THE REGULATION OF TYROSINASE SYNTHESIS

M. CHIRIŢOIU, S.M. PETRESCU

Institute of Biochemistry, Molecular Cell Biology Department, Splaiul Independenţei 296, Bucharest 17, Romania

Melanoma is one of the most aggressive forms of skin cancer, resulting as a consequence of the un-controlled growth and division of transformed melanocytes in the epidermis. One of the first factors involved in cancer development is hypoxia, a decreased availability of oxygen which favors tumor progression. It has been reported that in hypoxic conditions inside cells the hypoxia inducible factor-1 (HIF-1) is activated. HIF-1 consists of two subunits: a constitutively expressed β-subunit (nucleus) and an oxygen-sensitive α-subunit with cytoplasmic location in normoxia and nuclear in hypoxia. In melanocytes and melanoma cells a key enzyme of melanogenesis is tyrosinase, that catalyzes the conversion of tyrosine to 3,4-dihydroxyphenylalanine (DOPA) and DOPA to DOPAquinone, a chain of reactions that finally leads to melanin synthesis. Previous experiments have shown that microphthalmia-associated transcription factor (MITF) is involved in tyrosinase transcription. This factor is activated by α-MSH-cAMP pathway and it binds to the tyrosinase and TRP promotor increasing the expression of these genes. Recent studies have demonstrated that MITF induces HIF1α expression, through the same c-AMP dependent pathway, possibly contributing to angiogenesis and cell survival.

In this work we investigated the role of HIF-1 overexpression in cells expressing tyrosinase. HIF-1 was induced: a) with reagents that mimic hypoxia; b) with proteasome inhibitors; c) by transient transfection in melanocytic and non melanocytic cell lines. The activity and protein level of tyrosinase constitutively expressed in melanoma cells and overexpressed in non-melanocytic cells was investigated in conditions yielding HIF-overexpression. Further we analysed the intracellular trafficking of HIF-1 and tyrosinase in hypoxic cells. Our data showed that both tyrosinase and HIF-1 expression and trafficking were modulated by the absence of oxygen in melanoma cells.

3 RSBMM International Conference 2008: Poster session 1

45

[P3] PRELIMINARY STUDIES ON RAGE (ADVANCED GLYCATION END PRODUCT RECEPTOR) INTRACELLULAR

TRANSPORT AND BINDING PROTEINS: CLONING AND EXPRESSION

I. POPA, E. GANEA


Hyperglycemia is accompanied by an accelerated production of advanced glycation end products (AGEs), which represent a heterogeneous group of proteins covalently modified through nonenzymatic glycosylation reactions. AGEs accumulation over long periods of time in diabetes or aging exerts deleterious effects on wall vessel cells, leading to vascular dysfunction and progression of atherosclerotic lesions. AGEs promote perturbation of cellular activities partly by activating receptors on cell surface for AGEs, including RAGE. Besides AGEs, other known RAGE ligands are amphoterin, members of S100/calgranulin family, and amyloid fibrils, engaging RAGE in the pathologic cellular activation present in cancer and Alzheimer’s disease.

Commonly, early endosomes represent the entry station for internalized material, where the fate of cargo molecules is established by a complex mechanism of sorting and transport to other endocytic compartments. Since the intracellular distribution of RAGE is insufficiently investigated we are interested in the characterization of the intracellular transport route of the receptor and its ligand. Additionally, identification of new RAGE binding proteins is believed to bring an important contribution to the understanding of the mechanisms underlying its trafficking and signaling properties.

In the present work we describe the obtaining of basic experimental tools to initiate the characterization of RAGE intracellular transport and binding partners. The receptor and its intracellular tail were cloned into vectors for bacterial and mammalian expression. The glutathione S-transferase (GST) fusion protein of the receptor tail was produced in Escherichia coli DE3 strain, and purified on glutathione beads, to be used for GST pull-down assay. Antibodies against RAGE have been tested in cells by Western blot and immunofluorescence for the detection of endogenous and ectopically expressed RAGE.


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[P4] AGEs AND TGF-β1 COOPERATE IN THE INDUCTION OF TYPE I COLLAGEN PRODUCTION IN CCD-1070Sk

CELL CULTURE

I.A. ŞERBAN1, M.C. MUNTEANU2, V. TICĂ2, I. GÂJÂILĂ1, M. COSTACHE2, A. DINISCHIOTU2

1University of Agricultural Sciences and Veterinary Medicine, Faculty of Veterinary Medicine, Bucharest, Romania

2University of Bucharest, Faculty of Biology, Bucharest, Romania

Excessive accumulation of extracellular matrix proteins and skin aging are considered to be due to advanced glycation end products (AGEs) formation. The transforming growth factor (TGF-β1) seems to play an important role in these processes. The present study has examined the effects of soluble AGE-modified BSA (AGE-BSA) on TGF-β1 in relation to type I collagen gene regulation and protein expression in cultured human skin fibroblast cells (CCD-1070Sk).

The 70% confluent CCD-1070Sk fibroblast cultures (passage 3–5) were exposed to serum-free media containing BSA (control) and AGE-BSA at concentrations between 50 and 200 µg/ml for 12 h. The mRNA expression of procollagen α2 (I) and TGF-β1 was analyzed by quantitative real-time PCR. Type I collagen was assessed in culture media by Western immunoblotting. The TGF-β1 protein levels were determined with an ELISA kit. From all concentrations of AGE-BSA, that of 50 µg/ml up-regulated the mRNA expression of both procollagen α2 (I) and TGF-β1 with the highest increase of relative expression ratio (R) to 5.85± 0.14-fold (p<0.01) respectively 3.07± 0.32-fold (p<0.01) after 12 h of treatment. At 100 µg/ml AGE-BSA, R decreased to 2.92± 0.33-fold (p<0.05) for procollagen α2 (I) and 1.77± 0.35-fold (p<0.05) for TGF-β1, whereas at 200 µg/ml there was a trend for down-regulation of procollagen α2 (I) and TGF-β1 mRNA. The protein levels in the culture media correlated with the mRNA expression of procollagen α2 (I) and TGF-β1.

Thus, TGF-β1 may function as a key signaling intermediary in the AGE-up-regulated collagen gene expression pathway in CCD-1070Sk cells.


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[P5] INTERACTION OF XTP3-B WITH TYROSINASE MUTANTS

G. VASILE, Ş.M. PETRESCU

Institute of Biochemistry, Spl. Independenţei 296, Bucharest, Romania

Terminally misfolded or unassembled proteins in the early secretory pathway are degraded by a ubiquitin and proteasome dependent process known as ER-associated degradation (ERAD). How substrates of this pathway are recognized within the ER and delivered to the cytoplasmic ubiquitin-conjugating machinery is unknown. XTP3-B/Erlectin is a newly discovered ER-resident glycoprotein that binds to ERAD substrates.

To characterize the involvement of XTP3-B in ERAD and its interaction with degradation substrates we have generated the XTP3-B protein tagged at the C-terminus with V5 and Flag. The expression of the two proteins was validated using a cell-free system. Intracellular localization and interaction with ERAD substrates of XTP3-B were studied in Hek293 mammalian cells by transient expression using several techniques as metabolic labeling, immunoisolation, Western blot and immunofluorescence. As ERAD substrates we used tyrosinase and various mutants of this protein. The preliminary results showed that XTP3-B interacts with these substrates, but no effect on the degradation kinetics of these substrates could be detected.


48

POSTERS

Session 2: Protein Structure and Function

ROM. J. BIOCHEM., 45, 1, 49–62 (2008)

[P6] IDENTIFICATION OF AN IN VITRO SUBSTRATE OF PROTEIN TYROSINE PHOSPHATASE RPTPµ

R. BADEA, Ş.E. SZEDLACSEK

Institute of Biochemistry, Enzymology Department, Bucharest, Romania

RPTPµ is a type IIB receptor PTP containing an extracellular domain that mediates homophilic, cell-adhesive interactions, and an intracellular region containing the catalytic domains that modulate the phosphorylation state of cadherin/catenin complexes. It has been demonstrated that RPTPµ can interact with members of the cadherin family, catenin p120 and phosphatidylinositol-phosphate kinase type Iγ90 (PIPKIγ90), reducing the phosphorylation level of these molecules. Although RPTPµ plays an important role in some physiological processes like neurite outgrowth, little is still known about its physiological substrates and how they are involved in the cellular process. In our study, we focus on the identification of a new potential substrate of human RPTPµ. According to our previous studies, synthetic phosphopeptides, submitted to dephosphorylation by RPTPµ and analyzed by colorimetric and microarray assays, suggested preferential dephosphorylation of a phosphopeptide having its sequence identical to that surrounding tyrosine Y753 of human phospholipase Cγ2 (hPLCγ2). hPLCγ2 is highly expressed in platelets, B-cells and in some nervous cells. It is activated by phosphorylation of its tyrosine residues Y753, Y759, Y1197, Y1217 and Y1245. In this study we performed in vitro experiments to test PLCγ2 as a potential substrate of RPTPµ. Thus, the SH2-SH2-SH3 segment of hPLCγ2 containing Y753 and Y759 sites as well as mutants at each of the two tyrosine sites: Y753F and Y759F were cloned as His-tagged proteins in a bacterial expression vector. The three recombinant plasmids and a construct containing the active catalytic domain of RPTPµ fused with a GST-tag were separately expressed in E. coli and purified by affinity chromatography. The purified recombinant proteins were submitted to in vitro phosphorylation by Lck kinase and then tested for dephosphorylation by the catalytically active domain D1 of human RPTPµ. Dephosphorylation of recombinant hPLCγ2 constructs was monitored by immunoblotting using an anti-phosphotyrosine antibody. SH2-SH2-SH3 segment non-mutated and with Y759F mutation, but not the construct with Y753F mutation were efficiently dephosphorylated by different concentrations of D1 domain of RPTPµ. Our results demonstrated that, at least in vitro, PLCγ2 is a potential substrate of RPTPµ.

RSBMM International Conference 2008: Poster session 2 2 52

[P7] MOLECULAR DYNAMICS SIMULATIONS ON MODELLED FREE AND LIGANDED GP 120 STRUCTURES

O. CALBOREAN, D.F. MIHĂILESCU

University of Bucharest, Faculty of Biology, Bucharest, Romania

Entry of human immunodeficiency virus type 1 (HIV-1) into host cells requires its gp120 envelope glycoprotein to bind to two cell-surface receptors, CD4 and a co-receptor, either CCR5 or CXCR4. Any rational attempt to design anti-HIV inhibitors or vaccines must involve the characterization of the conformational states available to the gp120. The full-length HIV-1 gp120 has eluded structural analysis. However, core structures of gp120 from HIV-1 and from simian immunodeficiency virus (SIV) with deleted V1, V2 and V3 variable loops N- and C-termini have been crystallized.

Our studies’ aim is to derive the complete 3D structure of the free (unliganded) gp120 using experimental elements from different viral strains derived in different crystallization conditions. The reconstruction procedure is based on X-ray or NMR-derived primary data of certain structures: the unliganded, completely glycosylated, V3 loop-missing SIV structure; structures liganded to CD4 and antibody (HIV); the structure liganded to b12 antibody; the liganded structure containing V3 loop (HIV); the liganded structure with CD4 and CCR5; V3 loop and C5 domain in solution. The 3D structures were modelled by protein structure homology. Each of the manually adjusted alignments between the primary structures of known states and the ~100 strains modelled was used to generate the following 3D structures: the free gp120, the CD4-bound, the b12-bound, and CCR5-bound. All N-glycosylations were added in accordance with experimental data. The refinement of the ~400 structures and the determination of intermediary states were performed by molecular dynamics (MD) simulation computations. As a preliminary result, we present the extensive simulations on a complete and fully glycosylated structure of SIV gp120 in aqueous solution.

The present work proves the usefulness of the MD computer simulation method in providing information on the structure of proteins that are resistant to determination by experimental methods.

3 RSBMM International Conference 2008: Poster session 2 53

[P8] ANTI-INFLAMMATORY ACTIVITY OF LACTOFERRIN, RECOMBINANT LACTOFERRIN

AND LACTOFERRICIN DERIVED PEPTIDE

F. CHELU1, M. ICRIVERZI1, M. MOISEI1, M. TRIF1, I. MATTSBY-BALTZER2, A. ROŞEANU1

1Institute of Biochemistry, Ligand-Receptor Interaction Group, Splaiul Independenţei 296, Bucharest, Romania

2Department of Clinical Bacteriology, University of Göteborg, Guldhedsgatan 10, Göteborg, Sweden

Lactoferrin (Lf) is an iron-binding glycoprotein which exhibits a variety of properties including anti-inflammatory activity. The Lf derived peptide lactoferricin (Lfcin) retains the activities of the intact protein, being in many cases even more active. Given their biological effects, Lf and Lfcin are potential useful compounds for pharmaceutical and food-related applications. Due to the difficulty in obtaining high amounts of human milk Lf (hLf), different methods have been developed to obtain human recombinant Lf and synthetic peptides analogues to Lfcin.

In a previous study it has been reported the capacity of hLf to inhibit the LPS induced Il-6 secretion in a human monocytic cell line THP-1. Our aim was to extend this investigation by evaluating the anti-inflammatory activity of recombinant hLf (rhLf) and of a synthetic peptide corresponding to α helix region of hLfcin. Their effect was compared to that obtained with native protein. THP-1 cells stimulated with bacterial endotoxins (LPS) were used as experimental model of inflammation. We found that both rhLf and synthetic peptide inhibited the secretion of LPS-induced pro-inflammatory cytokines TNFα, IL-6 and IL-8. The level of inhibition of these cytokines was similar to the one resulted from the treatment with native hLf. As for hLf, the inhibitory effect was observed regardless whether rhLf or the peptide was added to the cells, before or after the stimulation with LPS. The iron content of protein does not affect its anti-inflammatory capacity.

The results are encouraging for a possible future application of rhLf and Lfcin analog peptides as anti-inflammatory agents. (This work was supported by a Swedish Institute Project and Romanian Academy Grant. We thank Ventria Bioscience for donating us the recombinant lactoferrin for this study).


[P9] COMPARISON BETWEEN THE FRACTAL CHARACTERISTICS OF MONOMER AND MULTIMER

PROTEINS

D. CRĂCIUN1, A. ISVORAN2, N.M. AVRAM3

1West University of Timişoara, Teacher Training Department, Blvd. V. Parvan 4, 300223 Timişoara, Romania

2West University of Timişoara, Department of Chemistry, Str. Pestalozzi 16, 300115 Timişoara, Romania

3West University of Timişoara, Department of Physics, Blvd. V. Parvan 4, 300223 Timişoara, Romania

The aim of this work is to compare the fractal characteristics of monomer and multimer proteins structures. We determine the surface fractal dimension (Ds) and the backbone fractal dimensions (D1 and D2) for a randomly chosen two unbiased sets of 30 proteins each: one for monomers and the other for multimers. The structural data necessary for calculations have been retrieved from the Protein Data Bank. The surface fractal dimension is calculated using an on-line free accessible tool, GETAREA, and the backbone fractal dimensions are calculated using an in house TurboPascal program. There are two fractal dimensions associated to the protein backbone length: one is obtained for a small interval of amino acids and it is correlated to the local folding (D1) and the other one is obtained for larger intervals of amino acids and it is correlated to the global folding (D2).

The mean value of the surface fractal dimension of monomers is 2.297±0.083 and that of multimers is 2.216±0.067, the two means being significantly different. The mean values of the backbone fractal dimensions are D1=1.345±0.006 and D2=1.147±0.014 for the monomers and D1=1.363±0.005 and D2=1.141±0.032 for multimers, respectively. These values are not significantly different. The surface fractal dimensions of monomer differ from those of multimers and it underlines the importance of the shape complementarity and the surface roughness for protein subunits association. The backbone fractal dimensions do not differ for monomer and multimer proteins being correlated to the protein folding not to the subunits interactions.


[P10] BIOLOGICAL PROPERTIES OF BOVINE LACTOFERRIN-DERIVED PROTEOLYTIC MIXTURE

OF PEPTIDES: IN VITRO STUDIES

P. FLORIAN1, F. CHELU1, M. ICRIVERZI1, V. CARNICELLI2, A.R. LIZZI2, A. ORATORE2, M. TRIF1, A. ROŞEANU1

1Institute of Biochemistry, Ligand-Receptor Interaction Group, Splaiul Independenţei 296, Bucharest, Romania

2University of L’Aquila, Department of Biomedical Sciences and Technologies, Via Vetoio, 67100 L’Aquila, Italy

Lactoferrin (Lf) is a multifunctional iron-binding glycoprotein present in almost all mammalian secretions. It has been reported that cationic peptides isolated after enzymatic digestion of bovine lactoferrin display higher antimicrobial property than the native protein and showed an inhibitory effect on tumor cells growth. The mechanism of their action is not fully understood yet.

Our aim was to investigate by in vitro studies whether the proteolytic mixture of peptides derived from iron-desaturated bovine Lf (ApoBLf) possess anti-inflammatory, antimicrobial and antitumoral activities.

ApoBLf was treated with trypsin and the enzymatic hydrolysate was then subjected to ultrafiltration with ultrafree-MC Microcentrifuge Filters NMWL 5,000 to separate peptides with high (>5000 kDA) and low molecular weight (<5000 kDA). We found that both fractions inhibited LPS-mediated production of pro-inflammatory cytokines IL-6, IL-8 and TNF-α in a human monocyte-derived macrophage cell line THP-1. The antimicrobial activity was present only in the lower MW fraction as demonstrated by the minimal inhibitory concentration (MIC) assay against bacterial strains from ATCC collection and clinical isolates. The same peptides fraction at a concentration below MIC value also demonstrated the bactericidal effect on E. coli CMC10106 revealing blebs on the bacterial surface and an appearance of emptied cells as shown by transmission electron micrographs. Both low and high MW mixture of ApoBLf-derived peptides significantly reduced the number of living B16-F10 murine melanoma cells.

Further studies are needed to define their mode of action. These peptides could be an interesting model for the design of new products useful against infections, inflammation, and cancer diseases.


[P11] NON SPECIFIC INTERACTIONS OF FIBRINOGEN UNDER MACROMOLECULAR CROWDING CONDITIONS

E. GANEA1, C. LASLO1, M. HILLEBRAND2 1Institute of Biochemistry, Bucharest, Romania

2University of Bucharest, Faculty of Chemistry, Romania

The interactions between proteins are important for many biological functions, such as assembly of cellular components, regulation of the enzyme activity or the mediation of signals from the exterior of a cell to the inside of that cell, activity called signal transduction. The nonspecific interactions, repulsive (steric, electrostatic) or attractive (electrostatic, hydrophobic) are normal consequences of crowding in most physiological fluid media, containing substantially greater total concentrations (50–400 mg/ml) of macromolecules than the test reactions. This is why, in order to understand molecular processes in such media, one must take into account the nonspecific interactions, rather than attempting to eliminate them.

Here we report the effect of the nonspecific interactions present in a crowded environment on structural and functional properties of fibrinogen. Three nonspecific high molecular mass polysaccharide crowding agents, polyethylene glycol 8000, Ficoll, and dextran 70 were used. The human native as well as reduced fibrinogen were studied in the presence and in the absence of crowding agents estimating the clotting activity, intrinsic fluorescence and circular dichroism spectra, SDS-PAGE and Western blot. In addition, the refolding process of the reduced fibrinogen under macromolecular crowding conditions was studied as well.

The results of this study showed that the polysaccharide crowding agents, although inert compounds, obviously influence protein function and structure by non specific interactions. We noticed a heterogeneity of various polysaccharide crowding agents effects, e.g., Ficoll did not modify the fibrinogen clotting activity during the first ten hours, whereas Dextran and PEG slightly decreased this activity. The shape and the molecular mass of these compounds might be involved. An important conclusion was also that protein folding/unfolding process is highly influenced by macromolecular crowding. Taken together, our findings suggest that macromolecular crowding could have an important role in understanding the protein function, folding and stability inside the cell.


[P12] GENETIC ANALYSIS OF ENDOPLASMIC RETICULUM ASSOCIATED DEGRADATION

IN CAENORHABDITIS ELEGANS

S. GHENEA, Ş.M. PETRESCU

Institute of Biochemistry of the Romanian Academy, Department of Molecular Cell Biology, Spl. Independenţei 296, Bucharest, Romania

Proteins that are unfolded or misfolded in the endoplasmic reticulum (ER) must be refolded or degraded to maintain the homeostasis of the ER. The unfolded or misfolded proteins are first retained in the ER, retrotranslocated to the cytoplasm by the machinery of ER-associated degradation (ERAD), and then degraded by the proteasome. A malfunction of the ER stress response could induce apoptotic cell death and cause various diseases such as diabetes, inflammation, and neurodegenerative disorders including Alzheimer’s disease, Parkinson’s disease, and for this reason elucidation of the ERAD machinery is under intense investigation.

C. elegans possesses all the components of the mammalian ERAD machinery, and we have used C. elegans as a genetic model system to dissect ERAD machinery in a multicellular organism. Thus, analysis of sel-1 gene deletion, highly similar to the human ER ubiquitin ligase SEL1L, and other mutants of ERAD pathway genes show degradation of intestine, and various degrees of developmental arrest at larval stage 2 (L2) upon ER-stress inductor, tunicamycin. We also show that upon ER-stress, a deletion of edem-3, the orthologue of the human EDEM3, is synthetic lethal with deletion of either sel-1 or cdc-48.2, both producing a developmental arrest at L2. Although the tissue-specific expression of EDEM-3 remains to be analyzed, this protein seems to be important in the axon guidance during nervous system development, because edem-3 mutant exhibits premature termination, interruption of the nervous dorsal cord, and misguidance of the axons. Interestingly, although with low penetrance, sel-1 and cdc-48.2 mutants also show similar axon guidance defects upon ER-stress induction.


[P13] LOCAL SLIDING OF GLYCOSYLATION SITE N371 IS INTERRELATED WITH HUMAN TYROSINASE

BIOLOGICAL CONFORMATION

A. MAREŞ, A.J. PETRESCU, Ş.M. PETRESCU


Unlike the rest of other glycosylation sites, the N-site seven of human tyrosinase (N371) is located in a highly conserved region with respect to the rest of other vertebrate tyrosinases suggesting a critical role of this site. Tyrosinase is a type I membrane glycoprotein localized in melanosomes. It is composed of 533 amino acids, presenting seven N-glycosylation sites, two cysteine rich domains and two copper-binding domains, with the seventh site residing in the second copper domain.

Molecular modelling suggests that N371 is located in a loop between the two alpha helices on which the second Copper binding site is located. In this study, we question the critical role of this site by performing a small shift, with only 2 or 7 positions downstream, to N373 (m1) and N378 (m2) – i.e., keeping the site within the loop – in order to assess if location changes do affect occupation, synthesis, maturation and enzymatic activity of tyrosinase.

The wild type and the mutant cDNAs were transiently expressed in HEK293T cells and analyzed for occupancy of the N-glycosylation site. The analysis of the mutants' molecular mass showed that both have the same number of occupied N-glycosylation sites as wild type, suggesting that the glycosylation efficiently occurs in the newly designed positions of the 7th site. In contrast, the DOPA-oxidase assay showed that all tested mutants are completely inactive. Endoglycosidase H digestion showed that all the mutants analyzed are completely retained in the endoplasmic reticulum, presenting only high mannose glycans. From these results we conclude that, in human tyrosinase, the occupancy and strict location at N371 of the seventh N-glycosylation site is crucial for native folding of tyrosinase and therefore for acquiring a conformation enzymatically active.


[P14] A 3D MODEL OF EPITHELIAL SODIUM CHANNEL BASED ON THE HOMOLOGY WITH ACID-SENSING

ION CHANNEL

M. MERNEA, O. CALBOREAN, D.F. MIHĂILESCU

University of Bucharest, Faculty of Biology, Department of Anatomy, Animal Physiology and Biophysics, Splaiul Idependenţei 91–95, Bucharest, Romania

Epithelial sodium channels (ENaCs) are highly selective ion channels that allow an amiloride sensitive flow of Na+ across polarized epithelial cells. The three-dimensional structure of ENaC is unknown. ENaC is a heteromultimeric channel, composed of three homologous subunits. The subunit stoichiometry of ENaC was controversial; ENaC was thought to be a tetramer or a nonamer. Both ENaC and acid-sensing ion channel (ASIC) are members of the ENaC/DEG family and share a ~70% amino acid sequence identity. This study presents a model of ENaC in closed and open state based on sequence homology with ASIC1 channel, whose crystal structure in closed conformation was recently solved. ASIC1 channel is a trimer and it is very likely that ENaC channels are also trimers, so ENaC was modelled as a trimer. The model was further refined using molecular dynamics methods. The proposed model is consistent with the functional data of wild-type and mutant ENaC channels. The extracellular “loop” of ENaC model appears as a compact structure that comprises α-helix domains and β-sheets. The extracellular region is stabilized by seven disulphide bonds from each subunit that involve cysteine residues conserved in ENaC/DEG family. Surprisingly, according to this model, the selectivity filter, previously thought to precede TM2, lies in the cytoplasmatic half of the lipid bilayer and has a helical conformation. One of the putative amiloride binding sites, that was thought to lie in the extracellular “loop”, is positioned in the present model on the TM2 helix, in the middle of the lipid bilayer. The other putative amiloride binding site from the extracellular “loop” was not included in the present model because it is positioned in an area with weak homology with ASIC. This study also presents the influence of ten mutations that are known to alter the channel functionality on ion passage through the channel.


[P15] ENZYMATIC CHARACTERIZATION OF A NEW ISOFORM OF HUMAN EYES ABSENT HOMOLOG 3

M. PASCARU, Ş.E. SZEDLACSEK

Institute of Biochemistry, Department of Enzymology, Spl. Independenţei 296, Bucharest, Romania

Eyes absent genes play an essential role in the events that induce the formation of Drosophila eyes. The lack of the expression of these proteins determines the programmed cell death in the precursor retinal cells, leading to small eyes or eyeless individuals. Four different eyes absent homologs (eyes absent 1–4) have been identified in humans, with one or more isoforms. We report the identification of a new isoform of human eyes absent homolog 3.

The new isoform was cloned by polymerase chain reaction (PCR) from a human cDNA library using specific primers for human eyes absent homolog 3. Sequencing of the resulting cDNA revealed that we obtained a new isoform of eyes absent homolog 3, not reported in NCBI GenBank, lacking a segment of 46-amino acids in the N-terminal domain. The PCR experiment was repeated several times and always the cDNA corresponding to the novel Eya 3 isoform was obtained. The cDNA of the new isoform was then inserted in a prokaryotic expression vector which enables the expression of the recombinant protein fused with an N-terminal tag of six histidine residues. The protein was purified using affinity chromatography and was further subjected to enzymatic tests.

The prokaryotic expressed protein was purified with high yield by affinity chromatography and used for enzymatic tests. The results showed that the new isoform displayed tyrosine phosphatase activity on two synthetic substrates: pNPP (para-nitrophenyl phosphate) and DiFMUP (6,8-difluoro-4-methylumbelliferyl phosphate). The measured values were comparable with those obtained for the catalytic domain of the human eyes absent 3 suggesting that the N-terminal domain of the new isoform has no influence on the enzymatic activity.


[P16] THE SULFITE REDUCTASES ACTIVITY IN THE PRESENCE OF DIFFERENT VIOLOGENS

A. PATRAŞ

University of Agricultural Sciences and Veterinary Medicine, M. Sadoveanu 3, Iaşi, Romania

The sulfite reductases are flavoenzymes which have the property to transfer electrons toward a substrate. Knowing that the sulfite reductase activity is inhibited in vivo by the methyl-viologen (which is the active substance of the herbicide Paraquat), we evidenced that in vitro, in certain conditions and in the presence of viologens, the sulfite reductase leads to the generation of superoxide radical anion and this O2

─ production is stimulated by the increasing concentration of viologen. The present study was conducted on two directions (anaerobiosis and

aerobiosis), in the presence of different viologens. Our sulfite reductase is emerged from E. coli. The work was done with the recombinant protein named SIRFP 43, with a molar mass of 43 kDa. The three viologens we used are: methyl-viologen, carbamoylmethyl-viologen and cyanomethyl-viologen. The sulfite reductase activity was measured by two methods: the spectrophotometric measure of the substrate consumption and the fluorimetric measure of the superoxide produced.

Our results show that in anaerobiosis, the MV2+ is acting as a second substrate of SIRFP 43, the NADPH being the first substrate. In the oxygen’s presence, the catalyzed reaction by the sulfite reductase is leading toward the production of the anion radical superoxide, obtained by MV2+ oxidation process, so, MV2+ has the catalyst role in producing O2

─. The NADPH consuming is correlated with the producing of superoxide and SIRFP 43 activity is stimulated by the increasing of methyl-viologen concentration.

In conclusion, the sulfite reductase can contribute to oxidative stress produced in plants by the methyl-viologen. Exactly the same results were obtained in the case of carbamoylmethyl-viologen. In the cyanomethyl-viologen case, we were confronted with an experimental problem, caused by the secondary reaction among the upper mentioned and the buffer, forming a product which is absorbing at the work wavelength.


[P17] MOLECULAR SIMULATION OF GRAM-NEGATIVE BACTERIA OUTER MEMBRANE AND ITS INTEGRATED

PORINES

R. TĂCUTU, L.N. SPIRIDON, M. MICLUŢĂ, A.J. PETRESCU


Understanding the molecular mechanisms of antibiotic resistance is very important due to the high frequency of this phenomenon and helps to improve the efficiency of the therapies against bacteria. In the case of Gram-negative bacteria, beta-lactamic antibiotics and fluoroquinolones penetrate the outer membrane through special transport proteins named porines. One of the methods that bacteria are using for resistance is impermeability by using modified porines.

In studying Gram-negative resistance mechanisms the in silico approach might prove useful not only for the understanding of the system behavior at atomic level, but also to assist the experimental studies. The in silico description includes both building the structural model of the system and the simulation of its dynamics, in order to assess its evolution in time.

In our case the model is composed of a porine (OmpF) included in a bilayer composed of lipopolysaccharide (LPS) molecules on the one side and phosphatidyl etanol amine on the other.

In order to build this model we first set the molecular dynamics parameters for a single lipopolysaccharide molecule using Glycam04 and Amber parameter DB force fields. The outer membrane was built by optimizing the geometry of a small bilayer (6 LPSs) and then multiplying it so that it can incorporate the porine protein in it. We used the in-house developed software Glyco-Pack to incorporate the porine with its channel perpendicular to the membrane plane. MD simulation was performed with Amber. This revealed sharply distinct dynamic regimes of the glucidic part of the LPS as compared to the lipid moiety. This is due to both the steric compactness of the glucidic part of LPS and the highly dense charge network formed by the LPS and its mobile counterions. The reference antibiotics were ceftazidime and ciprofloxacine.

POSTERS

Session 3: Clinical and Immunological Biochemistry

ROM. J. BIOCHEM., 45, 1, 63–91 (2008)

[P18] ANTIOXIDANT ENZYMES AND OTHER BIOCHEMICAL PARAMETERS IN ALCOHOLIC LIVER

DISEASES

M. BASA1, N. ROŞOIU2, I.G. VERMAN2 1Emergency Military Hospital Constanta, Clinical Laboratory,

96 Mamaia Blv. Constanţa, Romania 2“Ovidius” University Constanţa, Faculty of Medicine,

Biochemistry Department, 58 Ion Vodă St., Constanţa, Romania

The product of all alcohol metabolic pathways, acetaldehyde, is converted to acetate by mitochondrial aldehyde dehydrogenase generating hydrogen, which converts NAD to its reduced form NADH, increasing the redox potential in the liver. This replaces fatty acids as a fuel, lowers fatty acid oxidation and allows triglycerides to accumulate, causing fatty liver and hyperlipidemia. The increase of free radicals in the hepatocytes, including the hydroxyethyl radical, superoxide anion (O2

●–) and hydroxyl radical, determines the lipid peroxidation with diffuse lesions of cell and intracellular membranes, followed by progression to hepatocytes necrosis and fibrosis.

The aim of this study was to evaluate the production of ROS and TAS in comparison to other biochemical parameters in alcoholic liver diseases. Samples from 36 male patients were analyzed for ALT, AST, LDH, ALP, GGT, TB, CB, TC, TG, TAS and GR. The markers of the oxidative stress are varying with the evolution of the hepatic injury; TAS is normal in steatosis, reduced in alcoholic hepatitis and has the lowest value in the hepatopulmonary syndrome. GR is normal in alcoholic steatosis and increased in all patients with alcoholic hepatitis. In cirrhosis the values are increased, associated with the depletion of TAS. Elevations of aminotransferases are moderate, but AST/ALT ratio is more than 2 in 58% of cases. GGT increases in 71% of the patients. Bilirubin increases as the disease evolves. The cholesterol and triglycerides have increased the concentration in alcoholic steatosis; in hepatitis and cirrhosis the synthesis is decreased, and the values are in the reference interval. The activity of total LDH is increased in hepatitis and cirrhosis. We may conclude that the markers of the oxidative stress are useful in the evaluation of the hepatic biological balance as they vary depending on the evolution of the alcoholic hepatic damage.


[P19] THE EVOLUTION OF ANTIOXIDANT ENZYMES AND OTHER BIOLOGICAL MARKERS IN CHRONIC LIVER

DISEASES

M. BASA1, N. ROŞOIU2, I.G. VERMAN2

1Emergency Military Hospital Constanţa, Clinical Laboratory, 96 Mamaia Blv, Constanţa, Romania

2“Ovidius” University Constanţa, Faculty of Medicine, Biochemistry Department, 58 Ion Vodă St., Constanţa, Romania

The oxidative stress indicates an overproduction of reactive oxygen species (ROS), associated with an overproduction of precursors, a decreased efficiency of the antioxidant agents and a decreased efficiency of the inhibitory or scavenger systems. The liver is considered the key organ in free radical detoxification of the organism. A big part of the free radicals and peroxides are formed in the hepatic microsomes, and during hepatic insufficiency their plasma level is enhanced. The liver presents the highest concentrations of enzymatic and nonenzymatic antioxidants.

The target of the study was the estimation of total antioxidant capacity in the biochemical balance of the chronic hepatic pathology. We selected 30 patients who were analyzed for ALT, AST, LDH, iron, TB, CB, ALP, GGT, TC, HDL-C, TG, TP, ALB, A/G, CHE, creatinine, uric acid, PLT, TAS, GR. Decreased values of TAS were detected in all three chronic liver diseases, with the lowest value in cirrhosis. GR was normal in chronic hepatitis and increased in cirrhosis, associated with increased GGT activity, noticed in all the cases with increased activity of aminotransferases as a sign of hepatocytolysis. In the chronic hepatitis the iron absorption in the intestine was intensified. The siderosomes break and the iron set free will determine a noxious action over the hepatocyte. The presence of cholestasis indicates a severe necro-inflammatory activity. The degree of cell damage may be evaluated by hypoalbuminemia, A/G ratio decreased, CHE behavior parallel with the albumin concentration, indicating the alteration of protein synthesis. Hyper-γ-globulinemia is associated to cirrhosis. The low concentration of total cholesterol is an unfavorable indicator for the prognosis. The increased values of uric acid are able to inactivate the ROS and inhibit the PUFA peroxidation, protecting the hemoglobin against oxidative stress and the nucleic acids.


[P20] THE SIGNIFICATION OF IMMUNOLOGICAL METHODS IN PIG HERD HEALTH MANAGEMENT

S. BĂRĂIŢĂREANU, D. DANEŞ

University of Agronomical Sciences and Veterinary Medicine, Bucharest, Romania

The ELISA technique (immunoenzymatic assay) is a tool which penetrated and conquered the biotic agent’s surveillance methods market. Official programs for surveillance of animal health status as well as animal farm surveillance programs use ELISA technique in economic and sanitary politics.

We tested 32 lots of swine from nine farms, with 577 samples investigated by HerdChek Pseudorabies Virus gB Antibody Test Kit, CHEKIT APP-ApxIV Actinobacillus pleuropneumoniae Antibody Test Kit, HerdChek Mycoplasma Hyopneumoniae Antibody Test Kit, HerdChek PRRSV-Ab Test Kit, HerdChek Swine Influenza Virus Antibody Test Kits-H1N1 and H3N2. The positive immune status for Influenza virus H1N1: sucking piglets – 49% and sows – 78%. The positive immune status for Influenza virus H3N2: sucking piglets – 2.8%, pig for pork – 33% and gilt – 78%. The positive immune status for Mycoplasma hyopneumoniae: sucking piglets – 30%, pig for pork (mean growth period) 12%, pig for pork (final growth period) 17% and gilt – 69%. The immune status for PRRSv: sucking piglets – 49%, pig for pork 13% and gilt – 29%. The positive immune status for Actinobacillus pleuropneumoniae: sucking piglets – 43%, pig for pork (mean growth period) 20%, pig for pork (final growth period) 19.7% and gilt – 29%. The positive immune status for Pseudorabies virus: sucking piglets – 96.9%, pig for pork (mean growth period) 91%, pig for pork (final growth period) 88% and gilt – 99%.

The surveillance and monitoring of some infectious diseases by ELISA technique improved the programs of health control in investigated farms, identified the prevalence of some infectious agents in herds and evaluated the quality of immuno-preventive measures. The results of the investigations carried out on swine of different ages made business profitable.


[P21] EFFECTS OF LOW-DOSE NICOTINE ON NEURONAL OXIDATIVE STRESS STATUS

A. CIOBICĂ1, L. HRITCU1, V. ARTENIE1, M. PĂDURARIU2

1“Al. I. Cuza” University, Department of Molecular and Experimental Biology, Iaşi, Romania 2“Gr. T. Popa” University of Medicine and Pharmacy, Iaşi, Romania

Nicotine has been reported to be therapeutic in some patients with certain neurodegenerative diseases and to have neuroprotective effects on the central nervous system. However, nicotine administration may result in oxidative stress by inducing the generation of reactive oxygen species in the periphery and central nervous system. There is also evidence suggesting that nicotine may have antioxidant properties in the central nervous system. The antioxidant properties of nicotine may be intracellular, through the activation of the nicotinic receptors, or extracellular, by acting as a radical scavenger by binding iron. The possibility that nicotine might be used to treat some symptoms of certain neurodegenerative diseases underlies the necessity to determine whether nicotine has pro-oxidant, antioxidant properties or both.

In the present study we evaluated the effects of nicotine treatment (0.3 mg/kg b.w., i.p., SIGMA) on the activity of antioxidant enzymes. We assessed the activity of superoxide dismutase, glutathione peroxidase and malondialdehyde in the prefrontal and temporal cortex homogenates after 7 days of continuous nicotine administration.

The exposed animals had decreased levels of superoxide dismutase and glutathione peroxidase after nicotine treatment. The level of malondialdehyde was increased. This biochemical evidence suggested that exposure to a low dose of nicotine caused severe oxidative stress.


[P22] THE IMPLICATION OF OXIDATIVE STRESS IN A RAT MODEL OF PARKINSON’S DISEASE

A. CIOBICĂ1, L. HRITCU1, V. ARTENIE1, M. PĂDURARIU2

1“Al. I. Cuza” University, Iaşi, Romania 2“Gr. T. Popa” University of Medicine and Pharmacy, Iaşi, Romania

Parkinson’s disease (PD) stems from the loss of dopamine caused by the degeneration of the dopaminergic neurons of the substantia nigra. The nature of this degeneration remains unclear, although current theories suggest that reactive oxygen species are involved early in the disease development. The administration of 6-hydroxydopamine (6-OHDA) into rat brain produces a well-established model of PD. Many investigators have demonstrated that 6-OHDA induces oxidative stress which can lead to the induction of apoptosis and cellular loss. The effects of 6-OHDA are age-dependent as there is a greater effect in aged animals compared with young ones, particularly with lower doses of 6-OHDA.

The purpose of the present study was to determine the development of oxidative stress that is generated in substantia nigra and ventral tegmental area 6-OHDA lesion model of PD, by assessing the activity of antioxidant enzymes in the temporal and frontal lobes homogenates. Male Wistar rats, 22–23 month-old, were used for all experiments. The 6-OHDA lesions: substantia nigra (5.5 mm posterior to bregma; 2.0 mm lateral to the midline; 7.4 mm ventral to the surface of the cortex); ventral tegmental area (5.6 mm posterior to bregma; 0.5 mm lateral to the midline; 7.6 mm ventral to the surface of the cortex). The biochemical estimations: superoxide dismutase (SOD), glutathione peroxidase (GPX) and malondialdehyde (MDA). The data were recorded 2 weeks after neurosurgery. Lesioning of substantia nigra and ventral tegmental area with a low dose of 6-OHDA induced significant reduction in SOD and GPX specific activities and non-significant reduction of MDA concentration in the temporal lobe rather than in the frontal lobe homogenates, comparatively with the sham-operated control group. Also, the role of the substantia nigra was more prominent than that of the ventral tegmental area. Our results support that oxidative stress plays a role in the damage produced by substantia nigra and ventral tegmental area injection of 6-OHDA and that indices of oxidative stress could be important markers for evaluating therapeutic strategies.


[P23] CURCUMIN EFFECTS ON MAMMARY TUMOR CELL RESPONSE TO HYDROGEN PEROXIDE INDUCED STRESS

C. CIOFRĂNGEANU1, B. GĂLĂŢEANU1, R. CIUBAR1, V. MITRAN1, M.I. GRUIA2, L. BRAŞOVEANU2, L. PUIU3, A. CÎMPEAN1,

D. IORDĂCHESCU1 1University of Bucharest, Department of Biochemistry and Molecular Biology, Bucharest, Romania

2“Şt. S. Nicolau” Institute of Virusology, Bucharest, Romania 3“Prof. Dr. Alex. Trestioreanu” Oncology Institute, Bucharest, Romania

We explored the possibility that curcumin, a biologically active natural compound, could compromise the chemotherapeutic activity of some drugs which exert their effects through the induction of oxidative stress, by studying its ability to inhibit hydrogen peroxide (H2O2)-induced cell damage in MDA-MB-231 breast cancer cells. Cell viability was measured by MTT test; the level of lipid peroxides was evaluated by TBARS method; glutathione peroxidase activity was measured as described by Lawrence and Burk; the level of glutathione was determined using a Sigma kit; the percent of apoptotic cells was quantified by flow cytometry after annexin-FITC and propidium iodide staining; the invasive potential of tumor cells was evaluated by a chemotaxy assay. When added simultaneously with H2O2 (750 µM), curcumin (25 µM) resulted in an increase of about 10% in cell viability compared to the treatment with H2O2 alone, which induced a significant decrease in cell viability. This effect could be due to a direct reaction between curcumin and H2O2 or curcumin and free radicals derived from H2O2 in the cell culture medium, before they could react with the cells. For this reason we performed a pretreatment with curcumin for 4 h before H2O2 exposure and the viability increased with 15% compared to the treatment with H2O2 alone, suggesting that curcumin displays a free radical scavenger activity. These results were sustained by studies concerning the cellular oxidative status, apoptotic potential and in vitro migration capacity. However, when the cells were pretreated with curcumin for 12 h before H2O2

treatment, curcumin was unable to inhibit H2O2 induced cell damage. On the contrary, it caused an amplification of H2O2 cytotoxic effect. Our findings suggest that concomitant treatment with curcumin and drugs that induce a release of reactive oxygen species should be avoided in cancer therapy.


[P24] COMPARATIVE ANALYSIS OF PHYSICAL PARAMETERS OF JUNIOR FOOTBALL PLAYERS BY AGE

CATEGORIES. NUTRITIVE IMPLICATIONS

A. CIORSAC1, A. ISVORAN2, V. OSTAFE2

1Polytechnical University of Timişoara, Department of Physical Education and Sport, 2 Victoriei Sq., Timişoara, Romania

2West University of Timişoara, Department of Chemistry, 16 Pestalozzi, Timişoara, Romania

The aim of this study is to compare the physical parameters for junior football players grouped by age: 15–16 years and 18–19 years respectively. Two groups of different age of junior football players were tested in October 2007: group A consisted of 27 subjects of age 18–19 years and group B consisted of 21 subjects of age 15–16 years. The measurements consisted in: muscular power of legs (5 times consecutive horizontal jump), speed (50 m sprint) and commute (5 times 10 m). The mean values of the three measurements for every test were:

a) muscular power of legs: 12.449±0.157 m for the group A and 10.845±0.728 for the group B, the two means being significantly different at 0.05 level;

b) sprint: 6.486±0.338 s for the group A and 6.837±0.271 s for the group B, the two means being significantly different at 0.05 level;

c) commute: 11.821±0.316 s for the group A and 12.218±0.566 s for the group B, the two means being significantly different at 0.05 level.

Physical parameters are strongly correlated to the age and training experience of junior football players. These measurements may be used as a predictive tool for football player selection and for the rationalization and standardization of the training techniques. Also, it may inform about nutritive or effort supplements requirements to optimize physical performance of junior football players.


[P25] SERUM HOMOCYSTEINE IN LEVODOPA TREATED PARKINSON PATIENTS

M. DRONCA1, S.P. PAŞCA1, R. RUSU1, L. PERJU-DUMBRAVĂ2

1“Iuliu Haţieganu” University of Medicine and Pharmacy, Biochemistry Department, Cluj-Napoca, Romania

2“Iuliu Haţieganu” University of Medicine and Pharmacy, Neurology Department, Cluj-Napoca, Romania

Hyperhomocysteinemia is strongly associated with an increased risk of dementia and cognitive impairment, both of which are common in the course of Parkinson’s disease (PD). Although B vitamin status and genetic polymorhisms are important factors determining the degree of one-carbon metabolic disturbances in PD, the elevated levels of total homocysteine (tHcy) may be due to the methylation of levodopa by COMT to yield S-adenylhomocysteine, which is rapidly converted to homocysteine. The aim of this study was to determine whether hyperhomocysteinemia in levodopa treated PD patients is associated with the severity of disease and/or daily levodopa dose. Twenty five PD patients, receiving a mean levodopa dose of 404.0±34.63 mg/day, and 28 healthy control subjects, age and sex matched, were included in the study. Homocysteine levels were significantly higher in the patient group in comparison with the controls (14.01±0.95 vs. 11.44±0.80; p<0.05). There was a positive correlation between tHcy level and the severity of disease assessed by the Hoehn and Yahr staging (r=0.43; p<0.05). We also observed a positive correlation between tHcy and the daily levodopa dose, but only when we corrected for age (r=0.47; p<0.05). In conclusion, tHcy is increased in levodopa treated PD patients and the degree of hyperhomocysteinemia is correlated with the severity of disease and daily levodopa dose.


[P26] α-METHYLACYL-CoA RACEMASE: A NEW BIOMARKER FOR THE EARLY DETECTION

OF PROSTATE CANCER

R. DUMACHE1, B. BUMBĂCILĂ1, A. KAYCSA1, C. DEHELEAN2, F. MICLEA3, M. PUIU4

1“Victor Babeş” University of Medicine and Pharmacy, Biochemistry Department, Eftimie Murgu Sq.,Timişoara, Romania

2“Victor Babeş” University of Medicine and Pharmacy, Toxicology Department Eftimie Murgu Sq., Timişoara, Romania

3“Victor Babeş” University of Medicine and Pharmacy, Urology Department, 156 Iosif Bulbuca St., Timişoara, Romania

4“Victor Babeş” University of Medicine and Pharmacy, Medical Genetics Department, Eftimie Murgu Sq., Timişoara, Romania

The aim of our study is to prove that alpha-methylacyl-CoA racemase (AMACR) has a high sensitivity and specificity for the prostatic tissue in comparison with PSA (prostate specific antigen). AMACR is an enzyme that catalyzes the racemization of R-stereoisomers of branched-chain fatty acids to S-stereoisomers and plays a critical role in peroxisomal β-oxidation of branched-chain fatty acids. It is well known that the main source of branched fatty acids are the dairy products and red meat pork and beef, the consumption of which has been associated with an increased risk for prostate cancer.

In our study, we evaluated the AMACR gene expression in 35 patients aged 50 to 80, with total PSA values in the range 2 to 45 ng/ml. 28 out of the 35 patients were diagnosed positive for prostate intraepithelial carcinoma and they had PSA values greater than 10 ng/ml.

For these 28 men, PCR and microarray techniques on needle biopsies showed that they had an over expressed gene for AMACR. The other 7 patients with PSA values smaller than 10 ng/ml had normal expression of the AMACR gene.

According to these results we propose the use of AMACR as an important novel biomarker for the prostate cancer in the Urology Clinics, beside the PSA and morphopathological diagnostic.


[P27] OVEREXPRESSION OF CD 24 GENE IN PATIENTS WITH PROSTATE CANCER

R. DUMACHE1, B. BUMBĂCILĂ1, M. PUIU2, F. MICLEA3 1“Victor Babeş” University of Medicine and Pharmacy, Biochemistry Department, Eftimie Murgu

Sq., Timişoara, Romania 2“Victor Babeş” University of Medicine and Pharmacy, Medical Genetics Department, Eftimie

Murgu Sq., Timişoara, Romania 3“Victor Babeş” University of Medicine and Pharmacy, Urology Department, 156 Iosif Bulbuca St.,

Timişoara, Romania

The aim of this study is to correlate the high levels of PSA (prostate specific antigen) from patients with prostate cancer (PCa) with the overexpression of gene CD 24. We have collected prostate tissue samples and serum samples from patients from the Urology Clinic, Timişoara.

Patients were aged between 50 and 75 years. From a population of 50, 30 patients had prostate cancer (PCa) and 20 patients were diagnosed with BPH. All of them had PSA values higher than normal (6–48 ng/ml) and histopathological exams were performed on all 50 biopsies, to confirm the malignant or non malignant nature of the process.

The RNA was extracted from the tissue samples and using the RT-PCR method we have evaluated the expression of a novel biomarker, in course of validation, the CD24 gene. PSA levels were measured from the serum samples using the ELISA kit.

PSA levels were different between patients with PCa (PSA >15 ng/ml) and men with BPH (PSA 6–9 ng/ml). There is a strong correlation (93%) between the overexpression of CD 24 gene and the positive histopathological exam. 80% of the men with BPA had normal expression of the CD24 gene. Patients with increased PSA levels (>9 ng/ml) had an overexpression of the CD 24 gene. In most of them the malignant process was also diagnosed by the Histopathology Laboratory. The overexpression of CD 24 gene could be correlated with high PSA values.


[P28] IN VITRO MODULATION OF ENZYMATIC PROCESSES USING BIOLOGICALLY ACTIVE SUBSTANCES

WITH RELEVANCE FOR SKIN AGING

M.D. ENE, L. OLARIU, M. CONSTANTINOVICI, R. IONESCU

S.C. BIOTEHNOS S.A, Research Department,“In vitro” Testing Laboratory, Gorunului Str. 3–5, Otopeni, Romania

One of the most generally reliable and widespread clues observations on individuals’ age is the skin appearance during the aging process. The main visible changes are increasing irregularities such as scars, wrinkles, roughness, warts and moles. The visible signs are accompanied by a change in the skin texture due to cell and subcutaneous fat losses and to decreasing elasticity as the connective tissue elements are altered. The free radical theory of aging reveals the fact that intracellular redox homeostasis perturbation becomes progressively pro-oxidant during aging and therefore the organism becomes more susceptible to the oxidative stress.

In order to emphasize the potential aging preventing effects of some biologically active substances isolated and purified in our laboratories, enzymatic studies were done to determine their in vitro action on proteinases and free radicals and also cytotoxicity tests. Knowing that the collagen is a likely candidate for long-term deterioration because of its quite slow rate of turnover in the adult organism, tests were made to evaluate the influence of bioactive substances on collagenase activity and collagen fiber formation. Total antioxidant activity was evaluated by the DPPH indirect spectrophotometric method.

The experimental data demonstrated that some bioactive substances are favorable to form the collagen fiber, modifying the collagenase activity and presenting antioxidant activity. This study indicated that our bioactive substances can be included in cosmetic preparations, used individually or synergistically with other bio products in order to obtain a convenient efficiency/toxicity ratio.


[P29] MISCELLANEOUS CONSEQUENCES OF HYPERGLYCEMIA ON ERYTHROCYTES AND

ENDOTHELIAL CELLS

A. GLIGA, D. MARGINĂ, D. GRĂDINARU, N. MITREA

“Carol Davila” University of Medicine and Pharmacy, Faculty of Pharmacy, Biochemistry Department, Traian Vuia 6, Bucharest, Romania

The aim of this study was to evaluate apposite parameters of the erythrocytes redox status and NO synthesis in insulin dependent diabetes mellitus (IDDM) and non-insulin dependent diabetes mellitus (NIDDM), compared to healthy controls. We designed a clinical study of 42 patients, divided into three groups: control (n=10), NIDDM (n=18) and IDDM (n=14). À jeun blood samples were collected and processed to separate the erythrocytes from the platelets rich plasma. The erythrocyte suspensions (1g hemoglobin/ml) were used to evaluate the activities of glucose-6-phosphate dehydrogenase (G6PDH) and superoxide dismutase (SOD) and the erythrocyte susceptibility to peroxidation (ESP) (H2O2 induced). The platelet rich plasma samples were used to determine the nitrites and nitrates level (NOx) by global method (Griess reagent), as stable NO end products. Both IDDM and NIDDM patients had a significantly lower SOD activity (0.82±0.31 IU/g Hb for NIDDM and 0.45±0.17 IU/g Hb for IDDM patients compared to 1.41±0.14 IU/g Hb for controls, p<0.001) and a significantly higher ESP (712.67±202.80 mM MDA/g Hb for NIDDM and 1019.07±301.73 mM MDA/g Hb for IDDM vs. 166.86± 35.56 mM MDA/g Hb for controls, p<0.001) compared to the controls. Moreover, the G6PDH activity was higher in the NIDDM group (141.35±33.84 IU/ml, p<0.001) compared to the IDDM (47.67±24.62 IU/ml), and significantly lower in both groups compared to the controls (190.44±27.89 IU/mL). The NOx level was considerably increased in IDDM patients (2.17±0.90 µmoles NOx/l plasma) and NIDDM patients (2.09±0.50 µmoles NOx/l plasma) compared to the controls (1.19±0.50 µmoles NOx/l plasma, p<0.01).

Our results showed an impairment of the erythrocyte antioxidant systems (G6PDH and SOD) in both NIDDM and IDDM patients associated with an increased risk of red blood cell membrane peroxidation together with alterations in the NO synthesis.


[P30] ACTIVITY OF SOME ENZYMATIC SYSTEMS: EARLY CYTOLYSIS MARKERS OF INDUCED

HYPERCUPROSIS IN RAT AND SHEEP

G.V. GORAN, V. CRIVINEANU, C. PAPUC, M.D. CODREANU

Faculty of Veterinary Medicine, Bucharest, Romania

The objective of this work was to formulate a rapid diagnosis procedure of copper associated pathology on the basis of blood biochemical examination, regarding the activity of some enzymatic systems as suitable quick markers of copper induced cytolysis.

A number of 48 weaned, male Wistar and Sprague-Dawley rats were used in eight study groups, divided according to body weight. The study groups were randomly assigned to dietary treatments by copper source supplementary administered into feed (copper sulfate or proteinate) fed in two dietary levels (50 or 100 mg/kg). The animals were euthanized after one or three weeks from the beginning of the study and blood samples were collected to determine AST, ALT and alkaline phosphatase. 42 adult, not gestating Tsigai ewes were studied, during a period of six weeks, after treatment with supplementary copper doses in feed. The ewes were divided by body weight in seven groups. Six groups were randomly assigned to dietary treatments by copper source supplementary administered into feed (copper sulfate or proteinate) fed at one of three dietary levels (10, 20 or 30 mg/kg) and one was the control group. Each week blood samples were collected to determine the activity level of AST, ALT, ARG, SDH, LDH. In rats, hepatic and kidney toxicity was indifferent on the copper dose or source added in feed, and it was expressed by the increased activity of serum transaminases and alkaline phosphatase, associated with an increased level of blood nitrogen urea and creatinine. In sheep, the most sensitive marker of hepatic cytolysis induced by supplementary administration of different copper doses and sources was arginase, which indicated cellular destructions at least one week before the other studied enzymatic systems (sorbitol dehydrogenase, lactate dehydrogenase and aspartate aminotransferase), which had increased activities starting from the second week of oral administration of copper products.

The studied enzymatic activities indicated changes in the hepatic function, confirming the toxic action of copper in both species, but with a lower risk for organic copper compounds.


[P31] THE INFLUENCE OF A NEWLY SYNTHESIZED SALICYLATE PRODUCT ON BLOOD GLUTATHIONE

VALUE IN RATS

C. GRĂVILĂ1, L. STANA2, A.X. LUPEA3, M. PĂDURE3 1Faculty of Animal Sciences and Biotechnologies, Timişoara, Romania

2Faculty of Veterinary Medicine, Timişoara, Romania 3Industrial Chemistry and Environmental Engineering Faculty,

Timişoara, Romania

We have studied the comparative influence of salicylamide (SaA), sulfanilamide (SuA) and a newly synthesized salicylate product obtained from the amide of 5-chlor salicylic acid and sulfanilamide (P) on blood glutathione (GSH) level in rats. This study was carried out on 25 Wistar rats divided into 6 experimental batches (P, P’, SaA, SaA’, SuA, SuA’) and one control batch (C). The drug and the distilled water have been administered intraperitonally. GSH was measured quantitatively at a Perkin-Elmer spectrophotometer through the Beutler et al. method, at 412 nm. The first blood sample was taken 24 h after the 5th administration and the second sample after 24 h from the 7th administration. An important pathway for potentially toxic agents is the formation of glutathione conjugates that are ultimately converted into mercapturate derivatives, after other common conjugation reactions.

The result of our experiment indicates that the GSH blood level decreased more in the case of the new salicylate product, as compared with the GSH levels in rats exposed to SaA or SuA.


[P32] STUDY OF OXIDATIVE STRESS IN THE ELDERLY

M. GRIGORIAN1, C. FARCAŞ2, G. LILIOS1, Z. NICULESCU3, S. JURJA3, L. BOTEA4, R. BUICULESCU3

1“Ovidius” University, Faculty of Dental Medicine, Physiology Department, Ilarie Voronca 7, Constanţa, Romania

2“Ovidius” University, Faculty of General Medicine, Physiology Department, Ion Voda 58, Constanţa, Romania

3“Ovidius” University, Clinical Emergency County Hospital of Constanţa, Tomis Blvd 145, Constanţa, Romania

4Medical Analysis Laboratory, General Manu 58, Constanţa, Romania

Aging is characterized by a progressive decline in the function of cells leading to an increased generation of radical oxygen species (ROS). Oxidative stress is associated with certain cellular degenerative conditions. The objective of the present study was to analyze the involvement of free radicals in the elderly. We investigated the parameters of oxidative stress in 60 subjects divided into two groups of age: 30 healthy young subjects (25–35 years) and 30 healthy elderly subjects (55–75 years). All participants fulfilled the following criteria: no chronic health problems, nonsmokers, no cardiovascular, metabolic or respiratory diseases, no administration of antioxidant supplements within the past 6 months. We determined plasma ceruloplasmin level and erythrocyte Cu-Zn/superoxide dismutase (SOD) activity, as markers of free radical scavengers, in all subjects.

The results proved that Cu-Zn/SOD activity was significantly decreased in the elderly and no differences were found regarding plasma ceruloplasmin, showing the intensification of oxidative stress in elderly patients.


[P33] BIOCHEMICAL CHARACTERIZATION OF EXPERIMENTAL MALIGNANT MELANOMA INDUCED

FROM GENETICALLY TRANSFORMED CELL LINES

M.I. GRUIA, V. NEGOIŢĂ, L.E. SIMA*, G. NEGROIU*, Ş.M. PETRESCU*

“Prof. Dr. Alex. Trestioreanu” Institute of Oncology, Bucharest, Romania *Institute of Biochemistry of the Romanian Academy, Bucharest, Romania

The melanoma represents one of the most aggressive cutaneous cancer forms, characterized by malignant proliferation of melanocytes (cells of neural crest origin). Its aggressiveness is based on several mechanisms: high capacities of invasion and metastasis, expressed even in the early phases of tumor progression, remarkable genotypic and phenotypic heterogeneity, inter and intratumor (in the same tumors) highly resistive about classical therapeutic means (immunotherapy, radiotherapy, chemotherapy, etc.)

In our study we used C57Bl/6 mice, injected in the right flank with murine malignant melanoma tumor cells derived from B16F1 cell line. We have used previously generated sense (B16C+, B16D2+) and antisense (B16B-) stable transfectants of the murine GCP-2 cDNAs in B16 mouse melanoma cells. We followed the tumor appearance, the vital organs transformation, metastases appearance and the dynamic tumor volume growth as compared with the oxidative stress parameters determination (lipid peroxidation level, total albumin-thiol groups, copper-oxidase activity of ceruloplasmin, the ability of serum to reduce the iron). Our results showed an increased level of the investigated parameters in the first phase of the tumor development, followed by a plateau and then by a decreased level, without returning to the normal initial values.

The results obtained in this experiment do not emphasize significant modifications between the four cell types inoculated in the animals. It is possible that in vivo the cells react differently than in culture, a lot of immune protection systems being involved, which could be overtaken at one point, allowing the development of melanoma tumor.


[P34] EVALUATION OF THE OXIDATIVE STRESS PARAMETERS IN DIABETIC PATIENTS

V. GRUIA1, A.L. ARSENE1, D. MARGINĂ1, M. MOHORA2, D. GRĂDINARU1, N. MITREA1, B.Y. MANUEL3

1“Carol Davila” University of Medicine and Pharmacy, Faculty of Pharmacy, Department of Biochemistry, Bucharest, Romania

2“Carol Davila” University of Medicine and Pharmacy, Faculty of Medicine, Department of Biochemistry, Bucharest, Romania

3Antwerpen University, Endocrinology Research Unit, Antwerpen, Belgium

In order to assess the damages caused by oxidative stress in patients suffering from diabetes mellitus complications, we designed a clinical study to establish possible correlations between the time period from diabetes onset and some oxidative stress parameters expressed at plasma level.

We included 70 patients, 46 men and 34 women, aged between 45 and 75 years, hospitalized in “N.C. Paulescu” National Institute of Diabetes, Nutrition and Metabolic Diseases, Bucharest. The oxidative stress parameters evaluated were: the erythrocyte superoxide dismutase (SOD), the plasma malondialdehyde (MDA), total antioxidant plasma capacity (TEAC) and plasma vitamins C and E. Analyzing all these biochemical parameters, we noticed a simultaneous increase in fasting plasma glucose with the time period from diabetes onset. The SOD activity and the level of MDA were increased in patients with less than 10 years from diabetes onset. On the other hand, the plasma concentration of ascorbate was significantly higher in patients with more than 20 years from the diabetes onset. The values obtained for TEAC and vitamin E did not show any important modification depending on the diabetes onset time. Our results pointed out interesting correlations between diabetes onset and the redox status at systemic level and especially information on the need for an appropriate diet and/or antioxidant supplements for the improvement of antioxidant defence. For this reason, evaluating such parameters is useful in long term studies on diabetic patients who generally have cardiovascular complications and need an appropriate therapy.


[P35] THE CONNECTION BETWEEN SPATIAL MEMORY IMPAIRMENT AND OXIDATIVE STRESS STATUS

IN RAT HIPPOCAMPUS

L. HRITCU1, A. CIOBICĂ1, V. ARTENIE1, M. PĂDURARIU2

1“Al. I. Cuza” University, Department of Molecular and Experimental Biology, Iaşi, Romania 2“Gr. T. Popa” University of Medicine and Pharmacy, Iaşi, Romania

Impairments of cognitive performance have been observed in normal rats with beta-adrenergic and D1-dopamine receptors blockade, suggesting that these receptors have a facilitator role in learning and memory processes. In the present study we examined whether oxidative stress contributes to the memory deficits induced by beta-adrenergic and D1-dopamine receptors blocked by means of pindolol and SCH 23390. Male Wistar rats were divided into three groups: 1) sham-operated; 2) Pindolol; 3) SCH 23390. All drugs were stereotaxically injected in the rat hippocampus. Rats were treated for 12 days. Learning and memory tests began 2 weeks after the operation, and the ability of the rats to acquire the operant task was studied by means of Y-maze task and radial arm-maze task, respectively. We evaluated the antioxidant enzymes activity. Intrahippocampus injections of pindolol (10 µg/µl, 4.5 µl/site) and SCH 23390 (0.3 µg/µl, 4.5 µl/site) resulted in a significant impairment of both working and reference memory tested by means of radial arm-maze task, suggesting significant effects of spatial memory. Pindolol and SCH 23390 significantly decreased spontaneous alternation in Y-maze task, suggesting effects on spatial memory, especially on short-term memory. We observed that the levels of superoxide dismutase (SOD) and glutathione peroxidase (GPX) decreased in rats with beta-adrenergic (β AR) and D1-dopamine (D1R) receptors blockade by means of pindolol (10µg/µl, 4.5 µl/site) and SCH 23390 (0.3µg/µl, 4.5 µl/site), respectively, and the level of malondialdehyde (MDA) increase in same rats, compared with sham-operated rats. Learning and memory processes are coordinated with different brain regions. Since the oxidative damage may play a role in the aging process, including the associated decline, age-related impairment in spatial learning and memory may be alleviated by antioxidant treatment. Our results suggest that oxidative stress damage of hippocampus induced impairment in spatial memory of rats.


[P36] THE ALGESIC PROFILE OF SENSORY NEURONS DISSOCIATED FROM DORSAL ROOT GANGLIA

INS-HA+/–/TCR-HA+/– MICE COMPARED TO BALB/C MICE

A.D. IANCU1,2, B.M. MACRI1, C. STĂVARU2, D.L. RADU2

1University of Bucharest, Faculty of Biology, Center of Neurobiology and Molecular Physiology, Spl. Independenţei 91–95, Bucharest, Romania

2“I. Cantacuzino” INCDMI, Laboratory of Cellular Immunity, Spl. Indepedenţei 103, Bucharest, Romania

Diabetes mellitus is a global health problem, but the mechanisms implicated in the disease etiopathogenesis are not fully understood. Despite the increased prevalence of peripheral diabetic neuropathy, its causes are still unknown. The goal of our study was to reveal the molecular mechanisms of diabetic neuropathy in neuronal primary cultures.

Two transgenic mice lines are used: 1) the TCR-HA line, characterized by the presence of a receptor on the surface of T lymphocytes, and this receptor recognizes the hemagglutinin of the influenza virus A/PR8/34, and 2) the INS-HA line, presents the hemagglutinin of the same virus on the surface of the beta pancreatic cells. The offspring of these two lines are dTg mice. The presence of the transgenes, that are responsible for the etiopathogenesis of type I diabetes, is determined by the PCR technique. Blood and urine glucose and corporal weight are monitored after 4 and 6 weeks of life. Neuronal primary cultures are dissociated from dorsal root ganglia. Electrophysiological recordings of these neurons are performed by the patch-clamp technique, whole-cell configuration. There are significant differences between the electrophysiological parameters of sensory neurons from dTg mice compared to Balb/c mice. In our study we have recorded the intensity and amplitude of the action potential, the intensity and amplitude of the posthyperpolarisation and the effect of ATP, protons and capsaicin.

The algesic profile of peripheral sensory neurons from dTg mice is very different from that of the Balb/c mice. These results have a great impact in clinical immunology and open new perspectives in genetic therapy.


[P37] BETULIN AND BETULINIC ACID: AN IN VITRO ANTITUMOR EVALUATION

A. KAYCSA2, C. DEHELEAN1, C. ŞOICA1, B. BUMBĂCILĂ2, C. PEEV1, L. OLARIU3 1“Victor Babeş” University of Medicine and Pharmacy, Faculty of Pharmacy, Timişoara, Romania 2“Victor Babeş” University of Medicine and Pharmacy, Faculty of Medicine, Timişoara, Romania

3S.C. Biotehnos S. A., Bucharest, Romania

Betulin and betulinic acid are pentacyclic triterpenes used as potential antitumor compounds. Betulinic acid is now in preclinical trials and is considered a selective antimelanoma compound. Betulin is known as precursor in the betulinic acid synthesis and can develop an antitumor activity, too. The aim of this study was to demonstrate the antitumor activity of the two compounds if dissolved as complexes with cyclodextrin or PVP.

Betulin and betulinic acid were dissolved with hydroxypropyl gamma cyclodextrin and PVP by kneading procedure and coprecipitation. The complexes were tested for the antitumor activity on human metastasis melanoma cell lines. The stock solutions were 10 mM in water. For in vitro tests, increasing concentrations of the two compounds were added to the culture medium of the tumor cells.

The obtained data confirm the antiproliferative activity for both betulinic acid and betulin. Betulinic acid leads to major inhibition on melanoma cells. Betulinic acid develops a high antiproliferative activity on melanoma cells. The cyclodextrin incorporation of the compound increases its water solubility, allowing the use of higher concentrations and avoiding its solubilisation in organic solvents. (Acknowledgements for financial support to the grants: INOVARE 69/2007 and PN II 1257/2007).


[P38] c-Myc INDUCES ALTERATION OF TELOMERIC DNA AND OF ITS TRF2 ASSOCIATED PROTEIN

3-DIMENSIONAL ORGANIZATION

N. MIRANCEA1,2, D. MIRANCEA1,2, D. KRUNIC2, P. BOUKAMP2

1Institute of Biology of the Romanian Academy, Spl. Independenţei 296, Bucharest, Romania

2German Cancer Research Centre, Department of Genetics of Skin Carcinogenesis, Im Neuenheimer Feld 280, Heidelberg, Germany

Since the time when the cell destiny was essentially attributed to the chromatinian material, there was a constant and increased interest to know more and more about the relationships between structure, 3-D organization of the nucleus and genomic functionality. Telomeres, the nucleo-proteic terminals of each chromosome, play important roles in the context of genomic stability and functionality. Because of telomeres’ possible involvement in malignant transformation, a lot of interest was devoted to nucleoprotein complex of mammalian chromosome ends. In this context, we investigated telomeric DNA and its telomere repeat binding factor associated protein TRF2 3-D organization in immortal HaCaT keratinocytes and in HaCaT variant that constitutively expresses c-Myc protein. A digoxygenylated (CCCTAA)n Peptide Nucleic Acid (PNA) probe anti-(TTAGGG)n telomeric repeats followed by a gold labelled anti-digoxygenin antibody was used. A concomitantly immune detection of telomeric DNA and TRF2 was also applied. Investigations of 3-D distribution of telomeric DNA and TRF2 protein inside of interphase nuclei of immortal HaCaT cells showed that telomeres are distributed in distinct non-overlapping territories. In case of HaCaT c-Myc cells, the pattern is dramatically changed: telomeric aggregates coexist with telomeres of normal size. A telomeric aggregate appears to be formed by close association of a few telomeres. Taking into consideration that telomeric aggregates formation involved their respective chromosome aggregation and an abnormal pattern of their distribution inside of the interphase nuclei of HaCaT c-Myc cells, we suggest that these induce genomic instability and consecutively alterations of cell physiology.


[P39] PREDICTIVE MARKERS IN BREAST CARCINOMAS: AN IMMUNOHISTOCHEMICAL STUDY

D.M. NARITA1, D.A. IZVERNARIU2, A.M. CÎMPEAN2 1Department of Biochemistry, “Victor Babeş” University of Medicine and Pharmacy,

Timişoara, Romania 2Department of Histology, “Victor Babeş” University of Medicine and Pharmacy,

Timişoara, Romania

Breast cancer is a heterogeneous disease and there is a continuous effort to identify markers for disease prognosis and therapy management. To date, relatively few markers have established prognostic power, the estrogen receptor (ER) being the most powerful predictive marker in breast cancer. Progesterone receptor (PR) is also a widely used marker, although its value is less well established. HER2 status may predict response to chemotherapy, hormonal treatment and monoclonal antibody trastuzumab. In addition, it has been suggested that AR (androgen receptors) could be prognostic markers but their role in the pathogenesis of breast carcinomas is far to be clear. The purpose of this study was to assess the AR expression in female breast carcinomas and to correlate the results with the ER/PR status, HER2/neu status and some histopathological features of carcinomas, like histological type and grade, nodal and metastasis status.

We studied 156 surgical specimens from patients with breast cancer. The pathological diagnosis and grading were done on hematoxylin-eosin samples. Immunohistochemistry was performed to study the correlations between AR, ER, PR and HER2/neu expressions. AR was expressed in 71.8% of cases. Most of cases were represented by ductal invasive carcinoma (95.8%) and were well differentiated (78.8%); 48% of AR-positive carcinomas were nodal positive and respectively 7% were metastasis status positive. ER and PR were expressed in 54.5%, respectively 60.25% of carcinomas. Regarding the HER2 status, 37.18% of cases overexpressed HER2. 52.6%, respectively 60% of AR-positive breast cancers were ER, respectively PR positive; 63% of AR-positive tumors were HER2/neu negative. Most of AR-positive carcinomas were well differentiated, nodal and ER/PR status positive, HER2/neu and metastasis status negative. The assessment of AR expression could be useful for evaluating the prognostic outcome and for establishing new therapeutic strategies in patients with breast cancer.


[P40] EVALUATION OF SOME REDOX PARAMETERS IN THE SERUM AND TISSUES OF RATS UNDER CHRONIC

PSYCHOLOGICAL STRESS

V. NEGOIŢĂ1, I. GRUIA1, V.V. POPA2 1“Prof. Dr. Alex. Trestioreanu” Institute of Oncology, Bucharest, Romania

2Faculty of Veterinary Medicine, Bucharest, Romania

Psychological stress is incriminated as a risk factor in development of many human disorders such as cardiovascular, neurodegenerative, metabolic and immunological diseases. Persistent psychological stress is associated with increased oxidative stress in several tissues resulting from either high free radical production or decreased antioxidant/reparatory systems or both.

The study aimed to investigate the influence of chronic psychological stress on redox homeostasis in rat serum and tissues (liver, kidney, lung, brain, adrenals and skin). 12 male Wistar rats were exposed to chronic stress (open-field stress) for 30 days. 24 h after the last exposure, blood and tissues were collected for spectrophotometric determination of the content of thiobarbituric acid reactive substances (TBARS), total thiol groups (by Schosinsky test) and oxidase activity of ceruloplasmin (by Ravin test). The results showed an increased oxidative stress mainly in the serum and the brain of the stressed rats through elevation of TBARS level and concomitant decline of thiol level and ceruloplasmin activity.

The oxygen metabolites released during stress may function as signaling molecules, but also as cytotoxic mediators by oxidative attack of different cell targets and initiation of degenerative lesions in certain tissues. Antioxidants administration may be useful both in prevention and control of stress pathology, by limiting the detrimental effects of oxygen metabolites.


[P41] RHEUMATOID ARTHRITIS, REDOX STRESS PARAMETERS AND PERIPHERAL BLOOD MONONUCLEAR

CELL MEMBRANE FLUIDITY

C. NEGREI1, D. MARGINĂ3, A. BĂLĂNESCU2, M. ILIE1, A. GLIGA, D. BACONI1, D. BĂLĂLĂU1

1“Carol Davila” University of Medicine and Pharmacy, Department of Toxicology, Bucharest, Romania

2“Carol Davila” University of Medicine and Pharmacy, Research Center of Rheumatic Diseases, Sf. Maria Hospital, Bucharest, Romania

3“Carol Davila” University of Medicine and Pharmacy, Department of Biochemistry, Bucharest, Romania

Rheumatoid arthritis (RA) is associated with increased redox stress. The aim of the study was to evaluate the dynamics of some biochemical redox stress parameters (activity of erythrocyte glucose-6-dehydrogenase, G6PDH, susceptibility of erythrocytes to lipid peroxidation, ESP) and of peripheral blood mononuclear cells (PBMC) permeability in RA patients compared to healthy controls. The study group consisted of 10 RA patients that were compared to a control group of 10 healthy subjects. In red blood cells, separated from à jeun blood samples, G6PDH and ESP were assayed. PBMC were separated by differential centrifugation and were spectrofluorimetrically evaluated for the determination of membrane permeability (using TMA-DPH as a fluorescent probe). The results showed that G6PDH was significantly decreased (25.75±13.87 UI/g Hb vs. 190.45±27.89 UI/g Hb, p<0.0001) and the ESP was significantly increased (425.25±125.20 mM MDA/g Hb vs. 166.86±35.56 mM MDA/g Hb, p=0.0002) in RA patients as compared to controls. The membrane permeability parameter decreased significantly in the study group (0.052±0.041 vs. 1.36±0.76, p=0.0007). Our results show that RA is associated with a reduction in G6PDH activity and a subsequent decrease in NADPH production and antioxidant defense. The increase of the ESP illustrates the reduction of the liposoluble antioxidant content of the erythrocyte membrane. The RA patients are also characterized by pathological changes of the PMBC cell membrane, thus by a lower TMA-DPH permeation of the cell membrane and a subsequent reduction of the membrane fluidity, which could be due to the immune activation of these cells.


[P42] PREVENTION OF SALMONELLOSIS IN CHICKENS BY POTATO SHOOT LECTIN ADMINISTRATION

A. POP, G. DINESCU, I. TOGOE, M. MILITARU, C.P. CORNEA

University of Agronomical Sciences and Veterinary Medicine, Faculty of Veterinary Medicine, Spl. Independenţei 105, Bucharest, Romania

The restrictions in the use of antibiotics forced the poultry producers and researchers to look for other ways to effectively protect newly hatched chicks against Salmonella. Involvement of oligosaccharides in bacterial adhesion prompted us to investigate the in vitro interaction of some lectin preparations with Salmonella cultures and their in vivo protective effect.

Five lectin preparations obtained by affinity chromatography interacted with liquid cultures of Salmonella typhymurium and the degree of bacteria agglutination was determined. The most effective lectin was that isolated from potato shoots. Animals were treated as follows: control, not infected but lectin treated, infected with Salmonella, infected and lectin treated. Lectin was administered to 3 days old chicken, in the drinking water.

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

Orally administered lectin induced the immune system activation and also prevented the bacterial adhesion to the intestinal mucosa.


[P43] CORRELATION BETWEEN NITROSATIVE STRESS AND SEVERITY OF LIVER INJURY IN CHRONIC VIRAL

HEPATITIS C

C. STĂNCIULESCU, C. PISOSCHI, M. BANIŢĂ, V. COMĂNESCU, D. TACHE, N. MITREA*

University of Medicine and Pharmacy, Craiova, Romania *“Carol Davila” University of Medicine and Pharmacy,

Bucharest, Romania

The toxic action of nitric oxide is mainly mediated by peroxynitrite, a potent cytotoxic agent, resulted by its reaction with the superoxide anion, which promotes the nitrosylation of proteins and induces several tissue lesions.

We assessed the correlation between intrahepatic distribution of inducible nitric oxide synthase (iNOS) and nitrotyrosine (NT) in patients with chronic viral hepatitis C. Liver specimens from patients with hepatitis C were processed for paraffin embedding. 3µm slides were used to perform single immunohistochemical reactions using polyclonal anti-iNOS and anti-nitrotyrosine antibodies, streptavidin-biotin-peroxidase sequence to amplify the reaction and diaminobenzidine/H2O2 as revelators.

We found an intense hepatocellular expression of iNOS with a diffuse pattern in liver lobules, for the majority of specimens. The cells from the inflammatory infiltrate were negative for iNOS. Nitrotyrosine presented mostly similar distribution with iNOS but the positive hepatocytes were associated in clusters. We also performed correlations between intrahepatic iNOS and NT staining and the severity of liver disease was evaluated by Knodell and Metavir scores. Our results support the idea that the nitrosative stress shows a good direct correlation with the severity of liver injury in subjects with chronic hepatitis C.


[P44] THE PROTECTIVE ANTIOXIDANTS AND OTHER BIOCHEMICAL PARAMETERS IN BREAST CANCER

I.G. VERMAN1, N. ROŞOIU1, M. BASA2 1“Ovidius” University, Faculty of Medicine,

Biochemistry Department, Constanţa, Romania 2Constanţa Military Emergency Hospital, Constanţa, Romania

The main source for the reactive oxygen species (ROS) generated in the normal aerobic metabolism is mitochondria. The complex antioxidant systems delay or inhibit substrate oxidation (preventive antioxidants), remove the formed ROS preventing radical chain reactions (scavenging antioxidants) or catalyze the oxidation of other molecules (enzyme antioxidants). The imbalance between the production and degradation of ROS is considered to play an important role in cancer pathogenesis, because ROS determine oxidations, membrane damage, modification of proteins and DNA. The glutathione reducing power associated with other antioxidants protect the entire spectrum of biomolecules, regulate their function and facilitate the cell survival and optimal activity.

The study used 30 patients diagnosed with breast cancer, aged between 32 and 72 years. The biochemical data currently used for the diagnosis were completed with the assessment of GSH, GR, GPx and CPK concentration, of ALD activity and of the total antioxidant status (TAS). The metabolic changes determine a more pro-oxidant status during the cell differentiation process. The TAS is increased as a response to the inflammatory process associated to cancer. In the initial phases of chemical neoplasia or in nondifferentiated and very aggressive tumors, the increase of GSH was noticed mainly associated to tumor resistance to irradiation and chemotherapy. During tumor growth, the redox status of glutathione (GSH/GSSG) decreases, and GPx decreased as the neoplasia evolves. In the final phase the GSH decreases. The activity of GR and GSH were lower in the patients with cancer. These data reveal that antioxidative defense mechanisms might be impaired in cancer patients. Thus our study intended to establish the utility of the assessment of oxidative status and other biochemical markers for the diagnosis, prognostic and therapy monitoring of breast cancer.

POSTERS

Session 4: Advanced Methods in Biochemistry and Molecular Biology

ROM. J. BIOCHEM., 45, 1, 93–101 (2008)

[P45] CoMSIA APPLIED TO HMG-CoA REDUCTASE INHIBITORS

S. AVRAM1, D.M. DUDA-SEIMAN2, C. DUDA-SEIMAN3

1University of Bucharest, Faculty of Biology, Department of Physiology & Biophysics, Spl. Independenţei 91-95, Bucharest, Romania

2“Victor Babeş” University of Medicine and Pharmacy, Department of Medical Ambulatory, Medical Emergencies,

Blvd. C.D. Loga 49, Timişoara, Romania 3University of West Timişoara, Faculty of Chemistry-Biology-Geography, Department of Chemistry,

Pestalozzi 16, Timişoara, Romania

Statins are important in the modern concept of atherotrombotic cardiovascular disease prevention by their inhibitory role on hydroxyl-methyl-glutaryl-CoA reductase (HMG-CoA reductase). Here, the biological activities of tetrahydro-4-hydroxy-6-[2-(1H-pyrrol-1-yl]-2H-pyran-2-one as HMG-CoA reductase inhibitors were predicted by 3D-QSAR-CoMSIA (Comparative Molecular Similarity Indices Analysis).

The biological activities of HMG-CoA reductase inhibitors were selected from a published report. Molecular modeling of inhibitors was performed with the Sybyl 7.2 program. The minimum potential energy for statin derivatives was calculated by the conjugate-gradient method, convergence of 0.01, Tripos force field. In the end, Gasteiger-Marsili partial charges were loaded. Partial Least Squares (PLS) algorithm within Sybyl 7.2 was used to evaluate statistical parameters (q2, sPRESS, r2, Fisher test (F)). Also, the energetic contributions (e.g., steric, electrostatic, hydrophobic, hydrogen bond donor and acceptor) at HMG-CoA reductase inhibition were performed.

Leave-one-out cross-validated PLS analysis running with four principal components led to a high q2 cross-validated correlation coefficient of 0.60. Also, for a non-cross-validated PLS analysis, suitable fitted correlation coefficient (r2) of 0.98 was obtained. The contributions of steric, electrostatic and donor descriptors were just 0.154, 0.153 and 0.287 respectively, while hydrophobic contribution was found significantly higher (0.407). Also, the correlations between the predicted and observed biological activities were presented.

We believe that these results are very useful for the development of new HMG-CoA reductase inhibitors belonging to statins.


[P46] THE ACCURACY OF THE GC-MS DETERMINATION OF CAPSAICIN IN RAT BLOOD SAMPLES USING TWO

DIFFERENT DERIVATIZATION TECHNIQUES

B. BUMBĂCILĂ1, C. CANCIU2, A. KAYCSA1, R. DUMACHE1 1“Victor Babeş” University of Medicine and Pharmacy, Faculty

of Medicine, Biochemistry Department, Timişoara, Romania 2“Victor Babeş” University of Medicine and Pharmacy, Faculty

of Pharmacy, Pharmaceutical Biology Department, Timişoara, Romania

In this study we evaluated the levels of capsaicin in blood samples using a GC-MS (gas chromatography-mass spectrometry) technique and two different derivatization methods, after the intravenous administration of the capsaicin-hydroxypropyl beta cyclodextrin complex to eight adult Sprague-Dawley rats. Capsaicin (8-methyl-N-vanillyl-6-nonenamide) is a component of the Capsicum genus plants. It is an irritant for animals and produces a burning sensation on tissues. Still, there have been signalled anticancer properties of this compound. It appears that capsaicin is a specific apoptotic agent of the malignant cells, being harmless to the normal cell lines. Capsaicin has a very low solubility in water or in aqueous solutions, so we prepared its soluble complex with hydroxypropyl beta cyclodextrin (a derivative of a cyclic oligosaccharide composed of 7 α-D-glucopyranose units linked 1–4), in a 1:2 molar ratio. Immediately after the parenteral administration of complex, blood samples were analyzed. For increasing the capsaicin volatility, the blood samples were methylated and trimethylsilylized. The probes were determined by GC (DB5 capillary column)-MS (electron impact, ion trap). We observed that the trimethylsilylation technique with a mixture of hexamethyldisilazane and chlorotrimethylsilane in pyridine, for the derivatization of the capsaicin, offers a more accurate quantitative determination of capsaicin than the methylation method with methyl iodide, when performing GC.

The derivatization method used for increasing the volatility of capsaicin when analysing this compound by GC is very important if a quantitative determination of capsaicin is requested.


[P47] THE EFFECT OF SOME PLANT EXTRACTS ON THE GROWTH OF A S. CEREVISIAE STRAIN

CULTIVATED UNDER OXIDATIVE STRESS

L.A. GEORGESCU, A. NICOLAU, A.S. BOLOCAN

“Dunărea de Jos” University, Faculty of Food Science and Engineering, Str. Domnescă 111, Galaţi, Romania

Aromatic plants are rich in antioxidants, most of them being phenolic compounds. The food substances displaying antioxidant action protect against chronic diseases through the scavenging effect on the active oxygen radicals that are produced or introduced in the organism.

The aim of this research was to extract and quantify polyphenols from rosemary, thyme and basilicum, as well as to study their effect on the growth of S. cerevisiae under oxidative stress induced by H2O2. S. cerevisiae was chosen as model for an eukaryotic system. Knowing that the antioxidants have selective antimicrobial effects, we developed our study in order to understand their action on S. cerevisiae. The reducing capacity of hydro-alcoholic plant extracts was assayed using Oyaizu method and was correlated to the total polyphenolic content, which was determined according to Shahidi and Naczk. Plant extracts were obtained after 1 h of maceration at room temperature, using a mixture of 95% ethyl alcohol and water (1:1) and filtration through nylon filters. Growth kinetics of S. cerevisiae was tested in a medium containing various concentrations of H2O2 and plant extracts (1–10%) and was compared with that obtained in the same medium with or without extracts. The extracts and H2O2 were added in different developing stages of S. cerevisiae. The yeast growth was turbidimetrically assayed.

A beneficial effect on the growth kinetics of S. cerevisiae was noticed when adding up to 5% plant extract, both in a regular medium and in a medium containing H2O2. This protective effect was much more pregnant in the samples containing H2O2, while the catalase assessment proved that plant extracts rich in phenolic antioxidants improve the activity of this antioxidant enzyme.


[P48] RAPID VOLTAMMETRIC DETERMINATION OF VITAMIN C IN FRUIT JUICES

A.M. PISOSCHI1, A.F. DĂNEŢ2 1University of Agronomic Sciences and Veterinary Medicine,

Faculty of Veterinary Medicine, Bucharest, Romania 2University of Bucharest, Faculty of Chemistry, Bucharest, Romania

Ascorbic acid content of foodstuffs and beverages represents an important indicator of quality, which has to be carefully monitored. Many analytical methods can be used for ascorbic acid determination. Conventional techniques are represented by volumetric methods (titration with dichlorophenol indophenol, potassium iodate or bromate). Ascorbic acid was also determined by spectrophotometric and chromatographic methods. HPLC with electrochemical detection has turned out to be a selective and sensitive method for ascorbic acid assessment in foodstuffs and biological fluids. Electrochemical methods based on the construction of potentiometric or amperometric biosensors have the advantage of being sensitive, allowing analyte detection at concentrations as low as 10–8 M.

The aim of this work was to develop an accurate and sensitive voltammetric method for assessing ascorbic acid concentration in fruit juices. The voltammetric measurements were performed using a potentiostat-galvanostat, in a three-electrode cell, with a Pt disc working electrode, a Pt counter-electrode and a calomel electrode as reference. The anodic oxidation peak height increases with the analyte concentration. The obtained calibration graph shows a linear dependence between peak height and ascorbic acid concentration on the range 0.1–10 mM. The equation of the calibration graph was y=6.391x + 0.1903, r2=0.9995, r.s.d.=1.14%, n=10, Cascorbic acid=2 mM. The developed method was applied to ascorbic acid assessment in fruit juices. Therefore, the ascorbic acid content determined, ranged between 0.83 and 1.67 mM for orange juices and from 0.53 to 1.93 mM for lemon juices. The highest values were obtained for natural juices obtained by squeezing of fruits. These results obtained by cyclic voltammetry were in accordance to those obtained by the volumetric method with dichlorophenol indophenol and in previously published works. The results obtained in this study show that cyclic voltammetry can be successfully used in food industry, for assessing vitamin C content of natural fruit juices and soft drinks, being an accurate, rapid and relatively cheap method.


[P49] NEW EPIGENETIC METHOD FOR THE EARLY DIAGNOSIS OF PRADER WILLI SYNDROME

M. PUIU1, G. ANTON2, A. BOTEZATU2, M. ŞERBAN1, D. DAN3, N. CUCU4 1“Victor Babeş” University of Medicine and Pharmacy, Timişoara, Romania

2“Ştefan Nicolau” National Institute of Virology, Bucharest, Romania 3Prader Willi Romanian Association, Zalău, Romania


This work presents the preliminary results of a collaborative multidisciplinary project on Prader Willi and Angelman syndromes. These are distinct imprinting diseases, with a neurodegenerative component, both being caused by changes in parent contributions on the 15q11.2-13 chromosome, representing deletion, uniparental disomy and altered epigenetic marking through DNA methylation or chromatin modification. Due to high mortality and morbidity associated with PSW/AS, this project will be the ground for new clinical studies to establish guidelines for diagnosis and treatment in order to improve the quality of medical practice and an improvement of medical and social standard for affected patients. The usual method approached for the diagnosis, fluorescence in situ hybridization-FISH, is poor informal as it localizes the common lesion on the 15 chromosome; the proposed epigenetic method may, however, bring the key information regarding exactly the paternal or maternal allele affected, which differentiates the two syndromes, by the estimation of the DNA methylation profile on them. The optimization of the epigenetic method, methylation specific PCR–MSP, in order to complete the diagnosis strategy, has been performed, using a number of 20 clinically characterized cases, which have been administered by the Romanian PWS Association. The DNA methylation status on the affected allele confirmed 90% of cases which were analyzed also by classical karyotype and FISH methods. Specific primers for methylated and unmethylated DNA allele and bisulphite mutagenesis for targeting unmethylated cytosine residues have been used for the estimation of the DNA methylation profiles. The results indicated the detection of PWS, which is characterized by the paternal allele silencing through DNA methylation, as compared with AS, defined by maternal allele methylation. The implementation of such a new approach in PWS diagnosis in Romania is a priority of the National Plan for Rare Diseases which integrates this research direction into the FP7 programs.


[P50] CLONING AND EXPRESSION OF HISTIDINE-DOMAIN PROTEIN TYROSINE PHOSPHATASE IN ZEBRAFISH

C.A. TĂNASE

Institute of Biochemistry, Spl. Independenţei 296, Bucharest, Romania

Tyrosine phosphorylation of proteins is one of the most important cell signaling mechanisms involved in embryonic development. Thus, tyrosine kinases and tyrosine phosphatases, which regulate tyrosine phosphorylation are tightly controlled at transcriptional, translational and post-translational level. Histidine Domain Protein Tyrosine Phosphatase (HD-PTP) is a putative cytosolic tyrosine phosphatase whose functions are not yet known.

Our aim is to understand the role of this phosphatase in vertebrate development. In this study we sought to clone the zebrafish cDNA and to investigate HD-PTP expression at mRNA level. Total RNA isolation from adult fish and embryos, 5’/3’ RACE, cloning, sequencing, Northern blot and RT-PCR were the methods used. We partially cloned the zebrafish HD-PTP cDNA. The 2050 bp fragment cloned corresponds to the 3’end of the gene and is 70% identical to its human ortholog. The Northern blot analysis of total RNA isolated from adult fish with HD-PTP specific probes revealed a unique transcript with an apparent molecular mass of approximately 7 kb. RT-PCR amplification of total RNA isolated from adult tissues and from several early developmental stages showed that zebrafish HD-PTP mRNA is expressed in almost all the tissues analyzed (ovary, intestine, gills, liver, muscles, skin, brain, eye, but not in the heart), and it is present in early stages of development (i.e., cleavage period: 2 cell, 64 cell, gastrula period: shield, 75% epiboly, segmentation period: 4 somite, hatching period: 48 h, and not at 17 somite stage). We cloned the 3’end of the zebrafish HD-PTP cDNA. Zebrafish HD-PTP transcript is about 7 kb long. zfHD-PTP mRNA is expressed in most adult tissues and in almost all early developmental stages.

These patterns of expression suggest that this protein might have an important role in tissue homeostasis and in development. We will test this hypothesis by functional studies in zebrafish embryos.


[P51] OPTIMISATION OF AMOS PCR METHOD FOR DIFFERENTIATION OF BRUCELLA ABORTUS,

BRUCELLA OVIS AND BRUCELLA SUIS

M. VANGHELE, M. IONESCU, P. TAMBA, H. COSTE, I. SANDU

Institute for Diagnosis and Animal Health, Bucharest, Romania

The aim of our study was to test some reference strains in different amplification conditions for the PCR method optimisation, in order to discriminate among three species of Brucella (B. abortus, B. ovis and B. suis) involved in animal pathology.

The bacterial strains used in the present study are: B. abortus S99 (AFFSA Bq II), B. ovis REO 198 (AFFSA Bq II) and B. suis biovar2 1115 (IDSA). Preparation of cell lysate: DNA was obtained by thermic lysis, without purification. Briefly, cells were incubated at 95°C for 30 min in TE buffer pH 8.0. After that, the lysate was centrifugated for 10 min at 12,000 rpm and the supernatant was collected. Primers: Oligonucleotide primers were designed by B.J. Bricker and S.M. Halling (1994). PCR amplification of specific DNA: the amplification was performed on the serial dilutions of DNA (starting with 1 ng of DNA) from Brucella reference strains. The PCR conditions were different by the variation of the MgCl2 concentration (2–3.5 mM ), the dNTPs concentration (200–250 µM) and anneling temperature (Ta) (54–60°C). The PCR mix was performed in 50 µl final volume. The amplified products were separated in a 1.5% agarose gel in the presence of 0.5% TAE and visualized with ethidium bromide under UV light. As predicted, DNAs from each of the reference strains were evaluated as targets. DNA was amplified from B. abortus (498 bp), B. ovis (976 bp) and B. suis (285 bp) by using 1 ng of DNA, 1.5 mM MgCl2, 250 µM dNTPs and a Ta = 56°C.

The amplification results with the specific primers were positive for the Brucella reference strains. This PCR with a five-primer cocktail is a very good method for discrimination of Brucella species involved in animal diseases.

POSTERS

Session 5: Bio–Nano–Technologies

ROM. J. BIOCHEM., 45, 1, 103–127 (2008)

[P52] HPLC EVALUATION OF MURINE NEURONAL MONOAMINERGIC STATUS IN DIFFERENT

TYPES OF BEHAVIOR

A.L. ARSENE, C. ARAMĂ, A.N. CRISTEA, N. MITREA, C. DRĂGOI

“Carol Davila” University of Medicine and Pharmacy, Faculty of Pharmacy, Bucharest, Romania

Cerebral monoaminergic status can offer important data regarding patho-physiological susceptibility, providing clues for targeted therapy. Our study investigated the correlations between cerebral monoaminergic status, expressed as specific neuromediator (noradrenaline, dopamine and serotonin) concentration and different types of behaviour.

Experiments were performed on mice, divided into three behavioral groups according to their pain sensitivity: the adrenergic type (A type), the opioid type (O type) and the equilibrated N type. Neuronal monoaminergic status was evaluated both in basal state and after acute stress, by quantifying noradrenaline, dopamine and serotonin in the brain, using HPLC. Briefly, brain samples were deproteinized and prepared for HPLC reversed-phase separation with simultaneous UV detection for all three neurotransmitters. The results were expressed as µg/mg wet tissue. Basal state type A group developed lower neuronal concentrations for all neuromediators compared with O type group, as follows: noradrenaline 1.01±0.189 (type A) vs. 1.52±0.26 (type O), dopamine 0.87±0.2 (type A) vs. 0.1±0.001 (type O), serotonin 0.12±0.005 (type A) vs. 0.53±0.01 (type O). Following acute stress, type A mice showed significantly higher neuronal levels of noradrenaline (3.41±1.14) vs. type O animals (1.55±0.13, p<10–7) and also low amounts of dopamine and serotonin. As expected, the adrenergic type behavior developed higher concentrations of neuronal noradrenaline following acute stress (3.41±1.14), compared with the basal state (1.01±0.189, p<10–5). The same pattern was noticed for dopamine and serotonin. Type A possesses equilibrated neuronal mechanisms in basal state but develops high concentrations of neuronal noradrenaline under stress. Many studies speculated that the pathophysiological hallmark of type A individuals is the hyperactivity of the sympathetic nervous system, although the molecular bases of these findings have not been established yet.


106

[P53] THE INFLUENCE OF SUPERFICIAL CHARGE AND IONIC STRENGTH UPON THE INTERACTION BETWEEN

β-LACTAM ANTIBIOTICS AND OmpF PORINS

A. ASANDEI, L. MEREUŢĂ, R. CHIRIAC, T. LUCHIAN

“Al. I. Cuza” University, Faculty of Physics, Laboratory of Biophysics and Medical Physics, Blvd. Carol I, no. 11, Iaşi, Romania

The OmpF porin is an important constituent of Gram-negative bacteria outer membranes, believed to be the principal pathway for intracellular transport of the β-lactam antibiotics. The main purpose of this study was to investigate the interaction between the permeating ampicillin molecules and the OmpF porin, by quantifying the blockage events of the single channel conductance at different salt concentrations, and various lipid charge densities present on the supporting membrane. At moderately acidic pH values, ampicillin molecules are in the zwitterionic form and they can interact favorably with the constriction region inside the porin, and these interactions were found to potentiate the antibiotic binding and translocation. Substituting zwitterionic membrane lipids (DPhPC) with negatively charged ones (DPhPS), we found that the fixed charge of the lipid has a small effect on the antibiotic-induced conductance fluctuations of the channel, revealing the fact that the superficial charge of the membrane does little to the translocation process of the antibiotic molecules. However, we show that the number of blockage events per second is a strong function of the ionic strength. The interaction between the antibiotic molecules and the binding site at the constriction region are of electrostatic origin, so that it is conceivable that the rate of blockages would go up with the decreasing concentration, as we see in our experiments. Our experimental data confirmed that lower concentrations of the buffer solution favor the electrostatic interactions between antibiotic molecules and the porin itself, thus contributing to a more efficient transport of them across the lipid membrane.


107

[P54] RAFTS-INDUCED MODULATION OF TRANSPORT AND KINETIC PROPERTIES OF CERTAIN ANTIMICROBIAL

PEPTIDES

R. CHIRIAC, A. ASANDEI, L. MEREUŢĂ, T. LUCHIAN

“Al. I. Cuza” University, Faculty of Physics, Laboratory of Biophysics and Medical Physics, Blvd. Carol I, no. 11, Iaşi, Romania

Rafts are small (10–200 nm), heterogeneous, highly dynamic, sterol and sphingolipid-enriched domains that compartmentalize cellular processes. The term ‘raft’ refers to domains from the lo (liquid ordered) phase, which are relatively ordered lipid domains floating in a more fluid, ld (liquid disordered) lipid environment. Herein, we studied the effect of rafts-like microdomains on the alamethicin antimicrobial peptides embedded in a planar lipid bilayer, consisting of a ternary lipid mixture of palmitoyl-oleoyl-phosphatidylcholine (POPC), sphingomyelin (SPM) and cholesterol, at molar mixing ratio of 5:3:2. The electric properties of such mixed membranes containing lipid rafts were evaluated by quantifying their capacitance, which decreases in the presence of rafts domains (POPC-chol-bSPM (5:3:2) (lo+ld phase)), probably because these assemblies are more ordered and tightly packed than the surrounding bilayer. The difference in packing is due to the saturation of the hydrocarbon chains in raft sphingolipids and phospholipids, as compared with the unsaturated state of fatty acids of phospholipids in the liquid-disordered phase. Moreover, such experiments involving single alamethicin oligomers, have demonstrated an unexpected, non-monotonic dependence of the single channel electrical conductance in a different ternary lipid mixture. Specifically, our data prove that the electrical conductance of the second sub-conductive state of the alamethicin oligomer embedded in such membranes decreases in the presence of rafts domains (POPC-chol-bSPM (lo+ld phase)), as compared to non-raft mixtures (POPC-chol-bSPM (ld phase) and POPC-chol-bSPM (lo phase)). Our tentative conclusion derived from such experiments points to a possible involvement of lateral pressure effects within the lipid membrane, which lead to a prominent mechanical constriction of the alamethicin pore.


108

[P55] MATHEMATICAL EVALUATION OF STATISTICAL DATA OBTAINED BY HPLC ANALYSIS OF

ENROFLOXACIN AND CIPROFLOXACIN RESIDUES IN POULTRY MEAT

M. CRIVINEANU1, V. TRIFAN2, V. CRIVINEANU1, M.D. CODREANU1

1Faculty of Veterinary Medicine, Bucharest, Romania 2Crida Pharm S.R.L., Bucharest, Romania

The presence of pharmaceutical substances in alimentary products of animal origin arises from mixing these substances in feeds, for growth stimulation, or from their use for therapeutic purposes. The dimension of the phenomenon induced by residues present in alimentary products of animal origin is reflected by the numerous morbid entities expressed by the consumers and their descendents.

This work aims to establish an equation that obeys the residues depletion law, in order to determine the reasonable waiting period, taking no notice of the maximum imposed limits. To accomplish this purpose, enrofloxacin and ciprofloxacin residues in poultry meat were assessed and the results were expressed as exponential, logarithmic and polynomial curves. During the studied interval the enrofloxacin and ciprofloxacin decreasing rate in poultry meat was higher in the first days, drug’s elimination starting almost immediately after treatment interruption. Enrofloxacin and ciprofloxacin residual levels in poultry meat tended to zero on the 9th day after treatment interruption. The logarithmic equation reproduced the values better than the exponential and polynomial equations.


109

[P56] THE DETECTION OF ENROFLOXACIN RESIDUES IN PORK USING HPLC ANALYSIS

M. CRIVINEANU1, V.TRIFAN2, G. PARASCHIV2, V. NICORESCU1

1Faculty of Veterinary Medicine, Bucharest, Romania 2Crida Pharm S.R.L., Bucharest, Romania

The consequences of the presence of drug residues in animal organism and food products of animal origin represent an actual problem, due to the high diversity of drugs used for therapy.

The aim of this research was to compare enrofloxacin and ciprofloxacin residues depletion in pork, after the administration to pigs of two enrofloxacin pharmaceutical forms (oral solution 10% and powder 40%) for 5 days, in the manufacturer’s recommended doses. The pigs were slaughtered at various time intervals after treatment interruption; the samples were analyzed by high performance liquid chromatography (HPLC) method.

Considering the pharmacokinetics of enrofloxacin, residues level in pork was presented as sum of enrofloxacin and ciprofloxacin. During the surveyed interval of time, the drug was eliminated from the animal organism especially in the first days after treatment interruption. In the case of enrofloxacin, waiting interval or withdrawal period for pork is 6–8 days, depending on the pharmaceutical form and the concentration of the drug.


110

[P57] PHYTOPATHOGENIC FUNGI AND THEIR MYCOTOXINS

A. GHEORGHE1, F. CHELU2, A. ROŞU1, F. POPEA1, P. FLORIAN2, M. ICRIVERZI2, M. TRIF2, A. ROŞEANU2, L. JECU1

1National Research & Development Institute for Chemistry and Petrochemistry, Spl. Independenţei 202, Bucharest, Romania

2Institute of Biochemistry, Spl. Independenţei 296, Bucharest, Romania

Mycotoxins are natural products secreted as toxic secondary extracellular metabolites by filamentous fungi such as Aspergillus, Fusarium and Penicillium sp. The main mycotoxins are aflatoxins, citrinin, fumonisin, ochratoxin and zearalenone. Due to the toxic action of mycotoxins on human and animal health, various strategies including physical, chemical and biological processes have been developed to reduce/eliminate their harmful effects. Biological control, based on the ability of some microorganisms to prevent or inhibit the growth of mycotoxigenic fungi, is an alternative to synthetic chemicals. Agents such as Bacillus and Aspergillus sp. present the advantage to be more stable than chemicals, safer to use and less phytotoxic.

Our aim was to select microorganisms able to inhibit the growth of fungal pathogens. Using disc diffusion method, several Bacillus strains isolated from natural habitats were evaluated for their capability to affect the growth of Fusarium, Aspergillus, Penicillium and Trichoderma strains. The fungal strains were cultivated on Czapek-Dox medium, and after three days of incubation the content of ochratoxin A, afltoxin B1, citrinin and fumonisin in the culture medium was determined by ELISA method. A. niger 38 and A. niger 105 were found to produce ochratoxin and aflatoxin B1, the latter only in low quantity. A high level of citrinin was detected in A. niger 38 culture medium, a mycotoxin also produced by Penicillium sp. 41, A. oryzae 100, and Trichoderma sp. 103. Only a very low quantity of fumonisin was found in Fusarium 37 and Trichoderma 103 cultures. Dual solid cultures assay revealed an antagonism between several microbial strains. Thus, from the Bacillus strains tested, B. amylolichefaciens 1014 was found to be the most efficient biocontrol agent, leading to a total inhibition of Penicillium sp. 41, Fusarium sp. 37 and A. oryzae 100 growth.


111

[P58] CELL PROLIFERATION IS MODULATED BY ALCOHOLIC EXTRACTS OF GERANIUM ROBERTIANUM

AND HELLEBORUS PURPURASCENS

I. GHERASIM1, C. FILIPESCU1,2, C. ALIFANTI2, A.A. ATHANASIU2, M.T. NECHIFOR3, M. MOCANU1, A. BOANŢĂ4, M. LEABU1,4

1“Victor Babeş” National Institute of Pathology, Bucharest, Romania 2ICECHIM, Bucharest, Romania

3University of Bucharest, Faculty of Biology, Bucharest, Romania 4“Carol Davila” University of Medicine and Pharmacy, Romania

Various phytopreparations of Geranium robertianum and Helleborus purpurascens are currently used in therapy, despite the very scarce knowledge available on the mechanisms of action at cellular and molecular levels. Therefore, our goal was to obtain extracts of G. robertianum and H. purpurascens in order to investigate their biological effects on diverse cultured cells. Extracts in absolute and 70% ethanol were obtained and analyzed by TLC for their content in polyphenols and antioxidant activity. The effects of the extracts on cell proliferation were assessed for three cell lineages, at three extract concentrations. The extracts contained six major components. The polyphenol content was higher in hydro-alcoholic extracts for both plants. The antioxidant activity proved to be quite similar in G. robertianum for both types of extracts, but it was 7 folds higher in hydro-alcoholic extracts of H. purpurascens as compared with its absolute alcohol extracts. Moreover, the antioxidant activity of G. robertianum extracts was 15 times higher than that of H. purpurascens extracts. The effects on cell proliferation were different in terms of cell type and tested extracts. For all cell types G. robertianum extracts increased cell proliferation (by 30% for HeLa, by 25% for DOK and by 50% for HS27) in a concentration dependent manner. H. purpurascens extracts reduced cell proliferation by 25% for HeLa and Hs27, while DOK cells proved to be treatment refractory in our experimental conditions. In conclusion, our preliminary results suggest that the (hydro)alcoholic extracts of G. robertianum and H. purpurascens modulate cell proliferation in culture and the experimental approach is useful in current studies regarding elicited cellular mechanisms at molecular level. (Supported by the Romanian Ministry of Education and Research, Research of Excellence Program, Project CEEX125/2006).


112

[P59] MULTISTANDARD RT-PCR METHOD FOR THE STUDY OF DRUG METABOLIZING ENZYMES EXPRESSION

IN RAT CHOROID PLEXUS

D. GRĂDINARU, D. MARGINĂ, A.L. ARSENE, N. MITREA

University of Medicine and Pharmacy, Faculty of Pharmacy, Department of Biochemistry, Traian Vuia 6, Bucharest, Romania

The choroid plexus (CP), located in the lateral, third and fourth brain ventricles, is the site of elimination of xenobiotics and endogenous waste from the cerebrospinal fluid (CSF) together with convective flow associated with CSF turnover. The purpose of this study was to determine whether UDP-glucuronosyltransferases (UGTs) and NADPH-cytochrome P450 reductase are regulated by drug metabolizing enzyme inducers known to act as ligands or activators for the aryl hydrocarbon receptor (AhR: 3-methylcholanthrene, 3-MC), the constitutive androstane receptor (CAR: phenobarbital, PB) or the pregnane X receptor (PXR: dexamethasone, DX). We used a quantitative multistandard RT-PCR method that consists in mixing the total RNA samples extracted from rat tissues with a constant amount of total RNA prepared from mouse cerebral tissue, which brought both β-actin and UGT1A6 isoform sequences. Separate PCRs for UGT1A6 and β-actin amplification were performed by the use of specific and common oligonucleotide primers, able to hybridise with rat and mouse sequences with the same efficiency. Each amplification product was then distinguished by restriction site polymorphism, using Eco RI and Xba I restriction sites. For P450 reductase we found that the rate of amplification was exponential for 19 to 26 cycles, after which it decreased drastically and reached a plateau. So, kinetic patterns of control and xenobiotic treated animals were compared. The use of powerful RT-PCR measurements definitely established the UGT1A6 and P450 reductase mRNA expression in both the brain and choroids plexus. Moreover, it allowed us to show that UGT1A6 expression increased in the CP after an “in vivo” treatment of animals with 3-MC, but no changes were observed after PB and DX administration.

Results suggest that xenobiotics could modulate the biotransformation of exogenous compounds in the CP, and underline the role of detoxification enzymes in the maintenance of brain homeostasis.


113

[P60] SINGLE NUCLEOTIDE POLYMORPHISMS DETECTION BY AUTOMATED SSCP-BASED CAPILLARY

ELECTROPHORESIS IN AN ARABIDOPSIS THALIANA T-DNA INTEGRATION SITE

M.C. ICHIM1, A. MILCAMPS2, N. PAPAZOVA3, A. DEPICKER4, M. DE LOOSE3, G. VAN DEN EEDE2

1“Stejarul” Research Centre for Biological Sciences, Piatra Neamţ,Romania 2Institute for Health and Consumer Protection, Ispra, Italy

3Institute for Agricultural and Fisheries Research, Merelbeke, Belgium 4Ghent University, Department of Plant Systems Biology, Ghent, Belgium

We investigated the putative sequence changes of a T-DNA insertion site in the genome of the model plant A. thaliana (C24 ecotype) under high light (photo-oxidative) stress. The T-DNA insertion line was generated via Agrobacterium-mediated transformation and the corresponding insertion sites identified. Parallel analysis of the target site in wild-type plants, in both control and stress treated plants, will allow to define if and what kind of rearrangements are specifically induced by high light stress. Plants were grown under regular conditions (21ºC and 15h/9h photo-period) until the onset of flowering. A control set was brought to maturity in the same conditions, while a second set (SL25) was transferred in a phytotron for high light treatment. A high-throughput method (Single Strand Conformational Polymorphism, SSCP) was tested and optimized to detect even the minor (SNPs) changes that could occur. SSCP analysis was combined with capillary electrophoresis (CE-SSCP). PCR is carried out using fluorescent-dye-labelled sense and anti-sense primers specific for the DNA region of interest. Fragment M (264bp) spans a genomic region around the T-DNA insertion point in the transgenic line FH33 (clone F14M19, chromosome IV). For each set 100 plants were analyzed both individually as well as in pools of 5 plants. The SSCP profiles of genomic fragment M were obtained and no change in the SSCP pattern was detected. The results indicate that in wild-type plants, grown in standard conditions or exposed to high light stress, the sequence considered (which in the A. thaliana transgenic line FH33 spans the genomic region around the T-DNA insertion point) does not undergo any major or minor structural re-arrangement.


114

[P61] CELLULAR AND MOLECULAR MODIFICATIONS INDUCED BY ORGANIC SOLVENTS TO MODERATELY

HALOPHILIC MICROORGANISMS

M.M. LĂZĂROAIE

Center of Microbiology, Institute of Biology, Spl. Independenţei 296, Bucharest, Romania

Organic solvents are highly toxic to the majority of living organisms and can be tolerated only by relatively few species, causing serious environmental problems. Upon the release of organic solvents in the marine environment, the transformation of its constituents starts immediately, and microbial degradation is considered to be a major route for the breakdown of such components. Many bacteria capable to tolerate and degrade organic solvents have been isolated from seawater. However, their roles in the biodegradation of organic solvents in the marine environment have remained unknown. Halomonas sp. was isolated from seawater (Constanţa county, Romania) by the enriched cultures method. The use of mineral Halo-M medium with organic solvents as single carbon source allowed the selective development of the Halomonas sp. bacterial strain. Halomonas sp. was able to tolerate and degrade organic solvents such as n-hexane, n-heptane, toluene, styrene, xylene isomers, ethylbenzene and propylbenzene. Toluene, styrene, xylene isomers, ethylbenzene, with the logarithm of the partition coefficient in octanol-water mixture (log POW) between 2.64 and 3.17, were more toxic for Halomonas sp. cells than propylbenzene, n-hexane, n-heptane with log POW between 3.69 and 4.39. Cellular (cell viability, cell hydrophobicity) and molecular (lipid profile, protein profile, DNA) modifications induced by organic solvents to Halomonas sp. were revealed. These modifications differ according to the nature of hydrophobic substrate and culture conditions. This study shows the complex response of the bacterial cells to the presence of organic solvents in the culture medium.


115

[P62] OXYGEN METABOLISM AND AEROTAXIS IN MAGNETOSPIRILLUM GRYPHISWALDENSE

C. MOISESCU1, M. IGNAT2, M. CONSTANTIN3, I. ARDELEAN1

1Department of Microbiology, Institute of Biology of the Romanian Academy, Splaiul Independenţei 296, Bucharest, Romania

2National Institute of Research for Electrical Engineering ICPE-CA, Splaiul Unirii 313, Bucharest, Romania

3Institute IFIN-HH, IRASM Center, Atomiştilor 407, Măgurele, Romania

Magnetotactic bacteria are prokaryotes whose functional characteristic is magnetotaxis, i.e. the orientation along the Earth`s geomagnetic field lines. Magnetotaxis is determined by the presence inside the cell of particles named magnetosomes which are membrane-enclosed intracellular single-domain crystals of a magnetic iron mineral. In this study, we focused on: i) the intensity of cyanide-sensitive aerobic respiration in wild type M. gryphiswaldense strain grown under microaerobic conditions, either in iron depleted culture media (when no magnetosomes are synthesized) or in iron-rich culture media (when magnetosme synthesis occurs). This way we added an original contribution to the correlation between oxygen metabolism and magnetosome synthesis, one of the most important topics in magnetotactic bacteria research; ii) the quantification of water peroxide decomposition activity of intact magnetotactic bacteria, with special emphasis on specific contribution of enzymatic activity (catalase or peroxidase) or magnetite mediated water peroxide decomposition; (iii) fatty acid composition of intact bacteria and isolated magnetosome membranes, and iv) the study of the aerotactic behavior of wild type M. gryphiswaldense strain in the presence and in the absence of a magnetic field.


116

[P63] CC/HPLC-PDA ANALYSIS OF CAROTENOIDS FROM THE GREEN ALGAE MOUGEOTIA SP. AGARDT

E. MUNTEAN, V. BERCEA, N. MUNTEAN, D. NICOLAE

University of Agricultural Sciences and Veterinary Medicine, Faculty of Agriculture, Department of Chemistry, Cluj-Napoca, Romania

A procedure consisting in an open column chromatographic (CC) stage followed by reversed-phase HPLC which operates with gradient elution and photodiode-array detection (PDA) has been developed to analyze carotenoids from the green algae Mougeotia sp. Agardt (AICB 560 strain). These algae originated from the collection of the Institute of Biological Researches Cluj-Napoca. From this matrix, carotenoids were extracted using diethyl ether, then transferred to petroleum ether and subjected to CC on alumina. Three fractions were separated, using successively petroleum ether, petroleum ether:diethyl ether (1:1) and diethyl ether with 20% ethanol. Carotenoids from these fractions were transferred in ethyl acetate and subjected to HPLC. HPLC separations were achieved on a Nucleosil 120–5 C18 column, using the following mobile phases: A – acetonitrile:water (9:1) and B – ethyl acetate. The flow rate was 1 ml/min and the solvent gradient was as follows: initial conditions – 90% A, 10% B from 0 to 15 min – 30% A, 70% B from 16 to 22 min – 90% A, 10% B. Peak identities were established by comparing their HPLC retention times and their on-line recorded VIS spectra with those of known reference compounds.

Nineteen carotenoids were separated, from which nine were identified and also quantified: β-carotene, 9Z-β-carotene, 15Z-β-carotene, 5,6-epoxy-β-carotene, α-cryptoxanthin, echinenone, lutein, antheraxanthin and violaxanthin. HPLC alone revealed a simple chromatographic pattern, dominated by two major carotenoids: lutein and β-carotene. The use of column chromatography as a fractionation step before HPLC reveals a more complex chromatographic pattern of the studied matrix, as the limit of detection is significantly improved in this way. The obtained data demonstrate that the studied Mougeotia strain contains important levels of carotenoids to be considered as a raw material in obtaining carotenoid extracts.


117

[P64] CORRELATION BETWEEN CADMIUM, ZINC AND IRON IN RATS LIVER AND KIDNEY AFTER DEUTERIUM

DEPLETED WATER ADMINISTRATION

L. OLARIU, I. CHIŞU, M. PUP, C. TULCAN

Faculty of Veterinary Medicine, Biochemistry Department, Calea Aradului 119, Timişoara, Romania

The aim of this work is to investigate the influence of deuterium depleted water (DDW) administration on the toxic effects of Cd in rat liver and kidney. A 20 ppm Cd/kg b.w. single dose administration affects either Zn or Fe levels in the rat liver and kidney. The experiment was carried on 60 adult Wistar male rats, with a body weight of 220–240 g, one year old, maintained in good physiological conditions. The experiment took place during eight weeks on four groups. Each group included 15 rats which were treated after the following protocol: L1-control, DDW daily, ad libitum; L2 received a single dose of 20 ppm Cd/kg b.w.; L3 received DDW and after 4 weeks a single dose of 20 ppm Cd/kg b.w. was administered; L4 identically with L3, but after intoxication, rats received 4 weeks more, DDW ad libitum till the end of the experiment. Under general narcosis, blood was collected on heparin, by cardiac punction and then the rats were sacrificed and tissues collected. Cd, Zn and Fe from the liver and kidney were determined using a Shimadzu AAS-6200 atomic absorption spectrometer. Organs were digested with a MARS-CEM X digestor. The data are presented as means ± S.D. values. T-test was used to analyze mean differences between experimental groups for each parameter separately and between groups. Cd registered the highest values in L3, in the liver. A significant Cd decrease was registered in L4. The lowest values of Zn and Fe were registered for L2. After DDW administration in L3 and L4, the average values of Zn and Fe reached similar values as in L1. Cd competes with Zn and Fe for the SH groups but the DDW (preventive and treatment administration) recovers Zn and Fe normal values, after renal Cd excretion.


118

[P65] ANTIOXIDANT ACTIVITY OF SEA BUCKTHORN (HIPPOPHAE RHAMNOIDES) EXTRACTS: SCAVENGING

AGAINST DPPH AND SUPEROXIDE RADICALS, COMPARED WITH COMMON FOOD ADDITIVES

C. PAPUC, V. NICORESCU, G.V. GORAN, D. CRIVINEANU, C. DURDUN

Faculty of Veterinary Medicine, Bucharest, Romania

Sea buckthorn (Hippophae rhamnoides) is a widespread plant with diverse roles and uses, controlling soil erosion, in nutritious foods, drugs and skin-care products. This work was undertaken in order to examine the antioxidant activity of water-acetonic and alcoholic extracts obtained from sea buckthorn, by studying the scavenging of 1,1-diphenyl-2-picryl-hydrazil radical (DPPH), superoxide anion radical (O2

–) and the total antioxidant activity. The scavenging activity against DPPH radical was determined by the antioxidants induced decrease of its absorbance at 517 nm. The scavenging activity against superoxide anions (generated by the phenazin methosulfate/nicotinamid-adenin-dinucleotidphosphate, reduced form (NADPH) system) was detected within by the reaction with chloride of 2,2’-(di-p-nitrophenyl)-5,5’-diphenyl-(3,3’-dimethoxy-4,4’-diphenylene) ditetrazolium chloride (nitro blue tetrazolium). Total antioxidant activity of sea buckthorn extracts was determined by thiocyanate method. The antioxidant activities of sea buckthorn extracts were compared with standard antioxidants 2,6-di-tert-butyl-p-hydroxytoluene (BHT) and tert-butyl-hydroxyanisole (BHA).

The results indicate that water-acetonic extract and alcoholic extract prevent lipid peroxidation and radical chain reaction. Sea buckthorn extracts had more antioxidant capacity than BHT and BHA.


119

[P66] THE IN VITRO STUDY OF ANTI-CANCER ACTIVITY OF PLANE BUDS (PLATANUS ORIENTALIS L.)

C. PEEV1, S.C. PÎNZARU2, C. DEHELEAN1, A. KAYCSA1, C. ŞOICA1, Ş. FEFLEA1, B. BUMBĂCILĂ1

1“Victor Babeş” University of Medicine and Pharmacy, Timişoara, Romania 2“Babeş-Bolyai” University, Cluj-Napoca, Romania

The plane buds (Platanus orientalis L.) are a new category of vegetal products that are used as hydroglyceroalcoholic extracts in the modern phytotherapy. The plane is less known in the classic phytotherapy; fresh leaves being used for preparation of mother tincture in homeopathy. The bark contains triterpenic compounds such as betulinic acid, betulin and lupeol. The present study has highlighted the average content of triterpenic structures in foliar buds using TLC and FTIR and Raman spectroscopies and also the content of hemolytic saponins. The natural compounds, present in small quantities, develop a synergic anti-cancer activity. This was proved through the compared biotest with Lepidium sativum and, in vitro, on neuroectodermal cell types from skin carcinoma (A431) and breast cancer (MCF-7). The results show the presence of triterpenic compounds in foliar buds and a concentration of 1.95% hemolytic saponins. The 2% extract has an inhibition index of 90%; the plane foliar buds extracts show an in vitro antitumor activity on both cell lineages, causing the death of 65–95% of tumor cells.


120

[P67] KINETIC CHARACTERIZATION OF IMMOBILIZED ENZYMES INVOLVED IN A PROCEDURE OF XYLOSE

CONVERSION

G. PETRĂREANU1, M. BĂLAŞU2,1, Ş.E. SZEDLACSEK1 1Enzymology Department, Biochemistry Institute of the Romanian Academy,

Spl. Independenţei, 296, Bucharest, Romania 2Organic Chemistry, Applied Chemistry and Materials Science Faculty, “Politehnica” University,

Spl. Independenţei 313, Bucharest, Romania

Immobilized enzymes are very attractive tools for catalytic processes given their capacity of reutilization of the biocatalyst and an easier recovery of the products. In this study, xylose conversion to the biotechnologically important products acetyl-phosphate and glyceraldehyde-3-phosphate was carried out with an enzymatic system composed of xylose isomerase, xylulose kinase and phosphoketolase as immobilized enzymes. To this purpose, immobilized xylose isomerase was purchased from commercial source while immobilized xylulose kinase and immobilized phosphoketolase were obtained in our laboratory. Both xylulose kinase and phosphoketolase were expressed as recombinant proteins with (His)6 – tag, purified to high quality, then immobilized by chelating affinity on a His-Trap Sepharose column. Variation of some parameters like substrate concentration and flow rate on each enzymatic reaction were analyzed. Under the tested range of reaction parameters xylose isomerase reaction leads to 25% xylose conversion and phosphoketolase reaction leads to 85% xylose-5-phosphate conversion. Xylulose kinase reaction displayed the slowest conversion about 1% and therefore seems to be the critical step of the procedure. It is expected that our data will help to design and optimize a coupled enzymatic procedure for xylose conversion.


121

[P68] IN VITRO RUMINAL CELLULASES ACTIVATION BY LECTIN INTERACTIONS

E. ROTARU, C.P. CORNEA, G.P. NEGULESCU, C. FĂFĂNEAŢĂ, A. POP

University of Agronomical Sciences and Veterinary Medicine, Faculty of Veterinary Medicine, Splaiul Independenţei 105, Bucharest, Romania

The cellulose breakdown process in rumen is the result of the action of a large number of microbial cellulases. The main constraint for the activity of these enzymes is the pH decrease, as a result of simultaneous short chain fatty acids production. In order to improve the cellulose digestibility, in vitro interactions of ruminal fluid proteic extracts with different lectins were performed and the cellulases activities in the presence of acetic, butyric and propionic acids were studied.

Lectins from Raphanus sativus and Pharbitis purpurea seeds were purified by affinity chromatography. Ruminal fluid was collected from healthy slaughtered bovines and proteins were extracted by ammonium sulphate precipitation. Cellulases were assayed by enzymatic estimation of the released glucose, with Avicel substrate, at 39ºC, at three pH values: 4.5, 5.5 and 6.5.

Cellulases activities were reduced by 20% in the presence of propionate, butyrate and acetate (100 mM each). Supplementation of ruminal proteic extract with 1 µg Raphanus lectin/mg crude protein improved the cellulose digestibility at lower pH values. Pharbitis lectin addition did not present the same effect on the cellulolysis at pH below 6, but increased digestibility by 26% at pH 6.5.

Supplementation of ruminal fluid enzymes with lectins improved the cellulose digestibility in vitro, over a wider pH domain, and may represent a method for in vivo diminishing of the inhibitory effect of volatile fatty acids.


122

[P69] AN IN VITRO EVALUATION OF MAGNETRON SPUTTERED HYDROXYAPATITE CAPACITY TO INDUCE

OSTEOGENESIS

L.E. SIMA1, G.E. STAN2, C.O. MOROŞANU2, A.M. MELINESCU3, A. IANCULESCU3, Ş.M. PETRESCU1

1Institute of Biochemistry, Spl. Independenţei 296, Bucharest, Romania 2National Institute of Materials Physics, Atomiştilor 105, Măgurele,Romania

3“Politehnica” University, Polizu 1, Bucharest, Romania

In an attempt to improve the in vivo performance of metal surfaces, calcium phosphate ceramics have been used as coatings on metal prostheses. Synthetic hydroxyapatite, Ca10(PO4)6(OH)2, (HA) can directly bind to bones without infection and fibrous encapsulation, being thus considered as bioactive and biocompatible. In addition, it has been clinically evidenced that plasma-sprayed HA coating greatly enhances the early osseointegration and long term success rate of titanium prosthesis. An experimental lot of HA functional graded coating structures (FGM) were prepared by RF co-sputtering mode in argon atmosphere at low deposition temperature (150ºC). The deposition substrates were either transparent quartz or Ti6Al4V alloy shaped to an area of 1 cm2. Prior to biological tests the MS-HA structures were physico-chemical characterized by XRD, FTIR and SEM-EDX after a postdeposition annealing process at 550ºC in environmental air. To find out whether HA films could induce in vitro mesenchymal stem cells (MSCs) differentiation to osteoblasts, primary human MSCs were cultivated onto both biomaterial and standard surfaces for 3 weeks with or without osteogenic media. Cells were analysed by epifluorescence microscopy at 1, 7, 14, and 21 days after plating to study cell adhesion, spreading and proliferation index. The differentiation phases were described using osteoblast lineage specific markers. FTIR and XRD results obtained on the FGM structures showed the good reproducibility of the deposition method. After the heat-treatment at 550ºC a strong HA crystallization was evidenced. (002) crystallites with an average dimension of 100 nm and a degree of crystallinity of around 90% were noticed. SEM analysis revealed a rough surface morphology suitable for implant-type applications. Biocompatibility tests have proved that HA improves in vitro osteoprogenitor cell adhesion and function, being a promising coating material for implantology.


123

[P70] THE INFLUENCE OF NATURAL MEDIA ON THE CRYOPRESERVATION OF RAM SEMEN

E. ŞOGORESCU1, S. ZAMFIRESCU2, N. ROŞOIU2, A.H. ANGHEL1 1ICDCOC Palas, Department of Sheep and Goat Reproduction and Biotechnologies,

Constanţa, Romania 2“Ovidius” University, Department of Natural and Agricultural Sciences,

Constanţa, Romania

The objective of our research was the determination, within our pilot station for the gene preservation, of an optimal natural medium to be used in freezing of the ram semen. We tested 28 types of dilution media (predominantly natural, 90%). The diluters were tested regarding the cryo-biological indexes obtained by freezing-thawing the semen collected from three rams of active breeds: Karakul, Carabas and Merinos of Palas.

Diluted and undiluted fruit juices (apples and tangerines) were used to obtain 6 versions of diluters in whose composition was also used egg yolk (20%) and the cryoprotector glycerin (10%). The cryo-biological indexes were: motility (M6=30–50%), viability (V= 30.45–52.8%) and survival index (I.S.=35.82–70.4%). The highest values of these indexes were obtained with the diluted and undiluted apple juice: M6=50–50%, V=48.1–52.8% and I.S.=53.44–70.4% in Karakul and Carabas. Vegetable juices (carrots, celery, cucumbers), diluted or not, were used to obtain 9 versions of diluters without sugars for carrot juice and with sugars for the other juices, the semen cryobiological indexes being M6=20–40%, V=20.45–38.77% and I.S.=25.56–42.58%. The best results were obtained with undiluted carrot juice, 20% yolk, 10% glycerin: M6=35%; V=36.2%; I.S.=42.58%; with cucumber juice (with 1% sucrose) M6 = 40%, V=38.77%, I.S.=43%; with celery juice (with 1% sucrose) M6=35%, V=34.57%, I.S.=38.34%. The diluters based on soy milk, Aloe leaf juice and colostrum did not offer cryobiological indexes higher than 30%.


124

[P71] EFFECTS OF LIPID NANOSTRUCTURES ENCAPSULATING BIOACTIVE AGENTS ON HUMAN

ARTICULAR CHONDROCYTES

M. TRIF1, M. MOISEI1, A. ROŞEANU1, F. CHELU1, O. CRĂCIUNESCU2, L. MOLDOVAN2 1Institute of Biochemistry of the Romanian Academy, Bucharest, Romania

2National Institute for Biological Sciences, Bucharest, Romania

Many bioactive agents with therapeutic potential show in vivo limitations such as short circulation time, enzymatic inactivation or minimal localization to the action site. Their incorporation into lipid-based carriers may overcome such drawbacks. We showed that liposome entrapped anti-inflammatory biological agents had the ability to suppress joint inflammation and to modulate the cytokine response of lymph nodes T cells in mice with collagen induced arthritis. We extended the studies to another pharmacological active macromolecule, chondroitin sulphate (CS), which plays an important structural role in articular cartilage being used as chondroprotective agent in the treatment of osteoarthritis (OA). On the other hand, though it is one of the most widely used dietary supplements for OA, CS’s role in its treatment remains controversial. The incorporation of CS, a negatively charged biopolymer, into the hydrophilic core of liposomes, as well as its association with the positively charged lipid bilayers could be an additional benefit that increases liposome stability. We investigated the positive liposome ability to entrap CS and to deliver them efficiently to human chondrocytes (HC) in culture. The influence of free and liposomal CS on cell viability, proliferation rate and morphology was investigated. Both untreated and treated cells with liposome containing CS showed the same viability and morphology after 48h of incubation. Preliminary results suggested that liposome based CS were more efficient in reducing both IL β1 mediated inflammation in HC and the proinflammatory cytokines level (TNF-alpha and IL-6) in THP-1 cells stimulated with bacterial endotoxins. In conclusion, our studies indicate that liposomes could be used as efficient carriers for controlled cell delivery of CS and encourage their further use in the local treatment of rheumatic and inflammatory disorders.

(This work was supported by a grant from the Romanian Ministry of Research, ANCS-MATNANTECH, project CEEX-57/2006).


125

[P72] EVALUATION OF MOLECULAR BIOLOGY TECHNIQUES FOR PRIONIC PROTEIN GENETIC

VARIABILITY IDENTIFICATION IN SHEEP

M.A. TURCITU1, H. COSTE1, N. ALEXANDRU1, I. CODREANU2, M.D. CODREANU2, I. OLARU1, P. TAMBA1, I. ZYBACZYNSKI1

1Institute for Diagnosis and Animal Health, Bucharest, Romania 2Faculty of Veterinary Medicine, Bucharest, Romania

Allelic variant characterisation at prionic protein level, especially for codons 136, 154 and 171, and consequently identification of scrapie resistant or various susceptibility degrees genotypes is an important point in the selection program of sheep breeds. The purpose of this work was to evaluate two methods in order to establish a national protocol for scrapie eradication. Biological material was represented mainly by EDTA blood and to a lesser extent by brain homogenates, all originated from local sheep breed. Genomic DNA extraction was performed using MagNA Pure LC instrument along with MagNA Pure LC DNA Isolation Kit I, especially designed to purify DNA from whole blood and cell culture. Sample volume was 200 µl whole blood or brain homogenates, with nucleic acid elution in 100 µl buffer. The methods selected for investigation were melting curves analysis using primer and hybridization probes that span the region of codons 136, 154 and 171 and PCR amplification of a prionic protein segment together with direct sequencing of the amplicons, also containing codons 136, 154 and 171 and analysed with dedicated software. In conclusion, melting curves analysis is fast (124 samples processed in a 384 wells plate format, for all three codons), labourless (concerning processing time required for each sample to be analysed), low degree of contamination risk, due to reduced sensitivity compared to the second method. However, there are some drawbacks regarding the amount of DNA required (120–150 ng), being ideal for blood samples, due to the high levels of information carrier cells, but with a low performance for brain homogenates. Sequencing technique, due to the accuracy in determining the codons polymorphism, is used for exact characterization of prion protein, including the mutations that might occur at adjacent codons, situations that modify the melting curves format and consequently lead to the impossibility of validation.


126

[P73] PATHOGENICITY CHARACTERIZATION OF NEWCASTLE DISEASE THROUGH MOLECULAR BIOLOGY

TECHNIQUES

M.A. TURCITU1, H. COSTE1, R. CIORANU1, Ş. NICOLAE1, G. BĂRBOI, I. ONIŢĂ1, G. NEAGOE1, A. NEICUŢ1, A. SANDU2

1Institute for Diagnosis and Animal Health, Bucharest, Romania 2Institute for Control of Veterinary Biological Products and Medicines, Bucharest, Romania

Fusion gene cleavage site analysis at nucleotide level as well as amino acid translation offer important data regarding virus isolates pathogenicity. Pathotyping technique completes the diagnostic and characterization of Newcastle isolates, being used for outbrakes confirmation. This study consists in molecular characterisation of 47 virus strains isolated between 2002 and 2007, vaccinal strains used for disease control and challenge strain “Craiova” used for vaccine efficacy control. RNA extraction was performed using 200 µl sample and elution in 50 µl buffer. Reverse transcription and amplification of the F gene region was done by single tube procedure followed by gel purification of amplicons. Cleavage site for the fusion gene was characterised using direct sequence procedure and the obtained data were processed through dedicated softwares. Nucleotide sequences of the cleavage site revealed a specific pattern, being composed mostly by adenine and guanine. For mesogenic/velogenic strains, cleavage site consisting of a pair of essential amino acids (arginine-R and lysine-K) in 112/113 and 115/116 positions, respectively, and phenylalanine (F) in position 117 will determine the recognition by ubiquitary proteases, thus allowing the virus to replicate systemically and appearance of clinical signs of the disease. In contrast, for lentogenic strains the amino acids in positions 112/113 and 115/116, respectively, can include glycine (G) along with the two possibilities mentioned for the first category. Moreover, amino acid situated in position 117 is represented in this case by leucine (L). The majority of the studied isolates has 109SGGRRQKRFIG119 pattern, suggesting mesogenic/velogenic category, while for some isolates the sequence was 109SGGGRQG(K)RLIG119, belonging to lentogenic group (vaccinated flocks). Concerning “Craiova” strain, amino acid sequence 109SGGRRQRRFIG119 indicated a high degree of virulence.


127

[P74] DETERMINATION OF HONEY AND OLIGOSACCHARIDE EFFECT ON LACTOCOCCUS LACTIS

SE22 STRAIN

E. VAMANU1,2, A. VAMANU1,2, D. PELINESCU4, O. POPA1,2, S. NIŢĂ3, N. BĂBEANU1

1University of Agronomic Sciences and Veterinary Medicine, Faculty of Biotechnology, Bucharest, Romania

2Applied Biochemistry and Biotechnology Center, Bucharest, Romania 3National Institute of Chemical-Pharmaceutical Research-Development, Bucharest, Romania


The most recent studies showed that the human microbial flora may be controlled by prebiotics and probiotics administration. These products increase and intensify the activity of benefic microorganisms. The action of the probiotic microorganisms may determine an inappropriate medium for pathogen development, thus benefic for the host. The present study aims at the identification of the best prebiotic for the development of Lactococcus lactis SE22 strain. The MRS medium was supplemented with 1 and 2% inulin, lactulose and raffinose. The results were compared to those obtained for honey and 1% human milk. Honey was chosen because of its high content in oligosaccharides while human milk stimulates the probiotic strains of lactic bacteria and bifidobacteria in children. The experiments were done in Hungate flasks, at 37ºC, for 48 h and the lactic acid and glucide consumption were determined. The productivity, duplication time and growth rate were computed for each prebiotic, for honey and human milk, in order to obtain the maximum quantity of probiotic microbial biomass. The obtained results showed that inulin and lactulose were very good stimulants at 1% concentration. For raffinose, the optimum concentration was 2%. For honey the results were similar. The best results were obtained for human milk, but they were highly influenced by the culture medium composition. The results were better by approximately 10% compared to the other prebiotics and honey.


128

POSTERS

Session 6: Environmental Biochemistry

ROM. J. BIOCHEM., 45, 1, 129–132 (2008)

[P75] DOWNREGULATION OF HSP27 GENE EXPRESSION IN MANGANESE(II) INDUCED APOPTOSIS OF HUH7

CELLS

M.C. MUNTEANU, L. POSTOLACHE, M. COSTACHE, A. DINISCHIOTU

University of Bucharest, Faculty of Biology, Department of Biochemistry and Molecular Biology, 91–95 Splaiul Independenţei, Bucharest, Romania

The highly conserved heat shock proteins (Hsps) are closely related families of proteins that are rapidly induced by many stressors, including heavy metals, such as manganese, heat and anoxia. Manganese(II) is a Fenton type metal which generates reactive oxygen species. High concentrations frequently induce apoptosis by mitochondrial pathway. Bax is a proapoptotic member of the Bcl2 family mediating the dissipation of the mitochondrial membrane potential. Our aim was to study the correlation between the 5 mM Mn2+ induced apoptosis in human hepatocarcinoma cells (Huh7) and the expression of hsp27 and hsp70.

Synchronized Huh7 cells were treated with 5 mM manganese(II) and the level of gene expression of hsp27, hsp70 and bax was analyzed by quantitative Real Time PCR (qRT-PCR) after 30 min, 2, 6 and 12 h of exposure.

The two hours exposure to manganese(II) induced the overexpression of hsp27 by 750% compared to control. The hsp70 mRNA level was increased only after 30 min of intoxication, by 40% compared to zero moment. After 6 h, the two chaperone mRNA levels were lower than in controls and continued the trend until 24 h of exposure were reached. The bax mRNA profile presented a maximum after 6 h of treatment.

Our data suggest that the hsp27 gene product protects the Huh7 cells against 5 mM Mn2+ treatment during the first 2 h. Also, it seems that hsp70 gene products are less potent in this protection. At longer exposure, the hsp27 expression is inhibited and the induction of the mitochondrial pathway of apoptosis in Huh7 cells occurs.


[P76] INTERRELATIONSHIP BETWEEN MARKERS OF OXIDATIVE STRESS IN CHRONIC NITRATE/NITRITE

EXPOSURE

C. PISOSCHI, I. PREJBEANU, C. STĂNCIULESCU, O. PURCARU, D. TACHE, M. MANEA

University of Medicine and Pharmacy, Petru Rareş 2, Craiova, Romania

Nitrate and nitrite are naturally occurring ions involved in the nitrogen cycle. Contamination with nitrogen fertilizers raises the concentration of these ions in water, by decomposition of organic nitrogen into ammonia followed by oxidation, by the action of microorganisms in soil and water. Directly ingested nitrite and nitrite resulted from nitrate reduction are absorbed into the blood and stimulate oxidation of ferrous ion and initiate a wide range of toxic oxidative reactions.

The aim of this study was to evaluate the chronic nitrate/nitrite exposure on the antioxidant balance, assessing the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPX) in erythrocytes and the level of malondialdehyde (MDA) in the plasma. Blood samples were collected from 52 subjects living in various areas of Dolj county with increased water contamination. SOD activity in erythrocytes was measured by the rate of inhibition of 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride reduction. GPX activity was measured by a modification of Paglia and Valentine’s method. Hemoglobin was measured using Drabkin reagent and methemoglobin according their absorbance at 630 nm. MDA, marker of lipid peroxidation, was estimated as a thiobarbituric acid reactive substance.

We found an increased level of GPX activity in 78.85% samples. The level of SOD activity was decreased in 57.69% cases. Plasma MDA level was only slightly increased in subjects drinking polluted water compared to those who drank water without nitrate contamination (1.457±0.181 vs. 1.225±0.346 nmols/ml).

Our preliminary results suggest that chronic exposure to nitrate/nitrite could modify markers of the oxidative balance in a different manner and further assessments are requested to elucidate the mechanisms involved.

AUTHOR INDEX

ROM. J. BIOCHEM., 45, 1, 133–138 (2008)

ALEXANDRU, N. – P72 ALIFANTI, C. – P58 ANGHEL, A.H. – P70 ANTON, G. – P49 ARAMA, C. – P52 ARDELEAN, I. – P62 ARDELEANU, C. – O1 ARSENE, A.L. – P34, P52, P59 ARTENIE, V. – P21, P22, P35 ASANDEI, A. – O11, P53, P54 ATHANASIU, A.A. – P58 AVRAM, N.M. – P9 AVRAM, S. – O23, P45

BĂBEANU, N. – P74 BACALUM, M. – O2 BACONI, D. – P41 BADEA, R. – P6 BĂLĂLĂU, D. – P41 BĂLĂNESCU, A. – P41 BĂLAŞU, M. – O7, O21, P67 BALOTESCU, C. – O4 BANIŢĂ, M. – P43 BĂRĂIŢEANU, S. – P20 BĂRBOI, G. – P73 BAŞA, M. – P18, P19, P44 BELE, C. – O19 BERCEA, V. – P63 BOANŢĂ, A. – P58 BOLOCAN, A.S. – P47 BOTEA, L. – P32 BOTEZATU, A. – P49 BOUKAMP, P. – P38 BRAŞOVEANU, L. – P23 BUCUR, M. – O4 BUICULESCU, R. – P32 BUMBĂCILĂ, B. – P26, P27, P37, P46, P66 BUNEA, A. – O19 BUTUR, G. – O1

CALBOREAN, O. – P7, P14 CANCIU, C. – P46 CARNICELLI, V. – P10 CHELU, F. – P8, P57, P71 CHICHKOV, B.N. – O3 CHIRIAC, R. – P53, P54 CHIRIŢOIU, G. – P1 CHIRIŢOIU, M. – P2 CHIŞU, I. – P64 CHITANU, G.C. – O16

CÎMPEAN, A. – P23 CIMPEAN, A.M. – P39 CIOBICĂ, A. – P21, P22, P35 CIOFRĂNGEANU, C. – P23 CIORANU, R. – P73 CIORSAC, A. – P24 CIUBAR, R. – P23 CODREANU, I. – P72 CODREANU, M.D. – P30, P55, P72 COMĂNESCU, V. – P43 CONSTANTIN, M. – P62 CONSTANTINOVICI, M. – P28 CORNEA, C.P. – P42,P68 COSTACHE, M. – P4, P75 COSTE, H. – P51, P72, P73, CRĂCIUN, D. – P9 CRĂCIUNESCU, O. – P71 CRISTEA, A.N. – P52 CRISTEA, D. – O4 CRIVINEANU, D. – P65 CRIVINEANU, M. – P55, P56 CRIVINEANU, V. – P30, P55 CUCU, N. – P49

DAN, D. – P49 DANEŞ, D. – P20 DĂNEŢ, A.F. – P48 DE LOOSE, M. – P60 DEHELEAN, C. – P26, P37, P66 DELEANU, C. – O7 DEPICKER, A. – P60 DINCĂ, V. – O3 DINESCU, G. – P42 DINESCU, M. – O3 DINISCHIOTU, A. – P4, P75 DRACEA, O. – O4 DRĂGOI, C. – P52 DRONCA, M. – P25 DUDA-SEIMAN, C. – P45 DUDA-SEIMAN, D.M. – P45 DULF, F. – O19 DUMACHE, R. – P26, P27, P46 DURDUN, C. – P65 DWEK, R.A. – PL1, O14

ENE, M.D. – P28 EVANS, P. – O5 EVANS, R.W. – PL2

Author index 2

136

FĂFĂNEAŢĂ, C. – P68 FARCAŞ, C. – P32 FARSARI, M. – O3 FEFLEA, S. – P66 FETEA, F. – O19 FILIPESCU, C. – P58 FLORIAN, P. – P10, P57 FOTAKIS, C. – O3

GANEA, E. – P3, P11 GADHER, S.J. – O6 GĂLĂŢEANU, B. – P23, GEORGESCU, A. – O1 GEORGESCU, L.A. – P47 GHENEA, S. – O9, P12 GHEORGHE, A. – P57 GHERASIM, I. – P58 GÂJÂILĂ, I. – P4 GLIGA, A. – P29, P41 GORAN, G.V. – P30, P65 GOVERSE, A. – O20 GRĂDINARU, D. – P29, P34, P59 GRĂVILĂ, C. – P31 GRIGORESCU, A. – O14 GRIGORIAN, M. – P32 GRUIA, I. – P40 GRUIA, M.I. – P23, P33 GRUIA, V. – P34

HILLEBRAND, M. – P11 HÎRTOPEANU, A. – O7 HRITCU, L. – P21, P22, P35 HÜLSMANN, B. – PL3

IANCU, A.D. – P36 ICHIM, M.C. – P60 ICRIVERZI, M. – P8, P10, P57 IGNAT, M. – P62 ILIE, M. – P41 IANCULESCU, A. – P69 IONESCU, M. – P51 IONESCU, R. – P28 IORDACHE, C. – O4 IORDĂCHESCU, D. – P23 ISTRATI, D. – O7 ISVORAN, A. – P9, P24 IZVERNARIU, D.A. – P39

JECU, L. – P57 JURJA, S. – P32

KASOTAKIS, E. – O3 KAYCSA, A. – P26, P37, P46, P66 KRUNIC, D. – P38

LASLO, C. – P11 LAZĂR, C. – O8 LAZĂR, V. – O4 LĂZĂROAIE, M.M. – P61 LEABU, M. – P58 LILIOS, G. – P32 LIXANDRU, M.S. – O4 LIZZI, A.R. – P10 LUCHIAN, T. – O11, P53, P54 LUPEA, A.X. – P31

MACOVEI, A. – O8 MACRI, B.M. – O10, O23, P36 MANEA, M. – P76 MANUEL, B.Y. – P34 MAREŞ, A. – O9, P13 MARGINĂ, D. – P29, P34, P41, P59 MARIN, A. – O10 MATTSBY-BALTZER, L. – P8 MELINESCU, A.M. – P69 MEREUŢĂ, L. – O11, P53, P54 MERNEA, M. – P14 MICLEA, F. – P26, P27 MICLUŢĂ, M. – O22 MIHAI, M. – O1 MIHĂILESCU, I. – O16 MIHĂILESCU, D.F. – O13, P7, P14 MILICAMPS, A. – P60 MILITARU, M. – P42 MIRANCEA, D. – O12, P38 MIRANCEA, N. – O12, P38 MISEVIC, G.N. – O17 MITRAKI, A. – O3 MITRAN, V. – P23 MITREA, N. – P29, P34, P43, P52, P59 MOCANU, M. – P58 MOHORA, M. – P34 MOISEI, M. – P8, P71 MOISESCU, C. – P62 MOLDOVAN, L. – P71 MOMEU, C. – O15

3 Author index

137

MORARU, A. – O13 MORIARTY, R.M. – O7 MOROŞANU, C.O. – P69 MUNTEAN, E. – P63 MUNTEAN, N. – P63 MUNTEANU, M.C. – P4, P75 MUSTAŢĂ, R. – O14

NARITA, D.M. – P39 NEAGOE, G. – P73 NECHIFOR, M.T. – P58 NECULA, G. – O4 NEGOIŢĂ, V. – P33, P40 NEGREI, C. – P41 NEGROIU, G. – O1, O16, P33 NEGULESCU, G.P. – P68 NEICUŢ, A. – P73 NICHITA, N. – O8 NICOALE, D. – P63 NICOLAE, Ş. – P73 NICOLAU, A. – P47 NICOLESCU, A. – O7 NICORESCU, V. – P56, P65 NICULESCU, Z. – P32 NIŢĂ, S. – P74

OLARIU, L. – P28, P37, P64 OLARU, I. – P72 ONIŢĂ, I. – P73 ORATORE, A. – P10 OSTAFE, V. – P24 OVSIANIKOV, A. – O3

PĂDURARIU, M. – P21, P22, P35 PĂDURE, M. – P31 PAPAZOVA, N. – P60 PAPUC, C. – P30, P65 PARASCHIV, G. – P56 PAŞCA, S.P. – P25 PASCARU, M. – P15 PATRAŞ, A. – P16 PEEV, C. – P37, P66 PELINESCU, D. – P74 PERJU-DUMBRAVĂ, L. – P25 PETRĂREANU, G. – P67 PETRESCU, A.J. – O9, O20, O21, O22, P13 PETRESCU, Ş.M. – O1, O9, O14, O18, P1,

P2, P5, P12, P13, P33, P69

PINTEA, A. – O15 PÎNZARU, S.C. – P66 PISOSCHI, A.M. – P48 PISOSCHI, C. – P43, P76 PITICESCU, R.M.– O16 POP, A. – P42, P68 POPA, I. – P3 POPA, O. – P74 POPA, V.V. – P40 POPEA, F. – P57 POPESCU, A. – O7 POPESCU, O. – O17 POSTOLACHE, L. – P75 PREJBEANU, I. – P76 PUIU, L. – P23 PUIU, M. – P26, P27, P49 PUP, M. – P64 PURCARU, O. – P76

RADU, D.L. – P36 RADU, M. – O2, O10 RANELLA, A. – O3 RANGA, F. – O19 RĂUŢ, I. – O4 RAYMOND, D.A. – PL1 ROŞEANU, A. – P8, P10, P57, P71 ROŞOIU, N. – P18, P19, P44, P70 ROŞU, A. – P57 ROTARU, E. – P68 RUGINĂ, D. – O15 RUSU, R. – P25

SANDU, A. – P73 SANDU, I.– P51 ŞERBAN, I.A. – P4 SERBAN, M. – P49 SIMA, L.E. – O16, O18, P33, P69 SOCACIU, C. – O19 ŞOGORESCU, E. – P70 ŞOICA, C. – P37, P66 SPIRIDON, L.N.– O9, O20, O21, O22 STAN, G.E. – P69 STANA, L. – P31 STĂNCIULESCU, C. – P43, P76 STĂNICEANU, F. – O1 STĂVARU, C. – P36 SZEDLACSEK, Ş.E. – O21, P6, P15, P67 ŞVAB, I. – O13

Author index 4

138

TACHE, D. – P43, P76 TĂCUTU, R. – O22 TAMBA, P. – P51, P72 TĂNASE, C.A. – P50 TERZEA, D. – O1 TICĂ, V. – P4 TOGOE, I. – P42 TRIF, MIHAELA – P8, P10, P57, P71, TRIF, MONICA – O19 TRIFAN, V. – P55, P56 TULCAN, C. – P64 TURCITU, M.A. – P72, P73

UNGUREANU, C. – O4

VAMANU, A. – P74 VAMANU, E. – P74 VAN DEN EEDE, G. – P60 VANGHELE, M. – P51 VASILE, G. – P5 VASILESCU, F. – O1 VERMAN, I.G. – P18, P19, P44

WINTERHALTER, M. – O2, O10

ZAMFIRESCU, S. – P70 ZURAC, S. – O1 ZYBACZYNSKI, I. – P72

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