INTERNATIONAL CONFERENCE on BIOORGANIC CHEMISTRY, BIOTECHNOLOGY and BIONANOTECHNOLOGY, Dedicated to the 55Th Anniversary of the M.M

RUSSIAN ACADEMY OF SCIENCES RUSSIAN FOUNDATION FOR BASIC RESEARCH RUSSIAN BIOCHEMICAL SOCIETY “PARK-MEDIA”

INTERNATIONAL CONFERENCE ON BIOORGANIC CHEMISTRY, BIOTECHNOLOGY AND BIONANOTECHNOLOGY, dedicated to the 55th Anniversary of the M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences and the 80th Anniversary of Professor Yuri Ovchinnikov

Moscow, Russia September 15–19, 2014

SPECIAL ISSUE № 1 2014 | Acta naturae | 1 INTERNATIONAL CONFERENCE ON BIOORGANIC CHEMISTRY, BIOTECHNOLOGY AND BIONANOTECHNOLOGY dedicated to the 55th Anniversary of the M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences and the 80th Anniversary of Professor Yuri Ovchinnikov

September 15–19, 2014; Moscow, Russia

ORGANIZING COMMITTEE

CHAIRMAN Vadim IVANOV

CO-CHAIRS Alexander GABIBOV Sergei ZAVRIEV

MEMBERS Eugene GRISHIN Tatiana OVCHINNIKOVA Marina TRETYAK Vera KNORRE Alexandra ROGALSKAYA Victoria BUDKOVSKAYA

Contents Preface 3 Scientific Program 4 Invited Speakers and Chairmen 9 Oral and Poster Presentations 13 Young Scientists Competition 50 Author Index 61

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2 | Acta naturae | SPECIAL ISSUE № 1 2014 INTERNATIONAL CONFERENCE ON BIOORGANIC CHEMISTRY, BIOTECHNOLOGY AND BIONANOTECHNOLOGY dedicated to the 55th Anniversary of the M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences and the 80th Anniversary of Professor Yuri Ovchinnikov

September 15–19, 2014; Moscow, Russia

Dear Conference Participants, Dear Guests, This issue of the Acta Naturae journal contains abstracts of reports presented at the International Conference on Bioorganic Chemistry, Biotechnology and Bionanotechnology dedicated to the 55th Anniversary of the M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences (IBCh RAS) and the 80th Anniversary of Professor Yuri A. Ovchinnikov. We are grateful to everyone who accepted our invitation and joined us in these anniversary days to discuss contemporary problems of “life sciences”. Founded in 1959, the IBCh RAS has become the largest and unique research center of physicochemical biology and biotechnology in Russia, and we are justifiably proud of its success. The range of problems studied at more than 40 research departments of the Institute covers most of the modern trends in biomolecular science. At the Institute, along with research in traditional areas, research in frontier areas such as genomics and proteomics, molecular biotechnology and bioengineering, bioinformatics, problems of molecular oncology and immunology, and molecular modeling is conducted. The Institute employs about 1,100 people, including over 500 researchers (350 of them are doctors and candidates of sciences). At the IBCh, investigations in fields of physicochemical biology, biotechnology as well as studies on peptides and proteins, nucleic acids, and low- molecular weight bioregulators are at the top level worldwide and largely determine the level of this research in Russia and abroad. Last year, the International Advisory Board was established at the Institute, some Board members are speakers and participants of this forum. These are Professor Sidney Altman, the Nobel Prize Winner, world famous scientists Professor Angelo Azzi, Professor Ferdinand Huho, and Professor Joseph Schlessinger. Professor Burkhard Bechinger, Professor Ernesto Carafoli, Professor Norbert Dencher, Professor Jean-Luc Imler, and Professor Richard Pestel will participate in the conference. Along with the outstanding foreign researchers, heads of scientific units and key researchers of the IBCh will present their reports at the conference. Young researchers will have the opportunity to present their studies in the poster session. We are very pleased that many of our colleagues from other academic institutes and universities across the country are among the speakers. The conference will be held for five days only, and for me, as a chairman of the organizing committee, it was not easy to fit many reports into the strict limits of the program. I hope that the anniversary conference of the Institute will excite interest in the scientific community and will strengthen the position of Russian science in the international arena.

Vadim IVANOV Director, IBCh RAS

SPECIAL ISSUE № 1 2014 | Acta naturae | 3 INTERNATIONAL CONFERENCE ON BIOORGANIC CHEMISTRY, BIOTECHNOLOGY AND BIONANOTECHNOLOGY dedicated to the 55th Anniversary of the M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences and the 80th Anniversary of Professor Yuri Ovchinnikov

September 15–19, 2014; Moscow, Russia MONDAY, SEPTEMBER 15th 10.00 – 10.30 CONFERENCE OPENING Chairmen: Rem Petrov, Aleksey Rozanov Anatoly Grigoriev Welcome address of the Russian Academy of Sciences Vadim Ivanov Introduction 10.30 – 11.10 PLENARY SESSION Sidney Altman Antibiotics: present and future

11.10 – 11.30 Coffee break

11.30 – 12.45 PLENARY SESSION Chairmеn: Nikolay Nikolsky, Richard Pestell Eugene Sverdlov Academician Yury Ovchinnikov, RNA polymerases, interferons and we. Glimpse of today Angelo Azzi Gene regulatory functions of Vitamin E Konstantin Skryabin “Ancient” DNA

12.45 – 13.30 Conference Group Photo

13.30 –14.30 Lunch

14.30 – 15.30 YOUNG SCIENTISTS COMPETITION. THE FIRST AUDITION: POSTER SESSION

15.30 – 16.45 PLENARY SESSION Chairmеn: Michael Blackburn, Michael Ugryumov Michael Blackburn Recent advances in enzyme mechanisms: Active and passive roles for phosphates Viktor Tsetlin Three-finger toxins and four-helical receptors: from selective binding to new drugs Sergey Nedospasov TNF and lymphotoxin produced by distinct cellular sources have distinct functions: implications for anti-cytokine therapy

16.45 – 17.05 Coffee break

17.05 – 18.10 PLENARY SESSION Chairmen: Nadik Abdoulaev, Ernesto Carafoli Ernesto Carafoli Calcium signaling: function and dysfunction Mikhail Ostrovskiy Visual pigment rhodopsin and difference in spectral sensitivity between a lake/sea population pair of crustacean Mysis relicta Mikhail Kirpichnikov Bacteriorhodopsin of Exiguobacterium sibiricum – new type of the proton pump

18.30 – 20.00 Welcome reception

4 | Acta naturae | SPECIAL ISSUE № 1 2014 INTERNATIONAL CONFERENCE ON BIOORGANIC CHEMISTRY, BIOTECHNOLOGY AND BIONANOTECHNOLOGY dedicated to the 55th Anniversary of the M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences and the 80th Anniversary of Professor Yuri Ovchinnikov

September 15–19, 2014; Moscow, Russia

TUESDAY, SEPTEMBER 16th 10.00 – 11.15 PLENARY SESSION Chairmеn: Angelo Azzi, Georgy Georgiev Valentin Vlasov Nucleic acid therapeutics and targets Dmitry Chudakov Individual T cell receptor repertoires Pavel Georgiev Chromatin insulators and long-distance interactions in Drosophila genome

11.15 – 11.40 Coffee break

11.40 – 13.20 PLENARY SESSION Chairmеn: Michael Agadjanyan, Alexander Makarov Vladimir Shuvalov Primary processes of white energy conversion in reaction centers of photosynthesis Alexander Gabibov Mechanisms of antigen degradation Sergey Kozin, Alexander Makarov The role of the metal-binding domain of beta-amyloid in pathogenesis of Alzheimer's disease Michael Agadjanyan Active and passive vaccination strategy for Alzheimer’s disease

13.20 – 14.30 Lunch

14.30 – 15.30 POSTER SESSION

15.30 – 17.10 PLENARY SESSION Chairmеn: Alexander Boronin, Sergei Zavriev Valentin Stonik Secondary sea metabolites: some characteristic features and prospects of use Nikolai Myasoyedov New possibilities in development of peptide pharmaceuticals Sergey Kochetkov New blockers of socially significant infections Vladislav Deigin Peptide pharmaceuticals: from peptide extracts to oral peptidomimetics

17.10 – 17.30 Coffee break

17.30 – 18.45 PLENARY SESSION Chairmen: Norbert Dencher, Roman Efremov Vadim Govorun 3D-omics data integration: mycoplasma reveals regulation complexity Norbert Dencher How protein dynamics and protein-protein interactions contribute to energy conversion, healthy ageing, and neurodegenerative diseases Sergey Kostrov Pro-proteins and evolutionary adaptation

SPECIAL ISSUE № 1 2014 | Acta naturae | 5 INTERNATIONAL CONFERENCE ON BIOORGANIC CHEMISTRY, BIOTECHNOLOGY AND BIONANOTECHNOLOGY dedicated to the 55th Anniversary of the M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences and the 80th Anniversary of Professor Yuri Ovchinnikov

September 15–19, 2014; Moscow, Russia WEDNESDAY, SEPTEMBER 17th 10.00 – 13.00 YOUNG SCIENTISTS COMPETITION. FINAL AUDITION: ORAL PRESENTATIONS Chairmen: Tatiana Ovchinnikova

13.00 – 14.00 Lunch

14.00 – 17.00 RESUMPTION

THURSDAY, SEPTEMBER 18th 10.00 – 11.15 PLENARY SESSION Chairmеn: Burkhard Bechinger, Eugene Fesenko Alexander Arseniev, Eduard Bocharov, Konstantin Mineev, Kirill Nadezhdin, Sergei Goncharuk, Alexey Feofanov NMR view on transmembrane helix dimerization: cell signaling in human health and pathologies Burkhard Bechinger Biophysical investigations of peptide interactions with membranes Yuri Chizmadzhev Study of lipid-protein molecular machines responsible for viral infection of the cell

11.15 – 11.40 Coffee break

11.40 – 13.20 PLENARY SESSION Chairmеn: Dmitry Knorre, Sergey Lukyanov Lev Ovchinnikov Y-box binding protein 1 (YB-1): structural organization and biological functions Vladimir Popov Enzymes from extremophilic microorganisms: structural insight into the molecular basis of extremophilicity Vladimir Pletnev Three-dimensional structure of fluorescent proteins. Structure-functional relations Konstantin Lukyanov Fluorescent proteins: from understanding to advanced applications

13.20 – 14.30 Lunch

14.30 – 15.55 PLENARY SESSION Chairmеn: Jean-Luc Imler, Valery Lipkin Jean-Luc Imler Antiviral immunity in Drosophila Nikolai Modyanov Impact of ATP1B4 co-option on development of placental mammals Alexander Petrenko Subtle structural variations define the function of membrane proteins

15.55 – 16.15 Coffee break

Continued

6 | Acta naturae | SPECIAL ISSUE № 1 2014 INTERNATIONAL CONFERENCE ON BIOORGANIC CHEMISTRY, BIOTECHNOLOGY AND BIONANOTECHNOLOGY dedicated to the 55th Anniversary of the M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences and the 80th Anniversary of Professor Yuri Ovchinnikov

September 15–19, 2014; Moscow, Russia THURSDAY, SEPTEMBER 18th 16.15 – 19.00 REPORTS OF YOUNG SCIENTISTS FROM THE M.M. SHEMYAKIN AND Yu.A. OVCHINNIKOV INSTITUTE Chairperson: Tatiana Ovchinnikova Igor Deev pH-sensing properties of receptor-like tyrosine kinases Mikhail Matlashov Genetically encoded redox switches and pH sensors for neurobiology Alexandra Tsarkova A novel ATP-dependent bioluminescent system from the Siberian earthworm Fridericia heliota: structure elucidation of luciferin and its analogs Maria Tereshina Loss of Ag1 in ancestors of higher vertebrates correlates with the reduction of their regenerative capacity George Sharonov Cell guidance mechanisms: finding the only way through abundance of cues Victoria Shender Deciphering specific features of the ovarian cancer ascites proteome Mikhail Akimov N-acyl dopamines and other bioactive lipids in the organism’s anti-cancer defense system Natalia Kuznetsova Complement and liposomes loaded with lipophilic prodrugs of methotrexate and melphalan Zakhar Shenkarev Lipid-protein nanodiscs: multipurpose environment for cell-free production, folding and structure-functional studies of membrane proteins and membrane-active peptides Alexey Belogurov Ubiquitin-independent proteasome-mediated proteolysis of myelin basic protein and its role in health and disease Alexander Vassilevski Does investigation of toxin evolution have practical value?

SPECIAL ISSUE № 1 2014 | Acta naturae | 7 INTERNATIONAL CONFERENCE ON BIOORGANIC CHEMISTRY, BIOTECHNOLOGY AND BIONANOTECHNOLOGY dedicated to the 55th Anniversary of the M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences and the 80th Anniversary of Professor Yuri Ovchinnikov

September 15–19, 2014; Moscow, Russia

FRIDAY, SEPTEMBER 19th 10.00 – 11.40 PLENARY SESSION Chairmеn: Alexander Archakov, Valery Zubov Alexey Bogdanov Signal transduction in the ribosome: the key role of ribosomal RNA Alexander Spirin Polyribosomes: formation, conformational transitions, activity modulation and dormancy Olga Dontsova Unusual features of telomerase RNA Sergey Deev Multifunctional nanoconstructs for bioimaging and therapy

11.40 – 12.00 Coffee break

12.00 – 13.20 PLENARY SESSION Chairmеn: Vadim Ivanov, Ferdinand Hucho Joseph Schlessinger Cell signaling by tyrosine phosphorylation: from basic principles to cancer therapy Richard Pestell Cell cycle control in cancer

13.20 YOUNG SCIENTISTS COMPETITION: AWARD CEREMONY.CLOSING

8 | Acta naturae | SPECIAL ISSUE № 1 2014 INTERNATIONAL CONFERENCE ON BIOORGANIC CHEMISTRY, BIOTECHNOLOGY AND BIONANOTECHNOLOGY dedicated to the 55th Anniversary of the M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences and the 80th Anniversary of Professor Yuri Ovchinnikov

September 15–19, 2014; Moscow, Russia

Invited Speakers and Chairs

NADIK ABDOULAEV Institute of Biochem. Biophys. Research, University of Maryland Biotech. Institute, Rockville, MD, USA [email protected] MICHAEL AGADJANYAN The Institute for Molecular Medicine, USA [email protected] SIDNEY ALTMAN Yale University, USA [email protected] ALEXANDER ARCHAKOV V.N. Orekhovich Institute of Biomedical Chemistry, Moscow [email protected] ALEXANDER ARSENIEV M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow [email protected] ANGELO AZZI JM USDA-HNRCA at Tufts University, USA [email protected] BURKHARD BECHINGER University of Strasbourg/CNRS, Strasbourg, France [email protected] MICHAEL BLACKBURN University of Sheffield, U.K. [email protected] ALEXEY BOGDANOV M.V. Lomonosov Moscow State University [email protected] ALEXANDER BORONIN K.G. Skryabin Institute of Biochemistry and Physiology of Microorganisms, RAS, Pushchino [email protected] ERNESTO CARAFOLI University of Padova, Italy [email protected], [email protected] YURI CHIZMADZHEV A.N. Frumkin Institute of Physical Chemistry and Electrochemistry, RAS, Moscow [email protected] , [email protected] DMITRY CHUDAKOV M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow [email protected] VLADISLAV DEIGIN M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow [email protected] NORBERT DENCHER Technische Universität Darmstadt, Germany [email protected]

SPECIAL ISSUE № 1 2014 | Acta naturae | 9 INTERNATIONAL CONFERENCE ON BIOORGANIC CHEMISTRY, BIOTECHNOLOGY AND BIONANOTECHNOLOGY dedicated to the 55th Anniversary of the M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences and the 80th Anniversary of Professor Yuri Ovchinnikov

September 15–19, 2014; Moscow, Russia SERGEY DEYEV M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow [email protected] OLGA DONTSOVA M.V. Lomonosov Moscow State University [email protected] ROMAN EFREMOV M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow [email protected] EVGENY FESENKO Institute of Cell Biophysics, RAS, Pushchino [email protected] , [email protected] ALEXANDER GABIBOV M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow [email protected] GEORGY GEORGIEV Institute of Gene Biology, RAS, Moscow [email protected] PAVEL GEORGIEV Institute of Gene Biology, RAS, Moscow [email protected] VADIM GOVORUN Scientific Research Institute of Physical-Chemical Medicine, Moscow [email protected] ANATOLY GRIGORYEV Presidium of the Russian Academy of Sciences, Moscow [email protected] FERDINAND HUCHO Freie Universität Berlin, Germany [email protected] JEAN-LUC IMLER Institut de Biologie Moléculaire et Cellulaire du C.N.R.S., Strasbourg, France [email protected] VADIM IVANOV M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow [email protected] MICHAEL KIRPICHNIKOV M.V. Lomonosov Moscow State University [email protected] DMITRY KNORRE Institute of Chemical Biology and Fundamental Medicine, SB RAS, Novosibirsk [email protected] SERGEY KOCHETKOV V.A. Engelhard Institute of Molecular Biology, RAS, Moscow [email protected] SERGEI KOSTROV Institute of Molecular Genetics, Moscow [email protected]

10 | Acta naturae | SPECIAL ISSUE № 1 2014 INTERNATIONAL CONFERENCE ON BIOORGANIC CHEMISTRY, BIOTECHNOLOGY AND BIONANOTECHNOLOGY dedicated to the 55th Anniversary of the M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences and the 80th Anniversary of Professor Yuri Ovchinnikov

September 15–19, 2014; Moscow, Russia SERGEY KOZIN V.A. Engelhard Institute of Molecular Biology, Moscow [email protected] VALERY LIPKIN M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow [email protected] SERGEY LUKYANOV M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow [email protected] KONSTANTIN LUKYANOV M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow [email protected] ALEXANDER MAKAROV V.A. Engelhard Institute of Molecular Biology, RAS, Moscow [email protected] NIKOLAI MODYANOV University of Toledo College of Medicine Health Science, USA [email protected] NIKOLAI MYASOYEDOV Institute of Molecular Genetics, Moscow [email protected] SERGEY NEDOSPASOV M.V. Lomonosov Moscow State University [email protected] NIKOLAI NIKOLSKY Institute of Cytology, RAS, St Petersburg nnnik@mail.сytspb.rssi.ru, [email protected] MICHAEL OSTROVSKY N.M. Emanuel Institute of Biochemical Physics, RAS, Moscow [email protected] LEV OVCHINNIKOV Institute of Protein Research, RAS, Pushchino [email protected] TATIANA OVCHINNIKOVA M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow [email protected] RICHARD PESTEL Kimmel Cancer Center, Thomas Jefferson University, USA [email protected] ALEXANDER PETRENKO M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow [email protected] REM PETROV Presidium of the Russian Academy of Sciences Advisor, Moscow [email protected] VLADIMIR PLETNEV M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow [email protected]

SPECIAL ISSUE № 1 2014 | Acta naturae | 11 INTERNATIONAL CONFERENCE ON BIOORGANIC CHEMISTRY, BIOTECHNOLOGY AND BIONANOTECHNOLOGY dedicated to the 55th Anniversary of the M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences and the 80th Anniversary of Professor Yuri Ovchinnikov

September 15–19, 2014; Moscow, Russia VLADIMIR POPOV A.N. Bach Institute of Biochemistry, Moscow [email protected] ALEKSEY ROZANOV A.A. Borisyak Paleontological Institute, RAS, Moscow [email protected] JOSEPH SCHLESSINGER Yale School of Medicine, USA [email protected] VLADIMIR SHUVALOV Institute of Fundamental Problems in Biology, RAS, Pushchino [email protected] KONSTANTIN SKRYABIN “Bioengineering” Centre, RAS, Moscow [email protected] ALEXANDER SPIRIN Presidium of the Russian Academy of Sciences Advisor, Moscow [email protected] VALENTIN STONIK G.B. Elyakov Pacific Institute of Bioorganic Chemistry, FEB RAS, Vladivostok [email protected] EVGENY SVERDLOV Institute of Molecular Genetics, Moscow [email protected] VIKTOR TSETLIN M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow [email protected] MICHAEL UGRYUMOV N.K. Koltsov Institute of Developmental Biology, RAS, Moscow [email protected] VALENTIN VLASSOV Institute of Chemical Biology and Fundamental Medicine, SB RAS, Novosibirsk [email protected] SERGEY ZAVRIEV M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow [email protected] VITALY ZUBOV M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow [email protected]

12 | Acta naturae | SPECIAL ISSUE № 1 2014 Секция 3. Протеомика, пептидомикаOral and Poster и метаболомика Presentations

Acyl-dopamine-resistant glioma C6 cell line M.G. Akimov, N.M. Gretskaya, G.N. Zinchenko, V.V. Bezuglov M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Acyl-dopamines are a family of endogenous bioactive lipids. Several works of the last years demonstrated their anti-cancer activity on cancer cell lines and the absence or low toxicity for normal ones. However, as far as the selective target of these substances has not been yet identified the question of their specificity remains. The aim of this work was to develop an acyl-dopamine-resistant cell line in order to indirectly prove the existence of molecular mechanisms capable of recognition of these molecules. Rat glioma C6 cell line (Russian collection of cell cultures, IC RAS, SPb) was cultivated in DMEM medium supplemented with 10% of fetal bovine serum, 2 mM L-glutamine and antibiotics in 5% CO2 atmosphere at 37°C. To select resistant cells, the culture was repeatedly treated with increasing concentrations of docosahexaenoyl dopamine (DHA-DA) as a solution of DMSO (0.5%) in the growth medium for 20 h followed by a re-growth of survived cells on normal growth medium until confluence. The resistant cells (C6-DD) were maintained in the growth medium without acyl-dopamines and once a week subjected to a toxicity check with an acyl-dopamine panel. The result of the selection was a glioma cell line with an enhanced resistance to acyl-dopamines: LD50 for DHA-DA after 20 h incubation increased from 6±2 μМ to 50±5 μМ. According to relative resistance change, the analogues and metabolically related substances split in two groups: with 10x resistance increase (dopamine amides of arachidonic, docosahexaenoic and oleic acids and arachidonoyl 3-O-methyl dopamine) and with 20–30x resistance increase (amides of arachidonic acid with tyramine and norepinephrine, as well as stearoyl-dopamine). The resistance remained unchanged for at least three months of continuous cultivation. As a possible resistance cause, we analyzed mRNA levels of cannabinoid and vanilloid receptors, ABCG2 transporter, fatty acid amide hydrolase and plasma membrane NADH oxidase Ecto-Nox. The obtained data point to the existence of a genetically encoded mechanism of acyl dopamine resistance. At the same time, the toxicity of an acyl-dopamine analogue with the proglyprol moiety changed very little, which is an indication of action mechanism alteration after such modification. This work was partially supported by RFBR grant 13-04-40085-N.

Stress-protective effect of human lactoferrin G.M. Aleshina, I.A. Yankelevich, V.N. Kokryakov Institute of Experimental Medicine RAMS, Saint Petersburg, Russia Stress affects the interaction between nervous and immune systems changing a level of hormones and cytokines. It was previously shown that lactoferrin has regulatory effect on the number of antibody-forming cells in the spleen and the magnitude of delayed type of hypersensitivity modified by the psychic stress [1]. We have studied the influence of human lactoferrin on stress-induced changes of hormone levels, TLR4 and cytokines (IL-4, IL-6) gene expression in rats. The model of acute stress – swimming in cold (1°-4°C) water in 2 min has been used. Human lactoferrin (LF) from milk, ovalbumin (OA), served as nonspecific antigen control, and lipopolysaccharide (LPS) were administered intraperitoneally 5 min prior the stress application. Blood and spleen were collected in 30 min and 3 h after stress. Plasma corticosterone level was evaluated by IFA. mRNA expression in rat splenocytes was evaluated by real-time RT- PCR. Experiments were carried out in the same time period (11-00 am – 2-00 pm) to avoid the influence of circadian changes of corticosterone level. It has been shown that gene expression of IL-4 and TLR4 has been increased in 3 h after stress and gene expression of IL-6 was inhibited. Injection of human LF (unlike OA), abolished the stress-stimulated increase of IL-4 and TLR4 gene expression in rat spleen. Both proteins restore a gene expression of IL-6, acting apparently as nonspecific protein (antigen) molecules. As mentioned above stress affects on the immune response including LPS-induced changes expression of cytokines [2]. We have investigated the influence of LF on the gene expression of IL-4, IL-6 and TLR4 under stress condition and concurrent LPS administration. In 3 h after the LPS injection IL-6 gene expression demonstrated 300 fold increase which didn't change under stress and simultaneous LF administration. Meanwhile LPS injection doesn't influence on gene expression of IL-4 and TLR4 and stress-protective action of LF on the expression of these genes. We have demonstrated that LF had corticostatic activity. Injection of LF (unlike OA) reduced stress-induced the plasma corticosterone level in 30 min after stress. At the same time LF didn't change (LPS+stress)-stimulated corticosterone level. These results suggest that lactoferrin could be involved in the regulation of defense mechanisms during the development of the stress response. This work is partly supported by RFBR grants № 12-04-01498a and No 12-04-01573a.

1. Zimecki M. et al (2005). Pharmacol. Rep. Vol. 57(6). P. 811-817. 2. Diz-Chaves et al (2013). Brain Behav Immun. Vol. 28. P. 196-206.

Conformational properties of bactenecin molecule R. E. Aliyev Baku State University, Baku, Azerbaijan Bactenecin is a cationic antimicrobial peptide of the animal’s innate immune system. Extracted from the neutrophils of the cattle, bactenecin has a unique sequence R–L–C–R–I–V–V– I–R–V– C–R consisting of 12 amino acid residues (dodecapeptide) with one disulfide bond. Being the small- est of the known peptides, this molecule incorporates five different residues and is highly enriched by arginines. Apparently, the bactenecin’s original amino acid sequence is needed to formation of the particular spatial structure, which is essential for peptide binding to the membrane. The knowledge of the structural-functional and structural-conformational possibilities of the oligopeptide is required to understand the mechanism of action of the bactenecin’s stereo chemical features. In this paper, the bactenecin’s conformational possibilities are studied, using the fragmental approach to the calculation of the optimal spatial structures of oligopeptides and proteins on the classic base, proposed by E.M. Popov by the semiempirical method of atom-atom potentials. The calculations of the conformations were performed under the membrane and polar environment simulation. The investigation of the spatial structure of the bactenecin’s molecule showed that all its stable conformational states can be grouped in several forms of the backbone, which lead to the formation of the disulfide bond Cys3–Cys11. On the base of the received structural and conformational relationships of the bactenecin its advanced analogues may be constructed. As well as a new class of the drugs as an alternative or complement to the treatment of the various infectious diseases may be created.

Natural inhibitors of acid-sensing channels Y.A. Andreev, D.I. Osmakov, S.A Kozlov, E.V. Grishin M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Acid sensing Na+ channels ASIC are a group of proteins with extremely important regulatory and sensory function in neurons of peripheral and central nervous system. They are participating in both physiological and pathological processes. These channels are responsible for the sensitivity to pain that occurs during tissue acidosis in the muscles, cardiac ischemia, damage to the cornea, inflammation and local infection, that make them as a promising therapeutic target for the development of anti-inflammatory and analgesic drugs.

SPECIAL ISSUE № 1 2014 | Acta naturae | 13 Oral and Poster Presentations

We tested various natural samples for the presence of components that can modulate the activity of ASIC channels. Peptide π-AnmTX Hcr 1b-1 having a molecular weight of 4537Da was isolated from extract from sea anemones Heteractis crispa by multistage liquid chromatography. In electrophysiological experiments on channels expressed in Xenopus laevis oocytes peptide inhibited peak component of ASIC3 current. From the venom of sea anemone Urticina grebelnyi peptide Ugr 9-1 was isolated and shown to inhibit both peak and plateau components of the ASIC3 current. In animal models of pain Ugr 9-1 suppressed inflammatory pain and reduced pain response caused by the injection of acetic acid. Inhibitor of ASIC3 channel from an extract of thyme (Thymus armeniacus) named sevanol, was isolated and characterized in detail. Sevanol completely blocked the transient component of the ASIC3 current with an IC50 of 353±23 µM and partially inhibited the constant component of the current with an IC50 of 234±53 µM. Also this compound inhibits ASIC1a channel. Sevanol showed significant analgesic activity in vivo – in the dose of 1–10 mg/kg it significantly reduced thermal hypersensitivity caused by inflammation, and reduced the pain response to the acid. We can assume that sevanol is the one of the main components determining the known analgesic properties of thyme. Thus, we have found modulators of ASIC channels that could be the basis for development of a new generation of drugs.

Unique structural organization of the Lon family of AAA+ Proteases. The role of the LonA non-catalytic n-terminal region in the enzyme function A.G. Andrianova, A.M. Kudzhaev, N.I. Dergousova, T.V. Rotanova M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Lon proteases of AAA+ superfamily are key members of the quality control system of the cell proteome. They selectively hydrolyze a number of regulatory proteins and degrade abnormal and defective polypeptides by the processive mechanism. The single amino acid chain of these bifunctional enzymes comprises several domains that include a central AAA+ module formed by the nucleotide binding (NB) and α-helical (H) domains, a C-terminal serine-lysine endopeptidase (proteolytic, P) domain, as well as a non-catalytic domain, either N-terminal (in the LonA subfamily) or insertional (in the LonB subfamily). A unique feature of LonA proteases that distinguishes them from other AAA+ proteins is the presence of a second α-helical domain similar to the H domain of AAA+ module that includes a long coiled-coil (CC) region. This HI(CC) domain is located between the N-terminal domain and the ATPase module. Thus, the domain organization LonA proteases corresponds to the following scheme: N-HI(CC)-NB-H-P. HI(CC) domain is shown to display significant similarity to the H1(M) domain of AAA+-1 module of chaperone-disaggregase ClpB, whose subunit is formed by five domains: N-NB1-H1(M)-NB2-H2 and the M-region has a CC-conformation. A number of mutant, truncated and deleted forms of E. coli Lon protease (Ec-Lon, a model LonA enzyme) have been obtained in order to study the role of the N-terminal region (N-HI(CC)) in the function of LonA proteases and/or in maintaining their active structure. Enzymatic properties, the mechanism of proteolysis and oligomeric state of the modified Ec-Lon forms have been investigated and compared with the corresponding characteristics of the native enzyme. The N-terminal fragment (1–172) has been shown to be involved in the formation of the functional oligomeric structure of the enzyme required for processive degradation of target proteins. The key role of the HI(CC) domain in the ATP-dependent processive proteolysis and in enzyme stability under conditions of Ec-Lon protease dynamic activation has been revealed. Special significance of the C-terminal fragment of CC-region for the enzyme ATPase activity has been demonstrated. Participation of the N-terminal fragment of CC-region in protein substrate binding by Ec-Lon protease is suggested.

Original 3S-paradigm for preclinical testing of immunomodulators: specificity of in vitro step N.V. Antipova, D.A. Aronov, S.G. Semushina, S.D. Akhvlediani, D.Ch. Sultanov, E.V. Moiseeva M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Various standard mouse models exist to develop effective immunotherapeutic approaches to treat breast cancer (BC), however poor relevance of these models to human disease is well known. Preclinically revealed therapeutic efficacy is often not reproduced in BC clinics. Limited capabilities of standard mouse models and generally used preclinical schemes to test anti-cancer efficacy of immunomodulators compel to search for novel approaches. We propose original methodology for anti-cancer drug testing («3S-paradigm», E. Moiseeva. «Anti-breast cancer drug testing. Original approaches. Novel set of mouse models», 2009): 1. Set of complementary mouse models with various incidences of naturally arising inflammation and cancer development (mammary, ovarian/uterine cancers and lymphoma/leukemia); 2. Steps of drug testing procedure, including initial drug screening in vitro and revealing early treatment effects and distant consequences in vivo; 3. Stratification (instead of randomization in standard testing procedure) as methodology to form experimental groups before experiment start and to estimate results (selection of “beneficial” and “non-beneficial” subgroups comparing with corresponding control subgroups in each experiment). There are some specificities within anti-cancer activity of immunomodulator testing procedure within our novel conception: 1– initialin vitro step using both individual tumor cell line and short-term cultures of immune cells from mouse bearing the same tumor (as a rule, primary tumor effect of immunomodulators is lacking); 2– creation of collection of individual murine mammary cancer cell lines with various extent of aggressiveness and maintaining these lines both in vitro and in vivo. This approach permits carry out both steps using the same biomodel; 3– testing of prophylactic regimens in vivo using both transplanted and spontaneous mouse models. Described approaches were used to test anti-cancer effect of interleukin-2 (IL-2, “Novartis”) and Immunofan (“Bionox”) both in vitro and in vivo. Advantages of individualized 3S-paradigm (each mouse is monitored individually as a patient during the whole its life) was shown as an useful instrument to develop novel principles of preclinical testing procedure of anti-cancer drugs (including immune system modulating drugs) in agreement to modern 3P- conception of medicine (personalization, prognosis, and prevention).

Model of spatial structure of anaerobic methacrylate redox system O.V. Arkhipova1, G.V. Mikoulinskaia2, A.S. Galushko3, F.A. Kondrashov4,5,6 1Institute of Biochemistry and Physiology of Microorganisms, Pushchino, Moscow Region, Russia; 2Branch of Institute of Bioorganic Chemistry, Pushchino, Russia; 3University of Vienna, Vienna, Austria; 4Centre for Genomic Regulation, Barcelona, Spain; 5Universitat Pompeu Fabra, Barcelona, Spain; 6Institució Catalana de Recerca i Estudis Avançats, Barcelona, Spain The study of Geobacter species (Deltaproteobacteria) is of general and applied interest due to their significant role in the global cycles of metals and carbon and bioremediation of radioactive metals. Geobacter sulfurreducens AM-1 strain is unique: it is capable of using anthropogenic compound methacrylate (2-methylpropenoate) as the terminal acceptor of the bacterial reductase chain. Periplasmic methacrylate redox system G. sulfurreducens AM-1 consists of two chromoproteids: flavin-containing methacrylate reductase (50 kDa) and its physiological electron donor – cytochrome c (30 kDa). Genes of these proteins were identified in the genome of Geobacter sulfurreducens AM-1 and translated into amino acid sequences. The aim of this work was to analyze potential spatial structure of these components of methacrylate redox system and to identify the conservative amino acids participating in catalysis and substrate binding. Computer modeling of the methacrylate reductase and cytochrome c tertiary structures of methacylate redox system from G. sulfurreducens AM-1 showed high structural similarity with Shewanella’s soluble fumarate reductases. Sequence and structure of methacrylate reductase repeated two C-end catalytic domains of these fumarate reductases – FAD-binding and clamp. Cytochrome c had similarity with small N-end heme-containing domain of periplasmic

14 | Acta naturae | SPECIAL ISSUE № 1 2014 Oral and Poster Presentations

fumarate reductases of Shewanella. Furthermore, it was shown that methacrylate reductase has: 1) conserved for fumarate reductases amino acids participating in catalysis – histidine 461 and arginines 501 and 353; 2) typical for FAD-binding proteins Rossmann fold (β1α1β2α2β3); 3) conserved pyrophosphate binding sites: dinucleotide binding motif (xhxhGxGxxGxxxhxxh(x)8hxhE(D), where x is any amino acid, h is hydrophobic amino acid, and 11-amino acid’s segment T(S)xxxxxF(Y)hhGD(E).

Crystallization of the complex of translation initiation factors eIF2beta•eIF5 from eukarya V.I. Arkhipova, I.V. Mitroshin, E.A. Stolboushkina, M.B. Garber Institute of Protein Research, Pushchino, Moscow Region, Russia The initiation of protein synthesis in eukaryotic cells requires the coordinated activity of at least 10 eukaryotic initiation factors. The heterotrimeric translation initiation factor 2 (eIF2 alpha, beta, gamma) plays a key role in this process. In GTP-bound form eIF2 delivers initiator methionyl-tRNA to the 40S ribosomal subunit. The factor eIF5 (GTPase accelerating protein, GAP) stimulates hydrolysis of GTP by eIF2 upon AUG codon recognition and in complex with eIF2•GDP leaves the ribosome. In yeast this complex eIF2•GDP•eIF5 is proposed to represent a cytoplasmic reservoir of eIF2. Moreover, eIF5 in this complex plays a role of GDP dissociation inhibitor (GDI), necessary for tight control of translation initiation. Eukaryotic translation initiation factor 5 is a monomer protein and possesses two independently folded domains bound with a “linker region” (LR). The structures of both eIF5 domains are known. N-terminal domain (NTD) of eIF5 is responsible for the GAP function evidently through interaction of eIF5- NTD with the gamma subunit of eIF2. C-terminal domain (CTD) of eIF5 is known to mediate binding to eIF2beta and together with LR is responsible for GDI activity. Complex eIF2•eIF5 is functionally important component of initiation translation system and its structure is of a great interest. In this work we investigate the interaction between eIF2 and eIF5 from Saccharomyces cerevisiae, performed through the contacts between domains eIF2beta-NTD and eIF5-CTD. Nowadays the structure of eIF2beta is unknown. The recent physical-chemical studies showed that NTD of eIF2beta is disorder in the isolated state but folds into a definite structure when bound to eIF5. Therefore the complex eIF2beta-NTD•eIF5-CTD could be a perspective object for crystallization. In the frame of this work we screened crystallization conditions for the complex eIF2beta-NTD•eIF5-CTD and obtained microcrystals of the complex. We plan to optimize these crystallization conditions to obtain better quality crystals suitable for X-ray diffraction analysis. This research was supported by the Program of Russian Academy of Sciences on Molecular and Cellular Biology, and the Russian Foundation for Basic Research (project no. 14-04-31030 mol_a).

Biological activity of tryptophan-rich peptides A.Yu. Artamonov1, D.S. Orlov1,2, O.V. Shamova1,2, E.G. Rybakina1, N.I. Kolodkin3, M.P. Smirnova3 1Institute of Experimental Medicine, RAMS, St-Petersburg, Russia; 2Saint-Petersburg State University, Saint-Petersburg, Russia; 3State Research Institute of Highly Pure Biopreparations Saint-Petersburg, Russia Study of molecular and cellular mechanisms of anti-infective molecules nowadays is of utmost importance for forming a general conception of host defense. Antimicrobial peptides (AMPs) of animal origin represent the molecular base for the realization of defensive and adaptive reactions upon infections, stress, malignancy and immunodeficiency. AMPs and their synthetic derivatives also posses clinical potential beyond the treatment of antibiotic-resistant infection. The current study is dedicated to tryptophan-rich antimicrobial peptides. Indolicidin – the peptide from bovine leukocytes – is one of the most well studied compounds of this group of AMPs. Synthetic derivatives of indolicidin served as templates for a design of novel antibiotic drugs. Therefore the detailed investigation of tryptophan-rich peptides is a perspective direction of anti-infective therapy. In this work the biological activity of poorly investigated natural tryptophan-rich peptides 3B3, GSP-1a, PurA and tritripticine was explored. Peptides 3B3, GSP-1a, PurA and tritripticine, as well as indolicidin, demonstrated the potent antimicrobial activity against different microorganisms. Tritripticine, PurA, GSP-1a, 3B3 and indolicidin caused an increase of permeability of outer membrane of E. coli ML-35p. In spite of a potent antimicrobial effect, peptides GSP-1a and 3B3 showed a weak influence on the permeability of inner membrane of E. coli ML-35p. They had a lack of hemolitic effect on human red blood cells, unlike tritripticine, PurA and indolicidin that provided increase of permeability of cytoplasmic membrane of E. coli and also cased the hemolysis of erythrocytes at used range of concentration. Overall, our findings suggest that the peptides 3B3, GSP-1 and PurA are promising for the further detailed analysis directed to the invention of new drugs. These peptides are not hemolytic, but exert a potent antimicrobial activity against a broad spectrum of microorganisms. This work was supported by the RFBR grant # 13-04-02102а.

Expression and purification of the protein Jmjd6 from nematode Caenorhabditis elegans A.A. Artykov, N.B. Pestov M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia The proteins of the Jmjd family catalyze various oxidative reactions and possess a highly conservative JmjC domain that was initially described by Japanese scientists (the Japanese word jimonji stands for cruciform; the name was given because of the abnormal phenotype that emerges in neuroembryogenesis). Jmjd6 was earlier recognized as a phosphatidyl serine receptor (Ptdsr, PSR, PrdSeR) and was described as the receptor on the cell surface responsible for apoptotic cell recognition. The receptor function of the protein is currently disputed because it was shown that jmdj6 is localized in the nucleus, and this contradicts with its proposed function as a transmembrane protein. Jmjd6 contains oxoglutarate dioxygenase motives known for their ability to catalyze two types of post-translational modifications: hydroxylation of certain amino acid residues (proline, asparagine, tryptophane, asparagic acid) and the reaction of secondary dimethylation of methyllysine and methylarginine residues. Like other proteins containing JmjC domain, jmjd6 includes a dimethylase motive and is engaged in reversible dimethylation of histones, though it is not among its principal activities. In should be emphasized that the C-5 lysyl hydroxylation catalyzed by Jmjd6 results in the formation of the products with 5S stereometry, whereas collagen lysyl hydroxylase gives the products with SR-stereometry. This means that their mechanisms differ significantly, exactly because they are structurally unrelated to each other. Studies on mice with deactivated Ptsdr gene (knockouted mice) demonstrated the importance of the gene for normal growth. For example, defects of brain, eyes, lungs, kidney and intestine were observed on different stages of embryogenesis of the in knockout mice. The role of Ptsdr gene was shown in heart development, the loss of the gene’s function being associated with Tetralogy of Fallot. It is interesting that Jmjd6 function in ontogenesis is different in vertebrates and invertebrates. Orthologous genes inactivation in drosophila and C. elegans is not associated with embryo lethality and no substantial development defects because of the loss of protein function are observed. The goal of the work is cloning, protein expression, purification and characterization of catalytic properties of the lysyl hydroxylase/arginine dimethylase jmjd-6/psr-1 from C. elegans. For this purpose, we have synthesized cDNA of adult hermaphrodites, amplified and cloned the open reading frame of C. elegans jmjd-6/psr-1. At present, we are studying properties of the recombinant protein.

SPECIAL ISSUE № 1 2014 | Acta naturae | 15 Oral and Poster Presentations

Specific regulatory features of monocistronic operons encoding ribosomal proteins L25 and S20 L.V. Aseev, L.S. Koledinskaya, I.V. Boni M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Ribosome biogenesis requires coordination of synthesis of all ribosomal components -16S, 23S and 5S ribosomal RNAs (rRNAs) and more than 50 ribosomal proteins (r-proteins), – therefore it should be strictly controlled. Transcription of rRNA operons proceeds at high level during growth on rich media but it dramatically falls down upon starvation, the phenomenon known as a ppGpp-mediated negative stringent control. Genes encoding r-proteins, as a rule, are combined into clusters, polycistronic operons; however several genes (5 in E. coli) are independent transcription units. At the translation level, many r-protein operons are regulated by the mechanism of autogenous repression, according to which one of the operon products acts as a translational repressor of its own mRNA when produced in molar excess over rRNA. Up to now, regulation has been studied only for a half of r-protein operons (10 out of 21), and one cannot assert a priori that the principle of autogenous control holds true for the remaining cases. The goal of our work was to study regulation in vivo of two monocistronic operons, rplY and rpsT, encoding r-proteins L25 and S20, respectively. We show that transcription of rplY is under negative stringent control as in the case of rRNA operons, and at the translation level, the classic mechanism of autogenous repression is involved. Phylogenetic analysis revealed a high conservation of the specific structure of the rplY mRNA regulatory region (in particular, the absence of canonical Shine–Dalgarno sequence) in a subset of gamma-proteobacterial families. Using site-directed mutagenesis, we determined the role of conserved structural elements in high translation efficiency of the rplY mRNA and its regulation by L25 in trans. Our data also show that the rpsT operon (S20) represents a unique case when the synthesis of r-protein is not subjected to feedback regulation. The regulatory region of the rpsT mRNA lacks conserved structural elements; it is loosely structured in contrast to rplY and other feedback regulated r-protein mRNAs. The only highly conserved feature is a weak initiator codon UUG, whose change for a canonical AUG resulted in a 5-fold increase in translation yield. We suggest that the rpsT translation regulation in gamma-proteobacteria is based on utilization of a weak initiator codon that prevents the excessive synthesis of S20, which, as we have shown for the first time, is toxic for a bacterial cell. This work is supported by the RFBR grant 12-04-01138

Peptidome analysis for search serum markers of colorectal cancer I.V. Azarkin, R.H. Ziganshin, G.P. Arapidi, S.I. Kovalchuk, O.M. Ivanova, V.O. Shender, N.A. Anikanov, V.M. Govorun, V.T. Ivanov M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia According to the International Agency for Research on Cancer IARC colorectal cancer (CRC) is the third most common cancer in men and the second – in women. Depending on the type and location of the tumor, 50–80% of cases, because of the latent course of this disease, it is diagnosed only III or stage IV, which significantly reduces the chances of successful treatment. One of the major problems of monitoring and treatment of CRC is its early diagnosis. The aim of this work is to identify in the serum of patients with CRC peptide markers of the disease using the latest proteomic approaches. To work used serum samples of 50 patients with colorectal cancer and 50 healthy donors. To isolate peptides from serum used in series with the magnetic microparticle surface, and a weak cation exchange chromatography, anion exchange microcolumns. The resultant anion exchange column with an elution step with a salt gradient and desalted peptide fractions were analyzed by LC-MS/MS for quadrupole TOF mass spectrometer ABSciexTripleTOF +5600. Relative quantification identified serum peptides were performed by SWATH (sequential windows acquisition of all theoretical ion-fragments spectra). As a result of LC-MS/MS analysis of sera more than 6000 unique peptides originated from the almost 1000 unique proteins were identified. Among identified peptides 786 were unique for CRC samples, and 125 of those were originated from the proteins unidentified in the control samples. For the control group there were 1075 unique peptides, 259 of which were originated from the proteins unidentified in CRC samples. We believe that the presented data set contains valuable information which will enable interested researchers to identify of new potential biomarkers for colorectal cancer.

Side reactions occurring during the preparation of δ-aminO-γ-keto-α-alkylvaleric acid in the case of Dakin–West reaction usage and during the incorporation of the acids into the ketomethylene pseudopeptide chain V.N. Azev1, М. V. Molchanov2 1Branch of M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Pushchino, Moscow Region, Russia; 2Institute of Theoretical and Experimental Biophysics RAS, Pushchino, Moscow Region, Russia

The ketomothylene fragment -C(O)-СH2- is an isostere of the amide functional group -C(O)-NH-.[1] Ketomethyleme pseudopeptides (or peptidomimetics) that are the products of such substitution are used in medicinal or biological studies. [2] Ketomethylene isostere substitution in peptides is known for more than 30 years. Many methods for the synthesis of δ-amino-γ-keto-α-alkylvaleric acids, a core structural unit of ketomethylene pseudopeptides, have been published in literature. However, there is no much information available on the subject of side reaction that accompony synthesis of ketomethylene pseudopeptides. Herein, we report the results we obtained during the preparation of δ-amino-γ-keto-α-isobutylvaleric acids using the Dakin-West reaction approach. In particular, we demonstrated that a side product of Knoevenagel condensation could be isolated. This product is formed upon the reaction of the keto-group of the desired product with the methylene group of 3-phenyl-oxazolone-5, a substrate for the Dakin–West reaction. We found the reaction conditions that allowed tminimize the formation of the Knoevenagel condensation side product. We demonstrated that during the incorporation of the δ-amino-γ-keto-α-isobutylvaleric acid into the peptide chain another side reaction takes place. In this cyclization reaction, an amide group of the peptide chain interacts with keto-group of δ-amino-γ-keto-α-isobutylvaleric acid fragment. The structure of the side product is confirmed by physico-chemical methods. The data obtain in the present study could be potentially utilized in the preparation of another ketomethylene pseudopeptides in order to avoid the formation of similar side products.

[1] Gante, J. Angew. Chem. Int. Ed. 1994, 33, 1699-1720. [2] Watermeyer, J. M.; Kröger, W. L.; O'Neil, H. G. et al. Biochemistry, 2008, 47, 5942-5950.

The study of the new photochromic labelled human platelet aggregation inhibitors binding with their target N.E. Belikov1, O.V. Demina1, A.Yu. Lukin2, P.P. Levin1, S.D. Varfolomeev1, A.A. Khodonov1,2 1N.M. Emanuel Institute of Biochemical Physics RAS, Moscow, Russia 2M.V. Lomonosov State University of Fine Chemical Technologies, Moscow, Russia We have synthesized 3 new compounds (SP1-SP3) containing a “molecular address” in different spatial orientation to photochromic label fragment and studied their binding process with platelet membrane receptors to investigate the mechanism of action of new human platelet aggregation inhibitors class (pyridylisoxazoles).

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The formation of complexes in experiments on the binding of compounds (SP1-SP3) with human platelet membrane receptors has been detected spectrophotometrically by the measuring of the label’s merocyanine form photoinduced fluorescence level and parameters of its photoinduced absorption spectra. The compound (SP3), containing the complete pharmacophore fragment as a “molecular address”, has been shown to bind to the platelets much better, then the compound (SP2). The binding control of the compound (SP3) with the use of excess amounts of non-labelled

analog – 5-phenyl-3-(3-pyridyl)-4,5-dihydroisoxazole has shown that both compounds bind to the same target – the TxA2-receptor. This work was partly supported by the RFBR Grant (project № 14-04-01701a) and the Grant of the President of Russian Federation for Young Ph.D. Scientists (project No. МК-6901.2013.4).

Transmembrane dimerization interface of receptor tyrosine kinase FGFR3 and pathogenic mutations E.V. Bocharov, D.M. Lesovoy, M.V. Goncharuk, P.E. Volynski, A.S. Arseniev M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Four human fibroblast growth factor receptors (FGFRs) belong to the family of receptor tyrosine kinases (RTKs) and transduce diverse biochemical signals by lateral dimerization in the plasma membrane. FGFRs play an important role in human growth and development, and in the adult. Among the family, FGFR3 is known for the largest number of pathogenic mutations observed in human. At least eight pathogenic mutations, implicated in cancers and growth disorders, have been identified in single-span transmembrane (TM) domain of FGFR3. Structural-dynamic properties of dimeric FGFR3 TM fragment L357-R399 were characterized by heteronuclear NMR spectroscopy in membrane-mimicking micellar environment. In NMR structure, two long TM helices 369 398 (A -L )2 pack into a symmetric left-handed dimer with a small crossing angle. The helix-helix interactions are mediated by an extended heptad repeat motif YA374xxL377xxG380xxFF384xxIL388xxA391xxTL395. The central region of the dimer is characterized by intra- and intermolecular stacking interactions of 379 384 386 369 372 aromatic rings (Y –FF –F )2, whereas the N-terminal part of the FGRFR3 TM domain (A GSV ) becomes helical only upon dimer formation. Noteworthy, the most common pathogenic substitutions Gly380Arg and Ala391Glu causing the achondroplasia and Crouzon syndrome fall within the TM helix-helix interface. Nevertheless, several mutations are situated in weakly polar area rich in characteristic GG4-like motifs G370xxxA374xxxS378xxxG382 and S371xxxG375 – a natural candidate for alternative dimerization mode of the FGFR3 TM helix proposed by molecular modeling. This implies that while the observed NMR structure is important for FGFR3 signaling, the mechanism of FGFR3-mediated transduction across the plasma membrane is complex and not yet understood. We propose a FGFR3 signaling mechanism that is based on the NMR structure, the available structures of isolated soluble FGFR domains, and published biochemical and biophysical data. According to this mechanism the observed NMR structure corresponds to the basal phosphorylation receptor state, whereas the alternative dimeric conformation of the TM FGFR3 domain can be assigned to the fully active receptor.

Interaction of cholesterol with transmembrane domail of amyloid precursor protein APP O.V. Bocharova1, A.S. Urban1,2, K.D. Nadezhdin1,2, P.K. Kuzmichev1, E.V. Bocharov1, A.S. Arseniev1,2 1M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia, 2Moscow, Russia; Institute of Physics and Technology, Dolgoprudny, Russia Despite multiple data about pathogenesis of Alzheimer disease the initial steps of the pathogenesis remain unclear. More than half of mutations associated with familiar Alzheimer disease were found in transmembrane (TM) domain of amyloid precursor protein (APP) and are thought to affect its structural- dynamic properties and lateral dimerization in neuron membrane. It was shown that the cholesterol rich microenvironment is essential for the functioning and proteolitic cleavage of APP in the membrane. Using combination of the NMR spectroscopy and molecular modeling we characterized the interaction of cholesterol with APP fragment Gln686-Lys726, including the APP transmembrane domain with adjacent N-terminal juxtamembrane sequence. The cholesterol binding was studied in three membrane- emulating milieu composed of detergent micelles, lipid bicelles and model lipid bilayer. We found that the cholesterol weakly interact with APP in at least two sites. The major cholesterol-binding hydrophobic cavity is formed by weakly polar surface formed by residues Gly700, Gly704, Gly708 and Gly709 under the loop region where the juxtamembrane α-helix contacts with the membrane surface near N-terminus of the transmembrane α-helix of APP. These residues are employed in two alternative dimerization interfaces of APP, and thus the cholesterol binding can modulate the APP proteolytic cleavage by secretases.

Synergistic effect of antimicrobial peptide arenicin in combination with conventional antibiotics I.A. Bolosov, P.V. Panteleev, Yu.D. Ivanova, T.V. Ovchinnikova M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Spreading of multiresistant bacteria is one of the main medical and social problems. Low membrane permeability for antibiotics can cause bacterial resistance. A potential way to overcome the resistance is to use combination of antimicrobial compounds. In some cases simultaneous using of antibiotics having intracellular targets and membrane-active molecules allows to reduce the effective dose, and expand the spectrum of antibacterial activity. In recent years endogenous antimicrobial peptides (AMP) are considered as possible alternatives to conventional antibiotics. Being molecules that disrupt membrane integrity, AMP are supposed to facilitate delivery of hydrophobic antibiotics into the target cell. In this study, we investigated the synergistic activity of arenicin-1 in combination with conventional antibiotics, including ampicillin, azithromycin, gentamicin, lincomycin, and rifampicin against Escherichia coli and Staphylococcus aureus. Minimal inhibitory concentrations (MIC) of antibiotics are determined by the serial dilution method. The results were expressed as the fractional inhibitory concentration (FIC) indexes, which were assessed as follows: FIC = [A]/MICA + [B]/MICB, where MICA and MICB were the MICs of the individual peptides A and B, while [A] and [B] were the MICs of A and B when used in combination with each other. The FIC index of ≤0.5 is taken to indicate synergy (in the case of FIC=0.5 representing the equivalent of a fourfold decrease in the MIC of each compound in combination). Synergistic effects were observed when arenicin-1 was used in combination with ampicillin, azithromycin or gentamicin. The best effect (FIC=0.3) has been achieved using the combination of arenicin-1 with azithromycin against S. aureus. Apparently, the peptide significantly increases the membrane permeability for the hydrophobic molecule of azithromycin.

HSP70 levels and their associations with ROS production in human neutrophils in different age groups A.A. Boyko1, M.V. Grechikhina1, L.M. Kanevskiy1, G.V. Lutsenko1V.F. Semenkov2, A.M. Sapozhnikov1, E.I. Kovalenko1 1M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia; 2Centre of Gerontology of Russian State Medical University, Moscow, Russia One of the mechanisms of cell protection from the adverse consequences of ROS action is provided by highly conserved heat shock proteins (HSPs), with the most well studied proteins of family 70 kDa (HSP70). It is unclear whether HSP70 is involved in controlling the process of ROS generation in neu-

SPECIAL ISSUE № 1 2014 | Acta naturae | 17 Oral and Poster Presentations

trophils. In this work we have analyzed HSP70 content and ROS production in neutrophils as well as relationships of these parameters in three age groups of human individuals: 20–59 (young), 60–89 (elders) and 90 years and older (nonagenarians).

We did not reveal any remarkable age-dependent alterations in initial HSP70 content (HSP70basal) in the cells. Next we examined an ability of neutrophil HSP70 system to react to stress conditions and compared induced by heating (43°C, 10 min) elevation of intracellular HSP70 levels in different age groups.

The HSP70 levels registered immediately after heat shock (HSP70HS) did not differ between young, old donors and nonagenarians. At the same time, the HSP70 increase values estimated as differences between HSP70HS and HSP70basal (ΔHSP70HS) for each person in the groups were shown to be significantly increased with age supposedly indicating the functional importance of this parameter. It’s been shown, that ΔHSP70HS did not connect with HSP70 synthesis de novo but was associated with conformational changes in the HSP70 molecule, revealed with antibody recognizing C-terminal part of HSP70 molecule containing the substrate-binding domain. We suppose that this HSP70-related parameter may indicate indirectly the amount of HSP70 engaged in functional interactions with new proteins after heating. We demonstrated that some of ROS parameters in the group of nonagenarians (90 year and older) were similar to the parameters registered in the group of young volunteers in contrast to the group of elders Multiple relationships has been revealed between HSP70 and ROS production in all age groups, however they do not remain constant with age. Moreo- ver, in different age groups both negative and positive correlations between HSP70 and ROS parameters can be found, indicating that the relationships are indirect.

Morphology and content of microvesicles from urine of healthy donors and prostate cancer patients О.Е. Bryzgunova1, A.E. Grigor’eva1, Е.S. Morozkin1, М.М. Zaripov2, E.I. Ryabchikova1, V.E. Voytsitskiy2, V.V. Vlassov1, P.P. Laktionov1 1Institute of Chemical Biology and Fundamental Medicine SD RAS, Novosibirsk, Russia; 2Novosibirsk Regional Oncology Center, Novosibirsk, Russia Microvesicles of the different sizes, namely exosomes (30–100 nm), prostasomes (50–500 nm), oncosomes (50–500 nm) and other microparticles (100– 1000 nm) are found not only in blood, but also in urine. Exosomes from prostate cancer patients (PCa) can potentially contain cancer-specific nucleic acids, and thus represent the convenient source of diagnostic material. We have investigated morphology and DNA/miRNA content of microvesicles isolated from urine of healthy donors and PCa patients. Urine was clarified by two serial centrifugation at 400g and 17000g, 20°C, 20 min. Microparticles were precipitated from the resulting supernatant by high- speed centrifugation at 100000g, 18°C, 90 min, the pellet was re-suspended and re-pelleted by centrifugation under the same conditions. In order to isolate exosomes total microparticles were filtered through 0.1 micron pore filters and re-precipitated. The resulting pellets were re-suspended and investigated by transmission electron microscopy (TEM). DNA concentration was measured using a multiplex TaqMan PCR (À-satellite elements and LINE1 repeats). miRNA were quantified by TaqMan PCR after RT with steem-loop primers containing single strain overhang 6 nucleotides sequence complement to target miRNA. TEM demonstrate the presence of 20–300 nm microparticles in urine of healthy donors and PCa patients. 50–70% of all urine microparticles are represented by 30–100 nm exosomes and residual 30–50% by bigger particles. 0.1 micron filtration increase exosomes content up to 75–85%, and decrease maximum particle size to 120 nm. Microparticle preparations, isolated from the urine of healthy donors and PC patients do not differ in a protein concentration, which is in average 289 µg/ml of the total microparticles and 206 µg/ml in exosomes. The DNA concentration in the total microparticle and the exosomes do not differ (in average about 10 pg/ml of urine) and could not be concerned as cell-free DNA contamination: alpha-satellite concentration was 2–3 times higher than LINE1. MiRNA were isolated with efficacy no less than 60% from exosome preparations from 10 patients with prostate cancer and 20 healthy donors. Some miRNA like miRNA-126 were shown to be overrepresented in contrast to other like miRNA-16 which was shown to be under- represented in the microparticles and exosomes.

Whole-genome analysis of human chromatin structure by means of accessibility to methylation by DAM methylase S.S. Bulanenkova, O.B. Filyukova, A.V. Kudryavtseva, A.V. Snezhkina, S.B. Akopov, L.G. Nikolaev, E.D Sverdlov M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Eukaryotic genomic DNA with associated nuclear proteins forms chromatin. This structure serves not only for packaging of genomic DNA within nucleus but also plays a very important regulatory role influencing DNA accessibility to DNA-binding proteins. Open, transcriptionally active chromatin and silenced closed chromatin are distinguished. Local changes of chromatin structure, affecting regulatory regions such as promoters and enhancers, influence an expression of the corresponding genes. Large-scale changes of chromatin structure are responsible for regulation of transcriptional activity of multigenic loci and also influence chromosome localization within nucleus. We have proposed an approach for chromatin structure analysis in vivo at a whole-genome level. Assuming that DNA accessibility to Dam methylase depends on level of chromatin openness we expressed bacterial Dam methylase gene in HEK293 cells. After fragmentation of genomic DNA we selected only Dam-methylated fragments. In parallel, the library of all genomic fragments irrespective of the methylation status was prepared in a similar way. These whole-genome libraries were analyzed by next generation sequencing. As a result, we obtained the whole-genome distribution of Dam methylase accessible sites. The distribution profile was not uniform and contained regions of high and low density of Dam methylated sites. Regulatory regions of protein-coding genes had elevated level of Dam methylation. Moreover, the Dam methylation profile was also generally in agreement with distribution of other open chromatin markers. We concluded that the Dam methylase accessibility on whole-genome level is a good marker of chromatin openness in living cells and can be a good approach for identification of new regulatory regions and thus the important new tool for the whole-genome chromatin analysis in mammals. The work was supported by the Scientific School program of the Russian Federation President, the program of the Russian Academy of Sciences on Molecular and Cellular Biology and RFBR Grant № 12-04-32163-mol-a.

Sequence-specific transport of oligonucleotides into human endothelial cells V.S. Chernonosova, Zh.K. Nazarkina, I.A. Zaporozhchenko, P.P. Laktionov Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk, Russia It is known that nucleic acids (NA) are transported into eukaryotic cells mainly by the receptor-mediated endocytosis, thus binding of NA to cells surface is the major step of NA transport into cell [1]. Several DNA sequences which demonstrate high affinity to cell surface and readily penetrate into the cells were found using SELEX approach [2]. In a search for DNA sequences, tightly bound to the surface of human umbilical vein endothelial cells, we have developed an approach based on the isolation and identification of tightly bound short endogenous DNA from the nucleoprotein complexes exposed on the cell surface [3]. Using this approach, we have identified short consensus sequences CTACGT, GATCCA, CATGCAT with the frequencies 21.7%, 10.8% and 16.9% correspondingly in the total pool of isolated 6–12 nucleotides DNA. We investigated transport into endothelial cells and intracellular localization of 14 deoxyribooligonucleotides (ODN), from structure of genomic DNA and containing these motifs in different combination. Using radioisotope analysis, fluorescence microscopy, flow cytometry along with competitors and inhibitors of intracellular transport we have demonstrated that the process of ODN binding, intracellular transport and distribution among intracellular compartments depend on the sequence of ODN. The data obtained demonstrate that ODN are transported into the cells by different ways obviously in depend from their

18 | Acta naturae | SPECIAL ISSUE № 1 2014 Oral and Poster Presentations

binding with cell surface proteins as well as intracellular localization of ODN depend from sequence specific binding with different intracellular mediators.

1. Juliano R. et al. Mechanisms and strategies for effective delivery of antisense and siRNA oligonucleotides. Nucleic Acids Res. 2008, v. 36, p. 4158–4171. 2. Ohuchi S. Cell-SELEX Technology. Biores Open Access. 2012, v. 1, p. 265-272. 3. Mal'shakova V. et al. Isolation and sequencing of short cell-surface-bound DNA. Ann N Y Acad Sci. 2008, v. 1137, p. 47-50.

Identification, gene cloning and expression of new bacteriophage RB49 endolysin S.V. Chernyshov1, А.А. Zimin2, М.S. Shavrina1,3, G.V. Mikoulinskaia1,3 1Branch of M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Pushchino, Moscow Region, Russia, 2Institute of the Biochemistry and Physiology of Microorganisms RAS, Pushchino, Russia; 3Pushchino State Institute of Natural Sciences, Pushchino, Russia Virulent bacteriophages of Caudovirales order for the progeny escape from the host cell induce a system of lytic proteins with one of them, endolysin, hydrolyzing the cell wall peptidoglycan. The possibility for such proteins to be used “from without” as bactericidal agent causes great scientific interest to the search of new peptidoglycan hydrolyzing enzymes. Bacteriophage RB49 is virulent coliphage of Myoviridae family, belonging to T4-like phage group. Cytosine in its DNA is not hydroxymethylated as opposite to T-even phages; moreover, unlike T4, RB49 has ability for gene transduction. We carried out bioinformational RB49 genome analysis and found out an open reading frame coded a product with molecular mass of 14.7 kDa annotated as a hypopetical protein 102 (p102). Based on the sequence homology (38% similarity) with the previously characterized bacteriophage T5 lytic peptidase we have suggested that this protein was peptidoglycan hydrolase. Gene р102 396 bp in length was amplified by PCR with bacteriophage RB49 genomic DNA and cloned in рЕТ30b plasmid vector into the E.coli XL10 Gold cells using NdeI and XhoI restriction sites. The gene was expressed effectively in the E.coli BL21(DE3) and С41(DE3) cells; the target protein was well soluble and produced in amounts no less than 40% of total protein. Protein product activity analysis revealed that it efficiently lysed the permeabilized E.coli cells: the activity in crude extracts of producing strain amounted to 22.778±1.120 units per mg, what exceeded control one by two orders of magnitude. Addition of 1 mM EDTA to reaction medium led to specific activity inhibition to 2,966±0,342 U/mg. Thereby we suggested that studied endolysin could be a metalloenzyme. Endolysin RB49 activity checked on the Gram-positive cells of Micrococcus luteus (peptidoglycan of A2 type) and Streptococcus mutans (peptidoglycan of А3 type) revealed that their cell walls are not the substrate for this enzyme. We suppose that the enzyme is specific to peptidoglycan of Gram-negative bacteria refer- ring to A1 type.

SERS study of silver nanoparticles impact on bacteriorhodopsin A.A. Chistyakov, D.O. Solovieva, E.P. Lukashev, K.E. Mochalov, V.A. Oleinikov M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Silver nanoparticles (Ag NPs) have gained acceptance in a wide variety of applications, in particular, due to their potential antibacterial activity. Mechanisms of Ag NPs impact on biological objects and organisms are various and are intensively studied. One of promising research methods is the Surface Enhanced Raman Spectroscopy (SERS) [1, 2]. In this case, Ag NPs are used as the SERS-active surface. Under light irradiation and in surface plasmon resonance, the electromagnetic field near the Ag NPs surface increases essentially. It gives the SERS effect, however it also affects the investigated species characteristics thus resulting in incorrect data. There are three main groups of lines in SERS-spectra of bacteriorhodopsin (BRh) (near the 1000 cm-1, 1200 cm-1 and 1530 cm-1), characteristic of reso- nant excitation retinal. All three line groups are similar to the BRh Raman spectrum, recorded under the same wave length of excitation. It indicates the electromagnetic nature of the enhancement. Comparison of the optical spectroscopy and atomic force microscopy data allows to suppose that SERS-spectra arise from the BRh localized in so called “hot spots”, which are formed by Ag NPs aggregation caused by incubation with SERS-activator (sodium perchlorate was used). SERS-spectroscopy shows, that Ag NPs modify the BRh structure localized in the “hot spots”. Their structures close to dark-adapted BR548 form, contains 13-cis retinal with cis-C=N fragment. It was found that this type of Ag NPs impact suppresses the BRh’s photocycle. As the result, it was shown that Ag NPs generate two types of complexes with BRh. One of them is capable to change essentially the BRh molecules properties up to fixing the form of BRh, which is registered by SERS-spectra. The work was supported by RFBR 12-04-00779 and 13-04-00168.

1. Mochalov, K.E., Strel'tsov, S.A., Ermishov, M.A., Grokhovskii, S.L., Zhuze, A.L., Ustinova, O.A., Sukhanova, A.V., Nabiev, I.R., Oleinikov, V.A. (2002) Surface- enhanced Raman scattering spectroscopy of topotecan-DNA complexes: binding to DNA induce topotecan dimerization. – Optics and Spectroscopy, 93(3), 416- 423. 2. Feofanov A.V., Oleinikov V.A., Tuzikov A.B., Yanoul A.I., Kryukov E.Yu, Bovin N.V., Nabiev I.R. (1997) Study of sialylated neoglycoconjugates by surface-enhanced Raman scattering spectroscopy. - Russian Journal of Bioorganic Chemistry, 23(11), 810-817.

Genomos: an innovative automation system for controling genome expression C. Coral-Gomez Chelmsford, Massachusetts, USA We present a prototype of “GenoMoS”, an innovative automation system for monitoring, analysis and prediction of whole genome expression based on a novel representation of gene expression. In this new representation, gene expression is considered a dynamic state that cannot be observed directly but has to be estimated or “inferred” from uncertain and incomplete measurements such as DNA expression microarrays, or other high-throughput whole-genome expression measurement techniques. In this representation therefore, gene expression regulation is treated as what it truly is, a dynamic system, which can analytically be described by a linear dynamical system. The large dimension – small sample size problem, typical of high-throughput gene expression experiments is resolved in GenoMoS using a specially developed by the author algorithm. This proprietary algorithm transforms the sets of replicated gene expression measurements into a “compressive” domain computed by the principle of compressive sensing. Working in this new domain, GenoMoS assigns probability values to the belief of the initial expression state of the thousands of genes in the genome under study. Then, it applies a sequential Bayesian estimation method to update those probabilities recursively using additional information arriving from new replicated gene expression measurements obtained during whole-genome time-course experiments. This way, GenoMoS rapidly reduces the uncertainty of gene expression state estimations with the number of time-course experiments, reaching highly accurate estimates after a small number of experiments. High accurate estimates of gene expression allow the use of the “tensor of gene expression similarity”, derived by the author to quantitatively estimate relative changes in expression state among genes in time, or under different environmental conditions. It is expected that further development of GenoMoS will provide the scientific community with a key computational tool for advancing quantitative genomic research in different fields of biotechnology and biomedicine.

SPECIAL ISSUE № 1 2014 | Acta naturae | 19 Oral and Poster Presentations

How protein dynamics and protein-protein interactions contribute to energy conversion, healthy ageing, and neurodegenerative diseases Norbert A. Dencher Physical Biochemistry, Department of Chemistry, Technische Universitaet Darmstadt, Germany Biological membranes are the largest and most active organelles/organs in every cell/organism. They perform energy-conversion, e.g. oxidative phosphorylation in mitochondria and photosynthesis in chloroplasts, as well as signal-transduction, but are also involved in many diseases as primary trigger and target. A variety of aspects of energy-transducing membranes, from large protein complexes down to the level of protons and functional relevant picosecond protein dynamics, will be discussed. With the advent of the photon-driven proton pump bacteriorhodopsin (BR) in the purple membrane of Haloarchaea in the 1970’s, it quickly emerged as the prototype of an integral membrane protein, especially of the family of seven membrane-spanning α-helical proteins, such as the GPCRs. Due to the huge progress in the elucidation of its structure, function and dynamics, gathered also by the important contributions of Yuri A. Ovchinnikov and his co-workers at the Shemyakin Institute (1), at present BR is the best characterized active ion-translocating membrane protein and the first whose vectorial transport mechanism is unravelled at the atomic level (2,3). The first time for a membrane protein, we could demonstrate by the application of time-resolved X-ray and neutron scattering pump-probe techniques that changes in the picosecond dynamics and microsecond conformational changes of the tertiary structure drive the function of bacteriorhodopsin (2,3). The importance of water molecules for the function, structure, and dynamics of membranes could be established in this way. The single subunit BR with its molecular mass of only 26700 Da is relatively small and, although being arranged as a trimer in the membrane, the efficient functional unit is the monomer. However, bioenergetics and membranes like it large. Huge protein supercomplexes (up to 2.3 ×106 Da and larger) of the respiratory chain residing in the inner mitochondrial membrane power the ATP synthase, a rotary electro-chemical nanomotor, supplying the biological fuel ATP. ATP synthases and the other oxidative phosphorylation (OxPhos) complexes do form supramolecular assemblies that have an impact on cell metabolism, mitochondrial morphology, diseases, ageing and vice versa. These supramolecular assemblies that we examine by atomic force, single-particle and cryo-electron microscopy as well as by X-ray diffraction and native gel electrophoresis (3), play a crucial role not only in energy metabolism but also in lifespan control. Since enzyme activities of OxPhos-complexes assembled as supercomplexes (e.g. the OXPHOS super- complex I1III2IV1) are 2 to >15 times higher than those of the respective individual complexes, the amount, the proportion of the individual vs. specific supercomplexes and the activity of OxPhos (super)complexes will determine the overall performance of the respiratory chain / of ATP generation and have to be considered for a systems biological description of energy transformation as well as for elucidating the molecular basis of ageing and of the age-associated diseases Alzheimer’s and Parkinson’s Dementia. Changes in the amount of such superassemblies, as observed during ageing in fungi, worms, rats and human cells, affect mitochondrial structure and function (3). These dynamic alterations have a strong impact on energy metabolism and play a significant role in physiology and pathophysiology.

(1) Ovchinnikov Yu.A., Abdulaev N.G., Feigina, M.Yu., Kiselev A.V. and Lobanov, N.A. The structural basis of the functioning of bacteriorhodopsin: An overview. FEBS Letters 100, 219-224 (1979). (2) Dencher, N.A. Sass, H.J. and Büldt, G. Water and bacteriorhodopsin: structure, dynamics and function. Biochim. Biophys. Acta (Bioenergetics) 1460, 192-203 (2000). (3) Seelert, H. , D. N. Dani, S. Dante, T. Hauß, F. Krause, E. Schäfer, M. Frenzel, A. Poetsch, S. Rexroth, H. J. Schwaßmann, T. Suhai, J. Vonck, and N. A. Dencher. From protons to OXPHOS supercomplexes and Alzheimer’s disease: structure-dynamics-function relationships of energy-converting membranes. Biochim. Biophys. Acta 1787, 657- 671 (2009).

Model systems of animal blood serum N.A. Dovzhenko, I.V. Milaeva, V.I. Maximov, S.Y. Zaitsev Moscow State Academy of Veterinary Medicine and Biotechnology, Moscow, Russia Systems, modeled the blood serum, are important not only in the fundamental, but also in applied aspects [1]. Interfacial tensiometry method is important for veterinary biochemistry and biotechnology, allowing determine the parameters of the dynamic surface tension (ST) of biological liquids such as blood serum [1–3]. One of the major protein componente of human and animal serum is an albumin. For cattle, it is 38–50% of the total protein in serum (18-46 g/l) [1, 3]. The solutions of bovine serum albumin (BSA), as a model system, at a concentration of 1 g/l and 25–95 g/l were prepared and investigated for a better understanding of its effect on serum ST. The ST were determined using tensiometer BPA-1P (Germany, Sinterface Technologies): points at t→ 0 (σ0), t=0,02s (σ1), t=1s (σ2), t→ ∞ (σ3) and initial (λ0) and final (λ1) angles of tensiogramms. When the concentration of the BSA in solution increased, a gradual decreasing was observed with increasing time. Maximum of ST values for solutions in all concentrations were determined at t=0.02s whereas a minimum was at t → ∞. With an increasing of the surface time from 0.02s to ∞ in the solution with concentration of BSA 1 g/l ST decreased by 3%, at a concentration of 25 g/l – 12% at 35 g/l – 14% at 45 g/l – 15% at 55 g/l – 15% at 65 g/l – 16% at 75% – 17% at 85 g/l – 18% at 95 g/l – 18%. When the concentration of BSA in the solution increased, the values σ2 и σ3 decreased. With increasing concentration from 1 g/l to 95 g/l σ2 decreased by 7.8%, and σ3 – by 16.5%. When the concentration of BSA in the solution of from 1 to 55 g /l value λ0 increased by 4 times, at the concentration of 65 g/l decreased by 6%, in solutions at concentrations of 65–85 g/l, varies within the measurement error, when the concentration of BSA up to 95 g/l decreased by 13%. When the concentration of BSA in the solution was from 1 to 25 g/l, the values of λ1 increased by 6 times. At the concentration of 25–35 g/l of the values of λ1 varied within experimental error, from 35 to 75 g/l – increased by 28%, from 75 to 95 g/l – also varied within experimental errors. Thus, established patterns allow use these model systems to detect interactions of surfactants in serum of blood. This work was supported by Russian Scientific Foundation (grant 14-16-00046).

1. Zaitsev S.Yu. – LENAND, 2010. – 208 p. 2. Zaitsev S.Yu., Maximov V.I., Zarudnaya E.N., Dovzhenko N.A. // Nanotechnology and health protection – 2010. V. 2. №4(5). – p. 32-36. 3. Zaitsev S.Yu., Milaeva I.V., Zarudnaya E.N., Maximov V.I. // Colloids and Surfaces A: Physicochem. Eng. Aspects. - 2011. - V. 383. - P. 109–113.

5-alkyl-3-(3-pyridyl)isoxazoles with antiaggregatory activity O.V. Demina1, N.E. Belikov1, V.I. Shvets2, S.D. Varfolomeev1, A.A. Khodonov1,2 1N.M. Emanuel Institute of Biochemical Physics, RAS, Moscow, Russia; 2M.V. Lomonosov State University of Fine Chemical Technologies, Moscow, Russia The development of new heterocycles based antiaggregatory substances required for the prevention and treatment of cardiovascular system deseases is an important task for bioorganic and medicine chemistry. These substances are essential for the direct correction of the hemostasis, and the critical part of which are platelets preventing a loss of blood by the formation of a white plaque upon the blood vessel damage. The processes inside the platelet may be regulated selectively enough by the use of agonists and antagonists of receptors connected with arachidonic acid cascade, including TxA2 receptors, as well as inhibitors of the cascade first enzyme – cyclooxygenase. The TP receptor selective antagonists as well as the double action antiaggregatory compounds acting as on both TP receptor and thromboxane synthase are the most important development goals.

20 | Acta naturae | SPECIAL ISSUE № 1 2014 Oral and Poster Presentations

We were first to produce the series of new 5-alkyl-3-(3-pyridyl)isoxazoles with anitaggregatory activity. This series of compounds was synthesized by the polished scheme from the initial 3-pyridylhydroxymoyl chloride hydrochloride and terminal alkynes with the use of [3+2] cycloaddition reaction, and the structures of compounds were confirmed by NMR spectroscopy 1( H- and 13C-NMR) and mass spectrometry. We have studied the kinetic specificities of human platelet aggregation process induced by arachidonic acid with the use of these compounds as inhibitors, with the determination of IC50 values. These compounds are possible TxA2 receptor antagonists.

Study of influenza A hemagglutinin intramolecular interaction in two-peptide model system using MALDI-MS 1V.V. Egorov, 2Yu.P. Kozmin, 3N.V. Klopov, 4O.A. Mirgorodskaya 1Influenza Institute, St-Petersburg, Russia; Petersburg Nuclear Physics Institute, NRC Kurchatov Institute, Gatchina, Russia; 2M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia; Moscow, Russia, 3Petersburg Nuclear Physics Institute, NRC Kurchatov Institute, Gatchina, Russia; 4Influenza Institute, Saint-Petersburg, Russia Influenza A virus hemagglutinin structural transitions are essential for infection process. Discovering of intramolecular interactions key structural motifs in hemagglutinin is essential for finding of new drug targets for antiviral therapy. In our previous in silico studies we found two distant interacting regions in hemagglutinin. Interaction between these regions causes the C–C bond formation and was significantly different in H5N1 and H1N1 strains hemagglutinins. In this study we use two-peptide model and MALDI-MS for study such an interaction. We studied two peptide pairs with amino acid sequences identical to H5N1 (H5-N and H5-C) and H1N1 (H1-N and H1-C) hemagglutinins interacting regions. The interaction between peptides in pair could be detected by MALDI-MS because of intermolecular disulphide bond formation between (-N) and (-C) peptides was happened in experimental conditions close to physiological. MS/MS analysis showed that the peptides in (-N) - (-C) dimers have a mutual orientation identical to orientation of corresponding sites in hemagglutinin structure. The influence of environment, presence of an oxidants, and Triazavirine drug on such a dimers formation kinetics and structure was studied. The study results can be used for new hemagglutinin-targeted specific antiviral drugs findings. The work was supported by the Russian Foundation for Basic Research (grant no. 13-04-00168).

Plant NT-4/1 protein presumably involved in RNA long-distance transport T.N. Erokhina, S.S. Makarova, A.G. Solovyev, S.Y. Morozov M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia, Belozersky Institute of Physico-Chemical Biology, MSU, Moscow, Russia Arabidopsis thaliana 4/1 protein (At-4/1), initially identified as an interactor of Tomato spotted wilt virus movement protein in yeast two-hybrid system, is a plant-specific protein with no detectable sequence similarity to any known protein. In most plant species 4/1 is a single-copy gene with a characteristic exon-intron organization. Computer predictions indicated that the 4/1 protein is generally alpha-helical and has several coiled-coil domains. Using GFP- fused At-4/1, we demonstrated that the protein is capable of polar localization in plant cells and movement through plasmodesmata, showing therefore the properties consistent with a hypothesized 4/1 role in virus cell-to-cell movement. Further work was focused on the 4/1 protein encoded by Nicotiana tabacum (Nt-4/1). The Nt-4/1-GFP fusion protein was localized to cytoplasmic bodies, which were co-aligned with actin filaments and capable of actin-dependent intracellular movement. The Nt-4/1-GFP bodies, being non-membrane structures, were found in association with the plasma membrane, the tubular endoplasmic reticulum and endosome-like structures. Bimolecular fluorescence complementation experiments and inhibition of nuclear export showed that the Nt-4/1 protein was capable of nuclear- cytoplasmic transport. The Nt-4/1 nuclear export signal (NES) was mapped by site-directed mutagenesis. The Nt-4/1 NES mutant was localized to the nucleoplasm forming spherical bodies. A potential role of 4/1 protein in long-distance vascular transport of potato spindle tuber viroid (PSTVd) was analyzed. PSTVd was inoculated onto N. benthamiana plants where the level of endogenous 4/1 mRNA was down-regulated by virus-induced gene silencing. Despite the fact that accumulation of PSTVd in inoculated leaves of 4/1-silenced plants was significantly lower than in control plants, the level of viroid RNA accumulation in the 4th, 6th and 7th systemic leaves above the PSTVd-inoculated leaf was significantly higher in 4/1-silenced plants compared with control plants. These data point to the involvement of 4/1 protein in viroid transport in the vasculature. To test whether direct 4/1 interaction with viroid RNA could be involved, binding of bac- terially expressed Nt-4/1 protein to different RNA substrates was analyzed. Nt-4/1 exhibited weak binding to ssRNA and much more efficient interaction with viroid RNA. Assuming that the 4/1 protein normally functions in the absence of viroid infection, our data generally suggest a role for the 4/1 protein in RNA translocation in vascular tissue.

Pilot-scale intein-based biotechnologies for production of therapeutic proteins R.S. Esipov, V.N. Stepanenko, M.A. Kostromina, D.A. Makarov, Yu.A. Abramchik, T.I. Muravyova, L.A. Chupova, A.I. Miroshnikov M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Therapeutic recombinant proteins biotechnology is one of the most exciting and progressively burgeoning areas of science and industry in the contemporary world. Innovative medications broad use in humans is conditioned on the detailed studies of their specific pharmacologic activity and safety at the experimental stage. The prerequisite for the comprehensive studies of protein properties and medicobiological tests is the availability of the efficient methods for proteins production. The canonical small proteins production procedure includes their biosynthesis in hybrid proteins and follow-up extraction of the target polypeptide with chemical reagents or site-specific proteases. However, in a number of cases the conventional approach involves considerably high technology costs and makes further scaling up economically unfeasible. The alternative approach suggests the production of hybrid proteins capable of the controlled autocatalytic cleavage with the liberation of the target peptide. This alternative approach provides the following advantages: Ability to produce target proteins with the desired N-terminal amino acid; elimination of the stage of hybrid protein enzymatic hydrolysis by proteases; ability to chemically modify the target protein. Following the multi-year research and development effort focused on the intein-mediated process for recombinant polypeptides production we proceeded with the development of pilot- and industrial-scale biotechnological processes for production of therapeutic proteins. So far we have proved out the industrial-scale production of a number of recombinant pharmaceutical substances, namely: glycagon, oxyntomodulin, epidermal growth factor (EGF), pigment epithelium-derived factor (PEDF) fragment (44-77), tumstatin fragment (L69K-95), thymosin alpha-1 and beta-4, desirudin, lepirudin, purotoxin-1 from Geolycosa sp. and analgesic polypeptide APHC3 from H. crispa.

SPECIAL ISSUE № 1 2014 | Acta naturae | 21 Oral and Poster Presentations

Conjugate of chlorin E6 with a cobalt-bis(dicarbollide) overcomes multidrug resistance of MCF7 cancer cells A.V. Feofanov, A.V. Efremenko, D.E. Ermakova, M.V. Astapova, I.B. Sivaev, M.A. Grin, V.I. Bregadze, A.F. Mironov M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia, Biological Faculty, M.V. Lomonosov Moscow State University, Moscow, Russia, A.N. Nesmeyanov Institute of Organoelement Compounds, RAS, Moscow, Russia, M.V. Lomonosov Moscow State Academy of Fine Chemical Technology, Moscow, Russia

We have developed original conjugates of chlorin e6 with cobalt bis-dicarbollide nanoparticle, which can intensely accumulate in cancer cells and combine properties of highly active photosensitizer having absorption in the near IR region, effective neutronsensitizer and fluorescent contrast agent [1, 2]. We report on the ability of the most active nanoconjugate to overcome multi-drug resistance of the human breast cancer MDR_MCF7 cells and deliver in cytoplasm of 3.3×108 boron atoms/cell, providing the basis for successful boron neutron capture therapy. In addition, the studied nanoconjugate causes photoinduced death of resistant cells (LD50=150–280 nM depending on the light dose), that allows one to recommend it for photodynamic therapy. It is found that photocytotox- icity of the nanoconjugate is due to the generation of singlet oxygen in cells under illumination, which in turn causes the formation of superoxide anion. The analysis of features of intracellular distribution, accumulation and retention of nanoconjugate in resistant cells was performed as compared with the sensitive MCF7 cells. It is shown that the inhibition of P-glycoprotein activity in MDR_MCF7-cells with cyclosporine A leads to four times increase in intracellular accumulation of the nanoconjugate and to recovery of features of its intracellular distribution that are characteristic for sensitive MCF7 cells. This work was funded by the Russian Foundation for Basic Research (10-04-01436, 13-04-00670)

A. V. Efremenko, A. A. Ignatova, M. A. Grin, I. B. Sivaev, A. F. Mironov, V. I. Вregadze and A. V. Feofanov. Photochem. Photobiol. Sci., 2014, 13, 92-102. A.V. Efremenko, A.A. Ignatova, M.A. Grin, A.F. Mironov, V.I. Bregadze, I.B. Sivaev and A.V. Feofanov. In: Current microscopy contributions to advances in science and technology. Ed. A. Méndez-Vilas, Formatex Research Center, 2012, Vol. 1, P. 84-90.

Analysis of transgenic tobacco and duckweed plants containing 5’-end sequence of M2e peptide of avian influenza virus H5N1 in translational fusion with the gene subunit b of ricin castor (Ricinus communis) A.P. Firsov, I.V. Tarasenko, T.Y. Mitiouchkina, N.V. Gilyashova, L.A. Shaloiko, S.V. Dolgov M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia A promising direction in the development of modern biotechnology is the use of plant systems as bioreactor for the production of pharmaceutically relevant proteins including the edible vaccines. The purpose of this study was the cloning and expression analysis in tobacco and duckweed plants of M2e peptide M2 protein of avian influenza virus A/chicken/Kurgan/5/2005 (H5N1). The peptide M2e is the conservative influenza antigen, the possibility of creating on its basis vaccine broad spectrum has been repeatedly confirmed. The sequence of the 5'-end fragment of the gene M2, which includes M2e peptide, was synthesized by ligation of synthetic oligonucleotides with preliminary optimization of codons for expression in plants. We chose the sequence of ricin B subunit (RTB) - lectin from the castor bean (Ricinus communis) as ad- juvant, which can be used as an in the production of «edible» vaccines plant origin. The sequences of ricin B subunit and M130 were cloned in translational fusion based on a binary expression vector pBI121 under the control of the 35S promoter of cauliflower mosaic virus. The resulting plasmid, pBIspRM130, was used for transformation of plants Nicotiana tabacum and Lemna minor. Western blot using anti-RTB, and -M2e antibodies confirmed the presence of the fusion protein RTB-M130 in the three lines of transgenic tobacco plants and 12 transgenic lines of duckweed. The target fusion protein was detected as a single band around the 80 kDa that corresponded to protein expressed as a dimmer. The quantitative asialofetuin-binding ELISA of the crude protein extracts from transgenic duckweed lines confirmed expression of functional RTB-containing fusion protein ranged 1,0–2,2 μg of fusion protein per 1 g FW of duckweed plants. Thus, we first demonstrated the possibility of M2e peptide expression in stably transformed plants. Further we are going to assess the immunogenicity of the obtained fusion protein on the animals. The work was supported by a grant of the Ministry of Education of the Russian Federation №14.B25.310027

Identification of intracellular target of photoactivated fluorescent dye TMR-NN-813 A.A. Generalov1,2, S.Yu. Zaitsev1, E.V. Svirshchevskaya2 1Moscow State Academy of Veterinary Medicine and Biothechnology, Moscow, Russia; 2M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Fluorescent microscopy is a perspective and intensively evolving modern biological method of cell study. A new intensively developing direction is the application of photoactivated dyes which not only selectively label intracellular structures but are masked before photoactivation which gives an opportunity to stain samples using multiple probes with the same range of exitation lengh. In this work we have determined working concentrations and the conditions for photoactivation of a new dye TMR-NN-813, which has been recently synthesized in Max-Planck-Institute (Goettingen, Germany) [1]. The interactions of this precursor with the biomembrane lipids in monolayers at the interface were investigated [2]. The intracellular transport dynamics of TMR-NN-813 o o has been measured in MDCK cells incubated at physiological (37 С, 5% СО2) and stressed (+4 С, no CO2) conditions. A dynamics of TMR-NN-813 intracellular co-localization with different cell organelles (endoplasmic reticulum, lysosomes, Golgi apparatus, mitochondria, nuclei) was determined during 120 minutes. We have demonstrated a selective mitochondria labeling by TMR-NN-813. This work was supported by grant from the Russian Scientific Foundation (project №14-16-00046).

1. S. Yu Zaitsev, M. N. Shaposhnikov, D. O. Solovyeva, A.A. Rizvanov / World Applied Sciences Journal. 2013. - V. 26 (6). - P 712-718. 2. S.Yu. Zaitsev, M.N. Shaposhnikov, D.O. Solovyeva, I.S. Zaitsev, D. Moebius. / Cell Biochemistry and Biophysics, 2013, V. 67, Issue 3, pp. 1365-1370.

Submicron organic-inorganic hybrid particles with PH- and thermosensitive fluorescence A.N. Generalova, V.A. Oleinikov, M.V. Tretyak, Yu.P. Kozmin, V.P. Zubov M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Recently submicron organic-inorganic particles containing polymer matrix and inorganic nanoparticles, particularly luminescent semiconductor nanoc- rystals (QDs), attract considerable attention in biomedical applications. QDs possess excellent photostability, high quantum yield and a narrow emission peak which regardless of the excitation wavelength depends only on the QD size. In the past decades increasing attention has been paid to the preparation of “smart” hybrid particles reversibly responding to slight environmental changes, such as variations of temperature, pH, etc. [1–2]. Organic-inorganic hybrid particles with pH-sensitive fluorescence were prepared by the layer-by-layer deposition technique. Polyacrolein particles were used as cores for deposition of hydrophilic QDs/polyelectrolyte multilayers formed by electrostatic interactions. The outermost layer was essential for application of fluorescent hybrid particles in bioassay. For example, the fluorescence of such hybrid particles with bovine serum album in outer layer was sensitive to copper (II) ion while the fluorescence of these particles was practically insensitive to the other divalent ions. The detection limit of Cu2+ was about 15 nM. Hybrid particles with thermosensitive fluorescence consist of a solid poly(acrolein-co-styrene) core and a poly(N-vinylcaprolactam) (PVCL) shell doped with QDs. The temperature response of fluorescence was determined by the dependence of the QD fluorescence intensity on the distances between

22 | Acta naturae | SPECIAL ISSUE № 1 2014 Oral and Poster Presentations

them in the PVCL shell, which shrinks upon heating above its lower critical solution temperature. The organic-inorganic hybrid thermosensitive particles were assembled with protein molecules in such a way that they retained their thermosensitive properties, including the completely reversible temperature dependence of their fluorescence response. Bioanalytical applications of developed thermosensitive particles were illustrated by examples of their use for real-time remote monitoring the local temperature of a reaction mixture in the course of exothermic chemical reactions: enzymatic hydrolysis of protein and cross-linking of chitosan. The work was supported by RFBR 12-04-00779 and 13-04-00168 .

1. Generalova A.N., Oleinikov V.A., Zarifullina M.M., Lankina E.V., Sizova S.V., Artemyev M.V., Zubov V.P. Optical sensing quantum dot-labeled polyacrolein particles prepared by layer-by-layer deposition technique. - Journal of Colloids and Interfaces Science, 2011, 357(2), 265-272 2. Generalova AN, Oleinikov VA, Sukhanova A, Artemyev MV, Zubov VP, Nabiev I. Quantum dot-containing polymer particles with thermosensitive fluorescence. - Biosens Bioelectron., 2013, 39(1), 187-193.

A new approach to investigation of immunomodulators on condition of adaptive immunity N.M. Gevondyan1, A.I. Alehin2, I.B. Trofimova3, N.A. Didkovsky4, I.K. Malashenkova4, V.S. Gevondyan1 1M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia, 2Institution of Russian Academy of Science Centre Clinical Hospital, Moscow, Russia; 3Moscow State Medico-Stomatologic University, Moscow, Russia; Institute of Physical-Chemical Medicine, RAMS, Moscow, Russia The evaluation possibility research of the impact of weak radiations and low doses of peptide immunomodulators on the state of adaptive immunity, on body functional systems, and clinical manifestations of secondary immunodeficiency syndromes as well is shown. The research is based on changes of G1 antibody avidity, protective activity and quaternary structure. The approach is based on a new conception of single pathogenesis of secondary immunodeficiency due to suppression of defence mechanisms which are dependent on immunoglobulin G1, the leading part in formation of adaptive immunity. The new technologies of diagnostics, therapy, preventive health care, and prediction of secondary immunodeficiency development are developed on the basis of the new conception of single pathogenesis. Monitoring of apparently healthy donors and patients with various diseases associated with secondary immunological insufficiency showed that in health up to 80–100% secreted antibodies possess a high functional activity, i.e., are capable of polyvalent bind to the antigen and to exert a protective effect. On the contrary, in disease, up to 95% of the secreted antibodies have a low functional activity, possessing no protective properties. Comparative studies of functionally active and inactive antibodies made it possible to reveal structural differences in low avid and high avid IgG1, manifesting themselves in the presence of the reaction-capable residues important for functional activity of cysteine in health, and their absence in disease. There was shown G-class immunoglobuline ability to transform from low avid to high avid. There was determined that the transformation: is accompanied by the skipping enhancement of antibodies’ reactivity and connected with changes in the quaternary structure of antibodies. The comparative analysis of various ways of immunotherapy showed that the most effective is treatment by a way of selective impact on B-system of immunity and increase of its protective activity. The data we obtained do alter our concept on the structural organization of the IgG1 molecule and open new prospects for studying molecular mechanisms of functioning of antibodies and their role in formation of secondary immunodeficiencies.

Peptide antibiotic laterocidin: spatial structure and dimerization in membrane-like environment A.K. Gizatullina, Z.O. Shenkarev, A.S. Paramonov, M.Yu. Myshkin, Z.A. Yakimenko, E.I. Finkina, T.V. Ovchinnikova, A.S. Arseniev M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Laterocidin is a membrane-active peptide antibiotic isolated from gram-positive bacteria Brevibacillus laterosporus. Laterocidin belongs to the family of Tyrocidine antibiotics, which involves cyclic decapeptides produced by non-ribosomal biosynthesis (Tyrocidine, Gramicidin S, etc.). Spatial structure of laterocidin in methanol solution and SDS micelles was studied by NMR spectroscopy. In methanol solution (an isotropic membrane mimetic) laterocidin forms amphipathic antiparallel β-sheet is stabilized by four hydrogen bonds. Hydrophobic side-chains (Leu3, Val4, Leu6, Phe9, D-Phe10) are situated on the convex side of the β-sheet; the charged residues (Asp2; Orn5) and free carbonyls are located on the concave side. In the environment of SDS micelles (an anisotropic membrane mimetic) at low detergent concentration (detergent/peptide ratio ~15:1) laterocidin forms symmetric β-structural dimers. The increase in detergent concentration (detergent/peptide ratio ~60:1) leads to dissociation of the dimers in to the monomers. The spatial structure of the peptide undergoes only minor changes upon transfer form methanol to SDS micelles and dimerization. Obtained structural data are consistent with the “carpet-like” mode of the membrane-lytic activity of laterocidin.

Recoverin as a paraneoplastic antigen M.O. Golovastova, L.V. Tsoy, D.O. Korolev A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Moscow, Russia Paraneoplastic (onconeural) antigens are the proteins normally specific for the nervous system but it can also be aberrantly expressed in malignant tumors localized outside the immunoprivilege zone. A paraneoplastic antigen recoverin is the first retina-specific protein the aberrant expression of which has been detected in malignant tumors. In this study, we have carried out immunohistochemical investigation of renal tumors for the presence of recoverin expression and analyzed serial specimens of blood sera of patients with these tumors for the presence of autoantibodies against recoverin (AAR). In addition, we have determined the methylation status of the recoverin gene, including the region of the gene promoter up-stream of the first exon and the first exon itself, in renal tumors. We have carried out immunohistochemical investigation of paraffin sections of 49 renal tumors: cell carcinoma, oncocytoma, papillary adenocarcinoma, and chromophobe adenocarcinoma. The frequencies vary in the range between 50% (renal chromophobe adenocarcinoma) and ~92% (renal oncocytoma). Also, we have examined 42 serum samples from patients with renal cancer using immunoblotting with recombinant recoverin as an antigen. Autoantibod- ies against recoverin were detected merely in sera from one patient with papillary adenocarcinoma; while no recoverin-positive sera were found in control patients with urolithiasis. In addition, methylation analysis by sodium bisulphite sequencing was used to determine the level of DNA methylation in the recoverin gene in renal tumors. It has been demonstrated that demethylation of the certain CpG-dinucleotides in the recoverin gene is involved in the aberrant expression of this protein in renal tumor cells.

SPECIAL ISSUE № 1 2014 | Acta naturae | 23 Oral and Poster Presentations

Analysis of autocrine factors of cytotoxic T lymphocytes M.V. Grechikhina, G.V. Lutsenko M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Cell survival and energy metabolism in multicellular organisms are controlled by a great number of different cytokines, including autocrine factors produced by the cells of the same type. Previously we have shown that autocrine factors control survival of T lymphocytes through the regulation of ATP level in cells. The temporary drop of intracellular ATP content has been shown under the artificial autocrine factor deficit in the culture. ATP decrease in the cells led to strengthening of the cell stress (oxidative, hypothermic and alkalosis, pH 8.3) and increasing of the part of dead cells. In this study two autocrine factors of cytotoxic CTLL-2 cell line have described and analysis of their structural and functional distinctions has been carried out. Gel-filtration of CTLL-2 cell line condition medium was carried out on the column with “Bio-gel P-4” as a carrier for characterization of CTLL-2 autocrine factors. Two fractions (A and B) corresponding to distinct peaks on the chromatogram were obtained as a result of gel-filtration. Functional activities of obtained fractions were determined as their ability to stimulate ATP synthesis under chemical hypoxia conditions (with addition 10 mM NaN3 or 200 4 mcM CoCl2) or to promote cell survival in the absence of serum under low density culture conditions (2×10 cells/ml) for several days. The fraction A was shown to stimulate ATP synthesis under chemical hypoxia conditions but no to promote cell survival in the absence of serum into low density culture. On the contrary, the fraction B supported cell survival but no influenced on ATP intracellular level decreased under chemical hypoxia. With using of MALDI mass-spectrometry molecular mass of peptide from B fraction has been determined as 1157 Da. Further investigation has shown molecules of A fraction to be complexes of peptide 1157 Da with different quantity of lactate molecules. Thus, two autocrine factors of CTLL-2 cells were found out: one of them has properties of typical survival factor and is peptide with molecular mass of 1157 Da, the second autocrine factor has ability to regulate energy metabolism of cells and is the complex of peptide 1157 Da with lactate molecules.

Synthesis of bodipy labelled nootropic peptides PGP, HFPGP and MEHFPGP and their transport to pheochromocytoma cell line PC12 N.M. Gretskaya, M.G. Akimov, A.V. Lavrova*, V.V. Bezuglov M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia,*M.V. Lomonosov Moscow State University of Fine Chemical Technologies, Moscow, Russia Heptapeptide semax (MEHFPGP) is a drug for a broad range of disorders. It improves adaptation of organism to hypoxia, stroke and other damaging actions. Peptides HFPGP and PGP are the products of semax metabolism with a broad spectrum of biological activity. Recently the existence of specific binding sites for these peptides on plasma membranes from different parts of brain was shown. Altogether, these peptides are the part of the functional significant complex – synacton. Fluorescent labelled analogues of peptides were synthesized to investigate transport of these peptides to the cells. We proposed new fluorescent analogues of nootropic peptides PGP, HFPGP and MEHFPGP with various position of BODIPY probe at N- and C- terminus of peptide chain. BODIPY group was attached to the N-terminus of peptides by mixed anhydride technique. Before coupling peptides were treated with silylation reagent BSTFA to significantly improve yield of reaction. To attach BODIPY-label to the C-terminus we used ВОС-protected PGP bearing EDBE group (2,2'-(ethylendioxy)-bis(ethylendiamine) as a linker. Transport of new synthesized fluorescent peptides on rat pheochromocytoma cell line PC12 was investigated. It was shown that these cells accumulated BODIPY-PGP, and this uptake was bidirectional, saturable, time- and temperature-dependent process and was blocked with unlabelled PGP. Maximal 5 uptake of BODIPY-PGP was 27±2 fmol/cell, τ1/2=13±4 min, Vmax 14±2 pmol/min/10 cells, Км 0.8±0.5 μМ. Kinetic parameters of the BODIPY-PGP uptake mismatch to standard peptide transporter PEPT1 and PEPT2, but are similar to those of organic anion transporter (OAT1-4), whose capability to carry at least opioid peptide was demonstrated. The work was partially supported by the Russian Foundation for Basic Research, grant No 13-04-40085-Н.

Characterisation of M. Tuberculosis transcriptome in dormancy d. Ignatov1, E. Salina2, A. Kaprelyants2, T. Azhikina1 1M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia; 2A.N. Bach Institute of Biochemistry RAS, Moscow, Russia Mycobacterium tuberculosis can persist in the human host for decades after infection. During such latent infection the pathogen enters the dormant state, which is supposedly characterized by low level of metabolism and loss of culturability. An experimental model of dormancy involves cultivation of M. tuberculosis in a special K+ depleted broth, followed by addition of rifampicin in inhibitory concentration. In these conditions only dormant non-dividing cells survive. The advantage of this model is that the dormant cells are in “non-culturable” (NC) state and cannot grow on conventional broths. We used RNA-seq to study transcriptome of M. tuberculosis during growth in the K+ depleted broth: on logarithmic growth phase (“Log”) and stationary growth phase immediately before addition of rifampicin (“Stat”) ; and dormant cells: 10 days after addition of rifampicin (“Dorm_early”), and 30 days after addition of rifampicin (“Dorm_late”). We found that mRNA content per cell was 50–100 times lower in “Dorm” cells than in “Log” and “Stat” cells. This supports the hypothesis of low metabolic activity of dormant cells. M. tuberculosis transcriptome showed significant changes during cultivation in the K+ depleted broth: expression of genes coding F0F1 ATPase and NADH dehydrogenase subunits was down regulated, which indicates slowing of aerobic metabolism, and expression of PE-PGRS proteins was up regulated. PE-PGRS proteins are implicated in pathogenesis, but their function is yet unknown. The most significant change in transcriptome during transition from “Stat” to “Dorm_early” was down regulation of genes coding ribosomal proteins, which may indicate cessation of growth and protein sysnthesis. Interestingly, there were no changes in transcriptome between “Dorm_early” and “Dorm_late”. This indicates constant metabolic state of M. tuberculosis in dormancy. One of the main signatures of dormant cells was found to be cleavage of 23S rRNA. This cleavage occurs at position 592 (AAAG↓AGTA), and probably is the result of action of one of several dozen toxin-antitoxin systems of M. tuberculosis. The work was supported by the Program of the Presidium of the Russian Academy of Sciences ‘Molecular and Cellular Biology’ and Russian Foundation of Basic Research grants 13-04-40071-Н and 13-04-40072-Н.

Structure-functional organization of the glyprolin molecules l.I. Ismailova, R.M. Abbasli, N.A. Akhmedov Baku State University, Institute for Physical Problems, Baku, Azerbaijan One of the basic problems for molecular biophysics is investigating their structure–functional organization. Computer modeling helps to solve this problem. Peptides regulate all functions of a living organism. Using the regulatory peptides of the human body, you can create new and effective drugs. This work is devoted to study of the spatial organization, conformational possibilities of the glyproline molecules for the first time. Glyprolines are a new family of biologically active peptide drugs containing Gly (G) and Pro (P) amino asides in their structure. Glyprolines are fragments of collagens. These molecules modulate the nervous and immune system, possess antiulcer action. At present, the various synthetic analogues of natural glyprolines were founded simax (Met1-Glu2-His3-Phe4-Pro5-Gly6-Pro7) and selank (Thr-Lys-Pro-Arg-Pro-Gly-Pro).

Glyproline peptide family includes the simplest proline-containing linear peptides G-P, (G-P)2, (G-P)3, P-G, (P-G)2, (P-G)3, P-G-P, (P-G-P)2, semax( MEHFPGP) and silank (TKPRPGP). The biological functions of these peptides in living systems are related with their specific spatial structures.

24 | Acta naturae | SPECIAL ISSUE № 1 2014 Oral and Poster Presentations

To understand the mechanism by which the glyprolines function, it is necessary to know their spatial structures and the full complement of low-energy conformational states. The calculations were carried out by the method of theoretical conformational analysis and a special computer program. The potential energy of the each molecule was chosen as the sum of the non-valent, electrostatic and torsional interaction energies and the energy of hydrogen bonds. The low- energy conformations of this molecule, the dihedral angles of the backbone and side chains of the amino acid residues of each peptide, and the energies оf inter-residual interactions were determined. It is revealed that low energy conformations of these molecules have the folded type of backbone. Basing the calculation results the conformational mobility of the amino acid side chains are investigated and the amino acids with specific interplays with different receptors are founded. Comparing the results obtained, we can say that with repeated glyprolines fragments Gly-Pro essential for cytoprotective activity is the C-terminal dipeptide fragment Gly-Pro. This C-terminal dipeptide fragment Gly-Pro is present in all of the studed molecules. According to experimental data, the presence of this fragment is responsible for the protective action meet the following molecules per cell. The low-energy conformations of the semax and silank molecules were used as the initial structural states to explore the conformational possibilities of the artificial analogues.

Deceleration of glutamate-induced neuronal death under the influence of oligopeptide fragment from imidazoline receptor V.P. Ivanova I.M. Sechenov Institute of Evolutionary Physiology and Biochemistry, RAS, St. Petersburg, Russia In this work we studied the capacity of oligopeptide fragment from Imidazoline receptor to prevent glutamate (Glu)-induced neuronal damage. Primary cultures of neuronal cells prepared from cerebral cortex of 16-day-old rat embryos were used in the experiments after 7–10 days in culture. Neuronal cells were pretreated with the peptide in doses of 10-10–10-7 M for 30 min at 37°C. Then cells were exposed to Glu (1 mM) for 3–6 h at room temperature. In control primary cultures two groups of neuronal cells were observed to the 7 day of cultivation. The first one comprised differentiated cells having pyramidal or more elongated shape with numerous outgrowths and forming submonolayer. The second group included cell conglomerations with various cell numbers. Large conglomerations differed from small ones by the presence of dark membrane-like layer on the periphery of sphere. At the 3–4 day of cultivation cells mi- grated from conglomerations adhered to substrate and began to proliferate and differentiate. The maximum number of differentiated neuronal cells forming a dense monolayer was observed around the cell conglomerations. Differentiated neuronal cells were more resistant to toxic Glu dose then undifferentiated ones from conglomerations. Thus, the number of dead cells, in the first case, was nearly 50% and, in the second case – 80%. The pretreatment of neuronal cells with the peptide significantly diminished Glu-induced cell death. The rate of cell death was decelerated, especially, in the first 15 h exposure of cells to Glu. The results indicate that the peptide promotes the survival of Glu-treated cortical neurons. Perhaps, the peptide revealed effect may be associated with the partial allosteric inhibition of definite active sites in Glu (probably, NMDA-type) receptors due to conformation alterations under the peptide action in ecto- and/or transmembrane domains of receptor molecules restricting the “shock” opening of Ca2+-channels caused by the excitement of Glu receptors.

Peptidomic and proteomic analysis of cerebrospinal fluid of patients with Guillain–Barre syndrome O.M. Ivanova, R.H. Ziganshin, G.P. Arapidi, I.V. Azarkin, S.I. Kovalchuk, V.M. Govorun M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Guillain–Barre syndrome (GBS) is a very rare but highly dangerous immune-mediated inflammatory polyneuropathy disease with the most accepted mechanism involving autoantibody reaction against Schwann's sheath of the peripheral nerves leading to demyelinisation and damaging nerve signal transmission, in some cases involving respiratory muscles and autonomous nervous system. It was shown that autoantibody reaction might evolve as a side reaction of immune system response to bacteria or virus infection, when pathogen antigens mimic host Schwann cell membrane components, however in many cases exciting cause remains unknown. There are still a lot of questions concerning molecular mechanism of this disease and further studies are needed. Although GBS involves peripheral nerves only it is also known to be accompanied by increase in protein concentration in cerebrospinal fluid (CSF). To understand the composition change of peptidome and proteome we compared CSF samples from GBS patients and the control group of patients with non-neurological diseases. Endogenous peptides as well as tryptic protein fragments were analyzed by LC-MS/MS. Totally in all CSF samples 2562 unique endogenous peptides – fragments of 742 proteins –were identified. Among analyzed patient groups those identifications were distributed as follows: GBS patients – 2397 peptide fragments of 742 proteins; patients with non-neurological diseases – 689 peptide fragments of 182 proteins. Thus, in CSF of patients with GBS we found fragments of membrane proteins of Schwann cells. Proteome studies resulted in identification of 1541 unique proteins, 1050 of them in GBS samples and 1398 in CSF samples of control group. We analyzed the lists of the identified proteins from CSF of GBS patients and control by the STRING online service using the KEGG, GO cell component, and GO biological processes databases and reliably revealed indicative clusters of the interacting proteins.

Heterologous production of human lysyl oxidase homolog from Archaebacteria Haloterrigena turkmenica D.V. Kalinovsky, N.B. Pestov M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Lysyl oxidase (ЕС 1.4.3.13) is a copper-containing amine oxidase which catalyzes the oxidative deamination of ε-amino groups of lysine in collagen and elastin fibers. The subsequent condensation of modified lysine generates cross-linked fibrils in mature collagen and elastin. All members of the lysyl oxidase (LOX) family are characterized by a conservative amino acid sequence of the catalytic domain and display similar substrate specificities. Different types of cells secrete these proteins into the extracellular space where they participate in the post-translational modification of collagen and elastin thereby forming the structure of extracellular matrix and regulating cell behavior. Most of the animal genomes contain from one to five LOX genes, whereas plants and fungi lack LOX. It is also absent from the majority of prokaryote genomes, but the presence of LOX homologs in several species of bacteria makes it a very interesting object to study. It is obvious that the LOX gene has undergone repeated horizontal gene transfers and now it is often present in actinomycetes, occasionally present in other representatives of the Eubacteria domain and scarce in Archaea. This aspect is interesting not only from the phylogenetic point of view but also for possible biotechnological applications because catalytic properties of bacterial and archeal LOXes may be quite different from those of the human LOX genes. For this reason we have amplified and cloned a LOX gene from a halophile Haloterrigena turkmenica, the only arсheal species known to contain LOX. Subsequently, we successfully produced recombinant Haloterrigena turkmenica LOX and started to analyze its catalytic properties. Because H. turkmenica LOX is structurally rather diverged from the animal LOX, we expect a significantly different spectrum of substrate specificity.

SPECIAL ISSUE № 1 2014 | Acta naturae | 25 Oral and Poster Presentations

Methods to enhance the degradation of organic waste and mitigate the emission of methane at landfills A.Yu. Kallistova1, M.V. Kevbrina2, V.K. Nekrasova1, A.S. Savvichev1, A.N. Nozhevnikova1 1Winogradsky Institute of Microbiology, RAS, Moscow, Russia; 2OJSC Mosvodokanal, Moscow, Russia In Russia, landfilling is the only way of the municipal solid waste processing. Landfills are harmful objects which pollute the environment. A major part of the organic waste is slowly degraded at landfills by anaerobic methanogenic microbial community with the production of biogas (mainly a mixture of green- house gases – methane and carbon dioxide). The activity of microorganisms that cause the biogas production is a natural process. Attempts to artificially suppress this process can only lead to an increase of the duration of waste degradation. The promising method to enhance the degradation of organic waste and to accelerate the entry of landfills areas for recultivation is the in situ activation of anaerobic methanogenic microbial community. We showed in laboratory and field experiments a stimulating effect of digested sludge into the decomposition of organic waste and rate of methanogenesis. The intensification of methanogenesis at landfills equipped with biogas collection systems lead to an increase of methane release that represents an economic advantage. However the biogas is not extracted from landfills in Russia, therefore the development of «old» landfills areas for household needs is extremely difficult. Methane can penetrate into basements of buildings and underground engineering systems and create a fire and explosive situations. The promising method to mitigate methane emission from landfills is the activation of aerobic methanotrophic biofilter in landfill cover soil. The introduction of consortium of psychrotolerant methanotrophic bacteria oxidizing methane at low temperatures (15oC) into the landfill cover soil was carried out in field experiments at biggest landfill in Moscow province. The results demonstrated the ability of consortium to survive in the cover soil and cause a reduction of methane emission. This method could be used at «old» landfills when the cover soil layer is locally interrupted by its recultivation, as well as at the sites with a thin cover soil layer. The activation of methanotrophic bacteria in landfill cover soil can also be achieved by improving of soil aeration, loosening and planting.

Activation of human NK cells with Lipopolysaccharides L.M. Kanevskiy, M.A. Streltsova, S.A. Erokhina, E.I. Kovalenko M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Natural killer (NK) cells have been shown to play a regulatory role in sepsis. According to the current view, NK cells become activated via macrophages or dendritic cells primed by lipopolysaccharides (LPS). Recently, TLR4 gene expression was detected in human NK cells suggesting the possibility of a direct action of LPS on NK cells. In this study, effects of LPS on NK cell cytokine production and cytotoxicity were studied using highly purified human NK cells. NK cells were collected from peripheral blood of healthy donors by magnetic separation. CD3/CD14/CD19-negative, CD56-positive phenotype of the NK cells was confirmed by multicolor flow cytometry. In some experiments CD56brightCD57neg, CD56dimCD57neg, CD56dimCD57dim and CD56dimCD57bright NK cell subpopulations were then isolated by fluorescence-activated cell sorting using FACSVantage-DiVa instrument. The cells were incubated in the presence of IL-2 (500 U/ml) and LPS (5 μg/ml) for 18 h. IFN-γ content in culture supernatants was analyzed by ELISA. TLR4 expression was measured by surface and intracellular fluorescent immunostaining. NK cell cytolytic potential was assessed with a LAMP1-based degranulation assay. Isolated NK cells subpopulations differed in IFN-γ production and cytotoxicity: the maximal levels of IFN-γ production were detected in CD56bright- CD57neg or CD56dimCD57neg NK cells; the maximal cytotoxic activities in different donors were registered in CD56brightCD57neg, CD56dimCD57neg or CD- 56dimCD57dim subpopulations. LPS in combination with IL-2 was shown to increase IFN-γ production in CD56brightCD57neg and CD56dimCD57neg cells but not in CD56dimCD57dim and CD56dimCD57bright subpopulations. In the same time, in different donors LPS affected differently on NK cell cytotoxic activity in CD57neg NK cells. Both positive and negative effects of LPS on NK cell degranulation were registered. No LPS influence on NK cell cytotoxicity was revealed in CD56dimCD57dim and CD56dimCD57bright subpopulations. We did not detect any significant surface TLR4 expression in NK cells during 24 h following IL-2 stimulation whereas intracellular TLR4 amount increased gradually for 48 h after the stimulation. Thus, LPS modulate functional activity of the less mature NK cells. The results suggest the existence of mechanisms of LPS direct action on NK cells distinct from established surface TLR4-mediated signaling. Different LPS effects may be connected with differences of initial functional status of NK cells between individuals. This work is supported by RFBR (grants #13-04-02041 and #14-04-01842).

Highly efficient fluoropolymer- and polyaniline-containing composites for bioseparation, bioanalysis and medical diagnostics D.V. Kapustin, A.I. Prostyakova, V.P. Zubov M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia The most of modern methods of biopolymers isolation and separation are based on multistep procedure, when a mixture containing biopolymer (e.g. nucleic acids or proteins) contacts with the special adsorbent on the first step, then admixtures are removed with suitable buffer solution and finally the purified substance is eluted from the adsorbent surface. Multistepness of the procedure results in significant loss of the target substance, and such methods are difficultly to automate. The alternative conception, when the target biopolymer passes through the layer of polymer-coated composite in excluded volume while all admixtures are retained by the adsorbent, seems to be more effective. At least, two types of synthetic polymers demonstrate such chromatographic behavior, i.e. fluoropolymers and polyanilines. Those polymers themselves are not “good” as materials for adsorbents due to their poor physical-chemical properties. However, the formation of nano-layers of such polymers on the solid supports provides the obtaining of useful composites combining the defined mechanic properties and required sorption properties in the same material. We developed a number of techniques based on direct synthesis of the composites modified with fluoropolymers and polyanilines (including chemical activation of the support surface with polysulfonic acids or hydrophobic polymers, preliminary treatment of the support with ozone1 with following polym- erization of monomers from liquid or gas phase, etc). Developed materials were shown to be effective for one-step isolation of DNA from bacterial lysates, lysates of plant tissues, fungi and blood, in particular, for clinical PCR diagnostics of viral (including viral hepatitides HBV and TTV)2, different bacterial (including Mycobacterium tuberculosis complex) and mycotic human pathogens. Unique properties of the used immobilized polymer coatings allowed us to extend the field of their application, e.g. for separation ofss - and dsDNA3, for use as supports for solid-phase synthesis of oligonucleotides or supports for SELDI-TOF MS4 (without addition of energy absorber) as well as for isolation of water- and liposoluble vitamins from blood.

1. D.V. Kapustin, A.I. Prostyakova, D.Yu. Ryazantcev, V.P. Zubov, Nanomedicine, 2011, 6(2), 241-255. 2. Kapustin DV, Prostyakova AI, Zubov VP. Bioanalysis 6(7), 957–966 (2014). 3. Kapustin D., Prostyakova A., Bryk Ya., Yagudaeva E., Zubov V. In: Nanocomposites and polymers with analytical methods / Cuppoletti J. (Ed.) – Croatia: Intech, 2011. ISBN: 978-953-307-352-1, p.p. 83-106. 4. Vaczine-Shlosser G., Ribbing C., Bachman P.K., Zubov V.P., Kapustin D.V., Patent WO 2011004308 (A1).

26 | Acta naturae | SPECIAL ISSUE № 1 2014 Oral and Poster Presentations

Safe anti-allergic vaccines incorporating T-and B-cell epitopes of allerges, based on polymer nanoparticles E.I. Kashirina, P.D. Rechetov, D.Yu. Ryazantsev, L.G. Alekseyevа, E.V. Svirshchevskaya M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia The only pathogenetic treatment of allergy is allergen specific immunotherapy (ASIT), which consists of a prolonged subcutaneous course of allergen extract injections. However subcutaneous injections of full-length allergens activate the mast cell degranulation, which invariably entail the release of inflammatory mediators able to induce side effects including anaphylactic shock which can be fatal in some cases. The aim of this work was the development of a safe form of allergens for ASIT. In this study we developed a new approach in which full-size recombinant allergen proteins were packed into safe, biodegradable, biocompatible polymer nanoparticles (NPs). Packaging of allergens prevents their binding to the IgE antibodies however retains immunogenic properties required to gain the vaccination effect. Recombinant allergen of house dust mite proteins (HDM) Der f1 and Der f2 were packed into NPs developed from lauryl succinoyl chitosan (LSC) and alginate (ALG). LSC derivative was obtained from 40 kDa chitosan by carboxyacilation using anhydride method; lauric acid residues were introduced by the method of activated esters. NPs were formed by self-assembly. The immobilization of Der f1 and Der f2 on the NPs was fulfilled by carbodiimide method using the reaction of nucleophilic addition. The resulting LSC-NPs were coated by alginate membrane with the immobilized synthetic peptides Der f1 and Der f2. The resulting NPs were of 200–250 nm in size and +15 mV zeta- potential. Earlier we demonstrated that such NPs did not bind IgE from the sera of HDM patients. To study the immunogenic properties of the constructs mice were immunized either with pure Der f1, Der f2 proteins, LSC-Der-f-NPs, or with LSC-Der-f-ALG-peptides NPs. We demonstrated that the capsulation of full length allergens did not induce IgG formation while both pure allergens and LSC-Der-f-ALG-peptides NPs were effective in the induction of IgG specific to Der f proteins. Thus, capsulation of allergens into polymeric NPs completely protected the allergen from the contact with B-cells able to recognize allergens. The exposure of peptides from the allergen on the surfaces of NPs was effective in IgG induction while safe as the peptides do not trigger mast cell degranulation. Taken collectively we developed a new type of safe immunogenic vaccines for ASIT of HDM allergy.

The transcriptome analysis of the moss Physcomitrella patens R.A. Khazigaleeva, I.A. Fesenko, E.S. Kostrukova, T.A. Semashko, G.P. Arapidi, K.A. Babalyan, V.M. Govorun M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Increasing the power of genomic tools allows us to investigate changes in the expression of thousands of genes and to determine the transcriptional profile for the consistent development of the plant. Comparative analysis of transcription can be used for gene expression studies, gene function, biological pathways, and to identify functional homologues evolution transcriptome. As a model for the identification of regulatory genes in the development of moss we chose Physcomitrella patens. In order to characterize changes in gene expression during the development of dynamic Physcomitrella patens, we conducted transcriptional cell mRNA samples protonemal, gametophores and protoplasts. According annotations P. patens moss 32278 contains the coding portion (CDS) of which 17816 has been found-ti expression (more than one per million of chloride). We conducted a comparative analysis of differentially expressed genes in the two life forms and gametophores protonemal and stress protonemal and protoplasts. A comparative analysis of two life forms and protonemal gametophores was discovered 3170 DE genes, 1551 of which the expression was increased and decreased in 1619. When allocating protoplast cells moss P. patens experience severe stress associated with exposure to the enzyme preparation drayzelaza and as a result of the loss of the cell wall. Comparative analysis protonemal and protoplasts, we identified 1788 DE genes whose expression DE 1376 increased in protoplasts. Analysis of Gene Ontology (GO) terms showed that in protoplasts the transcriptional level of genes involved in responses to different stress factors increased, e.g. the response to abiotic stimulus (GO:0009628), response to cold (GO:0009409), response to temperature stimulus (GO:0009266), response to oxidative stress (GO:0006979; Supplemental Data Set 11 online). Severe stress in protoplasts could also be detected by the increased expression of genes participating in protection from reactive oxygen species (ROS), such as Pp1s152_86V6, putative ascorbate peroxidase 1. Besides the reactions to abiotic stress, which may be associated with the change in cellular form, we also observed increased expression of genes that re- spond to biotic stress result point to the complex nature of stress experienced by cells during protoplast isolation. We identified several genes that participate in jasmonic acid (JA) biosynthesis. JA is one of the stress hormones that coordinates plants’ responses to stresses such as wounding, pathogen attack.

The new model of conformational changes in the operation of ATP synthase E.A. Kasumov, R.E. Kasumov, I.V. Kasumova Research and Production Centre «KORVET», Domodedovo, Moscow region, Russia ATP is one of the main intermediates of chemical energy in the living organisms. It is mainly synthesized in ATP synthases by utilizing energy equal to energy of one foton either from oxidation of organic substances or from light via the process of oxidative- or photo-phosphorylation in energy transducing membranes of mitochondria, chloroplasts and bacteria. Functional activity of mitochondria in the cell is fully regulated by complex mixture of dissolved in water substances of the cytosol compound. The concentrations of these substances are different, which are important in processes such as osmotic regulation and signaling both of cell and of mitochondria for ATP synthesis. The structure of ATP synthase subunits undergoes several changes during their functioning. The details of the molecular mechanism of ATP synthesis are considered in the cyclic shrinkage-swelling of organelles. On the basis of structural and other data it is proved that ATP synthase is a pump(Ca2+/K+, Cl- ) pore – enzyme complex, in which γ-subunit rotates to 360o step by step 30о (30о, 90o rotations of the γ-subunit are associated with opening and closing of gate in channels – α-helical bundles of the α and β-subunits (DELSEED bead) both at synthesis, and at hydrolysis) owing to phosphorylation of positive charged amino acid residues of the N-terminal γ-subunit in electric field. The coiled-coil b2 subunits are the ropes shortening by phosphorylation of positive charged lysines or arginines and attract the α3β3-hexamer to the membrane in energization. ATP is synthesized during of γ-subunit rotation a back by the 2+ destabilization of the phosphorylated b2 subunits and C- and N-terminal positive residues of γ-subunit under action of Ca ions, which pumped over from storage – thylakoid (or intermembrane space) in swelling. So, main energy is consumed for the synthesis of ATP: for delivery of three phosphate ions and three protons into the hexamer with the help C-terminal α-helix of γ-subunit as on a lift against the energy barrier, a formation of phosphoryl groups, loading of three ADP molecules into hexamer and the release of three ATP molecules, synthesized from the hexamer.

The influence of apomyoglobin mutant forms stability on the propensity to form amyloids N.S. Katina, V.A. Balobanov, S.E. Permyakov, A.A. Vazina, A.D. Nikulin, V.D. Vasiliev, I.A. Kashparov, N.B. Ilyina, V.E. Bychkova Institute of Protein Research, RAS, Pushchino, Moscow Region, Russia Amyloid fibrils formation in organs and tissues causes serious human diseases, therefore investigation of the mechanism of protein aggregation is one of actual problems of medicine and biophysics. It was shown previously, that destabilization of protein native state is required for amyloid formation. This finding has led to an extensive investigation of amyloid formation under conditions of strong protein denaturation. However, the mechanism of amyloid formation under physiological condition remains poorly understood by now. Therefore the aim of our work was to study amyloid formation by apomyoglobin and its mutant forms under conditions, near to physiological ones (temperature 40°C, pH 5.5), where proteins retain their native properties.

SPECIAL ISSUE № 1 2014 | Acta naturae | 27 Oral and Poster Presentations

The structural properties of aggregates, formed by apomyoglobin mutant forms, have been studied with use of infrared spectroscopy, electron microscopy, X-ray diffraction and native electrophoresis. The protein stability has been estimated from pH-induced equilibrium unfolding data monitored by tryptophan fluorescence and far ultraviolet circular dichroism. It was shown that there is optimum of protein destabilization, where pronounced amyloid formation is observed. At the same time stable or strongly destabilized mutant proteins form insignificant amount of fibrillar aggregates. This work was supported by Programs “Molecular and Cellular Biology” of the Russian Academy of Sciences, “Fundamental Sciences – medicine” and INTAS.

An increase in organ-specific auxin activity enhances the tuberization of potato plants in vitro O.O. Kolachevskaya, V.V. Alekseeva, L.I. Sergeeva, E.B. Rukavtsova, I.A. Getman, Ya.I. Buryanov, G.A. Romanov K.A. Timiryazev Institute of Plant Physiology, RAS, Moscow, Russia; Branch of M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Pushchino, Moscow Region, Russia Phytohormones, auxins in particular, play an important role in plant growth and development, as well as plant productivity. Earlier studies in the laboratory of signaling systems of ontogenesis control at the Institute of Plant Physiology, RAS have shown that the auxin treatment of underground parts of potato plants accelerated tuberization and increased tuber biomass (Aksenova et al., 2000; Romanov et al., 2000). The next stage of this research was to create transgenic plants with increased levels of endogenous auxin in organs intended for reproductive development: stolons and tubers. To achieve this goal we have constructed the transforming binary vector pBinB33-tms1, harboring agrobacterial auxin biosynthesis gene tms1 encoding tryptophan-2-mo- nooxygenase, coupled with the promoter of the class I patatin gene (B33-promoter) of potato. More than 20 independent lines of transgenic potato plants (Solanum tuberosum L.) cv Desirée were obtained by Agrobacterium-mediated transformation. For this study B33:tms1-transformants were chosen which were not visibly different from control plants as regards phenotype parameters of vegetative growth. In the transformants, we investigated the consequences of transgene integration into the genome, including the tms1 gene expression pattern, auxin content and tuberization characteristics in different conditions of the in vitro cultivation. The level of the tms1 gene transcription in the transformants was as a rule significantly higher in tubers than in shoots, that was in accordance with the B33 promoter specificity. The IAA content in tubers and the tuber:shoot ratio of auxin content were also increased in the majority of transgenic lines as compared to non-transgenic ones. The organ-specific increase in auxin synthesis inB33:tms1 -transformants accelerated and intensified the process of tuber formation, reduced the threshold of carbohydrate supply required for tuberization and it dependence on the photoperiod. The revealed properties of B33:tms1-transformants indicate the important role of auxin in potato tuberization. At the same time this new biotechnological approach offers the perspective to increase the potato productivity by a moderate organ-specific enhancement of auxin level.

Characterization of intestinal amino oxidases of siberian sturgeon (Acipenser baeri) T.V. Korneenko, N.B. Pestov, M.I. Shakhparonov M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Biochemical processes underlying adaptation of aquaculturally important fish species to environmental pollution remain largely unknown. An important role is played by components of intestinal mucosa protective barrier, including amino oxidases that catalyze oxidative deamination of primary amines into corresponding aldehydes. Here we demonstrated that the gastrointestinal tract of Siberian sturgeon (Acipenser baeri) contains high levels of several amino oxidases, identified as Cu-dependent diamino oxidase (DAO) and mitochondrial monoamine oxidase (MAO). Tissues were homogenized in a sulfobetaine stabilizing solution and various amino oxidases were assayed fluorometrically using peroxidase-coupled reaction with Amplex Red. DAO is enriched in proximal intestine being especially abundant in pyloric gland and absent from stomach whereas monoamine oxidase has a more uniform distribution. Sensitivities to inhibitors such as berenil and aminoguanidine, as well pH profile, of the sturgeon intestinal DAO are quite similar to that of DAO from mammalian placenta (AOC1). Activities that may be attributed to other Cu-dependent amino oxidases (other AOC isoforms and lysyl oxidases) can be detected only at trace levels. Moreover, it was observed that sturgeon notochord contains inhibitors of lysyl oxidase. Intestinal diamino oxidase of the sturgeon is especially abundant in pyloric gland and its sensitivities to inhibitors as well pH profile resemble that of the mammalian placenta, which primarily corresponds to AOC1/DAO/histaminase. Also, D-amino acid oxidase is readily detectable and is enriched in the spiral valve of sturgeon colon.

Biotechnological production of pharmaceutical antithrombotic substances – lepirudin and desirudin M.A. Kostromina, R.S. Esipov M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia The introduction of direct antithrombotic agents, such as inhibitors of thrombin and factor Xa, in medical practice of treatment of venous thromboembolism is necessitated by the high risk of side effects associated with the use of heparin-based drugs in standard therapy. Desirudin and lepirudin, derivatives of the natural thrombin inhibitor hirudin-1 from the medicinal leech Hirudo medicinalis, have been extensively studied during multiple clinical research studies. At present, desirudin, as Iprivask (Canyon Pharmaceuticals), is approved for treatment of deep venous thrombosis in patients undergoing elective hip or knee replacement surgery. Lepirudin, as Refludan (Bayer Schering Pharma, Sanofi-Aventis), is approved for treatment of heparin-induced thrombocytopenia. We have developed the scheme of biotechnological production of analogues of these pharmaceutical substances. Desirudin is a complete analogue of the natural hirudin, and lepirudin contains two aminoacid substitutions [Val1Leu, Val2Thr]. Both polypeptides were produced as a part of intein-containing fusion proteins capable of autocatalytic self-cleavage with the release of target product. Artificial DnaB mini-intein from Synechocystis sp. tagged with the chitin-binding domain, acted as the N-terminal carrier protein. Based on the obtained expression constructs new producer strains were created, their culturing conditions were optimized and the fed-batch fermentation was carried out. A unified method for the production of both polypeptides, including isolation and cleavage of the fusion protein and purification of the target product by anion exchange chromatography, RP HPLC and SEC, was developed. The comprehensive analysis of the physico-chemical and biological properties was demonstrated that the antithrombotic agents obtained in the laboratory of biotechnology of the IBCH are completely identical to the pharmacopoeial standards of desirudin and lepirudin. After scaling up we produced pilot batches of desirudin and lepirudin. Based on the obtained results the technological regulatory protocols allowed to isolate at least 5 grams of the pharmaceutical substance of hirudin analogues per cycle of production were developed.

Advanced use of secreting bacterial proteases for prevention of the meningococcal meningitises O.V. Kotel’nikova1, A.P. Alliluev2, L.S. Zhigis1, A.A. Zinchenko1, V.S. Zueva, O.A. Razgulyaeva1, O.V. Serova1, E.Yu. Yagudaeva1, L.D. Rumsh1 1M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia; 2Medical Faculty, Russian University of Peoples’ Friendship, Moscow, Russia Bacterial meningitis relates to the socially important diseases especially for children and teenagers. Polycaccharide antimeningococcal vaccines era started by american E. Gotschlich in 1969 at first stage completed by introducing in practice А and С, than Х and W-135 vaccines. For several decades attempts to create B group antimeningococcal vaccine were made, which can’t be considered successful for the present. We have proposed to use as such vaccine the preparation of IgA1 protease from meningococcus – one of the main factors of virulence. High homology of this enzyme from different serogroups of meningococcus allows the hope for the possibility of creating universal vaccine against all representatives of this infection.

28 | Acta naturae | SPECIAL ISSUE № 1 2014 Oral and Poster Presentations

Currently the different variants of IgA1 protease preparations are generating and testing in order to find the optimal structure of protective antigene, the candidate of polyvalent antimeningococcal vaccine. High homology of primary structure of IgA1 proteases from other bacteria which pathogenicity caused by this enzyme (N. gonorrhoeae, H. influenzae, St. pneumoniae, St. sanguis etc.) suggests that such preparation will possess protective action against all of aforesaid infections. This work was supported by grant program of RAS “Fundamental sciences for medicine” 2012-2014

Developing a method for determination of spatial structure of γ subunit of aIF2 forming perfect twinned crystals O.V. Kravchenko, O.S. Nikonov, S.V. Nikonov Institute of Protein Research, RAS, Pushchino, Moscow Region, Russia Twinning is a crystal growth anomaly in which the specimen is composed of separate crystal domains whose orientations differ. Twinning refers to special case where some or all of the lattice directions in the separate domains are parallel. Hemihedral twinning is reported for macromolecules. In this case only two distinct orientations are assumed for individual twin domains. A twin fraction and twin law are needed to describe hemihedral twin crystals. The most difficult kind of twining is perfect twining (twin fraction about 50%). Now in the world such crystals practically aren't used for X-ray studies because work with them is extremely difficult and labor-consuming. However in some cases is not possible to grow crystals that are either untwined or have a sufficiently low twinning fraction and it is necessary to devise methods to work with an available material. The crystal structures of archaeal γ-subunit of initiation translation factor 2 (aIF2), aIF2 and it complex with tRNA forming perfect twinned crystals have been determined in our laboratory. Generally empirical approaches were used for determination such structures. Using as an example the perfect twinned diffraction data set from the γ-subunit of translation initiation factor 2 from archaea Sulfolobus solfataricus in complex with GDPCP at 2.5 Å resolution we made efforts to systematize and develop the previously used methods in order to determine an optimal way of the solution of such structures.

The first problem is the determination of a real space group of the crystal. Diffraction data show Р6221 space group. The availability of perfect twining assumes its reference to one of the following groups: Р62, Р64, Р3212, Р3112, Р3221, Р3121, Р31 и Р32. The second problem is concerned with the search of a start model for molecular replacement as requirements for this model is higher than in ordinary cases. We used the Normal Mode method to find the appropriate model. Now we referred the crystal definitely to space group P31, obtained a starting set of phases and start the structure refinement. Work is supported by a grant of the RFBR № 14-04-00571 A.

Studies on KV1 channel blockers with the use of novel bioengineering cell systems K.S. Kudryshova, O.V. Nekrasova, Y.V. Korolkova, A.V. Feofanov M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia; Biological Faculty of Moscow State M.V. Lomonosov University, Moscow, Russia Voltage-gated Kv1 potassium channels are pore-forming transport proteins that regulate membrane permeability for potassium ions in response to changes in membrane potential. Kv1 channels are broadly distributed in human body and perform various functions. Numerous diseases are associated with disorders of Kv1 channel structure or function. Kv1 channel blockers are considered as new drugs for treatment of a number of autoimmune diseases, asthma and periodontal disease. Previously we have developed bioengineering cell systems for search and study of new Kv1.x (x=1,3) peptide channels blockers [1, 2]. These systems are based on the expression of hybrid proteins containing Kv1.x (x=1,3) ligand-binding sites in E. coli inner membrane. Searching for new blockers is carried out by competitive inhibition of fluorescent ligand binding on the surface of spheroplasts (bacteria without cell wall) using confocal microscopy. These systems allow detection of Kv1.x pore blockers in the scorpion venoms [2]. The study of peptide blockers using new cell systems allowed us to identify peptides, whose interactions with potassium channels pore are mainly determined by electrostatic interactions. The contribution of electrostatic interactions to the binding of some peptides with Kv1.1 and Kv1.3 channels was found to be significantly different. The peptides were identified which are able to change pore conformation during their interaction with Kv1.x channels. This fact is recognized due to changes in ligand affinities in the competitive binding assay. New bioengineering cell systems allow understanding the essential features of peptide interactions with Kv1.x channels and will contribute to the development of highly selective channel blockers required in research and medical practice. This work was supported by the grant 14-14-00239 from Russian Science Foundation.

Nekrasova O.V. et al. J. Neuroimmune Pharmacol., 2009, 4(1), 83-91. Kudryashova K.S. et al. Anal. Bioanal. Chem., 2013, 405 (7), 2379-2389.

Production of VSTx1 toxin in cell-free expression systems D.S. Kulbatskii, M.A. Shulepko, E.N. Luykmanova, M.P. Kirpichnikov M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Neurotoxins are high-specific inhibitors of essential human receptors. Toxins are a convenient tool for structural and functional investigations of receptors, and toxin-based molecules are prospective diagnostic and therapeutic agents of directed action. Most of neurotoxins have dense spatial structure, which is stabilized by a complex of spatially-close disulfide bonds that considerably impedes their recombinant production. So, a search of new methods for recombinant production of neurotoxins is an actual scientific problem. The goal of presented work is to develop high-efficient methods for recombinant production of neurotoxins from animal poisons in cell-free expression systems (CFES). VSTx1 toxin from poison of Grammostola spatulata spider, an inhibitor of voltage-gated K+-channels (34 amino acids, 3 disulphide bonds) with membrane activity was chosen as a model object for this work. To improve the level of production of VSTx1 in CF, new 5’-terminal sequences in gene of target protein were introduced. According to our previous results [1], these sequences can considerably improve the protein outcome. To provide the correct formation of disulfide bonds we investigated the effects of various CFES modifications: formation of proper redox potential in CFES reaction media by addition of reduced and oxidized glutathione, dithiothreitol and 2-mercaptoethanol in various concentrations; attenuation of CFES media reductase activity by iodacetamide treatment of ribosomal extract to block free thiol groups; using of creatine phosphate-based system of ATP regeneration. This work was supported by RFBR grant № 14-04-32096.

1. Lyukmanova E.N., Shenkarev Z.O., Khabibullina N.F., Kulbatskiy D.S., Shulepko M.A., Petrovskaya L.E., Arseniev A.S., Dolgikh D.A., Kirpichnikov M.P. N- terminal fusion tags for effective production of g-protein-coupled receptors in bacterial cell-free systems. Acta Naturae (2012), 4(4), 58-64.

SPECIAL ISSUE № 1 2014 | Acta naturae | 29 Oral and Poster Presentations

The role of PARP-2 in DNA repair M.M. Kutuzov1, E.S. Ilina1, M.V. Sukhanova1, J.-C. Ame2, V. Schreiber2, S.N. Khodyreva1, O.I. Lavrik1 1Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, Russia, 2UMR7242, Biotechnology and Cell Signaling, Université de Strasbourg, CNRS, MEDALIS, ESBS, Illkirch, France The genome stability of living organisms is mainly maintained by functioning of DNA repair systems. Their correct functioning requires precise regulation of every stage. Poly(ADP-ribose)polymerase-1 (PARP-1) and poly(ADP-ribose)polymerase-2 (PARP-2) are among the other regulatory proteins. In response to DNA damages, PARPs synthesize polymer of ADP ribose (PAR) covalently attached to the protein acceptors including PARP themselves. The PAR is considered to be an intracellular signal about DNA damages while poly(ADP-ribosyl)ation (PARylation) leads to dissociation of protein-DNA complexes. In contrast to PARP-1, the role of PARP-2 in DNA repair is poorly characterized. In our work we established, that PARP-2 and PARP-1 display different selectivity when interacting with DNA structures that mimic intermediates of DNA repair including those with clustered damages in complementary DNA strands. Moreover, the ability of PARP-2 similarly to PARP-1 to form covalent adducts with DNA containing AP sites (AP-DNA) was shown. However, PARP-2, in contrast to PARP-1, is marginally active in AP site cleavage via mechanism of β-elimination. At the same time, PARP-2, with the efficiency similar to that of PARP-1, removes 5′-dRP residue that appears after AP site hydrolysis. The study of functional interactions of PARP-2 with several DNA repair proteins demonstrated that PARP-2, like PARP-1, can decrease the activity of such proteins as: DNA polymerase β, flap endonuclease-1 and AP endonuclease-1. The distinctive feature of inhibition caused by PARP-2 is preservation of its inhibitory effect under PARylation conditions. Noteworthy that under PARylation conditions the simultaneous presence of PARP-1 and PARP-2 results in preserving of PARPs’ inhibitory effect. Taken together, our results testify to additional regulation of DNA repair processes by PARP-2. Probably, the regulation caused by PARP-2 can be required under conditions of massive DNA damage or at dysfunction of some repair proteins. The work was partly supported by RFBR 14-04-31392, 13-04-93107, State contract 14.604.21.0018 and from Russian ministry of education for leading scientific schools 420.2014.4.

Studies on antitumor activity of beta-hairpin antimicrobial peptides D.V. Kuzmin, A.M. Surina, A.A. Emelyanova, M.B. Kalashnikova, I.A. Bolosov, P.V. Panteeleev, S.V. Balandin, T.V. Ovchinnikova M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Antimicrobial peptides are components of the innate immunity of multicellular organisms. A number of structural characteristics (positive net charge, amphiphilicity) allow them to act selectively against microbial cells. Some antimicrobial peptides possess antitumor activity. In vivo and in vitro studies revealed their selective cytotoxicity against cancer cells. They do not require special transporters for cellular uptake. Antimicrobial peptides are regarded as promising candidates for the development of new generation of anticancer drugs. In this work we investigated the therapeutic potential of three β-hairpin antimicrobial peptides: arenicin from polychaeta Arenicola marina, tachyplesin from horseshoe crab Tachypleus tridentatus, and gomesin from spider Acanthoscurria gomesiana. MTT test was performed using different cancer and normal cell lines to assay the cytotoxic effect of the above mentioned antimicrobial peptides. In order to estimate the non-specific cytotoxicity of arenicin, tachyplesin, and gomesin the hemolytic test was used.

IC50 values and therapeutic indices were determined for all the combinations of the peptides and cell lines. Comparative evaluation of the antimicrobial peptides cytotoxic activity against different cell lines, both tumor and normal, and human erythrocytes, was carried out. The cytotoxic activity of tachyplesin is the most evident: IC50 against tumor cell lines range between 34 and 68 μM. At the same time, the comparison of IC50 values shows that arenicin is the most specific antitumor agent. The cytotoxic profiles of antimicrobial peptides were found to depend on the presence of fetal bovine serum in the culture medium. The work was supported by the President of Russian Federation grant for young Russian scientists (МК-6023.2014.4).

Proteomics and rheological studies of blood plasma and platelets in patients with cerebrovascular disease V.B. Lantsova1, V.G. Ionovа1, R.H. Ziganshin2, V.O. Shender2, E.N. Tkatch1, M.Yu. Maksimova1, E.K. Sepp1, A.A. Ignatova1.2, A.V. Feofanov2, V.T. Ivanov2 1Research Center of Neurology, RAMS, Moscow, Russia; 2M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Acute stroke remains one of the most important medical and social problems. In Russian mortality in acute stroke is 35%, increasing to 15% by the end of the first year of the disease. Identification of new diagnostic markers in the pathogenesis of acute stroke and post-periods a stroke, development of additional modern test systems is a challenge. We examined 50 patients. As a control, studied 26 hypertensive patients and 10 patients with autoimmune myasthenia gravis. The study of the proteome of blood plasma, erythrocytes and platelets was performed in 9 patients with ischemic stroke in the acute phase of its development. Laboratory examination included assessment of platelet-vascular hemostasis. At the initial stage of the study to study protein spectrum of blood plasma, platelets and erythrocytes were determined by 1D-SDS РААG electrophoresis according to Laemmli pathological process dynamics. Study the content of basic blood plasma proteins of patients was performed by two-dimensional electrophoresis with differential staining with fluorescent dyes. In proteome analysis revealed that the spectrum of blood plasma protein includes proteins with MM 14–140 kDa range platelet – proteins weighing 13–120 kDa, while the spectrum of red cells – 15–66 kDa proteins. The increase of platelet aggregation and red cells were revealed. First results obtained from the analysis of 2D-gels, plasma, indicate that various pathological processes differently recognized by 2D-protein profiles. It was founded that the quantitative ratios of several proteins in the two groups were significantly different. The analysis of 2D-gel was formed PDQuest program report, which presents all of the protein spots, the protein content of which differs significantly (p<0.05) between the comparison groups, and which is supposed to identify further. Proteomic profiles of plasma and formed elements can be additional laboratory markers associated with the main pathophysiological processes, such as hemostasis, inflammation, immune activation, endothelial damage, and oxidative stress.

Establishment of iPSC lines from Parkinson diseased patients for generation of cellular model of disease 1O.S. Lebedeva, 2 E.D. Nekrasov, 2E.M. Vassina, 2I.V. Chestkov, 1E.V. Novosadova, 3S.N. Illarioshkin, 2S.L. Kiselev, 2M.A. Lagarkova, 1I.A. Grivennikov 1Institute of molecular genetics, RAS, Moscow, Russia; 2 N.I. Vavilov Institute of General Genetics, RAS, Moscow, Russia; 3 Research Center of Neurology RAMS, Moscow, Russia Parkinson’s disease (PD) is the second most common neurodegenerative disorder (after Alzheimer's disease). Parkinson’s disease is a gradually progressive, degenerative neurologic disorder. Although typically a sporadic disease, mutations in the PARK8 gene, encoding the leucine-rich repeat kinase 2 (LRRK2) have been identified as a cause of late-onset, autosomal dominant familial PD that is clinically and neurochemically indistinguishable from sporadic PD. Mutations in the PARK2 gene, encodes a E3-ubiquitin ligase Parkin, are associated with the development of the PD. Unlike the previous case, these mutations are inherited in an autosomal recessive mechanism. Although some progress on LRRK2 and Parkin involvement in the disease progression has emerged during last years an absence of adequate model complicates investigation of human diseases and development of novel therapies. Recent advances in cell reprogramming technologies facilitate the development of human cell models that allows precise mechanisms disease investigation. Using skin biop-

30 | Acta naturae | SPECIAL ISSUE № 1 2014 Oral and Poster Presentations

sies from PD patients with PARK8 (G2019S) and PARK2 gene mutation we reprogrammed dermal fibroblasts either with lentiviral constructs (integrated into the cell genome) or Sendai viruses (non-integrated into the cell genome) carrying Yamanaka’s factors (Oct4, Sox2, Klf4, c-Myc). Induced pluripotent stem cell (iPSC) clones were morphologically indistinguishable from human embryonic stem cells colonies. Complete characterization of iPSCs was carried out according to international standards (expression of basic markers of pluripotent state and the ability to differentiate into derivatives of all three germ layers). Using PD iPSCs clones we implemented a highly efficient directed differentiation into dopaminergic neurons. Generation of “diseased” iPSC lines of biopsies of different patients carrying common genetic alterations allows creation of cellular models to identify possible molecular pathways of disease development and to find differences in neuronal network formation.

HLDF-6 promotes NRF2 transcriptional activity А.P. Litvinenko, E.A. Surina, A.P. Bogachuk, I.V. Smirnova, E.V. Smirnova, V.M. Lipkin M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Previously, the endogenous differentiation factor HLDF was isolated from the HL-60 cells induced by retinoic acid and further characterized. 6-amino acid peptide Thr-Gly-Glu-Asn-His-Arg (HLDF-6) was identified in HLDF amino acid sequence, that peptide possess wide range of nootropic and neuroprotective activities. HLDF-6 neuropotective effects were observed in the experiments with the primary culture of hippocamp and cerebellum neurons. HLDF- 6 exerts anti-apoptotic activity by protecting neurons from β-amyloid, sodium azide, ceramide, and ethanol exposure, as well as cold stress and hypoxy. HLDF-6 activities have been well characterized in vivo. However, underlying molecular mechanisms still remain to be elucidated. We used mice glia primary cells to study the molecular mechanisms of HLDF-6 neuroprotection. HLDF-6 was shown to increase cell survival in oxidative stress and promote transcriptional activation of Nrf2 targets. These data suggest that HLDF-6 neuroprotective effects are mediated through Nrf2, which is the primary cellular defense factor against the cytotoxic effects of oxidative stress.

Cytokinin receptors in endoplasmic reticulum are functionally active and able to initiate hormonal signaling 1S.N. Lomin, 1Yu.A. Myakushina, 1D.V. Arkhipov, 2V.I. Popenko, 2O.G. Leonova, 3T. Schmülling, 1G.A. Romanov 1Institute of Plant Physiology, RAS, Moscow, Russia; 2Institute of Molecular Biology, RAS, Moscow, Russia; 3Institute of Biology / Applied Genetics, Free University of Berlin, Germany Cytokinin receptors were shown recently to be located mainly in the endoplasmic reticulum (ER). However, it remained unclear whether the ER-located receptors are active and the presence of a minor part of receptors in the plasma membrane (PM) could not be excluded. As ER and PM differ in composition and properties, the activity of receptors might depend on their local surrounding. We have, therefore, checked the functionality of receptors located in different membranes. Firstly, we tested receptor topology by a protease protection assay. The cytoplasmic part of the receptor AHK3 was shown to be located in the cytosol and the hormone binding domain in the ER lumen. This topology is consistent with signal transduction from ER membranes. To check the subcellular localization of receptor-phosphotransmitter interaction in planta we performed BiFC experiments. Receptor and phosphotransmitter genes were fused with split eYFP sequences, expressed in Nicotiana benthamiana leaves and the subcellular localization of protein interaction detected by confocal microscopy. We found that receptors interact with phosphotransmitters at the ER network and around nuclei, an interaction pattern being similar to receptor localization. Finally we tested the functionality of receptors in different membranes by an in vitro kinase assay visualizing the phosphorylation of phosphotransmitter proteins. Receptor genes were expressed in N. benthamiana leaves and ER and PM fractions were obtained from leaf homogenate by ultracentrifugation in a step sucrose gradient. Phosphotransmitters were obtained by expression of corresponding genes in E. coli followed by affinity column purification. Kinase assays were performed in a mixture of membranes, phosphotransmitters, and γ-32P-ATP as substrate in the presence of various trans-zeatin concentrations followed by electrophoresis and blotting onto PVDF membranes. We found that the bulk of cytokinin-dependent kinase activity belonged to ER fractions indicating that ER-located receptors are active. Together, the data led us to conclude that the ER is a major site of cytokinin signaling initiation.

Bioengineering cell-based system designed to search for high-affinity Kv1.6 potassium channel blockers E.A. Lyapina, O.V. Nekrasova, K.S. Kudryashova, Y.V. Korolkova, A.A. Vassilevski, A.I. Kuzmenkov, A.V. Feofanov M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Potassium voltage-gated Kv1.6 channel plays a role in conduction of nerve impulses, muscle contraction, maintaining of cellular membrane potential. Elevated expression of Kv1.6 channels in cortex interneurons largely contributes to the development of pathological neuronal conduction in the case of epilepsy. Inhibition of Kv1.6 is a useful approach to controlling epiactivity of neurons, and specific blockers of this channel may become promising therapeutic agents. Currently, there are no high-affinity selective blockers of Kv1.6 channel, which is one of the factors constraining the study of the functional activity of this channel, as well as its role in the pathogenesis of various neurological diseases. We report on the development of a bioengineering cell-based system designed to search for and study new Kv1.6 channel blockers. In the system, a recombinant hybrid channel KcsA-Kv1.6 is used as a receptor protein, which is expressed in the cytoplasmic membrane of E. coli. Oriented localization of hybrid channels in the membrane allows to detect binding of a fluorescent ligand to receptor molecules at the surface of spheroplasts (cells lacking cell wall) using laser scanning confocal microscopy. Developing a cell-based system, we have proposed an effective approach to determine the level of insertion of a membrane protein into cytoplasmic membrane. Further, data was obtained concerning general rules to optimizing the expression of recombinant proteins in E. coli and their targeting to plasma membrane. Significant increase in the expression level of Kv1.6 channels in the membrane resulted in generation of robust and sensitive system for detection of ligands with different affinity to Kv1.6 channels. Recombinant fluorescently labeled agitoxin was successfully used as a fluorescent probe. For system validation, various well-known Kv1 channel blockers were studied using the method of competitive binding, and values of equilibrium dissociation constants were estimated for agitoxin, OSK1, kaliotoxin, and tetraethylammonium. Using bioengineering fluorescent system, high-affinity Kv1.6 channel ligands were revealed in the venom of a scorpionMesobuthus eupeus, and identification of these peptide ligands is being carried out.

Using immuno-PCR for the detection of disaccharide LEc and the natural antibodies to it A.V. Maerle1, D.Yu. Ryazantsev2, K.L. Dobrochaeva2, O.E. Galanina2, D.Yu. Trofimov1, N.V. Bovin2, S.K. Zavriev2 1JSC «SPC DNA Technology», Moscow; 2M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia In the blood of both healthy and cancer patients are antibodies against different antigens associated with tumors, in particular LeC disaccharide. This disaccharide is a part of a well-known tumor marker – CA19-9. LeC specific monoclonal antibodies show high diagnostic value in breast cancer detection. The reduction of the level of LeC antibodies in serum of patients diagnosed with breast cancer has been previously shown. However, it is unknown whether tumor occurrence is responsible for the reduced antibody level or initially low levels of antibodies promote the tumor process. The study of antibodies levels against LeC in comparison with the analysis of the antigen expression on the tumor cell surface has not been performed yet. The study of this correlation will help to develop new topical approach to the tumor detection on early stages or identify the risk groups of the pathology. The ELISA assay traditionally used in such studies not always show significant sensitivity.

SPECIAL ISSUE № 1 2014 | Acta naturae | 31 Oral and Poster Presentations

The immuno-PCR method (iPCR) is a hypersensitive antigen detection method and includes the same steps as ELISA, except that the conjugate with a DNA label is used on the last step when the DNA label is amplified. The PCR amplification step significantly increases the sensitivity of the method, which is mainly determined by the affinity of antibodies used and can reach the antigen detection levels in a probe lower than fg/ml (10-15g/ml). The previously described supramolecular complexes formed by streptavidin and single-stranded 60 nt-long DNA fragments biotinylated at 5'- and 3'-ends were used for iPCR detection. This system is universal because it does not need to synthesize specific conjugates for different antibodies. In the model system the level of the LeC detection using biotinylated anti-LeC antibodies was 0.1 ng/ml. In the anti-LeC antibodies concentration measurement experiments where the immune complexes detection was performed using biotinylated antibodies against human immunoglobulins, the level of anti-LeC IgM detection was 10 ng/ml, what is more than 10 times higher than the sensitivity of ELISA tests.

Development of the acetylated peptides in vivo production concepts: case study of thymosin beta-4 D. A. Makarov, V. N. Stepanenko, R. S. Esipov M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia At present over two hundred types of post-translational modifications of proteins are known and the overwhelming majority of proteins can undergo such modifications. N-terminal acetylation is among the most common types of protein modifications. One of the proteins that undergoes post-translational modifications is thymosin beta-4, a peptide that is present in actually all human body fluids and tissues, and has a high potential as a remedy for treating trophic ulcers, wounds and burns, as well as for drug therapy in acute myocardial infraction. Earlier we have developed the thymosin beta-4 biotechnological production technique, where the limiting stage was N-terminal chemical acetylation of desacetylthymosin beta-4 with acetic anhydride. The acetylation reaction was exhaustive due to the high activity of acetic anhydride; however the target peptide yield upon the reaction completion constituted 60% of the total protein mass. Thus, although we have obtained positive results they were not optimal, since the balance 40% of the thymosin beta-4 precursor was lost in the reaction by-products. Development of a purely biotechnological method for production of thymosin beta-4 would eliminate the chemical acetylation stage and facilitate the target peptide extraction. For this purpose we have created a genetically engineered construct the induction of which leads to the formation of desacetyl thymosin beta-4 combined with C-terminal intein; the hybrid protein synthesis goes concurrently with the synthesis of the enzyme that acetylates in vivo the N-terminal of the thymosin beta-4 precursor. Ribosomal-protein-alanine acetyltransferase (rimJ) and ribosomal-protein-L7/L12-serine-acetyltransferase (rimL) were used as acetylating enzymes. In the in vivo experiment it was observed that in the course of the post-translational process the formylmethionine was completely removed from the hybrid protein N-terminal containing desacetylthymosin beta-4, and thereafter a high-degree acetylation of the N- terminal serine-containing peptide took place. Serine acetyltransferase (rimL) demonstrated the best efficiency, since, according to the experiment results, 75%, as a minimum, of the total hybrid protein mass became acetylated.

Kinetics of heat-induced aggregation of bovine serum albumin K.A. Markossian1, V.A. Borzova1, S.Yu. Kleimenov1, N.A. Chebotareva1, V.V. Shubin1, K.O. Muranov2, N.B. Polyansky2, B.I. Kurganov1 1A.N. Bach Institute of Biochemistry, RAS, Moscow, Russia; 2N.M. Emanuel Institute of Biochemical Physics, RAS, Moscow Russia Thermal aggregation of bovine serum albumin (BSA) has been studied using dynamic light scattering, asymmetric-flow field flow fractionation and analytical ultracentrifugation. The studies were carried out at fixed temperatures (60, 65, 70 and 80°C) in 0.1 M phosphate buffer, pH 7.0. Thermal denaturation of the protein was studied by differential scanning calorimetry. Based on the experimental data a mechanism of thermal aggregation of BSA has been proposed. The first step of the general aggregation process is unfolding of the protein molecule, which results in the formation of three forms of the non-native protein with different propensity to aggregation. One of the forms (Uhr) is characterized by a high rate of aggregation; aggregation leads to the formation of primary aggregates with the hydrodynamic radius of 9.6 nm at 60°C. The second form (Ulr) possesses a low ability for self-aggregation; however, this form is able to participate in the aggregation process by its attachment to the primary aggregates produced by the Uhr form. At complete exhaustion of the Ulr form, secondary aggregates with the hydrodynamic radius of 11.1 nm at 60°C are formed. Further aggregation of the protein is a result of the sticking of the secondary aggregates. It is established that this process proceeds in the regime of diffusion-limited cluster-cluster aggregation. The third form (Unr) shows no propensity to aggregate and remains in the non-aggregated state during prolonged heating. The relative portions of the Uhr, Ulr and Unr forms estimated by asymmetric-flow field flow fractionation at 60°C are 0.11, 0.36 and 0.53, respectively. The Unr form, showing no propensity to aggregate, was separated by size-exclusion chromatography and characterized by circular dichroism and fluorescence spectroscopy. This study was funded by the Program “Molecular and Cell Biology” of the Presidium of the Russian Academy of Sciences and the Russian Foundation for Basic Research (grants 14-04-01530-a and 12-04-00545-a).

The cytoskeletal protein Zyxin regulates dorsal-ventral patterning in the early Xenopus laevis embryo N.Y. Martynova1, F.M. Eroshkin1, N.A. Zhigalova2, E.B. Prokhortchouk2, E.E. Orlov1, A.G. Zaraisky1 1M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia; 2Center of Bioengineering, RAS, Moscow, Russia Zyxin is a low abundant LIM-domain protein that binds to alpha-actinin and initiates nucleation and assembly of actin filaments but is able to enter cell nuclei and interact with proteins involved in the transcription machinery. Due to such ambivalence, Zyxin is a good candidate for a mediator that couples, during the development, cell morphogenetic movements with gene expression. In support of this, we showed recently that in the early Xenopus embryo, Zyxin can modulate at least two important events that pattern early embryo. First, it suppresses activity of the homeodomain transcription factor Xanf1, which is the earliest regulator of the forebrain patterning (Martynova et al., 2008, Dev Dyn. 237, 736-49). Secondly, Zyxin modulates activity of sonic hedgehog signaling pathway through interaction with one of its regulator, the transcription factor Gli1 (Martynova et al., 2013, Dev Biol. 380, 37–48). Thus, basing on these results, one may suppose that Zyxin could be also involved in regulation of other pathways that regulate early development of the neural anlage. To reveal such pathways, we compared now by using high-throughput sequencing transcriptomes of the neural plate cells from the wild-type embryos and embryos, in which translation of Zyxin mRNA was inhibited by microinjection of anti-sense morpholino oligonucleotides. As a result of subsequent bio-informatic analysis of the differentially expressing genes, we have established that down-regulation of Zyxin functioning leads to suppression of the pathways responsible for the neural and skeletal muscle differentiation and, by contrast, to activation of the pathways that promote the epidermal and ventral mesoderm types of cell differentiation. These data demonstrate for the first time the role of Zyxin as a regulator of the dorsal-ventral patterning in early embryonic development.

Biotechnological production and uniform stable isotopes labeling of the recombinant dill lipid transfer protein D.N. Melnikova, M.E. Rychkova, E.I. Finkina, T.V. Ovchinnikova M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Plant lipid transfer proteins (LTP) are small basic multifunctional proteins. Most representatives of LTP display antimicrobial activity and possess an ability to bind and transfer lipid molecules in vitro. Identification and detailed characterization of LTPs can potentially lead to development of new drug delivery systems and production of new transgenic plants resistant to pathogenic microorganisms. In our previous study a novel plant LTP was purified from leaves and stalks of the garden dill Anethum graveolens. The protein, called Ag-LTP, possesses antifungal activity and is able to bind fatty acids. The complete amino acid sequence and cDNA structure of Ag-LTP were determined. In order to determine the spatial structure of the dill lipid transfer protein by heteronuclear NMR spectroscopy, the recombinant Ag-LTP and its 13C,15N-labeled analog

32 | Acta naturae | SPECIAL ISSUE № 1 2014 Oral and Poster Presentations

were obtained. Ag-LTP and its labeled analog were expressed in E. coli strain BL21(DE3) transformed by pET-His8-TrxL-Ag-LTP. The cells were grown 15 13 in LB or in a minimal medium, containing NH4Cl and [U- C6]-labeled glucose as the only nitrogen and carbon sources, respectively. Purification protocol of the recombinant proteins involved the fusion proteins separation by immobilized metal-chelate affinity chromatography (IMAC), dialysis, CNBr cleavage of the fusion proteins with the subsequent purification of the target proteins by IMAC and reversed-phase HPLC. Homogeneity of the obtained recombinant proteins and their identity to the natural protein were estimated by polyacrylamide gel electrophoresis, MALDI-TOF mass spectrometry, and N-terminal protein sequencing. А comparative analysis of the secondary structure of the recombinant Ag-LTP and 13C, 15N-labeled analog was carried out by CD spectroscopy. It was shown that both proteins have identical predominantly α-helical structure peculiar to plant LTPs. This work was supported by the Russian Foundation for Basic Research (projects Nos. 12-04-01224-а and 13-08-00956-a).

The limited proteolysis of оligopeptidase в from Serratia рroteаmaculans (PSP) А.G. Мikhailova1, М.V. Оvchinnikova2, V.A. Gorlenko2, L.D. Rumsh1 1M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia; 2Moscow State Pedagogical University, Moscow, Russia Oligopeptidase B (OpdB) is a trypsin-like serine peptidase that is present in ancient unicellular eukaryotes: trypanosomes and leishmania as well as Gram- negative pathogen bacteria. A characteristic feature of OpdB is a two-domain structure with localization of the catalytic triad in the C-terminal peptidase, or catalytic domain, and an unusual seven-bladed β-propeller N-terminal domain. The β-propeller allows oligopeptides to enter the active site, while exclud- ing large big globular proteins from it. It is suggested that the length of the peptide substrates of OpdB does not exceed 3 kDa. However, such a limitation is unlikely to be absolute; OpdBs are capable of hydrolyzing denatured proteins and proteins lacking the rigid secondary structures. In this work, we studied the high molecular substrates hydrolysis by the psychrophilic OpdB (PSP) found by us in Serratia proteamaculans. PSP did not hydrolyze recombinant human histone Н1.3. (22 kDа) and azocasein (23–27 kDа) even at prolonged incubation and high enzyme concentrations; the processing of recombinant human proinsulin was shown to be incomplete and ineffective. To use PSP for chimeric proteins processing we decided to design the modified forms of this enzyme potentially more effective toward high molecular subsyrates. On the basis of the theoretical calculations of the structures of such enzyme variants we for the first time obtained N-terminal truncated PSP, capable to hydrolyze azocasein efficiently. We used the limited proteolysis of PSP molecule by immobilized chymotrypsin. We determined the sites of PSP protein molecule chymotrypsinolysis and found that the main products are PSP 92-677 and PSP 101-677. These data confirm our hypothesis that the N-terminal region (≈100 residues) that, according to 3D structure envelops one side of the C-terminal PSP catalytic domain, prevents the high molecular substrates hydrolysis.

Molecular dynamics simulation of the structure of ribosomal protein L1 from halophilic archaeon Haloarcula marismortui А.О. Mikhaylina, V.G. Kljashtorny, S.V. Tishchenko, S.V. Nikonov Institute of Protein Research, RAS, Pushchino, Moscow Region, Russia First three-dimensional model of the 50S ribosomal subunit Archaeon Haloarcula marismortui was determined over a decade ago with a resolution of 2,4 Å followed by repeated refinement. However, as in the original, and in subsequent models some movable parts were missing, in particular L1-stalk of the large subunit. Two-domain structure of the ribosomal protein L1 from different organisms have been determined in isolated form and in complex with specific rRNA fragments. We have obtained a genetic construct and isolated protein L1 H. marismortui (HmaL1). In this work was made simulation of the structure of the isolated protein HmaL1, and compared the resulting model with the structures of ribosomal protein L1 from other organisms. The simulation was performed using the molecular dynamics method using the software package GROMACS 4.5.3 and Charmm27 force fields. Modelling was carried out in the orthorhombic water box with TIP3P water model at constant temperature (300 K) and pressure (1 atm.). The length of the molecular dynamics trajectory was 1.2 microseconds. Analysis of the results showed that the structures of the two domains are well preserved and have no significant difference compared with the known structure of the L1 protein of archaeon Methanococcus jannaschii. During modelling protein HmaL1 moved from the open conformation (domains apart) in a closed (domains are closed) and between domains formed close contact stabilized by several hydrogen bonds and hydrophobic interactions. Interestingly, earlier similar transition observed for the conformation of the L1 protein from the bacterium Thermus thermophilus: a closed conformation of the isolated protein goes into the open during the binding protein with RNA. For L1 protein from archaea Methanococcus and Sulfolobus was characterized as an open conformation in the isolated state and in complex with RNA. The simulations suggest that these two conformations can be characterized by the isolated ribosomal protein L1 from both bacteria and halophilic archaeon. This work was supported by the Russian Academy of Sciences, the Russian Foundation for Basic Research (grant number 14-04-00414-a) and the Pro- gram of RAS on Molecular and Cellular Biology.

TboIT1 – insect-selective toxin from Tibellus oblongus spider venom A.N. Mikov, I.M. Fedorova, E.V. Bocharov, E.E. Maleeva, D.B. Tikhonov, S.A. Kozlov, E.V. Grishin M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Spiders produce astounding diversity of bioactive compounds, trying to make venom more effective. Polypeptide molecules are the basic active power of venom, and have an influence on action of different molecular targets. From other side, main purpose of toxin action is killing insects. In this work, our object was studied from both sides. Spiders produce astounding diversity of bioactive compounds, trying to make venom more effective. Polypeptide molecules are the basic active power of venom, and have an influence on action of different molecular targets. From other side, main purpose of toxin action is killing insects. In this work, our object was studied from both sides. Tibellus oblongus – Middle East spider completely non-harmful to humans was chosen for an investigation. From its crude venom the most insect-toxic fraction was isolated using combination of different chromatographic methods and microinjections to insect larvae. Amino acid sequence was determined, and novel toxin was named TboIT1. Structurally, it belongs to Inhibitor Cystine Knot family. Recombinant production of the toxin and its several site- mutagenized analogs in E. coli provided sufficient amount for toxicity, electrophysiological and structural experiments. LD50 of TboIT1 was identified to be 12 μg/g (on M. domestica larvae). Toxin slowed down transmitter release in neuro-muscular junction of Calliphora vicina bottle fly. TboIT1 reversibly inhibited evoked Excitatory Post-Synaptic Currents (eEPSC), but did not affect frequency and amplitude of spontaneous Excitatory Post-Synaptic Currents (sEPSC). Interestingly, inhibition of eEPSC was not accompanied by significant change of rise time and decay of the eEPSC. Such parameters for sEPSC were also unaffected. Moreover, toxin did not affect any parameter of transmission in frog neuro-muscular junction. These data point out TboIT1 may be novel selective inhibitor of insect pre-synaptic calcium channels.

SPECIAL ISSUE № 1 2014 | Acta naturae | 33 Oral and Poster Presentations

Bacteriophage tailspike proteins as depolymerases of extracellular bacterial polysaccharides K.A. Miroshnikov1, M.M. Shneider1, S.A. Buth2, A.S. Shashkov3, Yu.A. Knirel3, P.G. Leiman2 1Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia; 2École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland; 3N.D. Zelinsky Institute of Organic Chemistry, RAS, Moscow, Russia Nosocomial infections caused by Gram-negative pathogens Acinetobacter baumannii and Pseudomonas aeruginosa are a major concern of clinical medicine. These bacterial species possess multiple antibiotic resistance, and secrete a thick layer of extracellular capsular polysaccharides thus preventing the penetration of antibacterial substances into the cytoplasm. Nevertheless, some bacteriophages that are able to disrupt the capsular protective layer during infection. The study of phage-encoded polysaccharide-depolymerases represents excellent targets for developing new ways to control the pathogens. A. baumannii bacteriophage АР22 (Myoviridae), and P. aeruginosa bacteriophages phi297 (Siphoviridae) and LKA1 (Podoviridae) infect their hosts forming negative colonies (plaques) with a broad semitransparent halo. This is a possible marker of a presence of proteins degrading cellular exopolysaccharides in phage virions. We have obtained and purified recombinant tailspike proteins of these bacteriophages, and studied their structure by X-ray analysis. Inde- pendent on phage morphology the structure of all three tailspike proteins is formed by trimeric β-helix involving domains of polysachcride binding and enzymatic degradation. High structural conservatism of tailspike structure reflects the possible evolutional relationship of tailed bacteriophages. NMR study of the A. baumannii 1053 and P. aeruginosa РАО1 capsular polysaccharides and products of their enzyme degradation have revealed that tailspike proteins cleave the link between polysaccharide residues by the mechanism of β-elimination and, thus are considered as specific polysaccharide lyases.

Impact of ATP1B4 co-option on development of placental mammals N. Modyanov University of Toledo, USA ATP1B4 genes represent a rare instance of orthologous vertebrate gene co-option (evolutionary changes of functions of the genes, which are present in a single unambiguous copy in all known vertebrate genomes) that radically changed functions of the encoded BetaM proteins, which are the Na, K-ATPase subunits in lower vertebrates. In placental mammals, BetaM completely lost its ancestral role and became the only currently known skeletal and cardiac muscle-specific protein of the inner nuclear membrane, which is expressed at the highest level during perinatal development and functions as a regulator of gene expression. Evolutionarily acquired new functions and a unique pattern of expression of BetaM proteins suggest that those new physiological functions are essential, might be necessary for survival of placental mammals in natural conditions, and provide an evolutionary advantage. To characterize BetaM function directly in vivo, the Atp1b4 knockout mouse model was generated. We have shown that loss of BetaM results in significantly lower body size and weight, growth retardation and high mortality of Atp1b4 knockout neonates. Transcriptome analysis by mRNA sequencing of skeletal muscle from neonatal wild type and knockout littermates revealed strong down-regulation of fast-twitch and up-regulation of slow-twitch muscle genes, and broad changes in expression of genes regulating lipid metabolism. These results imply that BetaM plays an important role during a critical period of perinatal and neonatal development of placental mammals and its damage or malfunction can cause birth and growth defects in humans. Most importantly, our experiments revealed a somewhat unexpected and profound beneficial effect ofAtp1b4 disruption on metabolic parameters in adult male mice. We have shown that Atp1b4 knockout males, which survived to adulthood, have a significantly lower percentage of body fat, exhibit enhanced metabolic rate and insulin sensitivity, and are resistant to high-fat diet-induced obesity. These robust changes in mouse metabolic parameters indicate a strong function of BetaM in energy expenditure pathways in muscle. Prevention of obesity and maintenance of insulin sensitivity suggests that knowledge of BetaM functions may have significant implications for a better understanding of mechanisms underlying metabolic diseases such as Western-style diet-induced obesity and type II diabetes in humans and might potentially lead to novel therapeutic approaches to metabolic and muscular diseases.

Functional characterization of pancreatic cells in 2D and 3D cultures Е.V. Myrsikova, М.V. Grechikhina, E.N. Kaliberda, Е.V. Svirshchevskaya M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Flat 2-dimentional (2D) cultures are routinely used for the in vitro estimation of antitumor activity of various drugs. However natural tumors represent 3D structures which can be reproduced in vitro by multicellular spheroids formed by cells grown on anti-adhesive films. Earlier we demonstrated that unlikely 2D cultures, cells in 3D cultures completely stopped proliferation as soon as spheroids are formed. The aim of this work was to study the functional activity of cells in 2D and 3D cultures. Spheroids from pancreatic cell lines AsPC-1, BxPC-3, Colo-357 and T3M4 were formed on anti-adhesive film PolyHEMA and incubated for 48 and 72 hrs. The next parameters were studied: i) ability to adhere to plastic in 2D conditions after cultivation in 3D conditions and consequent trypsinization; ii) production of matrix metalloproteinases (MMPs) analyzed by zymography; iii) synthesis of matrix proteins analyzed by anti-fibronectin staining and visualyzed by confocal microscopy; iv) production of cytokines analyzed by multiplex bead flow cytometry; v) induction of apoptosis and analysis of mitochondria membrane potential estimated by DiOC6 and propidium iodide (PI) staining and flow cytometry. We demonstrated that cells preserve their ability to adhere to plastic after 3D cultivation however they decrease their proliferation capacity in these conditions. At the same time 2D and 2D cells secret comparable types and amounts of MMPs and cytokines. Production of matrix protein fibronectin was comparable at 48 hrs while increased in 3D but not in 2D cultures to 72 hr. We found an increase in mitochondrial potential and a decrease in cell membrane permeability for PI in 3D cells over 2D cells. Starting from 72 h of incubation, a significantly higher fraction of apoptotic cells was found in 3D cultures in comparison with twin 2D cultures. Longer incubation of cells in 3D cultures (4 or 5 days depending on the cell type) culminated in a rapid death of all spheroids within several hours. Taken collectively we concluded that cells in 3D cultures demonstrate signs of differentiation and senescence induced by cell contact growth inhibition which result in cell death both by apoptosis and necrosis.

Structural investigations of amyloid precursor protein’s transmembrane domain with familial mutations V717I/G and L723P K.D. Nadezhdin1,2, O.V. Bocharova1, I.S. Chaplygin2, E.V. Bocharov2, A.S. Arseniev1,2 1M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia; 2Moscow Institute of Physics and Technology (State University), Dolgoprudny, Moscow Region, Russia Alzheimer’s disease (AD) is the most prevailing neurodegenerative disorder that affects elderly population all over the world. The hallmarks of AD are amyloid plaques and neurofibrillary tangles which were found in brains of patients. Plaques are made of beta-amyloid peptide – a product of sequential regulated intramembrane proteolysis of amyloid precursor protein (APP) by gamma-secretase complex. Many mutations found in APP transmembrane domain are associated with early onset of AD. Although these pathogenic (or familial) mutations are believed to influence the structure of APP, its dimerization capability and/or cleavage by gamma-secretase, the exact molecular mechanisms involved in these processes remain elusive. In this study we describe a highly efficient cell-free expression protocol for APP transmembrane domain with pathogenic mutations: V717I (also known as “London” mutation),

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V717G and L723P (referred to as “Australian” mutation). This method allows in short time to acquire 2 mg of pure isotope-labeled peptides for high- resolution NMR spectroscopy from 1 ml reaction mixture. Spectra of mutant peptides solubilized in micelles were assigned and compared with wild-type transmembrane domain of APP.

Octarphin – nonopioid peptide of the opioid origin E.V. Navolotskaya Branch of M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Pushchino, Moscow Region, Russia The peptide octarphin (TPLVTLFK) corresponding to the sequence 12–19 of β-endorphin was synthesized. It has been shown that it is a selective agonist of nonopioid (insensitive to the action of the opioid antagonist naloxone) β-endorphin receptor, Using tritium labeled the distribution of the receptor in the body was studied. It was found that it is available on immune cells (peritoneal macrophages, T and B lymphocytes of spleen and blood), endocrine (adrenal cortex, hypothalamus), cardiovascular (cardiomyocytes) systems. Characterization of the binding specificity showed that only un- labeled β-endorphin was able to compete with the labeled octarphin for the binding to receptor, tested in parallel α-endorphin, γ-endorphin, '[Met5] enkephalin, [Leu5]enkephalin and a number of peptide hormones were inactive. Investigation of the octarphin effect on the target cells was showed that it increases the mitogen-induced proliferation of human and mouse T and B lymphocytes in vitro, activates murine peritoneal macrophages in vitro and in vivo, stimulates growth of human T-lymphoblast cell lines Jurkat and MT-4, inhibits adenylate cyclase activity of rat adrenal cortex membranes and suppresses the secretion of glucocorticoids from the adrenal gland into the blood. Data on the molecular mechanism of octarphin action were obtained. It was shown that in a concentration range of 1-1000 nM the peptide increases the activity of inducible NO-synthase (iNOS), and the content of NO and cGMP in lipopolysaccharide-activated murine peritoneal macrophages. The octarphin activity was depended on the dose and was maximal at a concentration of 100 nM. Taking into account that NO acts as a primary activator of soluble guanylate cyclase (sGC), it can be assumed that the activating effect of octarphin on macrophages is realized in the following way: increase in the iNOS expression → increase in the NO production → increase in the sGC activity → increase in intracellular levels of cGMP.

Design and study of immunogenicity of polyepitope DNA vaccine against breast cancer Zh.K. Nazarkina1, M.V. Kharkova1, D.V. Antonets2, S.I. Bazhan2, L.I. Karpenko2, V.V. Vlassov1, A.A. Ilyichev2, P.P. Laktionov1. 1Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk, Russia; 2State Research Center of Virology and Biotechnology “Vector”, Koltsovo, Russia Breast cancer (BC) is the most common cancer among women and remains among the most dangerous diseases despite of enhancement of BC diagnostics and therapy. Induction of specific T cell immune response using anticancer DNA vaccines is a promising strategy of selective elimination of tumor cells. DNA vaccines may encode artificial antigens containing epitopes from various tumor antigens and due to intracellular expression induce effective T cell response against multiple antigens. Polyepitope DNA vaccine BC2 containing epitopesof two breast-cancer-associated antigens HER2 and Mammoglobin-1 (Mam) was designed using PolyCTLDesigner and TEpredict software. DNAs encoding polyepitope antigen and native antigens were synthesized and cloned into pcDNA3.1 vector. LPS-free DNA plasmids pBC2, pHER2 and pMam, encoding polyepitope antigen and full gene copies of HER2 and Mam, were produced and characterized. Dendritic cells (DCs) were generated from human peripheral blood monocytes. The protocol of DNA delivery into DCs was optimized. DCs obtained have morphology and phenotype of mature DCs as it was demonstrated by microscopy and flow cytometry. Mature DCs demonstrate 10–100-fold increase in IL-6 level and 2-fold decrease in IL-10 level in comparison with immature DCs. DCs stimulate proliferation of T cells and increase of IFNγ secretion by T cells 20–40-fold. DCs do not affect IL-4 secretion by T cells. Thereby, generated DCs stimulate Th1 polarization of T cells and induce cytotoxic immune response. Breast-cancer-specific T cell response was induced by DCs transfected with pBC2, pHER2 or pMam. T cells activated by pBC2-transfected DCs showed 2-fold increase in number of IFNγ-producing cells against MCF-7 cells in comparison with T cells activated by DCs transfected with pHER2 or pMam. These data demonstrate efficacy of polyepitope DNA vaccine as compared to DNA vaccines encoding full copies of antigens.

Study of molecular mechanisms of gene trim14 functioning in vivo V.V. Nenasheva, L.E. Andreeva, G.V. Kovaleva, I.V. Makarova, N.V. Khaidarova, V.Z. Tarantul Institute of Molecular Genetics, RAS, Moscow, Russia TRIM 14 belongs to the family of TRIM proteins typically containing the so-called TRIM motif (the tripartite motif). Many TRIM proteins are known to be involved in a wide range of biological processes, including transcription regulation, cellular proliferation and differentiation control, oncogenesis and apoptosis as well as the protection of cells against viruses, for example, against HIV. The functions of gene trim14 that was revealed by us as a gene specific for HIV-associated lymphomas have been hitherto scarcely studied. Earlier we obtained the lines of mouse embryonic stem cells (mESC) with elevated and declined expression of the human gene trim14 and the HEK293 line with overexpression of the gene, and found, by subtractive hybridization, several genes – hsp90ab1, prr13, hbp1, spi.1, junb, pdgfrb, baffr, taci, hlx1 – which expression was upregulated in trim14-transfected mESC and HEK293 as compared with the control cells. In order to study the properties of gene trim14 in vivo, we used an animal model of loach (Misgurnus fossilis L.) embryos transiently expressing human gene trim14 under the metallothionein (MT) and cytomegalovirus (CMV) promoters. An insignificant increase of the number of abnormal loach embryos as compared with the control was observed in six experiments at days 3-5 of loach embryo development. This fact probably indicates some negative effect of gene trim14 increased expression on the early growth of loaches. The analysis of hsp90ab1, hbp1, spi.1, junb, pdgfrb, hlx1 gene expression showed that all these genes were upregulated in the loaches with human gene trim14 transient expression as compared with non-transgenic loaches. We obtained a transgenic male mouse, which contained the human gene trim14 under the control of the CMV-promoter. Analysis of mRNA in different organs of the transgenic mouse showed the human trim14 overexpression, in descending order, in its blood, brain, testes, heart, muscles, spleen, liver, lungs. Expression analysis of previously revealed genes associated with trim14 upregulation showed that the transcription of genes hsp90ab1, junb, pdgfrb, baffr, hlx1 was increased in the blood and that of hsp90ab1, prr13, hbp1, spi.1, pdgfrb, baffr, taci, hlx1 was elevated in the brain of the animal. Thus, the analysis of the expression of the previously revealed genes associated with the elevated expression of the human trim14 in cultured cells confirmed, in two experimental models, the data obtained earlier.

The special features of colon epithelial microflora by colorectal cancer Nga Thi Nguyen1, I.G. Gataullin2, O.N. Ilinskaya1 1Department of Microbiology, Kazan Federal (Volga-Region) University, Kazan, Russia; 2Department of Surgery and Oncology, Tatarstan Regional Clinical Cancer Center, Kazan, Russia Microbial composition of the human intestine is importantfor health status.It might be due to the dietary habits in the developed nations; the composition of normal microflora in GIT, which is altered and causes dysbacteriosis consequently leading to several diseases including cancer. Even though intestinalmicro- biota play important role in human health, the abnormalities or changes of microbial composition in GIT are associated with the initiation and progression

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of different intestinal disorders such as inflammatory bowel disease, colorectal cancer (CRC) and obesity [Gurner et al., 2003].This study compares the microbial composition of malignant and non-malignant intestinal epithelial biopsies of individuals with confirmed colorectal cancer. The aim of this study was to identify the most significant differences in the composition, amount and some biochemical properties of the intestinal microflora isolated from malignant and non-malignant intestinal epithelial biopsies.Sixty eight cultivable microbial isolates were identified from biopsies of 18 patients by pattern matching with the libraries in BioTyper 2.0 software. MALDI-TOF mass spectroscopy (Escherichia coli, Serratiamarcescens, Bacillus, Pseudomonas, Klebsiella, Enterobacter). We did not reveal significant differences in the total number of bacteria between cultured samples of transformed and normal epithelial tissues.The current data also showed that an increased proportion of Gram-negative microorganisms were isolated from malignant epithelial biopsies compared to normal epithelium which is in accordance with the data obtained by Marchesi et al. Marchesi et al.[2011] demonstrated a general tendency, that more Gram-negative Bacteroidetes and less Firmicuteswas found from ‘ON-tumor’ tissue than ‘OFF-tumor’ mucosa. Isolated bacteria were sensitive to gentamicin, ciprofloxacin, chloramphenicol, and were resistant to penicillin, nalidixic acid, erythromycin, trimethoprim/sulfamethoxazole and tetracycline.8 of the 10 bacterial isolates, randomly selected, demonstrated α-and β-hemolytic activity on agar plates containing fresh blood. In addition to their antibiotic and hemolytic properties, we examined the ribonuclease(RNase) activities of isolated cultivable microflora towards high-polymeric yeast RNA.Particular attention was given to the activity of RNase secreted by bacteria, because it is known [Makarov, Ilinskaya, 2003], that RNases have antitumor effect.We established at the first time that RNase activity of microorganisms,isolated from malignant colorectal epithelium, is higher than ones ofmicroflora from normal tissue. It’s important to note that this phenomenon was characteristic of both gram-negative and gram-positive microorganisms, isolated from malignant epithelium.

Small angle x-ray and neutron scattering for structural studies of nanodiscs and membrane proteins Michael Nikolaev1,2, Dmitry Unuchek1, Alexander Kuklin2, Valentin Borshchevskiy1,2, Valentin Gordeliy1,2,4 1Moscow Institute of Physics and Technology(MIPT), Moscow, Russia; 2Institute of Complex Systems, Structural Biochemistry, Research Centre Jülich, Germany; 3Joint Institute for Nuclear Research (JINR), Dubna, Russia; 4Institut de Biologie Structurale, Grenoble, France Functional reconstitution of membrane proteins (MP) remains a significant barrier to their biochemical, biophysical, and structural characterization. These difficulties are caused by the MP requirement for a native lipid membrane-like environment in order to maintain structural and functional stability. One of the new and promising approaches of MP stabilization is the use of lipid-protein nanodiscs. Nanodiscs consist of discoidal patches of lipid bilayer, surrounded by two molecules of truncated human apolipoprotein A1, designated as membrane scaffold protein (MSP). The MSP molecules cover the hydrophobic perimeter of the bilayer [I.Denisov, 2004; T.Bayburt, S.Sligar, 2010]. Nanodiscs offer a high degree of flexibility to meet the integrated membrane protein requirements, since different lipids and scaffold proteins can be used, resulting in nanodiscs with variable diameters (from 9 to 17nm) and lipid composition. This technique makes possible to stabilize large MPs and their complexes in the nearly native state. One of the main advantages of the nanodiscs is their exceptional monodispersity and stability over a wide temperature range. We extensively use nanodiscs for cell-free expression of MPs, as well as for structural studies by small angle X-ray(SAXS) and neutron scattering(SANS). Here we focus on controlled insertion of monomeric and multimeric forms of MPs (sRII, monomers and trimers of pR and bR, pentamer of sodium ion-proton pump KR2) into nanodiscs of variable size. We have also studied the role of lipid environment in MP stabilization by changing lipid composition(DMPC, DPPC, POPC, DOPE, E.coli total lipid extract). Small angle scattering experiments were conducted at ESRF(Grenoble, France), IBR-2(JINR, Dubna, Russia) and at the X-ray HomeLab Rigaku system (MIPT, Moscow). Combination of these experiments made us possible to confirm the insertion of MPs and determine the size, shape and low-resolution structure.

Essential functions of CPEB Orb2 in spermatogenesis of Drosophila melanogaster G.A. Nosov1,2, L.V. Olenina2 1M.V. Lomonosov Moscow State University, 2Moscow, Russia; 2Institute of Molecular Genetics, RAS, Moscow, Russia Cytoplasmic polyadenylation is a one of main ways of protein biosynthesis regulation in gametogenesis, embryogenesis, immune response etc. For carrying out cytoplasmic polyadenylation requires the presence of cytoplasmic polyadenylation elements (CPE) in mRNA. These regulatory elements are known to bind CPEB proteins. In Drosophila melanogaster, there are two CPEB proteins: Orb and Orb2, the second is involved in spermatogenesis but not in the regulation of oogenesis. Orb2 also detected in brain, where it forms amyloid aggregates. In our work, we have studied Orb2 function at different stages of spermatogenesis. Using immunofluorescense staining with specific antibodies and confocal microscopy we revealed intracellular distribution of Orb2 at different stages of spermatogenesis. We showed that in spermatocytes Orb2 is located in germinal granules where it colocalizes with RNA helicase Vasa and other proteins involved in piRNAs-mediated silencing. Nevertheless, the loss of Orb2 does not disrupt piRNA-silencing and germinal granules. Loss of Orb2 causes meiosis arrest, approximately 60% of cysts are blocked in G2-phase, however, remaining 40% ones enter meiosis, but they fail to approach metaphase. Such arrests lead to the formation of two types of pseudospermatid: tetraploid pseudospermatids and pseudospermatids containing karyomeres – individual chromosome territories, surrounded by the nuclear lamina. In addition, we found using Western-blot analysis that a violation of Orb2 expression led to the accumulation of M-phase cyclines and other CPEB – Orb in the testes, without changing the level of their transcripts. We have shown for the first time a decreasing of polyadenilation of tails of M-phase cyclines mRNAs in mutants with the loss of Orb2 using PAT assay. Thus, in this study we investigated the intracellular localization of Orb2 in spermatocytes of Drosophila melanogaster and we showed that it is important for the transition through two phases of meiosis: prophase and metaphase. We founded an accumulation of M-phase cyclines and the decreasing of polyadenylation level of their transcripts in the testes with the loss of Orb2.

Heterologous expression and purification of avicin А, the antimicrobial peptide from Enterococcus avium E. K.-A. Nurmukhamedova, E.I. Finkina, T.V. Ovchinnikova M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Bacteriocins are ribosomally synthesized antimicrobial peptides and proteins, produced by Gram-negative and Gram-positive bacteria. They kill strains of closely related species providing their producers a competitive advantage in the struggle for the vital substrates. Bacteriocins have a narrow spectrum of antimicrobial activity displayed at nanomolar concentrations. That is why bacteriocins are proposed to be used as food preservatives or selective antimicrobials which inhibit pathogens but do not affect the normal flora. Earlier a new pediocin-like bacteriocin, termed avicin A (AvcA), was found in Enterococcus avium. This peptide consists of 43 amino acid residues, has molecular mass of 4290.74 Da and shows strong antimicrobial activity against Gram-positive bacteria, including Listeria monocytogenes. The aim of this study was the construction of heterologous expression system for avicin A production. The expression plasmid pET-His8-TrxL-Avc A was constructed with the use of pET32a (Novagen). The recombinant avicin A was expressed in E. coli BL-21 Star cells under control of the T7 promoter as the fusion protein contained the N-terminal histidine octamer and the thioredoxin A carrier protein. The BL-21 Star cells transformed with pET-His8-TrxL-Avc A were grown at 30°C in LB medium. Purification of the recombinant avicin A involved its isolation by immobilized metal chelate affinity chromatography (IMAC), dialyses with subsequent CNBr cleavage of the fusion protein His8-TrxL-Avc A, repeated IMAC and final reversed-phase HPLC. Homogeneity of the obtained recombinant avicin A and its identity to the natural peptide were demonstrated by SDS-PAGE, MALDI-TOF mass spectrometry, and N- terminal protein sequencing.

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Beta-hairpin host defense peptides, a template for the design of novel antimicrobials P.V. Panteleev, I.A. Bolosov, S.V. Balandin, T.V. Ovchinnikova M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Widespread use of antibiotics has given rise to a number of problems, the main one being the selection and extension of drug resistant pathogenic microorganisms. To overcome the current trend it is essential to search for new antimicrobial substances that have fundamentally different mechanisms of action in comparison to the conventionally used antibiotics. One class of such compounds are host-defense antimicrobial peptides (AMPs) – the key molecular factors of innate immunity of most multicellular organisms, including humans. The membrane-targeting mechanism of action and the ability to rapidly kill pathogens prevent the evolving of resistance to AMPs. Among the most active and stable to proteases AMPs of animal origin are the molecules that adopt a β-hairpin conformation stabilized by disulfide bridges. However, the relatively high cytotoxicity of natural peptides limits their application. Using methods of directed mutagenesis and heterologous expression in a bacterial system in this work we have obtained a wide range of mutant analogs of highly active β-hairpin AMPs: arenicin from polychaeta Arenicola marina and tachyplesin from horseshoe crab Tachypleus tridentantus. The mutations were introduced by amplification of expression plasmids using inverse PCR with mutagenic primers. The above mentioned AMPs are characterized by high content (about 50%) of hydrophobic amino acid residues, and also by considerable cytotoxic effect. It’s known that high hydrophobicity adversely affects the membrane selectivity of AMP, promoting interactions with zwitterionic phospholipids, thereby exerting toxicity against normal mammalian cells. The comparative evaluation of the impact of different hydrophobic amino acid substitutions on the level of antimicrobial and cytotoxic activity has been carried out. A number of positions in the polypeptide chain have been revealed where hydrophobic residues exert the greatest influence on the therapeutic index of arenicin and tachyplesin, and low-toxic analogs of these peptides have been produced. The work is supported by the Russian Science Foundation (the agreement No. 14-14-01036).

An influence of antimicrobial peptides on the functional activity of natural killer cells from patients with multiple myeloma T. Pazina1,2, K.S. Campbell2, O.V. Shamova1 1Institute of Experimental Medicine, RAMS, St-Petersburg, Russia; 2 Fox Chase Cancer Center, Philadelphia, USA Multiple myeloma (MM) is a malignancy of plasma cells. It is characterized by a monoclonal expansion of malignant plasma cells in bone marrow, accumulation of a monoclonal antibody produced by these plasma cells in blood, and occurrence of end-stage organ damage such as bone lesions, anemia and renal disease. The average survival of patients is 3–4 years, but with the currently available treatment (immunomodulatory drugs and proteasome inhibitors) the survival might be improved to 7–8 years. Antimicrobial peptides (AMPs) are the key components of innate immunity and are found among all classes of life. They play a critical role in prevention of invading microbial pathogens. There is an increased interest in developing antimicrobial peptides into anticancer peptides. Some AMPs including magainins, cecropins, and defensins were shown to have anticancer effects. They have varied mechanisms of malignant cell killing: in part due to the rapid disruption of cell membranes and in some cases by inducing apoptosis in target cells. In addition to killing bacteria/tumor cells directly AMPs exert a number of immunomodulatory functions: they act as chemokines and/or induce chemokine production, modulate dendritic cell responses and the functional activity of some other cells of the adaptive immunity, alter host gene expression. Considering the anticancer and immunomodulatory effects of AMPs we suggested that the peptides may serve as promising therapeutic agents for multiple myeloma treatment. It is known that natural killer (NK) cells are the key molecules in the antitumor host defense. In MM patients the functional activity of NK cells is impaired. We hypothesize that AMPs may have immunomodulatory activity toward NK cells. The goal of our work was an investigation of the influence of AMPs on the functional activity of NK cells from patients with MM. The effect of cathelecidin LL-37 and protegrin PG-1 on freshly isolated human peripheral blood lymphocytes from MM patients was studied. We incubated lymphocytes with various concentrations of peptides for 24 hrs and measured CD69 (early activation marker) expression using flow cytometry and fluorescent antibodies specific to lymphocyte markers. The activation of NK cells induces expression of CD69. NK cells were identified as CD3-CD56+ lymphocytes. We have shown that the level of CD69 was increased by 40% on the NK cells incubated with LL-37 as well as with PG-1 in comparison with untreated control. The molecular mechanisms of the observed effects will be studied in our further research. The obtained data support the idea that AMPs may serve as new therapeutic modalities for MM treatment.

Method for analysis of nonsense-mediated mRNA decay with fluorescent proteins A.P. Pereverzev, N.G. Gurskaya, N.M. Markina, E.I. Kudryavtseva, K.A. Lukyanov M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Nonsense-mediated mRNA decay (NMD) is a mechanism of degradation of mRNA species with a premature termination codon (PTC). mRNAs with PTC appear as a result of various events – gene mutations, alternative splicing, DNA rearrangements in immune cells, and others. In addition, many normal transcripts carry stop codons which are recognized as PTC and thus are targeted to NMD. Thus, NMD is not only a mechanism of cleaning but has an important role in global regulation of gene expression. NMD efficiency in a particular model can be evaluated by classical methods of RNA quantification. In addition, luciferase- or GFP-based reporter systems were designed to assess NMD efficiency by luminescence or fluorescence. Dual luciferase assay enables accurate quantification of the NMD-targeted transcript (which encodes Renilla luciferase) relatively to the control firefly luciferase-encoding transcript. However, the luminescence-based approach can not be used at single cell level. In contrast, GFP-based reporter makes it possible to assess NMD in individual cells by fluorescence microscopy and flow cytometry. At the same time, this reporter relies on single readout only (GFP green fluorescence) that does not allow applying corrections for gene expression level. Here, we developed a new NMD reporter system for quantitative evaluation of splicing-dependent NMD efficiency at single cell level by fluorescence microscopy and flow cytometry. It utilizes two fluorescent proteins, one of which is encoded by NMD-targeted transcript and the other serves as a control of expression efficiency. We tested the proposed method on mammalian cells and found lines with high (HEK293T and HeLa) and low (MEF and mouse ES cells) NMD efficiency. Generally, this agrees well with previously reported considerable difference in NMD efficiency in different cell lines. Moreover, dual-color analysis revealed that under certain conditions cells can display considerable heterogeneity even in the same dish. We proposed that decrease of NMD efficiency can result from cellular stress in areas with high cell density. It should be noted that such cell heterogeneity can not be detected by previous methods of analysis. This work was supported by Russian Foundation for Basic Research (12-04-00994-а).

X-ray study of green fluorescent protein WacCFP2. Bathochromic shift as a result of anionic state of the tryptophan- based chromophore N.V. Pletneva, K. Sarkisyan, E.A. Goryacheva, A. Mishin, K.A. Lukyanov, V.Z. Pletnev M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Fluorescent proteins (FPs) established themselves as efficient, noninvasive molecular instruments in cell biology and biomedicine, used for visualization and monitoring of internal processes within cells and whole organisms. Design of the new advanced biomarkers is strongly supported by X-ray studies which

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provide valuable information on relationship between structure and properties. The pH-dependent cyan/green-emitting (λem ~475/502 nm) fluorescent variant WasCFP2 with the tryptophan-based chromophore (Thr65-Trp66-Gly67) was developed from the cyan mCerulean introducing 18 point mutations with the key substitution Val61Lys nearby chromophore. WasCFP2 exhibits the dominant bright green fluorescence at physiological and higher pH and the weak cyan fluorescence at low pH. The main advantages for its practical application is the extremely high fluorescence lifetime and high quantum yield of the green form which could make it potentially useful tag for fluorescence lifetime imaging microscopy (FLIM), as well as excellent donor for fluorescence resonance energy transfer (FRET). The crystal structures of WasCFP2 at pH ~9.0 and pH ~4.8 have been determined at 1.35 and 1.18 Å resolution, respectively. The structure at high pH suggests the presence of the TWG chromophore in anionic state which is suggested to be responsible for the observed green fluorescence. At all pH the chromophore Trp66 exists mainly incis form with only ~20% contamination of trans form at low pH ~4.8. The key residue Lys61 proximal to chromophore demonstrates two major pH dependent conformations. At high pH, its side chain mostly extends towards chromophore mediating H-bonding linkage between indole nitrogen of Trp66 and the side chain of the proton acceptor Glu222 and, thus, promoting the Trp66 deprotonation. At low pH the side chain of Lys61 is directed apart from Trp66 making H-bond with Gln207 and the vacant mediating position of its ε-amino group near chromophore is occupied by the water molecule.

Open software development for T-cell receptor repertoire data analysis M.V. Pogorelyy1, V.I. Nazarov1,2, E.A. Komech1, I.V. Zvyagin1, I.Z. Mamedov1, Y.B. Lebedev1 1M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia; 2National Research University Higher School of Economics, Moscow, Russia Immune system interacts with great diversity of pathogens. Receptors involved in antigen recognition – BCR (B-cell receptors) and TCR (T-cell receptors) – are not encoded in genome due to its limited capacity, but generated by V(D)J recombination. Over a considerable period of time, no instrument to evaluate TCR diversity in individual organism has been proposed. Next generation sequencing methods (NGS) have created the possibility for deep TCR profiling. NGS generates huge amount of data – hundreds million short reads, hence it requires development of a new data analysis software. A software package for processing raw TCR sequencing data called MiTCR was introduced last year. It performs extraction of TCRs functional regions from raw reads and sequencing error correction. Here we introduce tcR, a new R package for MiTCR data analysis. tcR subroutines include, but not limited to, TCR segment usage comparison, customizable search of shared among repertoires clonotypes, spectratyping, random TCR data generation. It also includes various diversity measures, procedures for sequence analysis, clustering and different plotting tools. Using the package, researcher has access to the most of the known TCR repertoire analysis methods. tcR was used for data analysis in two our papers. The package is open-source, well documented and could be easily integrated with other data analysis tools written in R.

New silicone substrates for silicateins N.V. Povarova, K.А. Lukyanov, V.B. Kozhemyako M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Silicateins are the family of proteins, which play a major role in the formation of silica skeletal structures in marine sponges. Silicateins catalyze formation of amorphous silica from silicon-containing precursors under physiological conditions. These proteins are promising candidates for applications in biotechnology; new approaches for the formations of particles and surfaces different in shape and size using silicateins intensively are developing nowadays. The most popular silicon-containing substrate for silicateins is tetraethyl orthosilicate (TEOS). However, it has significant disadvantage: TEOS is insoluble in aqueous solution and forms emulsion, so it is poorly available for the protein. Here we tested interaction of the silicatein A1 from the sponge Latruncula oparinae and three silicone substrates – tetrakis(2-hydroxyethyl)orthosilicate (THEOS), bis(glycerol)orthosilicate (BGS) and tris(catechol) orthosilicate (TCS). All these substances are highly soluble in water. The use of BGS and THEOS as silicatein’s substrates was analysed for the first time. We characterized stability of the substrates in aqueous solution at neutral pH, efficiency of reaction with silicatein, and size of formed amorphous silica particles. Toxicity of THEOS, BGS and TCS for the mammalian cells was examined. We concluded that BGS and THEOS are useful substrates for silicateins.

Study of the 3D structure of bacteriophage T5 L,D-peptidase by high resolution NMR D.A. Prokhorov1, N.V. Molochkov1, G.V. Mikoulinskaia2, V.P. Kutyshenko1 1Institute of Theoretical and Experimental Biophysics RAS, Pushchino, Moscow Region, Russia; 2Branch of M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Pushchino, Moscow Region, Russia L-alanoyl-D-glutamate peptidase of virulent coliphage T5 (Endo T5) is peptidoglycan hydrolyzing enzyme specific to murein of Gram-negative bacteria. We have shown earlier that it is related to zinc metallopeptidases of M15 family and activated by Са2+ or Mn2+. All other known peptidoglycan hydrolases of this L,D-class are specific to murein of Gram-positive bacteria; they have modular organization and contain two domains – catalytic and cell wall binding one. Endo T5, as a majority of endolysins of lytic bacteriophages infecting Gram-negative hosts, has only one domain, combining catalytic and targeting functions. Because of difficulties with the crystallization of this class proteins up to date the only three-dimensional structure – of Listeria moderate phage L,D-peptidase Ply500 catalytic domain – was solved by crystallization with consequent X-ray diffraction analysis. Now we determined the spatial structure of the Endo T5 in presence of Zn2+ using high resolution NMR. The main structural domain contains three conservative α-helices (a.a. 7–14, 20–30, 87–104) stabilized by antiparallel β-sheet (a.a. 36–40, 71–75), joint with catalytic loop (a.a. 41–70). Cata- lytic loop contains α-helix (a.a. 44–51) and 310-helix (a.a. 66–68) with conservative His 66 inside. This residue together with Asp 73 and His 133 form Zn2+-binding site homologous to one of Ply500. α-helix (40%) and β-strands (9%) content agree with circular dihroism (CD) data. Apoenzyme according to CD contains 30% α-helical and 21% β-stranded structures. Occurence of Zn2+ leads to whole protein structure stabilization. Generally, EndoT5 in presence of Zn2+ could be characterized as relatively compact protein with strong intramolecular mobility.

Structure and functional aspects of the yeast alternative oxidase A.G. Rogov, E.I. Suchanova, R.A. Zvyagilskaya A.N. Bach Institute of Biochemistry of RAS, Moscow, Russia Most if not all the aerobic yeasts, in addition to the main cytochrome respiration chain, contain the cyanide-resistant alternative oxidase (AOX) branching from the main respiratory chain at the level of ubiquinol and is not implicated in energy conservation. The findings on the origin and biogenesis of AOX were sum up with special references on the peculiarities of the yeast AOX differing it from counterparts from plants. Data were collected on structure of AOX and its active centers, as well on structures of other well-known di-iron proteins. Using bioinformatics approaches, the gene sequences encoding AOX in the yeast Yarrowia lipolytica were identified. With these identified sequences and literature data on recently obtained x-ray structure of the AOX from Trypanosoma brucei [1], the spatial structural model of the yeast AOX was constructed, to our knowledge, for the first time. We succeed to recognize the location of the di-iron cluster and the ubiquinol molecule within the protein and to define

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the orientation of amino acid residues coordinating them. Also location of the ADP binding site in the yeast AOX during its activation was suggested. The constructed model was optimized by molecular dynamic simulations. This work was financially supported by RFBR (grant № 13-04-01530) and by the Presidium of RAS, Program on “Molecular and cellular biology”.

[1] Shiba, T., Kido, Y., Sakamoto, K., Inaoka, D.K., Tsuge, C., Tatsumi, R., Takahashi, G., Balogun, E.O., Nara, T., Aoki, T., Honma, T., Tanaka, A., Inoue, M., Matsuoka, S., Saimoto, H., Moore, A.L., Harada, S., Kita, K. 2013. Structure of the trypanosome cyanide-insensitive alternative oxidase. Proc. Natl. Acad. Sci. U S A, 110(12), 4580-4585.

Analysis of the immunogenicity of hepatitis B surface antigen synthesized in transgenic marker-free potato plants E.B. Rukavtsova, E.N. Puchko, N.S. Zakharchenko, N.V. Rudenko, Ya.I. Buryanov Branch of M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Pushchino, Moscow Region, Russia Transgenic plants are promising for the production of inexpensive and safe vaccines compared to traditional producers. In our work the marker-free transgenic potato plants synthesizing hepatitis B surface antigen (HBsAg) at the level up to 1 µg/g of raw tubers weight were obtained. Western blot analysis of plant-produced HBsAg was performed following its immunoaffinity purification. The molecular weight of purified potato-derived HBsAg is approximately 24 kD, which corresponds to a molecular weight of the main envelope protein of hepatitis B virus. Existence of a multimeric form of HBsAg in transgenic potato plants was demonstrated in the experiments on analytical gel filtration of the HBsAg. The HBsAg was detected in a narrow elution zone of high molecular weight compounds with molecular weights of approximately 2000 kD, which confirms formation in transgenic plant cells HBsAg multimers including not less than 70–80 monomers. Our data of analytical gel filtration of HBsAg synthesized by transgenic potato cells correspond to sedimentation and electron microscopy characteristics of antigen extracted from transgenic tobacco cells by other authors. During gel filtration, HBsAg is eluted in the zone, which is free from the presence of other proteins, which makes the gel filtration technique an efficient stage in the large-scale production of hepatitis B vaccine based on transgenic plants. The immunogenicity of HBsAg which produced in transgenic potato tubers was investigated on outbred mice NMRI. It was shown a significant increase in the number of antibodies to HBsAg in blood serum of immunized animals. Protective level of antibodies was maintained in the blood of the mice for more than a year after immunization. The data obtained show the prospects of using transgenic plants as a substance for the production of safe edible vaccine against hepatitis B virus. To date, no monitoring data of the immunity to hepatitis B virus of such duration and using transgenic plants as edible vaccine were performed. The obtained data confirm the promising way of transgenic marker-free plants use as a substance for the production of edible vaccines against hepatitis B. This study was supported by the Russian Foundation for Basic Research (project No 11-08-00413).

In vitro study effective combination antifungal drugs and enzymes of soil bacteria against resistant strains Candida albicans N.P. Sachivkina Peoples’ Friendship University, Moscow, Russia Purpose. Experimental study of the possible increasing antimycotics activity against drug-resistant fungi of the genus Candida using exoenzymes Cellulo- monas cellulans (E). Materials and Methods. Were examined 50 patients with chronic laryngitis in age from 21 to 66 years, some of them had a fungal nature of the disease – 37 people (74%). It is established that in the majority of pathogens fungal laryngitis (95%) were Candida albicans. In our experience, we took 12 out of 37 (32%) clinical isolates of Candida which have shown resistance to nystatin. The study was conducted in vitro, using the method of serial dilutions in microplates. Experiment was repeated three times. The control series did not contain a biological product E, but contained breeding nystatin suspension and resistant strains of Candida albicans. In the experimental series enzyme was added. Method of seeding wells with dilutions determined fungistatic and fungicidal action of antimycotics with or without E. Results. Our study found recovery effect of nystatin therapeutic action on yeast-like fungi Candida in the presence of E. Antifungal nystatin activity against clinical strains of C. albicans was increased in 2 times in the result of the application. In addition to restore sensitivity of Candida to antimycotics, also we saw a reduction in its current minimum concentration, in other words, the same effect was achieved at a lower dose of nystatin. Apparently, under the action of E changes happend in the cell wall of Candida albicans, and nystatin bioavailability increased, blocked the mechanisms of fungi resistance, thus providing therapeutic drug effect. Conclusions. Effective combinations of modern antimycotics and bacterial exoenzymes, which lyse fungi cell, potentially have high economic value. With their help it is possible to return to a life drugs for which there are resistance clinical strains, which may become one of the important directions in the fight against multidrug-resistant infections.

Investigation of the antimicrobial and antitumor activity of caprine bactenecin and its combined action with other antibiotic compounds O.V. Shamova1,2, M.S. Zharkova1, V.N. Kokryakov1,2, D.S. Orlov1,2 1Institute of Experimental Medicine, RAMS, St-Petersburg, Russia; 2Saint-Petersburg State University, St-Petersburg, Russia Antimicrobial peptides (AMPs) are stored in defensive cells of the host and provide its resistance to infections as well as participate in a variety of host defense reactions exerting immunomodulatory, antitumor activities and a spectrum of other biological effects. At present time AMPs are considering as promising templates for a development of novel anti-infective or anticancer drugs. An investigation of a mode of action of structurally different AMPs is an important goal of biological and medical research since provides a valuable information for a future design of AMPs synthetic variants with optimized for a practical usage properties. Another promising approach, directed to the optimizing of AMPs capabilities and decreasing their effective doses, is a search of combinations of the peptides with other antibiotic compounds aiming to revealing their synergistic effects, in particular the synergistic antimicrobial action. The current research was concentrated on the exploration of the antimicrobial and antitumor activity of a proline-rich peptide bactenecin ChBac3.4 that we previously have isolated from caprine leukocytes. It was shown that ChBac3.4 possessed the marked antimicobial activity towards Gram-negative bacteria including drug-resistant clinical isolates. In contrast to most of proline-rich peptides (PRPs) ChBac3.4 also demonstrated a significant activity against Gram-positive strains including some strains of staphylococci. The peptide also showed more distinct, in comparison with the typical PR|Ps, ability for damaging a cytoplasmic bacterial membrane. We have studied a combined action of ChBac3.4 with an array of conventional antibiotics and revealed the synergistic antimicrobial action upon an application of the peptide in combination with rifampicin, oxacillin ofloxacin. Unlike other PRPs, ChBac3.4 exerted a cytotoxic activity towards cultured tumor cells. In the range of concentrations of 10–20 microM the peptide induced apoptosis in target cells, while upon the concentrations exceeded 40 microM – necrosis. The obtained data point to the promising direction of the future research devoted to a design of synthetic variants of the caprine bactenecin ChBac3.4 for a future development of novel antibacterial or antitumor drugs. The work was supported by the RFBR grant № 13-04-02102а; 12-04-01573а.

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Characterization of proteolytic enzymes secreted by Fusarium anguioides micromycetes I.L. Shamraychuk1,2, M.A. Belozersky2, Y.E. Dunaevsky2 Moscow State University, Moscow, Russia; 2A.N. Belozersky Research Institute of Physico-Chemical Biology MSU, Moscow, Russia Microbial proteolytic enzymes are of great interest for their application in numerous industries. Fungal proteases may be preferred to the enzymes from bacteria, however, proteolytic activity of many species of mycelial micromycetes, including those belonging to Fusarium genus, remains poorly investigated. In the present work, secretion of the proteases with activity to the synthetic substrates for trypsin, subtilisin, phenylalanyl and leucyl aminopeptidases by the strains of Fusarium anguioides was shown. Total proteolytic activity of the studied micromycetes was detected under acidic, neutral and alkaline conditions; this fact indicates the broad spectrum of pH-activity of their extracellular proteases. The highest activity was achieved in alkaline, the lowest – in acidic conditions. Growth stage-dependent protease activity of F. anguioides was evaluated. Proteolytic enzymes were separated by ion-exchange chromatography on Mono Q and gel filtration on Superdex 200 FPLC. The characterization of extracellular proteolytic enzymes of F. anguioides and the data from comparative analysis of their activity and secretion by the studied strains expand understanding of the properties of proteolytic system of the given species, and also may contribute in assessment of biotechnological potential of F. anguioides strains. The work was supported by Russian Foundation for Basic Research (grants 04-12-01506 and 04-13-00970).

Preparation and study of the conjugate of recombinant histon H1 With photoactivated fluorescent dye M.N. Shaposhnikov1, S.Yu. Zaitsev1, A.A. Rizvanov2 1Moscow State Academy of Veterinary Medicine and Biotechnology, Moscow, Russia; 2Kazan (Volga Region) Federal University, Kazan, Russia Histone H1 is a cationic protein, capable of effectively penetration via the cytoplasmic membrane. Recombinant histone H1 can be used as a carrier for delivery of the drugs and nucleic acids into cells (histonefection). The aim of this study was to prepare and study of the conjugate of recombinant histone H1.3 and photoactivated fluorescent dye (PFD) on different cell cultures in vitro. The conjugate of histone H1.3 (provided of “Human Stem Cell Institute”) with PFD (tetramethylrhodamine derivative [1]), capable of bright fluorescence in the red region of the spectrum (after photoactivation [1]) was synthesized in our work. Succinimide group of PFD promotes formation of the covalent bonds with primary amino groups (lysine and arginine residues) of histone H1 to form the conjugate “Histone H1-PFD”. Details of the synthesis and characterization of the conjugate described [2]. The histone H1.3 and conjugate “Histone H1.3- PFD” are not toxic to the cell culture HeLa in amounts up to 0.25 mg per ml. The data on transportation and intracellular localization of the conjugate were obtained with the cell lines: HEK293, A431, HeLa, HBL-100 and MDCK (incubated for 1 hour with 30 μg/ml “Histone H1-PFD”) [1, 2]. The micrographs of the “native” and fixed cells were obtained in order to evaluate the intracellular distribution of the conjugate using the confocal microscope. The blue nuclear (Hoechst 33342) and green mitochondrial (MitoTracker®Green FM) dyes were used for better visualization of cell conjugate “Histone H1-PFD” and free PFD as controls. Thus, the resulting conjugate can be used for further study of drug delivery systems and nucleic acids into cells. The reported study was supported by RFBR research projects № 14-03-00154 and № 13-04-97099.

1. S.Yu. Zaitsev, M.N. Shaposhnikov, D.O. Solovyeva, A.A. Rizvanov / World Applied Sciences Journal. 2013. - V. 26 (6). - P 712-718. 2. S.Yu. Zaitsev, M.N. Shaposhnikov, D.O. Solovyeva, I.S. Zaitsev, D. Mobius /Cell Biochemistry and Biophysics, 2014 in press.

Cloning and expression in Escherichia coli of translationally fused protein DNA-methgyltransferase HhaI-EGFP T. Shevchuk, S. Tarlachkov, O. Dyachenko, N. Rudenko, Ya. Buryanov Branch of M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Pushchino, Moscow Region, Russia Translationaly fused proteins are widely used in molecular and cell biology as a convenient tool. Green fluorescent protein (GFP) of jellyfish Aequoria victoria is able to fluorescence in wide range of physiological conditions. At the same time it admits binding of other proteins retaining spectral characteristics unchanged. Thereby this allows observation of fused protein molecule in in vitro and in vivo conditions in real-time mode. We constructed, isolated and characterized translationaly fused protein М.HhaI-EGFP of CpG-methylation type. Being isolated from E.coli, plasmid expression vector carrying M.HhaI-EGFP fused protein demonstrated resistance to HhaI endonuclease hydrolysis, confirming M.HhaI-EGFP specific DNA-methylation activity in vivo. Homogenous preparation of M.HhaI-EGFP showed its activity on pBluescript by protection against HhaI endonuclease hydrolysis thus proving in vitro test of complete CGCG sites methylation. Spectroscopy studies showed minor distinctions of fluorescent characteristics between individual EGFP and fused M.HhaI-EGFP. The data presented demonstrate that fused M.HhaI-EGFP protein retained unaltered fluorescent and DNA-methyltransferase activities of parent molecules. Taken together this shows that fused protein M.HhaI-EGFP could be used in molecular biology experiments as analytical tool. Current work was supported by RFFR grant №14-08-00592

Integrated automated local genebank of human genome A.G. Shlikht, N.V. Kramorenko Far Eastern Federal University, Vladivostok, Russia The aim of reseach is to create a local genebank to improve performance when you work with the human genome. Genebank of the human genome is based on relational databases and is integrated through a system of hyperlinks with world-wide portals (NCBI, EBI, KEGG, OMIM, etc.). This approach provides a comprehensive analysis of human genes in a single query. In the local genebank store ontologically structured information on genome, transcriptome, exome, proteome, metabolome, diseases, enzymes, SNP, biomarkers, primers, integrated with the genes with related enzymes reaction and subsequent construction of expert systems and dynamic process models. The genebank stores a fully indexed DNA (GRCh38 Primary Assembly). Genebank data are not presented in horizontal text and vertically in the form of indexed entries in the database tables until a specific nucleotide numbering in the gene and amino acid in the protein. Metabolites and enzymes in the reactions are presented in the form of multiple indexed records database that allows you to automatically carry out numerous search and analysis a genome- wide correlation, proteome, metabolome. Inside the local part is automatic on all components of the system with an external access to the interactive world provides portals. Automatic program mode offers excellent performance and comprehensive analysis of genes, transcripts, introns, exons, encoded and encrypted part of the genome with database and knowledge base technology and statistical methods. This allows you to efficiently use the genebank to meet the challenges of the GENOTYPE-ENVIRONMENT-PHENOTYPE [1]. Created local genebank can serve as a portable tool (that is placed in the notebook, the size of the local indexed human genebank order 100 GB) for professionals as a source of gene-centric data to carry out research in the field of translational medicine, interpretation data, diagnosis, phylogenesis.

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Influence of deacetylation degree on chitosan molecular-mass characteristics S.V. Sizova1, A.A. Zubareva2, T.S. Shcherbinina2, V.P. Zubov1, V.A. Oleinikov1, E.V. Svirshchevskaya1 1M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia; 2Centre «Bioengineering», RAS, Moscow, Russia Chitosan is a N-deacetilated derivative of natural polysacharide chitin. A large number of reactive groups, the difference in the deacetylation degree (DD), polydispersity, and the ability to associate in aqueous solutions present difficulties in the characterization of chitosan molecular weight (MW). Average DD values affect on the solubility of chitosan in aqueous solutions, its ability to biodegradation and determine the biological activity of chitosan. The aim of this work was to analyze the effect of DD on the MW of chitosan, determined by different methods. For this purpose, we acetylated chitosan with 200 kDa MW and 89% DD (Chi1) in mild conditions (different concentrations of acetic anhydride, 5 min, RT) to obtain its derivatives with DD 50% and 40% respectively (Chi2 and Chi3) as was determined by 1H-NMR. MWs of Chi1-3 were determined by: i) Ubbelohde capillary viscometry (VU); ii) dynamic viscometry (DV); and a new method of asymmetrical flow field-flow fractionation (FFF) equipped with static light scattering (MALS) system. The MWs of the chitosan samples Chi1-3 determined by VU method were 177, 130 and 110 kDa, respectively. Viscosity values, obtained with DV method, were 11.2, 9.6 and 8.2 cP, which agreed well with the VU data with correlation coefficient (r) 0.99. When analyzed by FFF, the retention times of the first peaks (minus the void peak) which corresponded to the molecular fraction of chitosan, were 16, 14 and 10 min respectively, which also agreed directly with the VU data (r=0.97) and DV data (r=0.98). We have shown that the DD directly affects the conformation of chitosan molecules in solution leading to changes in the MW of chitosan determined by experimental methods. Data obtained by all the methods used to determine MM chitosan were in a good agreement. It is important to take into account the effect of DD on the chitosan conformation which can significantly modify biological effects. This work was supported by President Grants for Government Support of Young Russian Scientist, 2012-2014 (No. 90-2012-4).

Expression of short PIWIL2 isoforms in testicular germ cell tumors Yu.V. Skvortsova, I.V. Gainetdinov, S.A. Kondratieva, E.A. Stukacheva, T.L. Azhikina M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia PIWI family proteins appear to be essential contributors in numerous biological processes including germ cell development, stem cell maintenance and epigenetic reprogramming. Expression of some of the family members has been shown to be elevated in tumors. In particular, PIWIL2 has been routinely probed as a potential neoplasia biomarker in many cancers in humans. Previously, PIWIL2 was shown to be expressed in most tumors as a set of its shorter isoforms. In this work, we demonstrated the presence of its 60kDa (PL2L60) and 80kDa (PL2L80) isoforms in testicular cancer cell lines. We also identified the transcriptional boundaries of mRNAs and in vivo alternative promoter regions for these isoforms. Additionally, we found the 60kDa PIWIL2 isoform (PL2L60) to be predominantly expressed across testicular tumor samples. Importantly, the abundance of PL2L60 was found to vary across a range of differentiation stages on both mRNA and protein levels, i. e., PL2L60 isoform is more specific to undifferentiated testicular tumors. This pattern was further validated in the model system of retinoic acid induced differentiation in NT2/D1 cell line. Therefore, both PL2L60 mRNA and protein levels could serve as a marker distinguishing between highly curable undifferentiated seminomas and malignant non-seminomatous tumors with less favourable prognosis. The work was supported by the Program of the Presidium of the Russian Academy of Sciences “Molecular and Cellular Biology” and Russian Foundation of Basic Research grants 11-04-12071 and 14-04-32314.

Aggregation of human recombinant insulin induced by arginine and arginine-containing peptides: morphological characteristics of the aggregates E.Yu. Smirnova, B.Ya. Gurvits A.N. Bach Institute of Biochemistry, RAS, Moscow, Russia Protein aggregation represents a special tool in biomedicine and biotechnology to produce biological materials for a wide range of applications. The protein aggregates are very different morphologically varying from soluble amorphous particles to highly ordered amyloid-like fibrils. Fabrication of novel biomaterials resembling natural protein assemblies has awakened interest in identification of low-molecular weight biogenic agents as regulators of transformation of aggregation-prone proteins into supramolecular structures. Amino acid L-arginine (Arg) widely known as a chaperone-like agent effective in suppressing aggregation of partially folded proteins can be considered for this role. Paradoxically, in the present study, using dynamic light scattering, atomic force- and transmission electron microscopy and other methods, we have demonstrated a striking potential of Arg, the Arg-Phe dipeptide and the peptide fragment of human adrenocorticotropic hormone ACTH (1–24) containing 8 positively charged amino acid residues to generate aggregation and formation of hetero- genic supramolecular structures of a partially folded model protein, human recombinant insulin. Along with small-sized asymmetric granular units of 3–8 nm in apparent diameter, being associated into short chains of 20–50 nm in length, the larger chains of 100–200 nm were formed. This phenomenon was revealed under environment conditions where the aggregation of insulin taken alone was not observed. It is assumed that the effectors can induce changes in the physicochemical properties of an oppositely charged protein resulting in accumulation of ligand–protein complexes competent to self-assembly into species profoundly differing from those of the individual protein in type and size. As Arg and the peptides are inherent constituents of biological systems, they can potentially alter the aggregation behavior of proteins in vivo. The research is supported by the grants from the Presidium of the Russian Academy of Sciences, Program “Molecular and Cellular Biology” and the Russian Foundation for Basic Research, 14-04-01530-a.

Study of the contribution of catalytic properties of immunoglobulins to pathogenesis of schizophrenia L. Smirnova, V. Buneva, D. Parshukova, S. Ivanova, A. Semke, N. Fattahov, G. Nevinsky Mental Health Research Institute, RAMS, Tomsk, Russia; Institute of Chemical Biology and Fundamental Medicine, SB RAS, Novosibirsk, Russia Phenomenon of presence of catalytic properties in immunoglobulins has been actively investigated. Research of catalytic properties of antibodies in schizophrenic patients with various clinical manifestations of the illness has not been carried out previously. There is the observed dysregulation of schizophrenia between the nervousand immune systems, the causes of which may be changes in brain structure and dysfunction of immune cells. DNase activity of antibodies was identified according to degree of hydrolysis of supercoiled form of DNA of plasmid pBluescript per unit of time. Proteolytic activity was assessed according to degree of hydrolysis of basic protein myelin and its peptides. To study DNase and proteolytic activity of immunoglobulins G isolated from serum of blood of patients with schizophrenia. It has been shown that catalytic activity is an own property of antibodies. To assign the detectable catalytic activity directly to the antibodies was carried out a number of strict criteria: electrophoretic homogeneity of antibodies, gel-filtration in conditions «pH shock. We have identified that IgG of schizophrenic patients possess a higher (0.392±0.19 nM DNA /mgAB/h), than in healthy persons (0.029±0.06 nM DNA/mgAB/h) specific DNAse activity. Specific substrates for proteolysis by IgG from patients with schizophrenia are collagen, Human Serum Albumin (HAS), myelin basic protein (MBP). It has been found that proteolytic hydrolysis MBP of serum of blood of schizophrenic patients is 5 times as high as indices of hydrolysis in healthy persons. It has been revealed that patients with predominant negative symptoms show maximum high percent of protease hydrolysis MBP 66.9%, different from patients with leading positive symptoms (13.9%). Inhibition analysis of proteolysis toward 21 peptides MBP has shown that IgG from patients with schizophrenia are characterized by the type serineprotease activity.

SPECIAL ISSUE № 1 2014 | Acta naturae | 41 Oral and Poster Presentations

Increase of DNA-hydrolyzing properties of Ig G is likely to be associated with great number of extracellular DNA. Detection of high values of proteolytic activity may be associated with occurring in serum of blood of patients of large number of destructed or damaged proteins. Support by Grant of RSF № 14-15-00480 «The search for biomarkers of socially significant endogenous mental disorders»2014-2016.

Searching bacteriocin-like peptides in cell culture of lactobacillus plantarum 8PA-3 A.V. Soboleva, A.A. Kolobov, T.V. Grishina Saint-Petersburg State University, Saint-Petersburg, Russia There is a great interest last years to probiotic lactic-acid bacteria which are, because of their non-hazardous, high enzymatic and antimicrobial activity, in the focus of the interest of fundamental scientific investigations. Bacteriocins are bacterial antimicrobial peptides and proteins. Bacreriocins – bacterial antimicrobial proteins and peptides can be considered to be potential antimicrobial and antifungal agents which regulate bacterial population growth and provide colonization stability of human body. Sequencing of genome of commercial probiotic strain Lасtobacillus plantarum 8PA-3 found a locus that can be responsible for synthesis of two bacteriocins EF and NC8. For choosing an optimal method of purifying fractions of bacteiocins literature data analysis was performed. Two methods of purifying bacteriocins mentioned in this work are Mota-Meira’s method (Mota-Meira M. et al., 1997) and Todorov’s method (Todorov S.D. et al., 2004) Using of two methods mentioned above let us purify fractions of low molecular weight cationic peptides with molecular weights between 1 and 10 kDa which possess antnimicrobial activity against broad range of test microorganisms (E. coli, L. monocytogenes, St. epidermidis, C. albicans). During LC-MS analysis of bacteriocins fractions peptides with molecular weights between 2946.7–6280.4 kDa and 1535.9–4845.8 kDA were determined although during MW analysis no correlations between purified peptides and predicted bacteriocins EF and NC8 were found. The lack of correlation could be consequence of peptide degradation or modification during purifying or aggregation of bacteriocins with peptides from growth medium.

X-ray study of the red fluorescent protein from a lancelet. Chromophore covalently bound to a nearby tyrosine E.A. Souslova, N.V. Pletneva, E.A. Goryacheva, D.M. Chudakov, I.V. Yampolsky, K.A. Lukyanov, V.Z. Pletnev M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia In recent decades green fluorescent protein (GFP) and related fluorescent proteins (FPs) established themselves as efficient noninvasive molecular instruments in cell biology and biomedicine that are widely used for visualization and monitoring of internal processes within living cells and whole organisms. Design of new advanced fluorescent biomarkers is strongly supported by X-ray studies which provide valuable information on the structure-spectral relationships. The key property of GFP-like proteins is their ability to form chromophore autocatalytically by posttranslational modification of the internal amino acid triad. Green-emitting FPs can be further modified into red-emitting FPs. This study presents the results of an X-ray crystallographic and biochemical study of the wild-type FPs: green laGFP and red laRFP from the lancelet Branchiostoma lanceolatum (Chordata). Lancelet FPs are evolutionarily distant from extensively-studied cnidarian FPs (~20% sequence homology) and remain poorly characterized. It was shown that laGFP is characterized by narrow excitation and emission peaks which are typical for green FPs (λex/λem ~502/511 nm). Freshly purified laRFP demonstrates green fluorescence ex(λ /λem ~ 506/522 nm) which within several days slowly converts into red fluorescence (λex/λem ~521/592 nm). The structure of laRFP (the only known red FP outside phylum Cnidaria) has revealed three unique features: 1) unusual red chromophore-forming sequence Gly58-Tyr59-Gly60 2) presence of Gln211 instead of strictly conserved catalytic Glu crucial for the red chromophore biosynthesis. 3) absence of posttranslational modifications typical for the previously reported red chromophores and presence of an unusual covalent bond between Cβ-atom of the chromophore Tyr59 and hydroxyl of the proximal Tyr62. The impact of this covalent bond on red fluorescence emission of laRFP and its large Stokes shift (~ 70 nm) was further proved by extensive structure-based site-directed mutagenesis. We also performed stepwise mutagenesis aimed at the conversion of green laGFP to red-emitting variant by introduction of limited number of mutations in the chromophore-surrounding area. Based on the data above the mechanism of formation of the red chromophore from the intermediate green state has been proposed.

Four-locus analysis of the genetic polymorphism of enniatin-producing Fusarium species from different regions of Russia A.A. Stakheev, S.K. Zavriev M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Fusarium fungi are worldwide distributed agricultural pathogens. In addition to harvest quality and quantity decrease, Fusarium head blight (FHB) infection leads to accumulation of dangerous mycotoxins in grain and its products. One of the major types of mycotoxins produced by Fusarium species are enniatins – cyclic peptides which possess antimicrobial, insecticidal, and phytotoxic activities. The main producers of enniatins are Fusarium avenaceum, Fusarium tricinctum, Fusarium acuminatum, Fusarium torulosum. Nowadays, Fusarium taxonomy studies take into account data of the polymorphism of DNA sequences which allow estimating the rate of similarity between species and clearing the evolution history of the genus in whole. However, the number of highly polymorphic DNA sites with known sequence is relatively small and searching of novel variable loci proved to be a very relevant and important goal. In this study partial phosphate permease gene (PHO) sequences have been determined for four enniatins-producing Fusarium species: F. avenaceum, F. tricinctum, F. acuminatum, F. torulosum for the first time. Phylogenetic analysis of 27 strains of those species based on comparison of partial sequences of PHO, translation elongation factor 1 alpha (TEF1α), beta-tubulin (β-TUB) and enniatins synthase genes demonstrated that the PHO gene possesses the highest rate of both inter- and intraspecific variability among them. A dendrogram based on the analysis ofPHO sequences has revealed the presence of individual clusters of each of species studied. Moreover, F. avenaceum strains were divided into 5 separated clusters which appeared to be independent on their geographic origin and host plant. The polymorphism of PHO sequences have also been used to design primers and hydrolysis (TaqMan) probe to perform the highly specific identification of all four enniatins-producing Fusarium species studied.

Development of pilot-scale biotechnologies for production of analgesic peptide toxins for preclinical studies V.N. Stepanenko, T.I. Muravyova, D.A. Makarov, I.O. Zvereva, N.V. Oleynik, R.S. Esipov M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia The feature that unifies most diseases is inflammation that is usually accompanied by pain syndrome; therefore, pain relief is one of the key objectives of the medicine that strives to comfort the ill. The nature, location and etiology of pain vary from case to case, thus necessitating the search for new analgesics with a high specific activity. Earlier the Laboratory of Neuroreceptors and Neuroregulators (Institute of Bioorganic Chemistry of the RAS) was the first to extract and characterize Purotoxin-1 (РТ1) from the Geolycosa sp. venom and APHC3 from the sea anemone Heteractis crispa. Both toxins demonstrated a strongly pronounced analgesic effect and a high selectivity: PT1 in respect to the Р2Х3-receptor (isoform purinoceptor Р2Х), and APHC3 in respect to the TPRV1 channel. Therefore, it was reasonable to proceed with the development of the efficient technology for the above referenced toxins production for preclinical studies and, eventually, clinical trials.

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In the study addressed in the report we created 4 genetic constructs for each of the two toxins that provided for the production of the target toxins fused with different lead proteins. Two constructs used the carrier protein represented by the Chitin binding domain (CBD) sequences and Tioredoxin (Trx) and the classical protein cleavage by the site-specific protease of the tobacco etch virus. In the balance constructs the intein-mediated cleavage of the target product from the carrier protein was performed with the use of the SspDnaB and MxeGyrA mini-inteins. Producer strains based on the genetic constructs containing the SspDnaB mini-intein performed the best in terms of the recombinant peptide analgesics production. For each producer strain we have optimized fermentation conditions, proved out conditions for hybrid protein extraction and autocatalytic cleavage with the toxin release. The target product was purified by the two steps chromatographic technique. The developed lab scale technology was scaled up to the pilot scale, and test batches of PT1 and APHC3 (~5 g) were produced for preclinical studies.

Ethanol influences MICA/B surface expression and release from the immune cells M.A. Streltsova, L.M. Kanevskiy, E.I. Kovalenko M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Expression of proteins MICA and MICB (MICA/B), the ligands for immunoreceptor NKG2D, has been proposed to play an important role in the detection and elimination of tumor and other damaged cells. The result of the interaction of surface MICA/B with NKG2D is the increase of cytotoxic activity of NK and T cells and the subsequent killing of affected cells. On the other hand, soluble MICA/B can depress cytotoxic lymphocyte functions. We studied in several in vitro models the influence of ethanol on the expression of MICA/B and on their release from the cells. MICA/B expression was analyzed in a model of ethanol-induced cell stress using K562, Jurkat and THP-1 cell lines, as well as human peripheral mononuclear cells. Surface MICA/B expression was measured by flow cytometry. Significant levels of spontaneous surface MICA/B were registered in all lines. Surface expression of MICA/B increased under the influence of ethanol (0.5–1.5%) and decreased with ethanol dose increasing up to 2% and above. The later process was corresponded to cell death elevation. We did not find spontaneous surface expression of MICA/B in lymphocytes, but discovered it in CD14-positive cells, increased under the influence of ethanol-induced cellular stress (0.12–1%). A cause of the increase of MICA/B expression in cells may be the oxidative stress induced by ethanol resulting in DNA damage. ATM/ATR kinase inhibitor did not affect ethanol-induced changes in MICA/B surface levels. With confocal microscopy it was shown, that the inner pool of MICA/B decreased and the surface pool increased in comparison with the control cells. We can assume that under the influence of ethanol the MICA/B proteins move from the cytoplasm to the cell surface. It has been shown that ethanol induced the release of MICA/B from the cells as soluble proteins and in composition of microparticles and exosomes. The increase of soluble MICA/B level after cell treatment with methyl- cyclodextrin supports the role of lipid rafts in release MIC from the cells. Thus, we have shown that ethanol can alter MICA/B expression in cell lines and normal monocytes and have analysed mechanisms underlying this alteration. Changes in MICA/B surface and extracellular concentration induced by ethanol may have a modulating effect on the immune system. This work is supported by RFBR, grant № 14-04-32342.

Tri- and tetracyclic compounds application for GDNF production stimulation in retinal cells T. Sukhanova1, P. Baranov1, O. Yerov1, H. Lin2, C. James2, D. Morrow2, S. Wang2, P. Lei2, J. McNeish3, M.J. Young1 1Schepens Eye Research Institute, Boston, MA, USA; 2GlaxoSmithKline, King of Prussia, PA, USA;3GlaxoSmithKline, Boston, MA, USA Retinal degeneration and vision lost is one of the important problems of medicine. Probably one of the best ways of resolving this problem is a range of prophylactic measurements against retinal cell degeneration process. Glial derived neurotrophic factor (GDNF) is a member of the transforming growth factor-beta family [1]. It is secreted not only in various classes of neuronal and glial cells [2], but in other tissues and organs, for example kidney. It shows antiapoptotic properties, reduces damage from oxidative stress in retina [3] and increases retinal ganglion cell [4], photoreceptor [5, 6], especially rods [7] viability and it is responsible for cell proliferation in retina [7]. Application of various compounds is responsible for direct and indirect impact and protection of retinal cells by stimulating GDNF secretion. We studied an application of tricyclic (cyclobenzaprine) and tetracyclic (amoxapine and loxapine succinate) compounds for stimulation GDNF production in pigmental epithelial (ARPE-19), glial (SVG) and human progenitor retinal (hRPC) cells. Cells cultivated in UltraCulture Medium for 48 h, GDNF content has been determined by ELISA, cytotoxicity by MTT. It has been shown, 0.5–100 μM cyclobenzaprine stimulated GDNF secretion only in glial cells until 3.5 fold (5 μM), but had no significant effect on other studied cell lines. Amoxapine showed its impact on GDNF secretion in hRPC and glial cells (until 3 folds in the each case at 5 μM and 50 μM respectively). Loxapine succinate is effective for hRPC and glial cells too, GDNF secretion increased until 4.5 and 2 folds respectively at 10–100 μM. Inner content of GDNF in cell, determined in cell lisate, was increased by these 3 compounds too. As in case of secretion, cyclobenzaprine increased GDNF content only in glial cells (until 4 folds, 5 μM). Amoxapine is effective only for glial cells too (until 4 folds, 5–100 μM). Loxapine succinate had no effect on above mentioned cells except 100 μM (app. 2.5 folds).

Nosrat CA, Tomac A, Lindqvist E, Lindskog S, Humpel C, Strömberg I, Ebendal T, Hoffer BJ, Olson L. Cell Tissue Res. 1996;286(2):191-207. Duarte EP, Curcio M, Canzoniero LM, Duarte CB. Growth Factors. 2012;30(4):242-57. Dong A, Shen J, Krause M, Hackett SF, Campochiaro PA. J Neurochem. 2007;103(3):1041-52. Cui Q, So KF, Yip HK. Biol Signals Recept. 1998;7(4):220-6. Lipinski DM, Singh MS, MacLaren RE. Invest Ophthalmol Vis Sci. 2011; 52(10):7340-6. Del Río P, Irmler M, Arango-González B, Favor J, Bobe C, Bartsch U, Vecino E, Beckers J, Hauck SM, Ueffing M. Glia. 2011;59(5):821-32. Rothermel A, Layer PG Invest. Ophthalmol Vis Sci, 2003; 44(5):2221-2228.

Academician Yury Ovchinnikov, RNA-polymerase, interferons and we. Glimpse of today E.D. Sverdlov M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia The report will trace the historical links between the works initiated by Yury A. Ovchinnikov to create in the USSR the first genetically engineered interferon and to establish the world's first primary structures of bacterial RNA polymerases, their genes and mutations resistant to antibiotics and modern investigations of the Laboratory of Structure and Function of Human Genes to create gene therapy anti-cancer agents and to study the mechanisms of transcriptional regulation and tissue-specificity.

SPECIAL ISSUE № 1 2014 | Acta naturae | 43 Oral and Poster Presentations

Screening the pseudomonas aeruginosa and staphylococcus aureus – specific industrial phage preparations (microgen) by PCR test and electron microscopy, and isolation of individual phage species N.N. Sykilinda1, Z.V. Durmanova2, E.E. Kulikov3, M.M. Shneider1, V.A. Kadykov1, K.A. Lysko4, G.M. Ignatyev4, O.S. Darbeeva2, K.A. Miroshnikov1 1M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia;2Scientific Center for Expert Evaluation of Medicinal Products, Moscow, Russia; 3Winogradsky Institute of Microbiology, RAS, Moscow, Russia; 4Unitary Research and Production Association “Microgen”, Moscow, Russia Phage therapy is an important alternative to antibiotics in the current era of drug-resistant pathogens. Bacteriophage preparations are industrially produced and used successfully for diagnosis and treatment of many bacterial infections in Russia. However the phage content of industrial mixtures is selected mostly empirically according to the efficiency against current clinical strains of bacterial pathogens. The lack of genetic and molecular information on the components of the phage mixture obstructs the validation of the therapeutic preparation. We have designed the PCR-test based on conservative genes of different defined groups of P. aeruginosa and S. aureus phages. This test was used for screening of above 200 industrial therapeutic preparations produced by Microgen, with additional transmission electron microscopy (TEM) control. The study has revealed lytic P. aeruginosa KMV –like and phiKZ-like, and S. aureus K-like and AH44J-like phages as major components of the preparations. No known temperate P. aeruginosa phages were found. However, trace amounts of DNA belonging to B1 and B2 morphotypes of temperate S. aureus phages or prophages were detected by PCR. Also, disrupted and aggregated phages, and cell fragments were detected in minor quantities by TEM. About 40 individual lytic P. aeruginosa and S. aureus phages were isolated from single plaques for further analysis. The constructed PCR test system rapidly refers an unknown bacteriophage from the collection or isolated de novo to a certain genetic group. The result allows a conclusion on the feasibility of the application of such bacteriophage in therapy for сompliancy with modern molecular biology requirements. The work was supported by the Russian Foundation for Basic Research (project no. 120400765_a)

Creation of (cytosine-5)DNA methyltransferase inhibitor based on modified oligonucleotide duplexes S.V. Tarlachkov2, T.V. Shevchuk2, O.V. Diachenko2, A.M. Cherevatenko1, Ya.I. Burianov2 1M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia; 2Branch of M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Pushchino, Moscow Region, Russia Currently for chemotherapy of oncological diseases 5-azatsitidin is used for demethylation of tumor genes-suppressors. However, 5-azacytidine randomly incorporates into genomic DNA, which causes disturbance of the replication, transcription, DNA repair and leads to undesirable side-effects. Creation of new classes of pharmaceuticals will significantly reduce these side-effects and optimize chemotherapy process. Kinetic features of the enzymatic reaction of DNA methylation can be used to develop new classes of drugs for the epigenetic treatment. For this purpose, it was proposed to use asymmetrically modified oligonucleotide duplexes as an inhibitory substrate for DNA methyltransferase DNMT1. For binding and inhibition of DNA methyltransferases oligonucleotide duplexes were asymmetrically modified in recognition site by replacement of methylated cytosine to uracil. The presence of uracil in the recognition site causes formation of a stable complex under physiological conditions between oligonucleotide duplexes and DNA methyltransferase. To increase the affinity of the enzyme to substrate oligonucleotides, recognition sites were hemi- methylated. Experiments on the binding of the fusion protein M.HhaI-GFP with modified oligonucleotides, which contain recognition sites for HhaI DNA methyltransferase and target methyltransferase DNMT1 were performed. Formation of a specific stable complex between fusion protein M.HhaI-GFP and modified oligonucleotides was shown. This work supported by grant 14-08-00592 from RFBR.

Modulating effects of extracellular HSP70 on the level of ROS generation in phagocytes N.I. Troyanova, M.A. Shevchenko, A.A. Boyko, R.R. Mirzoev, M.A. Pertseva, E.I. Kovalenko, A.M. Sapozhnikov M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia The ability of professional phagocytes, in particular neutrophils and macrophages, to produce high level of reactive oxygen species (ROS) is an important feature that allows these effectors of innate immunity to protect the host organism from pathogenic bacteria. NADPH oxidases are the main source of ROS produced by phagocytes into extracellular medium and generated within phagosomes of the cells to destroy the infectious agents. Reception of the signals released by bacteria results in migration of neutrophils to the site of invasion and in activation of the cells, in particular in assembling membrane-associated NADPH oxidase complexes. Phagocytosis of the microorganisms in cites of inflammation is accompanied by avalanche increase of ROS production in neutrophils, so called “oxidative burst”, followed by accumulation of high local concentration of ROS. Such increased level of the oxygen radicals results in both killing of the bacteria and damage of the host tissues. For protection from these damaging factors eukaryotic cells have many different antioxidant components and mechanisms. However, it is obvious that another protective system directed to restriction of the level of ROS generation in phagocytes is very important for effective defense of the host tissue in cites of inflammation. We propose that a mechanism of such regulatory system is connected with extracellular HSP70 secreted in conditions of oxidative cell stress. Our previous works and results of other authors suggest this way of inhibition of ROS generation in populations of neutrophils. In this work we have screened several in vitro models allowing investigation of effects of exogenous recombinant HSP70 on processes of spontaneous and induced generation of ROS in populations of phagocytic cells. Besides, modulating action of some inhibitors of NADPH oxidase subunits on registered effects of HSP70 in used models was analyzed. The results did not confirm the supposition that HSP70 caused inhibition of ROS production in phagocytes was due to interaction of the protein with NADPH oxidase. Further investigations are necessary to clear the mechanisms of extracellular HSP70 effects on ROS generation level registered in our models. This work is supported by RFBR grant № 14-04-32203.

Spectral studies of derivative ditiacraun-ethers in the presence of metal cations M.S. Tsarkova, I.S. Zaitsev, S.Yu. Zaitsev Moscow State Academy of Veterinary Medicine and Biotechnology, Moscow, Russia Creating complex nanoscale structures with desired properties is one of the important directions «at the crossroads» Bioorganic and analytical chemistry, biotechnology and nanotechnology. One example of this is the supramolecular systems based membrane-active compounds (such as crown ether derivatives) and their complexes. The purpose of this work was to study the spectral properties derived dithiacrown-ethers (DTCE) synthesized CP RAS, salt solutions and monolayers to assess its interaction with various metal cations. In acetonitrile solution in the presence of Hg2 + cation was observed hypsochromic shift of the maximum absorption spectrum DTCE 22 nm. A comparative study of the absorption spectra of aqueous solutions in the presence of perchlorates DTCE alkali, alkaline earth and heavy metals (lithium, sodium, potassium, cesium, magnesium, calcium, strontium, barium, copper, zinc, lead, cadmium and mercury) different concentrations. DTCE extinction coefficient determined in aqueous solution, (ε=9329 -1M cm-1). The most prominent are the spectral characteristics of the solutions at a concentration of 10-4 M DTCE and concentration of metal salts 10-3 M. In the optical absorption spectra DTCE only in the presence of perchlorate mercury (II) showed significant shifts of the absorption maxima to shorter wavelengths by 29 nm. This is evidence of selective cation binding of mercury (II) with DTCE.

44 | Acta naturae | SPECIAL ISSUE № 1 2014 Oral and Poster Presentations

Confirmation of these results was obtained in the study of the absorption spectra of compounds DTCE monolayers transferred with double distilled 2 water and aqueous solutions of Hg (ClO4) at the surface pressure of 10 mN / m. Their main characteristic was the fact that the maximum absorption for -5 2 compounds DTCE monolayer transferred to 10 M solution of Hg (ClO4) , is at 406 nm and RLU is 0.00575, which is 209% higher than those for monolayer transferred from water. Shift of the absorption maximum in the presence of a monolayer DTCE mercury salts is 23 nm to shorter wavelengths. This is additional evidence of complex formation between DTCE monolayer cations and mercury(II) from the aqueous subphase, which is promising for the creation of chemosensory materials. This work was supported by RFBR grant 14-03-00154.

Changes in the composition of the lipofuscin granule fluorophores from human retinal pigment epithelium with age Marina A. Yakovleva1, Patimat M. Arbukhanova2, Tatiana B. Feldman1, Sergey A. Borzenok2, Mikhail A. Ostrovsky1 1N.M. Emanuel Institute of Biochemical Physics, RAS, Moscow, Russia; 2S.V. Fyodorov Eye Microsurgery Complex, Moscow, Russia The non-invasive method of fundus autofluorescence (AF) imaging is becoming more common for the diagnosis of age-related changes and degenerative diseases of the retina, primarily age-related macular degeneration (AMD). The fluorophores of lipofuscin granules (LGs) are the main source of AF. LGs accumulate in the retinal pigment epithelium (RPE) cells during the aging and particularly intense under degenerative diseases. It is known more than 21 fluorophores – bis-retinoids of LG, and A2E is most studied. The aim of this work was a comparative analysis of the spectral characteristics and changes in the composition of LG fluorophores with age. Cadaver human eyes were obtained from the Eye Tissue Bank of S.N. Fyodorov Eye Microsurgery Complex. LGs were isolated from RPE of individual donor cadaver eyes of different ages ranging from 17 to 74 years. The fluorophores of LGs were extracted into chloroform and studied by fluorescence spectroscopy, and high performance liquid chromatography (HPLC). It was shown that the relative content of the products of A2E photooxidation and photodegradation (A2Eox, deg) as a part of LG increases with age. The ratio A2Eox, deg/A2E increases with age of the donor from 0.64±0.03 to 1.31±0.04. The maximum position of the fluorescence spectrum of extracts did not changed and was in the range of 575±15 nm. Fluorescence of A2E oxidized forms in chloroform extract is more intensive than unoxidized A2E. Since the oxidized forms of A2E reveal photo/toxic properties, an increase in their relative content in LGs could be considered as a risk factor in aging retina and the origin and development of AMD. Assessment of their contribution to the fundus autofluorescence picture would improve the information value of this diagnostic method.

Study of the biological activity of cathelin-like protein of the rat leukocytes V.A. Yukhnev1, M.A. Shartukova2, O.V. Shamova1,3 1Institute for Experimental Medicine RAMS, St-Petersburg, Russia; 2Research Institute of Influenza, St-Petersburg, Russia; 3Saint-Petersburg State University, St-Petersburg, Russia Investigation of molecular mechanisms of functioning of the innate immune system is an important goal of biology and medicine since allows finding directions for the correction of a variety of pathological processes. Cathelicidins are the key effector molecules of the innate immunity. They are stored in specific granules of neutrophils as precursors, consisting of a conservative N-terminal domain – cathelin-like protein (CLP) and a variable C-terminal region that after a proteolytic cleavage releases an antimicrobial peptide (AMP). While the biological activity of the antimicrobial peptides-cathelicidins has been intensively explored, functions of CLP are still unknown. The aim of this work was an isolation of cathelin-like protein from the rat blood leukocytes for an investigation of its biological activity and the influence of this protein on the antimicrobial and cytotoxic activity of AMP. The neutrophil-rich fraction was isolated from blood of rats. The degranulation of neutrophils was stimulated by using two approaches – an incubation of the cells with the serum activated zymozan or with phorbol 12-myristate 13-acetate (PMA). In the obtained after the cell sedimentation supernatants we revealed CLP by using the specific antibody (Abcam, USA) and dot or Western blotting. High performance liquid chromatography (HPLC) was applied to receive a purified CLP. The CLP contained fractions were subjected to the additional cycles of HPLC. The best yield of the protein was achieved with using neutrophils stimulated with PMA. The purified CLP did not exert a significant antimicrobial activity. When we tested its combined action together with rat cathelicidin peptide rCRAMP it was shown that CLP does not affect the antibacterial activity of the peptide. On the other hand, the cytotoxic activity of rCRAMP towards cultured mammalian cells was decreased by 45% in the presence of CLP. The obtained results and our further investigation will contribute to the understanding the biological role of CLP in the functioning of the innate immune system. The work was supported by the RFBR grants 13-04-02102а, 12-04-01573а.

Horseradish peroxidase immobilization on ZnO-modified graphite electrodes A.R. Yunusov, I.A. Cherenkov, E.V. Haranzhevskiy, V.G. Sergeev Udmurt State University, Izhevsk, Russia Last decades feature rapid development of bioelectronics researches – biosensors, biofuel elements, logical units based on enzymes and cells are being widely studied. One of the perspective developments in the enzyme electrochemical biosensor field is based on applying of amphoteric metal oxides materials such as zinc oxide for improved enzymatic immobilization and signal transduction. ZnO-based electrode materials provide high biocompatibility, effective signal transduction, ability to form various nanostructures directly on the electrode. These materials could provide high surface to volume ratios and high surface activity, and thus possess unique advantages over other conventional materials. This study reveals the development of peroxidase biosensor model based on ZnO-modified graphite electrode. Research was based on graphite planar electrodes (Autocom, Moscow, Russia). ZnO suspension was prepared in 0.15 M Tris-HCl buffer, pH=7.2 using ultrasound. Obtained suspension was applied at the electrode surface and after that dried in dust-protected cell. Later ZnO-modified electrodes were cov- ered by horseradish peroxidase (HRP) enzyme solution and left incubating for 12 hours in 4°C cell for adsorption. For some electrodes the immobilization process passed in ZnO suspension volume before applying at the electrode surface. Modified electrodes were electrochemically studied using 1 mM toluidine blue solution in Tris-HCl buffer as an enzyme substrate. The solution strength of hydrogen peroxide as a second enzyme substrate varied. It was shown that the enzyme adsorption at ZnO modified electrodes provides an effective immobilization. It is expressed in its high catalytic activity that is 2–3 times greater than it was shown for unmodified electrodes. Voltammetric studies of modified electrodes shows the lack of toluidine blue oxidation peaks which is another sign of high enzymatic activity of immobilized peroxidase. ZnO-modified electrodes with HRP immobilized on electrode surface retain their enzymatic activity for 3 weeks in standard conditions. Results obtained in this study will be used for further researches of zinc oxide characteristics in terms of perspective construction material of bioelectronics based on enzymes and cells.

SPECIAL ISSUE № 1 2014 | Acta naturae | 45 Oral and Poster Presentations

Research of distribution peculiarities of boron containing conjugate chlorine E6 for the purposes of photodynamic and boron neutron capture therapy A.B. Volovetsky1, N.Y. Shilyagina1, I.V. Balalayeva1, A.V. Maslennikova1,2, M.A. Grin3, A.F. Mironov3, A.V. Feofanov4,5 1N.I. Lobachevsky State University of Nizhny Novgorod, Nizhny Novgorod, Russia; 2Nizhny Novgorod State Medical Academy, Nizhny Novgorod, Russia; 3Lomonosov Moscow State University of Fine Chemical Technologies, Moscow, Russia; M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia, Faculty of Biology of M.V. Lomonosov Moscow State University, Moscow, Russia The research objective was to examine biodistribution of conjugate chlorine e6 with a boron nanoparticle with an aminoalkyl linker (n=6). The means of control was conjugate chlorine e6 with an analogous linker but without a boron nanoparticle. The analysis of the biodistribution of the conjugate was made on the of Balb/c mice with implanted tumor Colo-26. The experiment was carried out on the 9–10th day, when the tumor node size was up to 5–7 mm. 10 mg/kg of nanoconjugate in 5% cremophor solution were injected in the animals’ caudal veins. Laser scanning microscopy ex vivo was performed by Axiovert 200M LSM510 META three hours after the injection. The fluorescence was excited with 543 nm, the recording of the spectra allowed to identify the signal conditioned by the accumulation of chlorine e6 conjugate in the tissues with strong autofluorescence. The injection of nanoconjugate with boron resulted in the emergence in the tumor tissue of a strong fluorescence signal of maximum of 670 nm range corresponding to chlorine e6 fluorescence. In skeletal muscles, as well as in the muscular walls of hollow organs and skin, the characteristic peak of 670 nm was not observed. Apart from tumor tissue, the accumulation of the conjugate is registered in the liver, kidneys and the spleen. According to the results of microscopic analysis, the content of the test compound in all of these organs did not exceed that in tumor tissue. The injection of chlorine e6 with an aminoalkyl linker (n=6) was characterized by uneven accumulation of the preparation in the tumor tissue. This work was supported by RFBR (project № 14-02-00715).

Gaponin regulates cellular response to oxidative stress by activating P53 E.E. Vorobyeva, E.V. Smirnova, T.V. Rakitina, V.M. Lipkin M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Oxidative stress (OS) is the cause of many human and animal pathological states, including cardiovascular and neurological disorders. Study of molecular mechanisms regulating cellular response to OS is a fundamental scientific problem, the solution of which will offer new targets for the development of therapeutic agents to protect cells from the adverse effects of reactive oxygen species (ROS). Previously we have shown that haponin localizes predominantly in the nucleus (O.V. Bogatova, 2009) and the sensitivity of cells to the OS correlates directly with haponin level in the cell: silencing of haponin expression in the cells reduces the cellular sensitivity to OS induced by peroxide hydrogen and, conversely, increase of haponin level enhances the cellular sensitivity to the action of ROS (E.V. Smirnova, etc. Doklady Biochemistry and Biophysics, 2011). Therefore we hypothesized that haponin is a positive regulator of apoptotic signal after OS. For further study of functional role of haponin in cellular response to the OS and its possible involvement in the apoptotic process we examined the expression of cellular proteins involved in apoptosis using genetically engineered HEK 293 cell lines with altered expression levels of haponin. We have found that haponin is a positive regulator of p53 activation under OS and by the method of co-immunoprecipitation using antibodies against p53 and haponin we have shown that haponin interacts with p53 directly. Further we examined the effect of changes in haponin level to the transcription of proteins involved in conducting of p53-dependent apoptotic signal (Вах, p21CIP1/WAF1, Noxa, MDM2) under normal conditions and under OS induced by hydrogen peroxide, and we showed that in cells with reduced expression of haponin the transcriptional activation of those proteins under OS was reduced comparing with the control lines in accordance with previously obtained data on the dependence of the cell viability in the OS with haponin expression level.

Molecular mechanisms of short pharmacologically active neuropeptides influence on GABA specific binding T.V. Vyunova, L.A. Andreeva, K.V. Shevchenko, N.F. Myasoedov Institute of Molecular Genetics, RAS, Moscow, Russia The purpose of the study was the investigation of key aspects of molecular mechanism of biological action of some pharmacologically active synthetic neuropeptides. We made detailed research at the molecular level of specific ligand-receptor interactions of radioactively labeled analog of GABA with specific receptors on the plasmatic membranes of rat brain nerve cells. In this research, we relied primarily on radioligand-receptor method of analysis of specific intermolecular interactions, on method “ex vivo” – to research set aside in time effects of the biologically active molecules introduction, on the methods of molecular detection analysis HPLC. In the work presented, we have studied molecular base of action of short synthetic neuropeptides with double psy- chotropic effects (nootropic and anti-anxiety) – Selank (used in clinical practice) and its C-terminal fragment (tetra peptide RPGP). Were also researched another class of biologically active neuropeptides – potential neuroleptics, representing analogues of neurotensin (WPYF and APYF). It was found that these neuropeptides are positive modulators of specific [3H]GABA binding to rat cortex cells plasmatic membranes. The quantitative assessment of regulatory peptides impact on specific binding of 3[ H]GABA shown the existence of two areas of peptides concentration, which is a sharp (almost two-fold) increase in the specific binding. So, for Selank it makes from 5 nM to 20 nM and from 5 mkM up to 50 mkM. In addition, it was shown that in the presence of ultralow concentrations of peptides (from 1 pM to 10 pM), exist the increase (in 1.5 times) in the number of binding places of tritium labeled GABA. The degree of influence on the specific binding of3 [ H]GABA of main ligands interacting with good known allosteric modulation sites of the GABA(A) receptors (diazepam, styrene, olanzapine, ethanol and other) was also tested in peptides friendly incubation conditions. At the next stage of research we tested the joint introduction of synthetic peptides and different GABA(A) receptors allosteric modulators (some of them use in clinical practice, like Diazepam). It is shown that in the presence of one of the neuropeptides (Selank and other) and one of known allosteric modulators of the GABA(A) receptor there is a formation of cooperative effects of specific [3H]GABA binding. Thus, we can conclude that neuropeptides with anxiolytic effects (Selank and other) able to make modulatory changing in specific GABA binding, and also can cause changes in functional activity of GABA receptors by starting cell biochemical mechanisms (when peptide molecules presence is need no more). The joint introduction of neuropeptides and drugs in medical practice will allow to reduce the dose of the drugs, as well as to neutralize the negative side effects that are common to many drugs.

Isotope exchange in the quantitative determination of the angiotensin-converting enzyme activity in the biological environment Y.A. Zabrodskaya, V.V. Egorov, A.V. Vasin, O.A. Mirgorodskaya, *Yu.P. Kozmin Influenza Institute, Saint-Petersburg, Russia; *M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Angiotensin-converting enzyme (ACE) hydrolyses the decapeptide angiotensin I (Ang I) to angiotensin II (Ang II). ACE participates in a wide variety of physiological processes. The ACE quantitative determination in biological fluids, such as serum and bronchopulmonary lavage, may serve as a marker for the diagnosis and monitoring of treatments for pulmonary disease. Quantitative measurements in bronchopulmonary lavage are difficult because the lavage-containing samples from patients have different dilutions and need to be standardized.

46 | Acta naturae | SPECIAL ISSUE № 1 2014 Oral and Poster Presentations

This study demonstrates the practical application of our patented method of quantitative analysis by mass spectrometry (MALDI-MS) using isotope- labeled standard products of oxygen O18 carboxyl groups partial exchange in analyzed polypeptides. In this study it is once demonstrated both possible ways for preparing isotope-labeled standards: (1) the sample of synthetic Ang II with a known concentration and the experimentally determined value of the isotopic forms with varying degrees of substitution; (2) the randomly chosen albumin tryptic products sample for standartization of biological fluids samples from patients. It is demonstrated that ACE activity increases in influenza virus infected mice serum and (more dramatically) bronchopulmonary lavage. ACE activity decreases in the experimental drug treatment subjected infected animals compared with control infected animals. Special experiments showed that the experimental drug added in vitro to blood serum also leads to a decrease in enzyme activity. The work was supported by RFBR project № 13-04-00168.

Interaction of the novel precursors of two fluorescent dyes with model phospholipid in monolayers S.Yu. Zaitsev1,2, M.N.Shaposhnikov1,2, D.O.Solovyeva1,2, I.S.Zaitsev1, D.Moebius2 1Moscow State Academy of Veterinary Medicine and Biotechnology, Moscow; 2Max Planck Institute for Biophysical Chemistry, Goettingen, Germany Photoactivated (“caged”) fluorescent dyes are modern tools for structure and function studies of cell membranes and subcellular organelles. Recently synthesized precursors of rhodamine fluorescent dyes (abbreviations PFD813 and PFD814) important for microscopic probing of biological objects have been studied in solution. In order to characterize their behavior at interfaces, monolayers of PFD813 and PFD814 have been formed and investigated. The interactions of these precursors with the biomembrane lipid dimyristoylphosphatidyl-ethanolamine (DMPE) in monolayers at the interface and after transfer to glass plates have been studied by measuring monolayer parameters and spectroscopic properties (before and after photo-chemical formation of the fluorescent rhodamine dyes Rho813 and Rho814, respectively) [1]. The novel dye PFD813 forms stable monolayers with collapse pressure of around 30 mN/m whereas PFD814 does not. This is due to the presence of carboxyl group in the side ring of PDF814 (that increases the “hydrophilicity” of the molecules) as compared to the methyl-ester group of PDF813. However, PFD 814 may be stabilized at the air-water interface by interaction with the lipid such as DMPE, a synthetic analog of the common natural membrane phospholipid. The collapse pressures of the mixed monolayers PFD814:DMPE and PFD813:DMPE (molar ratio dye:lipid = 1:1), are 65 mN/m and 45 mN/m, respectively. The pressure-area isotherm of PFD813:DMPE is shifted to much larger areas as compared to PDF814:DMPE, indicating that PFD813 can be inserted into the DMPE matrix to a larger extent than PFD814. Obviously, the presence of methyl ester in PFD813 increases its membrane-active properties. The spectral investigations of PFD813 and PFD814 demonstrate that these precursors of fluorescent dyes can indeed be photo-activated to the fluorescent dye in an environment similar to biomembranes. Therefore, PDF813 and PDF814 should be adequate for studies of cells and organelles by fluorescence microscopy [2]. This work was supported by grant of the Russian Science Foundation (project №14-16-00046).

1. S.Yu. Zaitsev, M.N. Shaposhnikov, D.O. Solovyeva, I.S. Zaitsev, D. Moebius. Cell Biochemistry and Biophysics, 2013, V. 67, Issue 3, pp. 1365-1370. 2. S.Yu. Zaitsev, M.N. Shaposhnikov, D.O. Solovyeva, I.S. Zaitsev, D. Moebius. Cell Biochemistry and Biophysics, 2014 in press.

Enhanced resistance to phytopathogenic microorganisms in transgenic tobacco plants with gene of antimicrobial peptide arenicine 2 N. S. Zakharchenko1, E.I. Finkina2, S.V. Balandin2, O.V. Furs2, Ya. I. Buryanov1, T.V. Ovchinnikova2 1Branch of Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Pushchino, Russia; 2Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, RAS, Mosсow, Russia Expansion of the spectrum of antimicrobial peptides in the plant cell is a promising strategy of plant defense against phytopathogenic microorganisms. Antimicrobial peptides are the integral part of the innate immune system of all multicellular organisms having a wide spectrum of bactericidal and fungicidal activity. Arenicin 2 (2.7 kDa) isolated from Arenicola marina, was cloned in a binary vector of pSS controlled by the dual 35S RNA promoter of cauliflower mosaic virus. As a gene source, we used plasmid pG-Ar2S containing the gene antimicrobial peptide arenicine 2 (are2). This gene was synthesized again by PCR with the goal of flanking its ends by convenient sequences for cloning in vector to be used in plant transformation. Strain Escherichia coli HB101 was transformed with design of constructoion. The vector pSS::are containing the gene antimicrobial peptide arenicine 2 in a straight orientation relative to the promoter was transferred to cells of Agrobacterium tumefaciens GV3101(pMP90RK). The resulting Agrobacterium strain was used for genetic transformation of tobacco plants (Nicotiana tabacum L.). Direct regeneration of sprouts was observed after two weeks on transformed tobacco leaf explants cultivated on a selective regeneration medium MS. The presence of gene are 2 in genomes of transgenic tobacco plants was confirmed by PCR. The expression of the gene are 2 in plants are shown for antimicrobial activity of plant extracts. The obtained transgenic plants exhibit enhanced resistance to bacteria (Erwinia carotovora) and phytopathogenic fungi (Sclerotinia sclerotiorum). The obtained construct with the antimicrobial peptide are2 gene can be further used in studies on the effect of this gene in various transformed plants. The work was supported by RFBR grant № 12-08-00131, 13-04-00636.

Immunotherapeutic activity of prion proteins synthetic fragments on alzeimer’s disease animal model y.V. Zaporozhskaya, T.D. Volkova, A.V. Kamynina, D.O. Koroev, N.I. Medvinskaya*, N.V. Bobkova*, E.V. Ponomareva**, S.I. Gavrilova**, O.M. Volpina M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia; *Institute of Cell Biophysics, RAS, Pushchino, Moscow Region, Russia, **Mental Health Research Center, RAMS, Moscow, Russia Alzheimer's disease (AD) is a serious neurodegenerative disease of the central nervous system, which can described in terms of progressive intellectual disability, cognitive deterioration, derangement of memory and behavioral changes. Recently was proved, that a prion protein is one of the membrane targets of an amyloid beta peptide – the main AD neurotoxic agent. The role of the prion 95–110 region in the amyloid beta peptide binding and antibodies protective role to this site were determined. At the same time, protein N-terminal fragment involvement in an interaction with the amyloid beta peptide was revealed, but significance of this fragment wasn’t studied in concrete. The aim of the research was to find out an immunoprotective fragments of the prion protein N-terminal part. Two fragments adequate to the regions 17–33 and 22–33 of the prion protein sequence were selected and synthesized. The 17–33 region contained theoretically- calculated T-helper epitope, which was needed for immunostimulation without a conjugation of the peptide to a carrier protein. The 22–33 region without T-helper epitope required the career protein conjugation for the immunological potency efficiency. Immunotherapeutic activity of synthesized peptides was studied on bulbectomised mice, which were a valid model of a sporadic form of the disease. Synthesized fragments immunization of the animals caused a high level antipeptide antibody formation. The experiments in a Morris water maze represented the prevention of spatial memory derangement of bulbectomised animals. Immunization significantly reduced the amyloid beta peptide level in bulbectomised mice brain and restored the hippocampus and cortex morpho- functional state. The study of binding of serum with synthetic fragments of the prion protein and synthetic fragments of the other target proteins of the amyloid beta peptide revealed the peptides and statistically significant difference of antibody levels in patients and healthy donors. The research was carried out with the support of grant of RFBR COMFY № 13-04-40106-Н and RFBR 14-04-31232

SPECIAL ISSUE № 1 2014 | Acta naturae | 47 Oral and Poster Presentations

Influence of acyl dophamines on inflamatory response in macrophages RAW264.7 culture D.A. Zayara, M.G. Akimov, N.M. Gretskaya, V.V. Bezuglov M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia N-acyl dopamines are a class of the endogenous compounds representing conjugates of long-chain fatty acid and dopamine. Literature data show the possible involvement of these substances in the regulation of inflammatory response. However, a detailed study of this process was not conducted. The aim of this study was to investigate the influence of natural N-acyl dopamines on the inflammatory response in murine macrophage cell culture RAW264.7. Inflammatory response was estimated by the accumulation of NO signal molecule in the culture medium. N-acyl dopamines (0.1; 1; 10 and 100 μM) were added as a solution in DMSO (final concentration 0.5%) in medium with 2.5% fetal bovine serum and 2 mM L-arginine. To determine the anti-inflammatory activity RAW264.7 were additionally stimulated with lipopolysaccharide (1 mg/ml) and phorbol 12-myristate 13-acetate (0.5 μg/ml). After incubation for 20–24 h, the concentration of NO was measured using the Griess colorimetric reaction. Evaluation of toxicity was conducted using the MTT assay. In this study, we investigated the effects of dopamine amides of arachidonic, docosahexaenoic, oleic and stearic acids, arahidonoyl noradrenalin, anandamide and prostamide E2. It was found that in the anti-inflammatory model the effective dosage of dopamine and noradrenaline derivatives (IC50 7–10 μM) is substantially lower than of anandamide and prostamide E2 (IC50 60–80 M). At the same time, substances with an unsaturated fatty acid residue were more efficient than saturated fatty acid derivative (IC50 7–10 μM and 18 μM respectively). However, dopamine derivatives were toxic to RAW264.7 (LD50 2–10 μM), and so the reason for the decline of the NO concentration in the environment requires additional studies. At concentrations of about 0.1 μM proinfalmmatory effects of examined derivatives of dopamine and noradrenaline (8–14% of positive control) were revealed, which were virtually absent at concentrations of 100 μM. Thus, depending on the conditions and concentration, N-acyl dopamines can both stimulate and inhibit the generation of NO and inflammatory response. The work was partially supported by the RFBR grant 12-04-00608а.

Compression is the possible inactivation factor of Escherichia coli K 12 cells under the action of TiO2 nanoparticles in acidic conditions L.V. Zhukova A.N. Bach Institute of Biochemistry, RAS, Moscow, Russia Investigation into the impact of nanoparticles on the prokaryotic and eukaryotic cells is relevant in connection with the growing use of nanoscale materials in technology. Clarifying the mechanism of nanoparticles activity will help to minimize their possible negative impact on the environment and human health, or, conversely, to increase their destructive effect on pathogens and abnormal cells of eukaryotes.

It was established that the activity of TiO2 nanoparticles upon the near ultraviolet (UVA) irradiation and at certain conditions in the dark resulted in a reduction in the number of different microorganisms. The mechanism of this action remains unclear. The most common hypothesis is that the cell death occurs as a result of the cell walls and membranes damage by the strong oxidizers (electron vacancies and reactive oxygen species), formed by TiO2 nanoparticles upon UVA irradiation. In the absence of UVA, supposedly, also the disruption of the cell membrane as a result of its contact with nanoparticles leads to the cell death.

To test these hypotheses in this study the physiological activity of E. coli K12 bacterial cells after exposure to TiO2 nanoparticles in the absence or under UVA irradiation was investigated using fluorescent staining with acridine orange. To determine the role of the contact between the cells and nanoparticles in cell inactivation the experiments were carried out at pH values of 4.0–4.5, providing the contact: at these conditions the surfaces of cells and nanoparticles are maximum oppositely charged. It turned out that even in conditions providing a sorption of the nanoparticles on the cell surfaces, all cells are not inactivated. Under the irradiation as well as without UVA inactive cells were detected only within the composition of the aggregates formed as a result of nanoparticles action, predominantly in their inner part. Cells on the surface of the aggregates, which were under the most intense UVA irradiation, were inactivated with increasing duration of exposure in the least, that is, after the cells inside of the aggregates, which were shielded by the external cells. These results cannot be explained by the aforementioned hypotheses. The results obtained in this work make it possible to propose another explanation for the nanoparticles action: compression of the cells coated with nanoparticles due to the mutual attraction between such cells is the possible inactivation factor of the cells by TiO2 nanoparticles under irradiation or without UVA, and irradiation enhances this process. The results were partially published (Nanotechnologies in Russia, 2013, V. 8, N. 9–10, pp. 678).

Symmetry infringement in mathematical metrics of hydrogen atom as illustration of ideas by V.I. Vernadsky concerning origin of life and biosphere A.E. Zlobin V.I. Vernadsky State Geological Museum RAS, Moscow, Russia The metrics was suggested by the author of abstract earlier (A.E.Zlobin, 2-nd International Congress “EurasiaBio-2010”, April 13-15, Moscow). Latest paper was presented in 2013 (arXiv:1402.1408 [physics.gen-ph]). The author deduced mathematical metrics of atom of hydrogen on the base of Fibonacci sequence of numbers. Accurate view of the metrics is: (PxF)/(exp(F))=j. This mathematical expression links between themselves four constants: Ludolph number (P=3.1415...), Napier number (e=2.7182...), golden ratio (F=1.6180...) and so-called “irrational one number” (j=1.0079...). The term “irrational one number” was suggested for the first time by the author. The most wonderful property of this mathematical expression is that the factor j=1.0079… simultaneously coincides to the value of atomic mass of hydrogen with high accuracy. It is strongly demonstrated that the metrics of atom of hydrogen con- nects between each other characteristics of substance, form and number. Thus the expression may be used as universal system of measurements (metrics) for analysis of atoms. In 1931 V.I.Vernadsky mentioned some aspects which are connected to problem of initiation of life and biosphere on the Earth. Among these aspects were ocean water, gas functions, pressure and temperature, climate etc. Also three interesting ideas were mentioned by V.I.Vernadsky. The first idea is known for a long time: “omne vivum e vivo” (it means that every living thing descends from living thing). The second idea means that all living things do not have strict symmetry, and left and right side of every living thing are different. Thus, all living things are characterized with the property of asymmetry. The third idea is that this asymmetry may be described mathematically as infringement of symmetry. If to take into consideration the paper by V.I.Vernadsky, we can note good correspondence between obtained metrics of atom of hydrogen and V.I.Vernadsky’s ideas concerning origin of life. In particular, infringement of symmetry is clearly expressed in such mathematical metrics. The metrics reflects the rule “omne vivum e vivo” too. The author of abstract considers that the metrics makes possible the working of pattern recognition algorithm at the level of atoms. This algorithm seems effective for transformation of inert organic substance into living things. Also the fact of deduction of the metrics from Fibonacci sequence of numbers must be taken into consideration. This may be theoretical hint on complicated internal logical structure of atom and reflection of logical properties of the atom too.

48 | Acta naturae | SPECIAL ISSUE № 1 2014 Oral and Poster Presentations

Multifunctional biodegradable microparticles with improved surface chemistry for tissue engineering M.A. Zlobina1, M.G. Drozdova1, A.M. Privalova1, T.S. Demina2, T.A. Akopova2, A.N. Zelenetsky2, E.A. Markvicheva1 1M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia; 2N.S. Enikolopov Institute of Synthetic Polymer Materials, RAS, Moscow, Russia Microcarriers are promising scaffolds for tissue engineering due to significant advantages over other types of matrices, namely injection by non-invasive technique and large surface for cell attachment. Moreover, biodegradable microcarriers can play a dual role, namely they can serve both as delivery systems of growth factors and as supports providing cell attachment and growth. The aim of the study was to develop multifunctional biodegradable poly (lactide) (PLA) microparticles with improved surface chemistry to promote cell adhesion and loaded with bioactive peptide to stimulate cell growth and proliferation. Cell adhesion and spreading on microcarriers can be significantly influenced by the microparticle surface chemistry. In order to promote attachment and growth of cells, surface of these microparticles was modified with chitosan by physical sorption. The obtained results showed that the modification of microparticles surface indeed enhanced growth of L929 cells. However, this modification cannot be used for preparation of peptide loaded microparticles, since peptide can be completely released from the microparticles during their incubation. Therefore, one-step technique has been proposed to obtain the surface-modified microparticles using emulsion solvent evaporation technique with usage of copolymers of chitosan with poly (lactide). This approach allows obtain microparticles with different surface properties (topography, chemical composition) and encapsulated biologically active peptides to stimulate the growth and proliferation of cells in a single step. Different microscopy techniques (fluorescent and SEM) have been used to study the effect of these modification. The impact of the modification on cells growth was evaluated using the MTT assay on day 4 and 7 of cell cultivation on microcarriers. It was shown that the proposed surface modification promotes cell adhesion and spreading on the surface of the microcarriers as well as improves cell growth, compared to cell growth on the unmodified microparticles.

Multifunctional nanoconstructs for bioimaging and therapy S.M. Deyev M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Nowadays nanobiotechnologies open up new possibilities for diagnostics and treatment of oncological, cardiovascular, autoimmune, and other diseases. А novel strategy for design of targeted imaging and therapeutic heterostructures based on the ribonuclease barnase and its inhibitor, barstar, was suggested. The barnase and barstar are small, stable, very soluble, resistant to proteases proteins. The complex between them is extremely tight with a Kd~10-14 M. The N- and C-terminal parts of both proteins are localized outside of the barnase∙barstar interface and are therefore accessible for fusion with targeting, visualizing or toxic compounds. We developed a modular engineering concept using proteinaceous ‘molecular glues’, most notably, the barnase−barstar system (BBS). It seems particularly well suited to the production of heterooligomeric constructs because the extremely specific barnase∙barstar interaction eliminates reliably the mispairing problems. The important advantage of barnase∙barstar over the majority of other dimerization modules is that their interaction ratio is precisely 1:1, and neither of the partners is aggregation prone. This universal platform provides a straight-forward technology to design a multifunctional nanoheterostructures “when the whole is greater than the sum of the parts”. It could potentially be applied to create theranostic agents for complex stimuli-controlled targeted drug delivery [1]. Results on bioimaging applications of BBS [2].with important types of the nanoparticles (NPs), including luminescent nanodiamonds (LNDs) [3], colloidal gold [4], magnetic NPs, luminescent upconversion NPs [5] quantum dots (QDs) [6] as well as delivery of radioisotope, pseudomonas exotoxin A and phototoxic fluorescent proteins [7] to the HER2/neu overexpressing human adenocarcinoma cells are reviewed. This study was supported by the Ministry of Education and Science of the Russian Federation (# 14.Z50.31.0022), the RFBR (# 13-04-40228).

1. Nikitin M.P., Shipunova V.O., Deyev S.M., Nikitin P.I. Nat. Nanotech. 2014. doi: 10.1038/nnano.2014.156. 2. Aghayeva U.F., Nikitin M.P., Lukash S.V., Deyev S.M. ACS NANO, 2013, 7(2), 950-961. 3. Sreenivasan V.K., Kelf T.A., Grebenik E.A., Stremovskiy O.A., Say J.M., Rabeau J.R., Zvyagin A.V., Deyev S.M. Proteomics, 2013, 13(9), 1437-1443. 4. Ivanova J.L., Edelweiss E.F., Leonova O.G., Balandin T.G., Popenko V.I., Deyev S.M. Biochimie, 2012, 94, 1833-1836. 5. Grebenik E.A., Nadort A., Generalova A.N., Nechaev A.V., Sreenivasan V.K.A., Khaydukov E.V., Semchishen V.A., Popov A.P., Sokolov V.I., Akhmanov A.S., et al. J. Biomed. Opt., 2013, 18(7):76004. doi: 10.1117/1.JBO.18.7.076004 6. Zdobnova T.A., Stremovskiy O.A., Lebedenko E.N., Deyev S.M. PloS One, 2012, 7(10), e48248. 7. Mironova K.E., Proshkina G.M., Ryabova A.V., Stremovskiy O.A., Lukyanov S.A., Petrov R.V., Deyev S.M. Theranostics. 2013. 3(11):831-840. doi:10.7150/ thno.6715.

SPECIAL ISSUE № 1 2014 | Acta naturae | 49 Young Scientists CompetitionYoung Scientists Competition

Study of plant cell рН using Pt-GFP expressed in Arabidopsis thaliana (L.) plants M. Ageyeva, I. Makarov, L. Katicheva, A. Yudintsev Lobachevski State University, Nizhny Novgorod, Russia Regulation and stability of intracellular compartments pH is an essential condition for plant cell vital activity because all metabolic processes, protein structure, ion channels activity and membrane throughput depend on the acidity. The main flaw of exogenous fluorescent probes used for cell H+ ions determination of concentration has damaging influence on cell when making measurements. GFP-based proteins (Green Fluorescent Protein) is integrated into plant genome and expressed in cell enabling cell рН measurement in vivo without any external actions. The aim of this work is evaluation of Arabidopsis thaliana (L.) cell рН using ratiometric рН-sensor Pt-GFP with the help of fluorescent spectroscopy and laser scanning microscopy. There were used transgenic A. thaliana plants expressed рН-sensor Pt-GFP (Notthingham Arabidopsis Stock Centre, UK) and control A. thaliana plants (ecotype Columbia) grown in vitro on Murashige-Scoog medium. The excitation and emission spectra for genetically modified and nontransgenic plant leaves and roots were obtained using spectrofluorimeter Shimadzu RF-5301 PC (Shimadzu, Japan) with annex for luminescent measures of solid objects “Lyagushka” (Granat, Russia). The highest intensity of transgenic plant fluorescence was registered in 490–540 nm range with 510 nm maximum (excitation – 488 nm). Fluorescent images of control and transgenic plant leaves and roots were obtained with the help of laser scanning microscopy system Carl Zeiss LSM 710 based on inverted microscope Axio Observe Z1 (Carl Zeiss, Germany) when excitation was 488 nm and emission – 500–540 nm. It was prepared calibra- tion fluorescence intensity ratio-intracellular pH curve in 4–8 units range using plant suspensions made on buffer solutions with different pH. The obtained accuracy of approximation was 0.962. Thus genetically coded protein sensor Pt-GFP, that is susceptible to cell H+ ions concentration, can be successfully applied for investigation of different factors (temperature, salinization, drought) influence of plant organisms. The research was supported within the framework of “UNN competitive recovery Programme. Establishing scientific-investigational laboratories jointly with high tech organizations”.

Development and investigations of innovative gene therapy drug AntioncoRAS-M for cancer treatment I.V. Alekseenko, E.V. Snezhkov, G.S. Monastyrskaya, R.I. Yakubovskaya, E.D. Sverdlov M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Gene-directed enzyme prodrug therapy (GDEPT) consists in targeted delivery into tumor cells of a suicide gene responsible for in situ conversion of a prodrug into cytotoxic metabolites. Major limitations of GDEPT that hinder its clinical application include inefficient delivery into cancer cells and poor prodrug activation by suicide enzymes. We tried to overcome these constraints through combination of the suicide gene therapy with immunomodulating therapy. We developed gene therapy drug AntionkoRAS-M for cancer treatment. The AntioncoRAS-M consists of a gene construct containing the suicide gene herpes simplex virus thymidine kinase (HSVtk) and a cytokine gene granulocyte-macrophage colony-stimulating factor GM-CSF. The gene construct is packed into a low toxic chemical carrier – a PEI-PEG copolymer (polyethylenimine-polyethylene glycol copolymer). Coexpression of HSVtk and GM-CSF genes in tumor cells leads to the death of cancer cells due to the function of HSVtk and release of these tumor antigens, which are effectively presented by GM-CSF activated antigen-presenting cells to T cells of the immune system and ensure the activation of specific antitumor immunity. As a result the mortality of tumor cells significant increases and the probability of their metastasizing decreases. During pre-clinical studies we have shown a high antitumor activity of AntioncoRAS-M. The therapeutic potential of the drug was evaluated in mouse grafted with tumors S37, C26, CSC5, Hep2, HeLa and HT1080. The data obtained showed that treatment with AntioncoRAS-M plus ganciclovir had a biologically significant antitumor effect in animals. On day 35 in the case of mouse sarcoma S37, tumor growth inhibition (TGI) and extension of animal lifespan (EAL) were 88 and 83%, respectively, metastasis process inhibition was 82%. On day 26 with mouse carcinoma C26, TGI and EAL were 68 and 42%, respectively. Administration of the AntioncoRAS-M plus ganciclovir caused a statistically significant inhibition of tumor growth in immunodeficient mice. TGI in nude /c mice was 66–78%, depending on the type of tumor. In the experiments on the general toxic properties of the preparation, we have shown that the drug is safe.

Interaction of antitumor liposomes equipped with tetrasaccharide selectin ligand with vascular endothelial cells A.S. Alekseeva1, N.R. Kuznetsova1, M.R. Kapkaeva2, O.N. Shcheglovitova2, I.A. Boldyrev1, N.V. Bovin1, J.G. Molotkovsky1, E.L. Vodovozova1 1M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia; 2Gamaleya Research Institute of Epidemiology and Microbiology, RAMS, Moscow, Russia Liposome-based drug delivery systems hold the leading positions in the field of nanomedicine. We have designed stable 100-nm liposomes based on natural phospholipids loaded in the lipid bilayer with the lipophilic prodrug of an alkylating agent melphalan (Mlph), indispensable in treatment of multiple myeloma and metastasizing tumors. Liposomes equipped with a tetrasaccharide selectin ligand SiaLeX (SiaLeX-L-Mlph) produced antivascular effect and surpassed the non-targeted formulation by the antitumor effect in a Lewis lung carcinoma model [Kuznetsova et al. J. Drug Target. 2014]. E-Selectins are expressed on activated endothelial cells (EC) at sites of inflammation and infection, as well as in tumor blood vessels. In the work, we studied the specific features of interactions between the liposomes and EC using the primary culture of human umbilical vein endothelial cells (HUVEC). According to the data of confocal laser scanning microscopy, E-selectin expression on cells increased exponentially with the increase of the concentration of tumor necrosis factor alpha (hTNF-α) in the medium. To visualize the liposomes, fluorescently labeled phosphatidylcholine was introduced in the bilayer. We used flow cytometry to demonstrate that the increase in SiaLeX content in liposomes from 2 to 10 mol. % leads to 5-fold increase in SiaLeX-L-Mlph binding with hTNF-α-activated cells, while non-activated HUVECs retain low level of liposome binding. In the presence of excess anti-E-selectin antibodies, binding of SiaLeX-liposomes with activated cells was inhibited by 70%. According to fluorescence spectroscopy data, both processes – inding and internalization by activated HUVECs – were 40–50% more efficient in the case of SiaLeX-L-Mlph if compared to the non-targeted liposomes. Endocytosis of SiaLeX- liposomes occurred via clathrin-independent pathways, which does not contradict the available literature data on E-selectin localization in plasmalemma. Using SiaLeX-L-Mlph liposomes with double fluorescent label, we found that they are internalized by activated HUVECs within minutes through membrane fusion, while non-activated HUVECs consumed negligible dose of liposomes for at least 1.5 h. Thus, the data evidence the selective effect of SiaLeX- L-Mlph on activated EC and agree with our previous results obtained in vivo. The work was supported by the RFBR project №№ 12-04-00168 and 13-04-00069.

Protein-protein interactions in the system of cytokinin signal transduction D.V. Arkhipov Timiryazev Institute of Plant Physiology, RAS, Moscow, Russia Cytokinin signal transduction in plant cell is provided by a multistep phosphorelay system (Lomin et al., Acta Naturae, 2012), where “hot” phosphate moves from one protein to another. This signaling system includes a number of different proteins: receptor – hybrid sensor histidine kinase (HK), phosphotransfer protein (Hpt) and transcriptional factor – response regulator. The objective of our work was to study interactions of these proteins at

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the early stages of cytokinin signal transduction, namely dimerization of receptors and phosphotransfer proteins, as well as interaction between HK and Hpt. Previously, using the BiFC method (Bimolecular Fluorescence Complementation) in combination with confocal microscopy, we showed that receptors as well as phosphotransfer proteins were able to dimerize in vivo, dimers of these proteins being differently localized in cell compartments: HK’s in the endoplasmic reticulum (ER) while Hpt’s in the cytoplasm and nucleus. Also, the interaction between receptors and phosphotransfer proteins registered mainly in the ER has been revealed. To study the molecular basis of interaction between signal proteins, we employed the computer modeling method. First, using the Modeller software, three-dimensional structural models of HK’s and Hpt’s of Arabidopsis thaliana were built by a homology modeling method, followed by validation based on PROCHECK and ProSA-web software. To study the interfaces of interaction of HK’s themselves and with Hpt’s, we used as templates three-dimensional dimer structures of homologous proteins based on X-ray crystallography and taken from the PDB (Protein Data Base). Hpt dimerization was studied by protein-protein docking using ClusPro and PatchDock services. The work allowed us to suggest for a first time a structure of Hpt-Hpt dimer, in which the interfaces of interaction between phosphotransfer proteins and receptors remained free. This allowed us to demonstrate how Hpt-dimer can interact with the receptor dimer and accept «hot» phosphate. As a result of studying the interaction interface between various HK’s and Hpt’s a number of bonds, including one hydrogen bond (Ser-Asn) that was strictly conservative, and another semi-conservative one (Ser-Asn or Gln-Asn), were deduced. Thus, the modeling allowed us to give a detailed description of the most likely mode of interaction between cytokinin receptors and phosphotransfer proteins. This hypothesis can be verified experimentally by a site-directed mutagenesis of amino acid residues presumably

Detachment of key adhesive proteins from membrane skeleton is observed in phosphatidylserine-positive activated platelets and regulates their adhesive properties E.O. Artemenko1,2, A.O. Yakimenko1,2, F.I. Ataullakhanov1,2,3,4, M.A. Panteleev1,2,3,4 1Center for Theoretical Problems of Physicochemical Pharmacology, Moscow, Russia; 2Federal Research and Clinical Center of Pediatric Hematology, Oncology and Immunology, Moscow, Russia; 3National Research Center for Hematology, Moscow, Russia; 4Faculty of Physics, M.V. Lomonosov Moscow State University, Moscow, Russia The attachment of membrane glycoproteins to the cytoskeleton in activated platelet subpopulations has been poorly studied. In resting platelets, adhesive membrane glycoproteins are attached to the cytoskeleton with special proteins, and their degradation is supposed to be controlled by calpain, a calcium- dependent protease. Upon strong physiological activation, two activated platelet subpopulations (phosphatidylserine (PS)-positive and PS-negative) are formed, which have different proaggregatory and procoagulant properties and thus may play distinct roles in hemostasis and thrombosis. We developed a new flow cytometry-based single-cell approach to investigate the attachment of membrane glycoproteins to the cytoskeleton in platelet subpopulations. Platelets were isolated from the whole blood of healthy donors by centrifugation and gel-filtration. Then they were activated with physiological agonists such as thrombin and collagen-related peptide. Subpopulations of PS-positive and PS-negative platelet were separated by flow cytometry due to their different binding of PAC-1, an antibody against the fibrinogen-binding site of activated integrin αIIbβ3. The attachment of membrane glycoproteins (integrin

αIIbβ3, glycoprotein (GP)Ib and P-selectin) to the cytoskeleton was estimated by staining of platelets with fluorescently-labeled antibodies (CD61, CD42b or CD62P, respectively), fixing with paraformaldehyde and treating with detergent. We observed a release of membrane surface glycoproteins in only the PS- positive platelets, but not in the PS-negative activated platelets or resting platelets, and it was prevented by calpain inhibitors (calpeptin and MDL 28170). This effect correlated with the degradation of talin and filamin, as detected by protein electrophoresis. These results suggest that degradation of cytoskeletal proteins that provide attachment of membrane surface glycoproteins to the cytoskeleton occurred only in PS-positive platelets. Detachment of key adhesive proteins from platelet surface should affect on adhesion of PS-positive platelets. We investigated the ability of PS-positive platelets to resist shear-induced detachment from the immobilized fibrinogen. It was increased more than 10-fold in the presence of calpain inhibitor MDL 28170 compared with control platelets. Inhibition of calpain resulted in a significant decrease in the proteolytic degradation of cytoskeletal proteins, suggesting that calpain is involved in the regulation of the attachment of adhesive proteins to the platelet membrane skeleton. Our data suggest that detachment of membrane glycoproteins play a role in the regulation of the adhesive properties of PS-positive activated platelets. This work was supported by the Russian Foundation for Basic Research (RFBR) grant 13-04-00401.

Differential effect of the level of superhelicity on divergent promoter activity in regulatory region of appY Escherichia coli Z.Sh. Babayeva1, S.V. Chernyshov2, O.N. Ozoline1, I.S. Masulis1 1Institute of Cell Biophysics, RAS, Pushchino, Russia; 2Branch of M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Pushchino, Russia In the genome of E. coli near 25% of genes have divergent orientation so that their regulatory regions are partially overlapped or tightly stacked “head-to- head”. Accounting that transcription is usually accompanied by DNA structural transitions and local variations in superhelical density one may assume that additional mechanism in regulation of gene expression mediated by interference of global and local superhelicity may be involved in the case of divergently oriented genes. Promoter islands having high density of potential transcription initiation sites on both DNA strands (Shavkunov KS et al., NAR, 2009) may be considered as natural model for studying of mutual interference of divergent promoters under conditions of modulated superhelicity. To monitor transcriptional activity of such promoters a fragment of E. coli intergenic locus tfaX/appY containing a part of appY regulatory region (‑260 ~-65 relatively to ATG) was cloned in promoterless reporter plasmid between divergently placed genes of EGFP and mCherry. Computational predictions revealed enormously high density in distribution of potential promoters on template as well as on nontemplate strands in this genomic region. High level of synthesis of EGFP and mCherry was detected for analyzed genome fragment indicating bidirectional transcription. In spite of high probability of multiple transcription initiation sites within region studied only single reverse transcription product was detected in the direction of appY (started at -81 relatively to ATG) and single one – for opposite direction (started at -147). Nalidixic acid in the range of concentrations 0.1–2.0 mcg/ml caused differential effect on transcriptional activity suppressing RNA synthesis in the direction of appY. Opposite promoter, which function yet remains to be understood, appeared to be more resistant to the decrease in negative supercoiling under nalidixin treatment. The effect observed is considered in frame of twine-supercoil domain model assuming that transcription imposes negative and positive superhelicity in the vicinity of DNA-bound RNA-polymerase. The work was supported by grants of RSCF (14-14-00985) and RFBR (13-04-00997).

Development of HPLC analysis method for retinoid derivatives N.E. Belikov1, M.A. Yakovleva1, O.V. Demina1, T.B. Feldman1, A.A. Khodonov1,2, M.A. Ostrovsky1 1N.M. Emanuel Institute of Biochemical Physics, RAS, Moscow, Russia; 2 M.V. Lomonosov State University of Fine Chemical Technologies, Moscow, Russia In present study we determined the chromophore identity (retinal A1 or A2) of the visual pigments of two populations of opossum shrimp (M. relicta) living deep in the dark, brown peat lake and in the relatively brightly lit, greenish water of Gulf of Finland. Their dominant visual pigments differ markedly in spectral absorbance (530 nm for sea population and 566 nm for lake population). Among hypotheses of mechanism of visual pigments’ spectral differences of different populations of M. relicta we made an assumption that visual pigment chromophores – vitamins A1 and A2 – can reversibly interchange.

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To verify this hypothesis we have perfected the method of synthesis, isolation and purification of retinal A2, proposed the procedure for extracting retinoids from shrimp eyes preparations, carried out the study of composition of retinoid mixes from shrimps eyes extracts by analytical HPLC with help of synthesized retinals A1 and A2.

It has been shown that retinoid mixes extracts from both sea and lake populations of M. relicta do not contain traces of all-E-3,4-didehydroretinal (A2). Thereby one can make a conclusion that the light adaption process of given species is not due to reversible pigment interchange (A1 – A2) [1]. This work was partly supported by RFBR Grant for young scientists (project № 12-04-31190).

References [1] Belikov N., Yakovleva M., Feldman T., Demina O., Khodonov A., Lindström M., Donner K., Ostrovsky M. Visual pigments of lake and sea populations of Mysis relicta with different absorption maxima contain the same A1 chromophore. // PLoS ONE, 2014, v. 9, № 2, e88107.

Phenolic compounds content in Oxycoccus macrocarpus (Ait.) Pers. calluses and approaches to its improvement E.V. Berezina, Yu.S. Noskova N.I. Lobachevsky State University, Nizhny Novgorod, Russia Biologically active substances are widely used in food and pharmaceutical industry. They can be obtained with the help of plant cell cultivation in vitro , and their production can be improved by basal nutrient medium modification. Among substantcies with high biological activity there are polyphenols, that is why investigation of their improved synthesis is quite urgent. The ultimate goal of this work was to study the influence of nutrient medium composition on phenolic compounds content in cranberry calluses. The research objects were American cranberry (Oхуcoccus macrocarpus (Ait.) Pers., cv. Howes) calluses. It was analysed influence of mineral, carbon and hormonal medium components and elicitors addition as well. Calluses grown on Anderson’s nutrient medium with 30 g/l sucrose and auxins and cytokinins in 0.5/0.5 mg/l concentration were used as control. Quantitative analysis of total soluble phenolic compounds (TSPC), flavonoids and catechins was conducted spectrophotometrically (Shimadzu UV-1700). Analysis of polyphenols content in calluses after 0, 1, and 5 subcultures revealed cranberry cells ability to synthetize secondary metabolites; besides, TSPC quantity in cultures from media with α-naphthylacetic acid was up to 24 times bigger than from media with 2.4-dichlorophenoxyacetic acid. Improved production of investigated metabolites was promoted by increased sucrose concentration (TSPC, for example: 5.3 mg/g of fresh weight while culturing with 30 g/l sucrose and 9.2 mg/g of fresh weight while culturing with 50 g/l sucrose), substitution of glucose or fructose for sucrose, and pectin addition. Nutri- ent medium supplementation with fungal elicitors led in some cases to calluses biosynthetic ability enhancement. Stimulating effect was demonstrated for commercial preparation “Glyokladin” (flavonoid content increase up to 8.5 times towards control), Trichoderma virens mycelium, and its culture broth. Thus there were revealed the most promising approaches to enhanced cranberry callus cultures synthesis of biologically active phenolic substances. The study was supported by “UNN competitive recovery Programme (task 4.3)”.

Novel lipid-transfer protein from pea Pisum sativum I.V. Bogdanov, E.I. Finkina, T.V. Ovchinnikova M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Lipid-transfer proteins (LTP) is an ubiquitous family of small cationic proteins in the plant kingdom. A biological role of these proteins in plants is still obscure. However, they are believed to be involved in angiosperms fertilization, embryogenesis, lipid metabolism, formation of the cuticle, and protection of plants against biotic and abiotic stress. The known plant LTP have a compact spatial structures stabilized by four disulfide bonds. Some LTP were identified as strong allergens. In this work we isolated a novel protein from the pea Pisum sativum seeds, designated as Ps-LTP1. The N-terminal amino acid sequence of Ps-LTP1 has a high degree of homology with other plant LTP. Isolation and purification of Ps-LTP1 was carried out according to a scheme comprising extraction, dialysis, heat treatment, two-stage ultrafiltration, cation exchange chromatography, and two-stage reverse phase HPLC. In order to determine the complete amino acid sequence of Ps-LTP1, the total RNA was isolated from the germinated pea seeds and reverse transcription, and rapid amplification of the 3’-and 5’- cDNA ends were performed. As a result of cloning and sequencing of the obtained fragments, we determined the structure of one partial and two full-length cDNA of the new LTP precursors. All of them contain typical for LTP signal peptides of 24–25 amino acid residues and a mature protein of 95 amino acid residues. Complete concordance of the amino acid sequence of the isolated Ps-LTP1 with the one translated from the corresponding cDNA was confirmed by tryptic hydrolysis of the reduced protein with subsequent mass spectrometry analysis of the obtained fragments. The isolated Ps-LTP1 has a molecular mass of 9399 Da and includes eight conservative cystein residues forming four disulfide bonds. Circular dichroism spectrum of Ps-LTP1 is typical for LTP. This work is supported by the Russian Foundation for Basic Research (project No. 13-08-00956-a).

Application of massive parallel sequencing for identification of circulating epigenetic prostate tumor DNA-markers A.A. Bondar1, E.S. Morozkin1,2, A.M. Kurilshikov1, I.A. Zaporozhchenko1, M.R. Kabilov1, M.M. Zaripov3, V.E. Voytsitskiy3, E.D. Chikova1, V.V. Vlassov1, P.P. Laktionov1,2 1Institute of Chemical Biology and Fundamental Medicine, SB RAS, Novosibirsk, Russia; 2Novosibirsk State Research Institute of Circulation Pathology, Novosibirsk, Russia; 3Novosibirsk Regional Cancer Center, Novosibirsk, Russia Aberrantly methylated DNA fragments of RNF219, KIAA1539, ZC3H4 genes were found as potential cancer DNA-markers in our previous comparative study of cell-free DNA (cfDNA) from prostate cancer (PC) patients, patients with benign prostate hyperplasia (BPH) and healthy donors (HD) using HCGI12k microarrays [Cortese et al., Hum. Mol. Genet., 2012]. Pyrosequencing and preliminary Sanger’s sequencing of target loci have shown high variability in methylation levels of individual CpG dinucleotides within one target and presence of different types of aberrantly methylated DNA patterns in cfDNA. Thus in order to identify reliable markers for cfDNA-based tumor diagnostics methylation profiles of all individual target genes DNA molecules circulating in blood must be analyzed. Present study aimed to identify all methylated DNA patterns of RNF219, KIAA1539, ZC3H4, GSTP1 genes and calculate their frequency in blood plasma of HD, BPH and PC patients using massively-parallel sequencing (MPS). Pilot study included 5 HD and 5 PC patients. Fragments of analyzed genes were amplified using barcoded primers after bisulfate conversion of cfDNA (Zymo Research), and sequenced using MiSeq Platform (Illumina). Four loci were sequenced with 10 000–105 000 × coverage. A novel approach was elaborated for analysis of MPS data, namely Representation of Indi- vidual Types of Molecules (RITM) analysis. RITM analysis calculates occurrence of molecules with the identical methylation profiles and their portion in

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whole pool of cfDNA for each patient. Several individual unique molecule variants were found in PC patients, but not in HD for GSTP1 gene. No such unique molecule variants were found for RNF219, KIAA1539 and ZC3H4 genes, but cfDNA pools of RNF219 and KIAA1539 genes contained age- dependent individual methylation patterns. The data obtained demonstrate usefulness of the approach for detection of the methylated DNA patterns inherent only for cancer patients, however, a further comparative analysis of extended groups of HD, BPH and PC is needed to obtain statistically significant data.

Anti-aggregation activity of alpha-crystallin and its quantification V.A. Borzova A.N. Bach Institute of Biochemistry, RAS, Moscow, Russia A new approach to the quantification of anti-aggregation activity of protein chaperones (small heat shock proteins) was developed. For the dem- onstration of the applicability of that approach test-systems based on dithiothreithol-induced aggregation of target proteins (bovine serum albumin (BSA), pH 7.0, 45°C and α-lactalbumin, pH 6.8, 37°C) were used. The aggregation kinetics was registered from the increase in light scattering intensity at 632.8 nm. The comparison of the anti-aggregation activity of intact, cross-linked with glutaraldehyde and UV-irradiated α-crystallin was 1/n performed in the {(v/v0) ; x} coordinates, where v0 and v are the initial rates of the target protein aggregation without and in presence of α-crystallin, respectively, n is the aggregation order with respect to the target protein and x is the ratio of molar concentrations [α-crystallin]/[target protein]. 1/n The dependence of (v/v0) on x for cross-linked α-crystallin is linear. For intact α-crystallin the dependence includes the initial linear part and the 1/n following plateau at higher α-crystallin concentrations. It is supposed that this shape of the dependence of (v/v0) on x for intact α-crystallin is due to dynamic character of the α-crystallin quaternary structure. The reciprocal value of the length cut off on the abscissa axis by the extrapolation of 1/n the initial linear part of the dependence of (v/v0) on x is the characteristic of adsorption capacity (AC0) of α-crystallin with respect to the target protein. The AC0 parameter was used for the quantification of the chaperone-like activity of α-crystallin. It has been shown that cross-linking with glutaraldehyde results in the 12-fold decrease of AC0 value for α-crystallin with respect to BSA. The decrease in the AC0 value for UV-irradiated α-crystallin is mainly caused by irradiation-induced denaturation of α-crystallin. The additional decrease in the AC0 value is due to cross-linking of subunits in α-crystallin oligomers. This study was funded by the Program “Molecular and Cell Biology” of the Presidium of Russian Academy of Sciences and the Russian Foundation for Basic Research (grants 14-04-01530-а and 12-04-00545-а).

Borzova V.A. et al., PloS One 2013, 8(9):e74367; Intern. J. Biol. Macromol. 2014, v. 68, p. 144-150.

Biofilm formation by anammox bacteria community during flow cultivation in anaerobic bioreactor E.A. Botchkova, Yu.V. Litti, A.N. Nozhevnikova Winogradsky Institute of Microbiology, RAS, Moscow, Russia Microorganism community with the prevalence of anammox-bacteria was enriched during more than 4 years of flow cultivation in lab-scale upflow anaerobic bioreactor. Nitrogen load has been gradually increased, nowadays nitrogen load is 5.4 g N/l day. Biomass was immobilized at specific polymer carriers – ersch. Biomass immobilization helps to avoid unwanted washout of biomass. The aim of this work was to study morphology, microbial community composition and biofilm formation dynamics in bioreactor. Mature biofilms formed by immobilized microorganisms on ersch carriers, are bunch-shaped, 1–5 mm in diameter with rigid cover. Thin long layers with small round-shaped aggregates embedded into them are formed on the reactor walls. Both types of biofilms are formed by anammox cells microcolonies and their satellites. According to molecular phylogeny, anammox bacteria in bioreactor belong to three different species. The dominant phylotype is 98% similar to species Candidatus ‘Jettenia asiatica’. Two other species belong to the genus Candidatus ‘Brocadia’. There are other various groups of bacteria in bioreactor community, among them are uncultured filamentous forms – Chloroflexi. In such conditions, type two biofilms formation de novo (on embedded into bioreactor glasses) starts at 15–17 day. Anammox bacteria and filamentous bacteria, presumably, Chloroflexi, are involved in biofilm formation. Further biofilm growth is connected with increasing of involved cells number and intracellular polysaccharide matrix synthesis.

Functional definition of amino acid residues involved in pH sensitivity of IRR receptor (insulin receptor-related receptor) N.A. Chachina, O.V. Serovа, I.E. Deyev, A.G. Petrenko M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Tyrosine kinase receptors play a key role in the functioning of the organism: the regulation of cell proliferation and differentiation, cell migration and metabolism, and participate in cell cycle control. Now it is known about 20 families of these receptors, including the insulin family. This family includes the insulin receptor, insulin-like growth factor and insulin receptor-like receptor (IRR). In our laboratory, we were able to show and prove the IRR as a sensor of slightly alkaline, and to identify the functionally important part of the recognition domains. However thoroughly tell which amino acid sequences in these domains are responsible for the recognition of the extracellular pH of the medium we could not at that time. Therefore, we have been tasked with mapping the amino acids involved in the pH sensitivity of the receptor IRR, and for this we have developed a modified technique of receptor autophosphorylation in vitro. We obtained the IRR mutated sequences with single point mutations to alanine amino acids M406, V407, D408, P436, V437 and T582, as well as mutations evolutionarily conserved sequence of seven amino acids (P727, W728, K729, V730, T731, N734 and K735) at the end of the alpha-subunit of receptor IRR. These mutations were introduced into the functionally important domains of the receptor: in the fibronectin repeats and L2 domain. We showed triple mutation M406, V407, D408 causes a drop in activity to 28±10% of the activity of wild-type and single replacement T582 reduced activity by 40%. The remaining substitutions did not cause a large negative effect. However, a complete replacement of either the first repeat of fibronectin FnIII-1 led to a drop in activity by 60%, whereas the replacement once the second and third repeat of fibronectin FnIII-2 & 3 led to a complete loss of pH sensitivity. Thus, we have shown that the pH-sensitive sites are scattered along the entire extracellular domain of the receptor IRR. This work was supported by RFBR (grant 13-04-01359_a).

Inhibition of dsRNA-induced immune response in primary and transformed human cells by deoxyribonucleotides A.V. Cherepanova1, Zh.K. Nazarkina1, V.V. Vlassov1, P.P. Laktionov1,2 1Institute of Chemical Biology and Fundamental Medicine, SB RAS, Novosibirsk, Russia; 2Novosibirsk State Research Institute of Circulation Pathology, Novosibirsk, Russia DNA inhibits poly(I:C)-activated production of pro-inflammatory interleukins (IL-6 and IL-8) in human primary fibroblasts (Cherepanova et al., Im- munobiology, 2013) and inhibiting efficacy depends on DNA sequence. The possible approach to identify the mechanisms of immunoinhibiting action of DNA is investigation of DNA inhibiting effect on poly(I:C)-induced immunomediators, produced via signal transduction pathways different from IL-6/ IL-8 as well as evaluation of poly(I:C)/DNA effect in various cell types, which may differ in expression of pattern-recognizing receptors and downstream pathways efficacy.

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The goal of this work is to investigate the influence of specific ODNs on production of pro-inflammatory molecules in poly(I:C)-activated human primary endothelial cells from umbilical cord vein (HUVEC), gingival fibroblasts (GF), immature dendritic cells from blood monocytes (DC) and transformed cell lines (cervical carcinoma (Hela) and epidermoid carcinoma (A431)). IL-6 production was measured using ELISA (Vector-Best, Russia), the expression of interferon beta1 (ifnb1) and antibacterial peptide beta-defensin 2 (hbd2) was evaluated using SYBR Green Real-time PCR. Poly(I:C) activate IL-6 production in all cell types under investigation. Previously shown inhibition of poly(I:C)-induced interleukins production by specific ODNs is also shown in HUVEC, Hela and A431 cells, but is not observed in DC. ODN inhibit poly(I:C)-induced expression ofhbd2 in A431 cells and expression of ifnb1 in GF. Inhibition of defensins expression confirms the influence of ODNs on NFκB and АР-1 transcriptional factors, whereas inhibition of interferon beta1 indicates the involvement of IRF3 and IRF7 transcriptional factors. The data obtained indicate that inhibition occurs on the early stages of signal transduction cascade: before the recruitment of TRAF6, which activate NFκB via TAB2 polyubiquitinilation and TRAF3, responsible for IRF3 and IRF7 phosphorilation by TANK-binding kinase 1 (TBK1). The absence of ODN-dependent inhibition of IL-6 production in dendritic cells indicates that the effect is not universal for all cell types. Whether the difference of receptors/signal molecules expression or the efficacy of ODN transport into dendritic cells are responsible for the effect remains to be investigated.

DNA-methyltransferase in mammalian cells A.M. Cherevatenko1, O.V. Diachenko2, S.V. Tarlachkov2,, N.V. Rudenko2, T.V. Shevchuk2, Ya.I. Burianov2 1M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia; 2Branch of M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Pushino, Russia In eukaryotic cells DNA methylation occurs in CpG-, CpHpG- and CpH-sequences (H=A, T or C). The greatest attention is paid to studying CpG-type while non-CpG-type is poorly described. Disruption of normal methylation pattern of the mammalian genome accompanies different epigenetic pathologies, including cancer. Chromomethylase CMT3 performs methylation of plant DNA in CpHpG- sequences while interacting with chromatin elements. The HEK293 cell line which express the gene of DNA-methyltransferase CMT3 was obtained. New morphological phenotype of transformed cells was identified. These cells differed from control with more round shape and demonstrated lower proliferation rate then control ones. HEK293 cells which express cmt3 gene shows protection of it’s DNA from restriction by CpHpG-specific methylsensitive endonuclease and differed with lower efficiently of colony formation from control line. These results may be used in studying of new strategies of antitumor therapy. This work supported by grant 12-08-00731 from RFBR.

Novel modified biomaterials based on polyvinyl alcohol for tissue engineering M.G. Drozdova1, V.A. Lebedeva2, R.A. Akasov1, D.S. Zaytseva-Zotova1, A.S. Golunova2, A.A. Artyuhov2, M.V. Maslova3, A.N. Sonina3, G.A. Vihoreva3, M.I. Shtilman2, E.A. Markvicheva1 1M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia; 2D. Mendeleev University of Chemical Technology of Russian, Moscow, Russia; 3Moscow State University of Design and Technology, Moscow, Russia Polyvinyl alcohol (PVA) is a biocompatible polymer, which has been widely used in various fields of biomedicine. However, PVA-based scaffolds do not provide good adhesion and cell spreading, because PVA is bioinert material. Since cell adhesion, growth and proliferation are strongly influenced by the surface properties, in order to enhance cell attachment and spreading on the support, PVA could be modified either chemically by introduction of co-monomers, or physically by preparing mixtures of various natural polymers. The aim of the research was to prepare and to study new biomaterials based on PVA modified by the introduction of charged (diethylaminoethyl methacrylate and acrylic acid) and hydrophobic (hydroxyethyl methacrylate) co-monomers or by the introduction of natural polysaccharide chitosan into the scaffold for their further use in tissue engineering. This study was focused on investigation of two types of scaffolds: 1) macroporous hydrogel prepared by copolymerization of acrylated PVA and corresponding co-monomer and 2) micro-and nanofibers obtained by electrospinning of PVA/chitosan mixtures. Confocal and scanning electron microscopy as well as MTT-assay were used here to evaluate the structure of the obtained scaffolds and their cytotoxicity. The mouse fibroblast cell line (L929) and mesenchymal stem cells derived from human adipose tissue (MSC) were used in the study. It has been shown that the introduction of charged and hydrophobic groups into PVA scaffold (in case of hydrogels) and the introduction of chitosan to the moldable mixture (in case of fibers) substantially affected the structure of the obtained scaffolds. Cytotoxicity studies in vitro (extract and contact tests) showed that the proposed modifications did not exhibit cytotoxic effect. The analysis of the growth of different cell types on/in hydrogels, micro-and nanofibers showed that these modified scaffolds promoted cell adhesion and spreading on the polymer surfaces, and thereby they supported cell growth and proliferation at long-term cultivation.

Possibility of pathogenic regulation of oxidative stress by catalytic antibody of patients with schizophrenia E.A. Ermakov, L.P. Smirnova, Y.N. Borodyuk, V.N. Buneva, S.A. Ivanova, G.A. Nevinsky Novosibirsk State University, Novosibirsk, Russia; Mental Health Research Institute, SB RAMS, Tomsk, Russia; Institute of Chemical Biology and Fundamental Medicine, SB RAS, Novosibirsk, Russia Involvement of the immune system in the pathogenesis of schizophrenia was shown in many articles. It is known that glutamate and dopamine hyper- stimulation of the postsynaptic neurons leads to transport increase of Ca2+ ions into cells, activation of calcium-dependent processes, calcium-dependent phospholipase A2, and the formation of large number of reactive oxygen species, which in turn leads to the development of generalized oxidative stress in patients with schizophrenia. Recently antibodies (Ab), having a catalytic activity and named abzymes, were detected in serum of patients with autoimmune, infectious diseases and in milk of healthy mother’s. It was shown in the works of E. Ihmyangan that Ab from the blood of mammals possess oxidoreductase activities. In this work we investigated oxidoreductase activities of IgG, isolated from serum of patients with schizophrenia and healthy donors. It was shown that the Abs in serum of patients with schizophrenia have catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GP) activity. Based on use affinity sorbents, homogeneity of isolated IgG and gel filtration in pH-shock conditions, we proved that the activity is intrinsic property of studied Ab. It was established that IgG of patients with schizophrenia possess CAT-activity, and at healthy donors it wasn’t tested. It was interesting that activity of erythrocyte catalase decreased in 2 times in patients with schizophrenia with compared of healthy people. Inhibition of CAT-activity IgG using an irreversible catalase inhibitor, 3-amino-2,4-triazole, indicated the presence of histidine in the catalytic region of the active center of IgG and suggested a similar mechanism of catalysis. Km values were determined to be significantly smaller than that of the native enzyme catalase. SOD- and GP-activity of Ab in schizophrenic patients was higher in 2–4 times with compared to healthy individuals. Similar activity of cellular enzymes in patients with schizophrenia was significantly reduced. Based on these results we can suggest that IgG may play a role in organism protection from oxidative stress. This work was supported by a grant from RSF № 14-15-00480.

54 | Acta naturae | SPECIAL ISSUE № 1 2014 Young Scientists Competition

New hybrid fluorescent proteins for vizualization of inflammation markers S.Sh. Gapizov1,2, L.E. Petrovskaya1, L.N. Shingarova1, E.V. Svirshchevskaya1, D.A. Dolgikh1,2 1M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia; 2Lomonosov Moscow State University, Moscow, Russia Tumor necrosis factor alpha (TNFα) is the main human proinflammatory cytokine involved in the development of several chronic inflammatory diseases such as Crohn's disease, rheumatoid arthritis, cerebral malaria, psoriasis, etc. Endothelial cells represent one of the targets of TNFα activity during the inflammation. As a result of endothelial cell activation by TNFα, an increased surface expression of different adhesion molecules and receptors including αvβ3-integrin and vascular endothelial growth factor receptor 2 (VEGFR2) is observed. Visualization of these molecular markers of endothelial cell activation is a promising approach for the early diagnostics and monitoring of inflammatory diseases and neoangiogenesis. We have developed a new class of hybrid fluorescent proteins for the imaging of cell markers of neoangiogenesis. Recombinant proteins comprising the fluorescent protein mCherry and αvβ3-integrin or VEGFR2-binding proteins on the basis of 10Fn3 were obtained by expression in E. coli cells in a soluble form. We have demonstrated that the expression products are in monomeric form, and their fluorescence spectra are identical to mCherry. The resulting proteins Ch-3JCLI4 and Ch-CT322 were tested in vitro using cell lines HL-60, MDCK, EA.hy926 and primary endothelial cells (HUVEC). It was shown that the fusion proteins bind the same targets at the cell surface as monoclonal antibodies to αvβ3-integrin and VEGFR2, respectively. EA.hy926 and HUVEC were selected to develop a model of inflammation induced by TNF-α and VEGFA. Activation of the cells by the addition of 25 ng/ml TNFα and VEGFA for 5 hours resulted in the increased expression of cellular adhesion molecules, which were identified using specific antibodies. We have demonstrated that the expression of αvβ3-integrin and VEGFR2 increases on the surface of activated EA.hy926 and HUVEC. The work was supported by RFBR grant 13-04-12405 OFI_m and Russian Academy of Sciences program “Molecular and Cellular Biology”.

The influence of fragilysin active site and zinc-binding motif structure on E-cadherin cleavage E.N. Grafskaia1,2, D.D. Kharlampieva1, V.A. Manuvera1, Lazarev V.N.1,2 1Scientific Research Institute of Physical-Chemical Medicine, FMBA, Moscow, Russia; 2 Moscow Institute of Physics and Technology (State University), Dolgoprudny, Russia Fragilysin is a toxin secreted by enterotoxigenic Bacteroides fragilis strain termed BFT (B. fragilis toxin). Three different isoforms have been identified. The catalytic domain of each isoform contains Zn2+-binding motif specific to metalloproteinases. According to the literature, the main effect of BFT on intestinal epithelial cells is a disruption of tight junctions caused by E-cadherin cleavage. To test a hypothesis that E-cadherin is a natural substrate for the BFT, recombinant E-cadherin was obtained in Escherichia coli and Expi293 cells. It was shown that BFT do not cleave any recombinant E-cadherins in vitro. BFT is the Zn2+-dependent metalloproteinase, it was necessary to examine whether the BFT proteolytic activity is necessary for E-cadherin cleavage of human colon carcinoma cell line - HT-29. In this research, the influence of amino acid substitutions on the biological activity of recombinant BFT-1, 2, 3 obtained in E. coli heterologous system was studied. Mutant recombinant BFT-1, 2, 3 were obtained by site-directed mutagenesis. We replaced glutamic acid residue to alanine residue at the BFT-1, 2, 3 active site and three histidine residues chelating a zinc ion to tyrosine at the BFT-2 zinc-binding motif. To check the biological activity of the obtained recombinant proteins we performed the test on HT-29 cells. Within 1 h of HT-29 cells treatment with wild-type BFT-1, 2, 3 at a concentration greater than 0.06 µg/ml the alteration of intestinal epithelial cell lines morphology (cells rounding) was exhibited. Mutant proteins did not show such effect. These results were confirmed by Western blot hybridization with E-cadherin antibodies as well. E-cadherin cleavage was observed only at HT-29 cells treatment with wild-type proteins. Thus, amino acid substitutions prevent E-cadherin cleavage and cells rounding. The native structure of the active site and the zinc-binding motif of BFT is required for E-cadherin cleavage of HT-29 cells.

Comprehensive analysis of clonal TCR repertoire in patients with ankylosing spondylitis and HLA-B27+ healthy donors E.A. Komech, I.V. Zvyagin, V.I. Nazarov, M.V. Pogorelyy, I.Z. Mamedov, Y.B. Lebedev M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Nowadays immunology is highly concerned with defining the factors that affect adaptive immune system formation and homeostasis. One of the most informative characteristics of adaptive immunity status is clonal T-cell receptor (TCR) repertoire. TCR is an geterodimer with each chain having one hy- pervariable CDR3 region generated via independent recombination of V-, D- and J-gene segments and insertion of nontemplated nucleotides, that helps to recognize a diverse array of antigenic peptides in the complex with major histocompatibility complex (MHC). Introducing NGS techniques for TCR repertoires analysis allows to quantitatively and qualitatively define the structure of the repertoire and thus reveal distinctive properties of repertoire in immunopathological conditions, particularly in autoimmune diseases (AD). One example of those is ankylosing spondylitis (AS, Bechterew's disease) – chronic inflammatory disease of the axial skeleton that was shown to be strongly associated with MHC class I al- lele – HLA-B27. The purpose of this study is revealing the distinctive properties of TCR repertoires of patients with idiopathic AS in contrast to HLA-B27+ healthy donors. To reconstruct individual T-lymphocyte repertoires of HLA-B27-positive donors (healthy individuals and patients with AS), and HLA-B27-negative controls, we used developed previously original technique, that includes: (i) RNA-based preparation of mature TCRβ genes PCR-libraries with insertion of molecular barcodes in course of reverse transcription, (ii) sequencing of obtained PCR-libraries on Illumina and (iii) processing of sequencing results with original software. In this study we have performed a comprehensive analysis of T-lymphocyte repertoires in patients with AS and healthy HLA-B27+ donors. Within each group shared (including public) clonotypes were found and their frequencies calculated. We analyzed structural diversity of high-frequency clonotypes. Also we compared common characteristics of HLA-B27+ donors repertoires with those of HLA-B27- individuals and revealed several differences in usage of a number of V- and J-segments, peculiar to repertoire of HLA-B27+ individuals.

Two-phase nutrient medium for isolation and maintenance of microorganisms of the genus corynebacterium V.P. Korchagina, E.L. Alutina, G.G. Harseeva Rostov State Medical University, Rostov-on-Don, Russia Individual colonies of Corynebacteria diphtheriae show growth after 24 hours incubation on typical nutrient media (Tellurite blood agar, Clauberg II medium, Tinsdale-Sadykova serum, etc.). Following 48 hours incubation, the number of colonies formed is enough for proper bacteriological research conduct. The aim of this research project has been to develop nutrient medium with enhanced cultivation properties for isolation and maintenance of C. diphtheriae. The two-phase nutrient comprises solid medium (pancreatic hydrolysate of fish protein, hemophilic bacilli growth promoting substance, sodium chloride, yeastrel, glucose, microbiological agar, potassium tellurite solution 2%) and nutrient broth (pancreatic hydrolysate of fish protein, peptone fermentation mixture, sodium chloride, yeastrel, glucose, bovine serum). The nutrient medium cultivation properties (growth rates, C. diphtheriae germination period, serum sensitivity) were calculated in accordance with PG 4.2.2316-08 «Quality Control of Microbiological Culture Media» (Moscow, 2008). Tellurite blood agar was used as test medium.

SPECIAL ISSUE № 1 | Acta naturae | 55 Young Scientists Competition

Obtained nutrient sensitivity amounts to 10-8, the test one being 10-7. Corynebacteria growth rates on said medium vary from 13.5 to 14 hours, while the test one's are in the range from 24 to 48 hours. C. diphtheriae germination percentage in observed medium (254–270%) is reliably higher (р≤0.05) than in test medium (38–39%). The potassium tellurite solution 2% added to the medium inhibited the growth of attached microbes abundant in starting material. Consequently, the two-phase nutrient medium for cultivation of C. diphtheriae possesses enhanced bacterial growth properties when compared to tellurite blood agar, effectively improving sensitivity 10 times, increasing colony count more than twice and rates of diphtheritic microbe growth – 1.7 times.

Role of RNA helicase Belle (DDX3) in germline stem cell maintenance, proliferation and differentiation in the testes of D. melanogaster A.A. Kotov, L.V. Olenina Institute of Molecular Genetics, RAS, Moscow, Russia DEAD-box RNA helicases are involved in all aspects of RNA metabolism in the eukaryotes. They provide ATP-dependent local RNA-unwinding and re- modeling of ribonucleoprotein complexes. The members of subfamily DDX3 of family DEAD-box RNA helicases take part in transcription, mRNA nuclear export, translation initiation, cell cycle control and apoptosis. In our work we investigated a role of RNA helicase Belle (DDX3) in spermatogenesis process in Drosophila melanogaster. A failure of expression of homologous protein DBY (DDX3) in humans leads to a germ cell loss in the testes, while maintaining somatic cells of niche and is known as Sertoli cell-only syndrome (SCOS) (Foresta et al., 2000; Lardone et al., 2007). We have found that a strong deficiency of Belle expression in mutant flies also led to a lack of germ cells, including stem cells, in the testis but not in the ovaries. Testes of mutant flies were significantly reduced in size, but they maintained somatic hub cells and somatic cells of cysts. Using TUNEL assay and confocal microscopy, we have shown that the way of a germ cells loss is apoptosis. RNAi-induced knockdown of Belle in germ cells but not in somatic cells, led to the same phenotype as in the case of belle mutants. Thus, the function of Belle is needed intrinsically for maintaining germline stem cells. Analysis of testis morphology of third instar larvae with belle mutation showed that the laying of precursors of germ tissues in the mutants proceeds normally and all types of premeiotic germ cells presented in the testes. However, it was found a reduced number of germline stem cells (6.6±3.8 in mutants versus 15.2±4.3 in wild type flies) without disturbing their adhesion to the hub. In the belle mutants we detected germ cells with abnormally large cellular and nuclear volumes. This indicates a delay of the cells in G2 phase and their inability to enter into mitotic division. Western blot analysis revealed that in the case of belle mutations it was observed a significant decline of expression of mitotic cyclins A and B in the gonads but not of cyclin-dependent kinase Cdc2 and the S-phase cyclin E. Using real time PCR, we found a reduced levels of transcription of mRNAs of mitotic cyclins in the testis, indicating the transcriptional level of their regulation mediated by Belle. Ectopic expression of additional copy of cyclin B but not cyclin A in the background of RNAi-induced knockdown Belle in the germline led to a partial rescue of germ cells and restoration of male fertility. Thus, we have shown for the first time that a role of RNA helicase Belle in maintenance of germline stem cells and regulation of their mitotic divisions is associated with transcriptional control of mitotic cyclins. Our results support an important role of mitotic cyclins regulation for survival and functioning of germline stem cells taking into account presumptive conservative mechanisms of this regulation in humans. This work was partially supported by the Molecular and Cellular Biology program of Russian Academy of Sciences and RFBR (project # 13-04-00699-a).

Investigation of pattern of genomic DNA fragmentation during apoptosis E.M. Loseva, E.S. Morozkin, A.M. Kurilshikov, E.Y. Rykova, V.V. Vlassov, P.P. Laktionov Institute of Chemical Biology and Fundamental Medicine, SB RAS, Novosibirsk, Russia Factors that influence the formation of the pool of extracellular DNA such as packaging of genomic DNA and susceptibility of different DNA regions to nu- clease hydrolysis can result in a different representation of a genome region in the pool of extracellular DNA as compared to genomic DNA of parent-cells. Since apoptosis is the main mechanism of generation of extracellular DNA, a study of composition of apoptotic and genomic DNA has been performed with massively parallel next-generation sequencing platform (SOLiD 3). Apoptotic DNA isolated from culture medium of 5 HUVEC cell lines with induced apoptosis and genomic DNA from untreated cells from same lines was sequenced using SOLID 3 platform. Analysis of the data (peak calling, MACS14) has identified 764 DNA sequences (peaks) overrepresented in at least 4-apoptotic DNA libraries. Cross-reference with UCSC Genome Browser data has shown that the identified peaks are enriched in CpG islands areas and sites of hypersensitivity to DNase I. The verification of computed peaks with TaqMan PCR has shown that concentration of specific peaks' sequences is 2 to 12-fold higher in apoptotic DNA as compared with genomic DNA of HUVEC. By comparing the representation of the sequences of interest in plasma DNA from healthy donors and primary lymphocyte genomic DNA it has been found that selected DNA sequences are 1 to 61-fold more frequent in plasma than in genomic DNA. These data suggest that DNA fragmentation by apoptosis is not random, but the composition of overrepresented sequences among biological replicas is varied.

Detection of active promoters in genomic fragment libraries C.A. Lubimova, D.A. Didych M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Promoters are important regulatory elements of the genome that initiate transcription of all genes. Promoters ensure cellular specificity of gene expression, including expression of cancer-specific genes. Detecting of promoter active in tumor cells is a necessary step in creation of safe anti-cancer gene therapy preparations. We employed the reporter gene approach for detection of promoter activity in human genome fragments library inserted into a plasmid vectors. The vectors contained genes of green and red fluorescent proteins in head-to-head orientation. Two vectors for detecting promoter activity were prepared; one of them contained a strong cytomegalovirus enhancer. The genome fragment library was cloned in both vectors between reporter genes and two plasmid pools containing approximately 400000 fragments each obtained. These pools were used for transient transfection of cancerous A-431 cells and non-cancerous 293T cells and percentage of fluorescent cells was determined with the help of flow cytometer. Both analyzed libraries contained promoters active in A431 and 293T cells. Moreover, we showed that the cytomegalovirus enhancer increase percentage of the fluorescence-positive cells in both cell lines analyzed. The use of well-known tissue-specific enhancers could allow for detecting of enhancer-inducted promoters with various cellular specificities. The research may become a base for constructing human genome libraries enriched with cell-specific promoters.

Search of ligands of galectins -4, -8 and -9 on endothelial cells M.A. Malchevskaya, E.M. Rapoport, E.Yu. Korchagina, I.S. Popova, I.M. Ryzhov, H.J. Gabius, N.V. Bovin M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Recently we have shown that tandem-repeat galectins -4, -8 and -9 display high binding to histo-blood group ABH-antigens (Vokhmyanina et al., Gly- cobiology, 2012, 22, 1207–1217); the biological significance of this interaction is not known yet. Blood group ABH determinants are present on various glycopeptides and glycolipids, which are widely distributed in human tissues in particular on endothelial cells. We suggested that tandem-repeat galectins

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expressed on lymphocytes induce their migration through endothelial barrier via binding to ABH-antigens. To this end we studied the binding of galectins -4, -8 and -9 to EC. As a model of EC cells EA.hy 9 were used; this cell line was obtained by immortalization of human umbilical endothelial (HUVEC) and lung carcinoma A-549 cells (referred further as iHUVEC); it conserves phenotype of primary EC but unlike those it can be grown in large quantities and does not lose viability in the course of propagation. To determine whether galectins bind H-antigen cells were treated with glycosidase digesting terminal fucose. Defucosylation provides decrease of binding of galectin-9 but not of galectins -4, -8 to the iHUVEC. Since HUVEC do not express A and B-antigens we inserted neoglycolipids onto the cells and studied the binding of galectins to modified cells. ABH-neoglycolipids with different length between glycan (Glyc=A or B tetrasaccharides) and lipid parts were synthesized for cell modification. The neoglycolipids Glyc-Ad-DOPE with short (1.9 nm) spacer were obtained by condensation of 3-aminopropyl glycosides (Glycβ1-O(CH2)3NH2) with activated derivative of 1,2-O-dioleoyl-sn-glycero-3- phosphatidylethanolamine (DOPE-Ad-ONSu). The neoglycolipids Glyc-Ad-CMG2-DOPE with long carboxymethylglycine (CMG2) based spacer (7.2 nm) were synthesized as follows: coupling of Glycβ1-O(CH2)3NH2 with activated adipinic acid and subsequent condensation of activated glycans (Glycβ1- O(CH2)3NH-Ad-ONSu) with amino-terminated CMG2-DOPE. It was shown: 1) insertion of B (type 2)-Ad-DOPE provided the increase of binding of galectin-4 and galectin-8 and not of galectin-9 to the cells. On the contrary, modification of cells by B (type 2)-Ad-CMG2-DOPE did not affect the binding capacity of galectins; 2) insertion of neoglycolipids carrying A (type 2) – tetrasaccharide led to increase of binding only of galectin-9. Thus, modifying of glyco-landscape of cells allowed us to conclude that galectins -4 and -8 could induce adhesion to EC via binding to B-glycans whereas galectin-9 – to H- and A-ones. The work is supported by the grant of Russian Foundation for Basic Research № 13-04-00096.

Synthesis and spectral properties study of spiropyran derivatives with ionogenic groups I.A. Melnikova1,2, A.Yu. Lukin1, N.E. Belikov2, O.V. Demina2, S.D. Varfolomeev2, A.A. Khodonov1,2 1M.V. Lomonosov State University of Fine Chemical Technologies, Moscow, Russia; 2N.M. Emanuel Institute of Biochemical Physics, RAS, Moscow, Russia Photochromic ionophores and artificial receptors which allow to control the cation-binding process and the properties of the resulting products using irradiation by light with selected wavelengths, are of particular interest for researchers in different fields [1]. In present study a new type of photochromic systems containing ionogenic or fluorophoric groups, capable to effective interaction with metal cations, has been developed. For their preparation we applied direct modification of the formyl group at the 5'-position of the photochromic molecule with using of well-known and efficient organic synthesis experimental procedures: reductive amination or Wittig olefination. The spirobenzopyran derivatives have been prepared in pre- parative amounts; their structure has been characterized by a number of physicochemical analysis methods. Their photochromic behavior has been studied in toluene and ethanol solutions.

This work was partly supported by the Grant of President of the Russian Federation for Young Ph.D. Scientists (project No. МК-6901.2013.4).

References [1] Chambers J.J., Kramer R.H. Photosensitive Molecules for Controlling Biological Function. In: Neuromethods, v. 55, Springer Science+Business Media, LLC, 2011.

The role of the transmembrane domain in the VEGF receptor 2 activation K.S. Mineev1, S. Manni2, D.R. Usmanova1,3, E.N. Lyukmanova1, M.A. Shulepko1,4, X. Deupi2, K. Ballmer-Hofer2 , A.S. Arseniev1,3 1M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia; 2Paul Scherrer Institute, Villigen, Switzerland; 3Moscow Institute of Physics and Technology (State University), Dolgoprudnyi, Russia; 4Lomonosov Moscow State University, Moscow, Russia Vascular Endothelial Growth Factor receptors, VEGFRs, regulate blood and lymphatic vessel development and homeostasis and belong to receptor tyrosine kinase (RTK) family of integral membrane proteins. Transmembrane signaling by RTKs entails ligand-mediated dimerization and structural rearrangement of the extracellular domain. RTK activation also depends on specific orientation of the transmembrane domain (TMD) helices, as suggested by pathogenic, constitutively active RTK mutants. Such mutants carry polar amino acids in the TMD, promoting stable transmembrane helix dimerization, which is essential for kinase activation. Here we investigated the spatial structure of the TMDs and the activity of VEGFR-2 constructs, where we mutated the wild-type sequence by replacing specific amino acids in the TMD with glutamate residues. Our data revealed that single-point mutations V769E, I767E and L768E are capable to activate the VEGFR-2 receptor with deleted extracellular domain, but not the full-size receptor. On the other hand, double mutations G770E/ F777E and T771E/F778E triggered the ligand-independent activation of the full-size VEGFR-2. With this respect we then investigated the spatial structure of the dimers and the dimerization propensities for three transmembrane peptides, corresponding to the wilt-type TMD of VEGFR-2 and to V769E and G770E/F777E mutant TMDs with solution NMR spectroscopy. As a result we found that three major aspects distinguish the G770E/F777E TMD dimer from the wild-type: (1) dimerization propensity is enhanced; (2) dimerization occurs via a completely different interface; (3) the spatial structure of this dimer is flexible. Further molecular dynamics simulations confirmed the structural interpretation of the NMR data. The obtained structural and functional data on the VEGFR-2 TMD, both wild-type and carrying various mutations, were then summarized to propose the new mechanism for the VEGFR-2 activation, involving the ligand-induced reorientation of TMDs in the pre-formed VEGFR-2 dimers.

Anaerobic microbial digestion of organic waste and methane production under different temperatures A.A. Nikitina, A.N. Nozhevnikova Winogradsky Institute of Microbiology, RAS, Moscow, Russia Municipal solid waste (MSW) and sewage sludge are the main organic wastes of big cities. In Russia, MSW and sewage sludge are disposed at landfills, which have long-term negative impact on the environment and health of the population. Anaerobic microbial digestion of organic waste in bioreactors is

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an alternative way to landfill disposal. This biotechnology increases the waste transformation rate hundreds of times and produces an alternative energy source – methane. Thermophilic digestion (50–55°C) accelerates the reaction kinetics, provides the stabilization and disinfection (reduction of pathogens) of digested biomass that is necessary for its further agricultural use as bio-fertilizer, therefore, it is more effective than digestion under mesophilic conditions (30–35°C). Complex trophic interactions with «bottlenecks» that cause disruption of the stability of the anaerobic digestion exist between different microbial groups in methanogenic communities. Therefore, careful experimental study is essential for full-scale implementation of this technology. The anaerobic microbial degradation of OF-MSW was studied under ambient air temperature (20±2°C) and under thermophilic conditions (50°C). Digested sludge and soil samples taken from landfill anaerobic zone were used as inoculants. The highest rate of methanogenesis was observed at both temperatures when landfill soil was used as inoculum together with thickened digested sludge. Acidification due to the excessive formation and accumulation of volatile fatty acids (VFA) was the main problem of microbial decomposition of concentrated organic waste. Addition of active inoculum up to 30–50% of volatile solids (VS) promoted the transformation of VFA to the methane and stabilized the thermophilic anaerobic digestion. After that the VS reduction 3 -1 increased up to 75–85%, the methane yield reached 0.2 dm g of VSfed and its content in biogas was 60–65%, which is close to the maximum values. Ther- mophilic syntrophic bacteria and methanogenic archaea from inoculum adapted to high concentrations of VFA (10 g L-1) were enriched. These consortia will be used to develop a stabilizing microbial inoculum in order to solve the problem of acidification during the digestion of food waste.

Bright fluorescent dyes based on oligo-bodipy fluorophores A.A. Pakhomov, I.A. Boldyrev, V.I. Martynov M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Fluorescent labels are widely used for visualization of processes occurring in live systems. Here we tried to generate extrabright fluorescent organic compounds by combining several fluorophores in one molecule with dendritic scaffold. It is known that high local concentration of chromophores often leads to its reciprocal quenching. However synthesized oligo-BODIPY derivatives showed immaterial decrease in quantum yield (0.75 in case of tetramer). Four times extinction grow resulted in three times increasing of brightness. Taking into account outstanding properties of BODIPY, generated fluorescent dyes exceed usual commercially available compounds such as AlexaFluor, Cy and other BODIPY derivatives. Main conditions for generation of extrabright compounds appeared to be high quantum yield of fluorophore and steric hindrance of its interactions. Further development of new fluorescent labels based on these principles could significantly increase sensitivity of fluorescent methods.

Study of the synergystic effect of bacteriophage T5 endolysin and membrane permeabilizing agents on E. coli and P. aeruginosa cells М.S. Shavrina1,2, А.А. Zimin3, S.V. Chernyshov2, G.V. Mikoulinskaia1,2 1Pushchino State Institute of Natural Sciences, Pushchino, Moscow Region, Russia; 2Branch of M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Pushchino, Moscow Region, Russia; 3Institute of Biochemistry and Physiology of Microorganisms, RAS, Pushchino, Moscow Region, Russia Peptidoglycan hydrolyzing enzymes (PHE) including bacteriophage endolysins are capable of causing lysis of bacteria cells “from without” and therefore can be considered as a probable alternative to antibiotics. Multi drug resistance, especially relevant for nosocomial infections, provided the great surge of interest in PHE during last 13 years. Until recently PHE were considered as lytic agents only against Gram-positive micro-organisms, because their murein is not protected by outer membrane. Bacteriophage T5 endolysin is PHE specific to murein of Gram-negative bacteria but unable to penetrate through their cell wall outer membrane. We investigated the bacteriolytic action on the E.coli and P.aeruginoza (PAO1) live cells of the homogeneous enzyme preparation combined with agents increasing membrane permeability. For this purpose bacteria cells at a concentration of 2·108 CFU/ml were incubated overnight at 37°С in the presence of 40 µg/ ml enzyme and permeabilizing agents in various concentrations (control did not contained the enzyme). Then a culture volume corresponding to initial 107 CFU were grown tonight on agar plates and next morning the arisen colonies were counted. At the result of the work it was shown that at E. coli cells’ lysis bacteriophage T5 endolysin displayed strong synergism with diverse permeabilizing agents: peptide antibiotics polymyxin B (5 µg/ml), gramicidin D (4 µg/ ml) and 50 mM hydroxyamine (Tris). In all cases the number of survived CFU ranged from 0 to 14 and the differences with control were no less than one order of magnitude. In addition, synergism was observed when the enzyme was applied together with cationic peptide (polylysine) and cationic detergent (cetyl trimethylammonium bromide). At the P. aeruginoza (PAO1) cells’ lysis expressed synergistic effect of endolysin was observed with polymyxin B at concentration of 40 µg/ml. Introducing approach can reduce the concentrations of antibiotics commonly used by an order of magnitude and more.

Immunogenic properties of proteins encapsulated into polymeric nanoparticles T.S. Shcherbinina1, V.P. Varlamov1, E.V. Svirshchevskaya2 1Centre “Bioengineering”, RAS, Moscow, Russia; 2M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Induction of immune response with predetermined properties requires the development of the constructions which can stimulate humoral and cellular immune responses independently. The aim of this work was the analysis of immunogenic properties of the proteins encapsulated into polymeric matrix forming nanoparticles (NPs). Lactoferrin (Lf) was used as a model antigen. Succinoyl-chitosan (sChi) with substitution degree 70–75% was obtained from low molecular weight chitosan and used to coat Lf. NPs were formed from Lf or Lf and sChi by the controlled heat treatment. NPs developed were of 200±50 nm in diameter and had ζ-potential 20±5mV. CВА/CaJ mice were immunized via intraperitoneal rout either with Lf-NPs or Lf-sChi-NPs. Serum was collected at days 0, 10 and 21. At day 21 mice were sacrificed, lymph nodes and spleens collected and lymphocytes stimulated with different doses of Lf in vitro . We demonstrated that Lf encapsulated into sChi shell (Lf-sChi-NPs) was unable to stimulate humoral response while Lf-NPs effectively induced IgG production as was measured by ELISA. Proliferation of T-cells was estimated at days 3–6 using either CellTiter Blue test or flow cytometry. We found that starting from day 4 lymphocytes from both lymph nodes and spleens proliferated. No differences in the intensity of proliferative responses between T-cells obtained form the mice immunized with Lf-NPs or Lf-sChi-NPs were found. Analysis of CD4+ and CD8+ Т-cells demonstrated that there was a difference between groups. T-cells from mice immunized with Lf-NPs were mostly CD4+ while CD8+ T-cells proliferated in Lf-Chi-NPs group. Thus, we demonstrated that the encapsulation of proteins into polymeric matrix resulted in the different outcomes of immunogenic properties. Immunization with free protein of NPs developed from free protein induced both cellular and humoral immune response while coating of protein with polymeric shell led to the prevention of B-cells from the contact with the antigen leading exclusively to the cellular but not humoral immune response. Moreover, protein encapsulation resulted in the formation of CD8+ cytotoxic cells which can be used for antiviral vaccine development.

Deciphering specific features of the ovarian cancer ascites proteome V. Shender1, M. Pavlukov1, G. Arapidi1, S. Kovalchuk1, N. Anikanov1, R. Ziganshin1, V. Govorun1,2 1M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia; 2Scientific Research Institute of Physical-Chemical Medicine, FMBA, Moscow, Russia Ovarian cancer ascites is a native medium for cancer cells that allows investigation of the secretome of cancer cells in their natural environment. On the one

58 | Acta naturae | SPECIAL ISSUE № 1 2014 Young Scientists Competition

hand, this medium is of interest as a promising source of potential biomarkers, on the other hand, as a medium for intercellular communication. Studies of the ascites with the use of omics technologies can help understand the peculiarities of cancer cell activity in the organism and elaborate new therapeutic approaches. The aim of this study was to elucidate specific features of malignant ascites proteome. Comparison of malignant ascites with those of cirrhosis allowed the revealing of the components specific for malignant ascites and omitting those that belong to systemic response to the ascites formation. A novel combined approach to protein fractionation prior to mass spectrometry analysis allowed the identification of 1632 and 1139 proteins in ovarian cancer and cirrhosis ascites, respectively, 663 proteins were specific for malignant ascites. Importantly, this value was several times higher than the earlier published data. Interesting results were obtained by the functional analysis of proteomic data. We demonstrated that the major differences between cirrhosis and malignant ascites were observed for the cluster of spliceosomal proteins as well as large number of RNA-binding proteins was found in the malignant ascites. Also we showed that several splicing RNAs were exclusively detected in malignant ascites, where they probably exist within protein complexes. In summary this study extended our knowledge of the protein composition of the ovarian cancer ascites and revealed its specific features which were associated with the function of the ascitic fluid as a medium of interaction between the malignant cells and their environment.

Novel immunotoxin based on HER2-specific darpin and pseudomonal exotoxin A E.A. Sokolova1, T.A. Zdobnova1,2, O.A. Stremovskiy2, I.V. Balalaeva1, S.M. Deyev1,2 1Lobachevsky State University, Nizhny Novgorod, Russia; 2M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Recombinant immunotoxins consisting of targeting and toxic moieties are promising agents for targeted antineoplastic therapy. HER2 (Human Epidermal growth factor Receptor 2), a member of the epidermal growth factor receptor family, presents one of the well-known molecular targets for targeted therapy of solid tumors. Overexpression of HER2 is associated with rapid progression and metastasis of tumor as well as its resistance to chemotherapy. The goals of the work were to create novel immunotoxin consisting of HER2-specific DARPin as a targeting moiety and fragment of pseudomonal exotoxin A as a toxic moiety, and to evaluate its properties. Binding of the created immunotoxin with cells was estimated by immunocytochemistry. Cytotoxicity of the immunotoxin against cell lines expressing different levels of HER2 was studied using MTT assay. Therapeutic effect of the immunotoxin in vivo was tested by fluorescence bioimaging using fluorescent model of human cancer overexpressing receptor HER2. The xenograft tumor model was established by intraperitoneal inoculation of human ovarian carcinoma cells transfected with red fluorescent protein Katushka (SKOVip-kat) in immunodeficientnude mice. Dynamics of tumor growth was estimated on the basis of integral intensity of fluorescence signal measured in peritoneal cavity area. The created immunotoxin was shown to bind to HER2-positive cells and display a specific cytotoxic effect on them. The monitoring of growth of HER2- overexpressing intraperitoneal tumor in vivo revealed that the immunotoxin causes tumor growth inhibition as compared to the control. These preliminary results testify to therapeutic efficacy of novel immunotoxin against tumors overexpressing receptor HER2 and to expediency of further study of the immunotoxin properties and the optimal dose selection. This study is supported by the Ministry of Education and Science of the Russian Federation (project no. 14.Z50.31.0022), the Russian Foundation for Basic Research (project no. 13-04-40228-Н).

3d matrixes for vascular grafts engeneering: influence of the structure and polymer composition to functional state of endotheliocytes A.O. Stepanova1,2, A.Yu. Dementieva1, P.P. Laktionov1,2 1Insitute of Chemical Biology and Fundamental Medicine, SB RAS, Novosibirsk, Russia; 2Research Institute of Blood Circulation Pathology, Novosibirsk, Russia In the first instance vascular grafts of small diameter are necessary for replacement of the coronaries and blood vessels of lower extremities. Materials commercially available for production of large and medium diameter prostheses are less convenient for production of the vascular grafts less than 6 mm in diameter and small diameter grafts produced from such materials apt to thrombosis, neointimal hyperplasia, sclerotization, which can lead to complete oc- clusion of prosthesis. Electrospinning is one of well convenient method to produce 3D matrix for vascular grafts, providing production of the materials with appropriate structure and characteristics. The tasks of the current study was to investigate mechanical properties of 3D matrixes electrospun produced from synthetic polymers and their compositions with proteins and functional state of primary endotheliocytes, cultivated at the surface of such 3D matrixes. Basic biodegradable polymers namely polylactic-co-glycolic acid and polycaprolactone along with nonbiodegradable polymer - Nylon 6, as well as their mixes with gelatin were used for production of 3D matrixes. Young's modulus of the 3D matrixes produced from PCL is 6 MPa, 12 MPa from PCL with the addition of gelatin, 37 MPa from Nylon 6 and 3 MPa from polylactic-co-glycolic acid. The data obtained demonstrate excessive stiffness of all 3D matrixes as compared to native blood vessels. In the investigations of cell viability and proliferation rate of primary endotheliocytes demonstrate, increase of the adhesion and the proliferation rate of the cells cultivated on the 3D matrixes produced from basic polymers in the mixture with gelatin. Investigation of the expression of genes encoding adhesion proteins, structural proteins and the proteins of extracellular matrix proteins in endotheliocytes cultivated on the surface of different 3D matrixes, demonstrate that expression of genes listed, does not depend from the microstructure of 3D matrix surface, but depend from the basic polymers used for 3D matrix production. The optimal polymer for production of the inner layer of vascular grafts is polylactic-co-glycolic acid.

Nuclear translocation of lysyl oxidase is promoted by interaction with transcription repressor p66β A.Z. Sukaeva, I.A. Okkelman, N.B. Pestov M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Lysyl oxidases comprise a family of copper-dependent amino oxidases that includes five members in mammals: lysyl oxidase proper (LOX) and four lysyl oxidase-like proteins (LOXL1, LOXL2, LOXL3 and LOXL4). Lysyl oxidases share the conserved C-terminal catalytic domain and catalyze the oxidative deamination of ε-amino groups in peptidyl lysine residues. Lysyl oxidases are well known to be responsible for cross-linking of collagen, elastin and other ECM proteins. The extracellular lysyl oxidase interactome includes fibronectin, fibulins, placental lactogen, TGF-β and these interactions may be important for ECM mediated signaling. Less characterized is the role that LOX plays among various nuclear proteins, and molecular mechanisms of its transport to the nucleus are currently unknown. Since LOX functions as an oncosuppressor in ras-transformation, it is quite intriguing whether it interacts also with any nuclear proteins. Indeed, both LOX and LOXL2 were reported to be present in the nucleus where they regulate gene expression, putatively through oxidative modification of histones. Here we employed yeast two hybrid library screening and found that LOX catalytic domain interacts with transcription repressor p66β. This interaction was confirmed in vitro and was found to be accomplished through CR2-containing domain of p66β. Moreover, co-expression of p66β and LOX in living tumor cells leads to nuclear accumulation of LOX. Interestingly, p66β could also bind to catalytic domains of other lysyl oxidase isoforms, (least human LOXL3 and LOXL1) indicating that conserved surfaces of the catalytic domain are responsible for binding with p66β. In conclusion, p66β may be important for the regulation of lysyl oxidase in the nucleus.

SPECIAL ISSUE № 1 | Acta naturae | 59 Young Scientists Competition

Search of the promoters for cancer gene therapy of tumors with high stroma content D.V. Tyulkina, V.V. Pleshkan, E.D. Sverdlov M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia Gene therapy offers unique opportunities for anticancer therapy. As part of this, a new approach based on tumor stroma influence is developed. Tumor stroma elements include fibroblasts, immune, endothelial, mesenchymal stem cells, cytokines, etc. All of these elements are involved in crucial processes such as maintaining, survival and spread of the tumor cells. Therefore, impact on the tumor stroma is becoming increasingly important, especially for tumors with high content of stroma component. Promoters of genes with increased expression in the stroma should be used for expression of the transgene in the cells of the stroma microenvironment. We have selected few gene promoters (IGFBP2, SDF1, JAG1 and CTGF) to generate reporter constructs which allows us to evaluate perspectives of used promoters for further design of an anticancer gene therapy constructs. Comparative analysis of activity of the promoters within reporter constructs were tested on a panel of human cancer cell lines consisting of PANC-1, MIA PaCa-2, AsPC-1, Calu-1, endothelial cell line HUVEC and pancreas tumor stroma fibroblasts IVP-9TS. Thus, we have developed constructs containing a reporter gene under the control of fragments of promoters IGFBP2, SDF1, JAG1 and CTGF, which thought to have a high level of expression in the tumor stroma cells. A comparative analysis of promoter activity on a panel of human cell lines showed that CTGF promoter has the highest level of activity among the studied promoters in all cell lines tested. For the first time we had cloned a JAG1 promoter fragment [-1708; 19] and demonstrated its activity in vitro . CTGF and IGFBP2 promoters are promising for use in gene therapy of tumors with a high content of the stroma component.

Interaction of antimicrobial peptides with complement protein C1q E.S. Umnyakova, M.N. Berlov, V.N. Kokryakov Institute of Experimental Medicine NWB RAMS, Saint-Petersburg, Russia; Saint-Petersburg State University, Saint-Petersburg, Russia Antimicrobial peptides (AMPs) are important molecular components of innate immune system. They are known to act not only as molecules, that kill pathogens directly, but also as immunomodulators. In particular, the influence of AMPs on the activation of the complement system through the interaction between AMPs and receptor molecules of the complement cascade (C1q, MBL) has been described, but these data are not so numerous and they are rather contradictory. That is why our research work is connected with studying of protein-peptide interactions of human C1q molecules with some AMPs. In our work there were investigated representatives of major groups of human AMPs – α-defensin (HNP-1), β-defensin (HBD-2) and cathelicidin (LL-37). We also used porcine protegrin-1 (PG-1) in this work, which is a member of the cathelicidin family, but has a lot in common in structure with defensins. To estimate the ability of C1q to interact with AMPs we used conjugate of C1q with horseradish peroxidase and polyclonal anti-C1q C subunit antibodies. The results of the experiments with conjugate confirmed some literature facts about the interaction of HNP-1 and HBD-2 with C1q. We also discovered strong interaction of PG-1 with C1q and found that LL-37 doesn’t interact with this protein. Protein complexes, consisting of AMPs and C1q, are stable in 0.5 M NaCl. The experiments with antibodies revealed only PG-1 – C1q complex that is stable in 0.5 M NaCl. We can assume that the important role in the formation of HNP-1 – C1q and HBD-2 – C1q complexes play the C subunit of this receptor molecule or the antigen determinants that are common for different subunits, because the peptide – protein complexes can be destroyed by the antibodies that we use for С1q identification. Possibly, this С subunit is not so important for PG-1 – C1q complex formation. We also showed that HNP-1 and HBD-2 (in concentrations 10–40 micrograms per milliliter) can activate complement cascade, while LL-37 has no influence on this process. These data we received via the permeabilization test of cytoplasmic membrane of E. coli with 5% serum. It appeared to be impos- sible to estimate the influence of PG-1 on the cascade activation with this method because of high membrane lytic activity of porcine cathelicidin. We can propose that defensins play an important role of physiological complement activators. Probably, the activation occurs as a result of interaction of C1q and antimicrobial peptides. This investigation is supported by grant №12-04-01573а of Russian Foundation for Basic Research.

Novel antiviral polyaromatic compounds A.V. Ustinov. А.А. Chistov, P.P. Streshnev, А.V. Guz., S.V. Kutyakov, V.А. Korshun M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia A variety of antiviral nucleosides has been prepared over the last fifty years. Most of them are inhibitors of DNA-polymerase. However analogs bearing bulky substituters, such as 5-(phenylethynyl)-2’-deoxy-uridine 1, showed lower activity. Nevertheless, in 1996 we obtained the first nucleoside 2 with pyrene moiety, which, surprisingly, exhibited high activity against Herpes simplex virus 1 (HSV-1), including acyclovir-resistant strains. Then some, even more active, analogs 3–5 were synthesized. As we noticed, a rigid linker between bulky aromatics and nucleoside core is necessary for the compounds to be active. For the new class of compounds we propose a “non-nucleosidic” mechanism of action involving inhibition of virion-cell fusion. Viruses can not encode their envelopes, so selection of resistant strains is less likely. The synthetic route to nucleosides involves Sonogashira coupling of protected 5-iodouridines and arylalkynes. In such manner, we have obtained about dozen of novel nucleosides and can derive their structure-activity relationships.

This research was supported by Russian President scholarship for young investigators (project SP-2494.2012.4).

60 | Acta naturae | SPECIAL ISSUE № 1 2014 AUTHOR INDEX

Abbasli R.M. 24 Boni I.V. 16 Feldman T.B. 45, 51 Abramchik Yu.A. 21 Borodyuk Y.N. 54 Feofanov A.V. 22, 29, 30, 31, 46 Ageyeva M. 50 Borshchevskiy V. 36 Fesenko I.A. 27 Akasov R.A. 54 Borzenok S.A. 45 Filyukova O.B. 18 Akhmedov N.A. 24 Borzova V.A. 32, 53 Finkina E.I. 23, 32, 36, 47, 52 Akhvlediani S.D. 14 Botchkova E.A. 53 Firsov A.P. 22 Akimov M.G. 13, 24, 48 Bovin N.V. 31, 50, 56 Furs O.V. 47 Akopov S.B. 18 Boyko A.A. 17, 44 Gabius H.J. 56 Akopova T.A. 49 Bregadze V.I. 22 Gainetdinov I.V. 41 Alehin A.I. 23 Bryzgunova О.Е. 18 Galanina O.E. 31 Alekseenko I.V. 50 Bulanenkova S.S. 18 Galushko A.S. 14 Alekseeva A.S. 50 Buneva V.N. 41, 54 Gapizov S.Sh. 55 Alekseeva V.V. 28 Burianov Ya.I. 44, 54 Garber M.B. 15 Alekseyevа L.G. 27 Buryanov Ya.I. 28, 39, 40, 47 Gataullin I.G. 35 Aleshina G.M. 13 Buth S.A. 34 Gavrilova S.I. 47 Aliyev R. E. 13 Bychkova V.E. 27 Generalov A.A. 22 Alliluev A.P. 28 Campbell K.S. 37 Generalova A.N. 22 Alutina E.L. 55 Chachina N.A. 53 Getman I.A. 28 Ame J.-C. 30 Chaplygin I.S. 34 Gevondyan N.M. 23 Andreev Y.A. 13 Chebotareva N.A. 32 Gevondyan V.S. 23 Andreeva L.A. 46 Cherenkov I.A. 45 Gilyashova N.V. 22 Andreeva L.E. 35 Cherepanova A.V. 53 Gizatullina A.K. 23 Andrianova A.G. 14 Cherevatenko A.M. 44, 54 Golovastova M.O. 23 Anikanov N.A. 16, 58 Chernonosova V.S. 18 Golunova A.S. 54 Antipova N.V. 14 Chernyshov S.V. 19, 51, 58 Goncharuk M.V. 17 Antonets D.V. 35 Chestkov I.V. 30 Gordeliy V. 36 Arapidi G.P. 16, 25, 27, 58 Chikova E.D. 52 Gorlenko V.A. 33 Arbukhanova P.M. 45 Chistov А.А. 60 Goryacheva E.A. 37, 42 Arkhipov D.V. 31, 50 Chistyakov A.A. 19 Govorun V.M. 16, 25, 27, 58 Arkhipova O.V. 14 Chudakov D.M. 42 Grafskaia E.N. 55 Arkhipova V.I. 15 Chupova L.A. 21 Grechikhina M.V. 17, 24, 34 Aronov D.A. 14 Coral-Gomez C. 19 Gretskaya N.M. 13, 24, 48 Arseniev A.S. 17, 23, 34, 57 Darbeeva O.S. 44 Grigor’eva A.E. 18 Artamonov A.Yu. 15 Dementieva A.Yu. 59 Grin M.A. 22, 46 Artemenko E.O. 51 Demina O.V. 16, 20 Grishin E.V. 13, 33 Artykov A.A. 15 Demina O.V. 51, 57 Grishina T.V. 42 Artyuhov A.A. 54 Demina T.S. 49 Grivennikov I.A. 30 Aseev L.V. 16 Dencher N. 20 Gurskaya N.G. 37 Astapova M.V. 22 Dergousova N.I. 14 Gurvits B.Ya. 41 Ataullakhanov F.I. 51 Deupi X. 57 Guz А.V. 60 Azarkin I.V. 16, 25 Deyev I.E. 53 Haranzhevskiy E.V. 45 Azev V.N. 16 Deyev S.M. 59 Harseeva G.G. 55 Azhikina T.L. 24,41 Deyev S.N. 49 Ignatov D. 24 Babalyan K.A. 27 Diachenko O.V. 44, 54 Ignatova A.A. 30 Babayeva Z.Sh. 51 Didkovsky N.A. 23 Ignatyev G.M. 44 Balalaeva I.V. 59 Didych D.A. 56 Ilina E.S. 30 Balalayeva I.V. 46 Dobrochaeva K.L. 31 Ilinskaya O.N. 35 Balandin S.V. 30, 37, 47 Dolgikh D.A. 55 Illarioshkin S.N. 30 Ballmer-Hofer K. 57 Dolgov S.V. 22 Ilyichev A.A. 35 Balobanov V.A. 27 Dovzhenko N.A. 20 Ilyina N.B. 27 Baranov P. 43 Drozdova M.G. 49, 54 Ionovа V.G. 30 Bazhan S.I. 35 Dunaevsky Y.E. 40 Ismailova L.I. 24 Belikov N.E. 16, 20, 51, 57 Durmanova Z.V. 44 Ivanov V.T. 16, 30 Belozersky M.A. 40 Dyachenko O. 40 Ivanova O.M. 16 Berezina E.V. 52 Efremenko A.V. 22 Ivanova O.M. 25 Berlov M.N. 60 Egorov V.V. 21, 46 Ivanova S.A. 41, 54 Bezuglov V.V. 13, 24, 48 Emelyanova A.A. 30 Ivanova V.P. 25 Bobkova N.V. 47 Ermakov E.A. 54 Ivanova Yu.D. 17 Bocharov E.V. 17, 33, 34 Ermakova D.E. 22 James C. 43 Bocharova O.V. 17, 34 Erokhina S.A. 26 Kabilov M.R. 52 Bogachuk A.P. 31 Erokhina T.N. 21 Kadykov V.A. 44 Bogdanov I.V. 52 Eroshkin F.M. 32 Kalashnikova M.B. 30 Boldyrev I.A. 50, 58 Esipov R.S. 21, 28, 32, 42 Kaliberda E.N. 34 Bolosov I.A. 17, 30, 37 Fattahov N. 41 Kalinovsky D.V. 25 Bondar A.A. 52 Fedorova I.M. 33 Kallistova A.Yu. 26

SPECIAL ISSUE № 1 2014 | Acta naturae | 61 Kamynina A.V. 47 Lagarkova M.A. 30 Moebius D. 47 Kanevskiy L.M. 17, 26, 43 Laktionov P.P. 18, 35, 52, 53, 56, 59 Moiseeva E.V. 14 Kapkaeva M.R. 50 Lantsova V.B. 30 Molchanov М. V. 16 Kaprelyants A. 24 Lavrik O.I. 30 Molochkov N.V. 38 Kapustin D.V. 26 Lavrova A.V. 24 Molotkovsky J.G. 50 Karpenko L.I. 35 Lazarev V.N. 55 Monastyrskaya G.S. 50 Kashirina E.I. 27 Lebedev Y.B. 38, 55 Morozkin Е.S. 18, 52, 56 Kashparov I.A. 27 Lebedeva O.S. 30 Morozov S.Y. 21 Kasumov E.A. 27 Lebedeva V.A. 54 Morrow D. 43 Kasumov R.E. 27 Lei P. 43 Muranov K.O. 32 Kasumova I.V. 27 Leiman P.G. 34 Muravyova T.I. 21, 42 Katicheva L. 50 Leonova O.G. 31 Myakushina Yu.A. 31 Katina N.S. 27 Lesovoy D.M. 17 Myasoedov N.F. 46 Kevbrina M.V. 26 Levin P.P. 16 Myrsikova Е.V. 34 Khaidarova N.V. 35 Lin H. 43 Myshkin M.Yu. 23 Kharkova M.V. 35 Lipkin V.M. 31, 46 Nadezhdin K.D. 17, 34 Kharlampieva D.D. 55 Litti Yu.V. 53 Navolotskaya E.V. 35 Khazigaleeva R.A. 27 Litvinenko А.P. 31 Nazarkina Zh.K. 18, 35, 53 Khodonov A.A. 16, 20, 51, 57 Lomin S.N. 31 Nazarov V.I. 38, 55 Khodyreva S.N. 30 Loseva E.M. 56 Nekrasov E.D. 30 Kirpichnikov M.P. 29 Lubimova C.A. 56 Nekrasova O.V. 29, 31 Kiselev S.L. 30 Lukashev E.P. 19 Nekrasova V.K. 26 Kleimenov S.Yu. 32 Lukin A.Yu. 16, 57 Nenasheva V.V. 35 Kljashtorny V.G. 33 Lukyanov K.A. 37, 38, 42 Nevinsky G.A. 41, 54 Klopov N.V. 21 Lutsenko G.V. 17, 24 Nguyen Nga Thi 35 Knirel Yu.A. 34 Luykmanova E.N. 29 Nikitina A.A. 57 Kokryakov V.N. 13, 39, 60 Lyapina E.A. 31 Nikolaev L.G. 18 Kolachevskaya O.O. 28 Lysko K.A. 44 Nikolaev M. 36 Koledinskaya L.S. 16 Lyukmanova E.N. 57 Nikonov O.S. 29 Kolobov A.A. 42 Maerle A.V. 31 Nikonov S.V. 29, 33 Kolodkin N.I. 15 Makarov D.A. 21, 32, 42 Nikulin A.D. 27 Komech E.A. 38, 55 Makarov I. 50 Noskova Yu.S. 52 Kondrashov F.A. 14 Makarova I.V. 35 Nosov G.A. 36 Kondratieva S.A. 41 Makarova S.S. 21 Novosadova E.V. 30 Korchagina E.Yu. 56 Maksimova M.Yu. 30 Nozhevnikova A.N. 26, 53, 57 Korchagina V.P. 55 Malashenkova I.K. 23 Nurmukhamedova E.K.-A. 36 Korneenko T.V. 28 Malchevskaya M.A. 56 Okkelman I.A. 59 Koroev D.O. 47 Maleeva E.E. 33 Oleinikov V.A. 19, 22, 41 Korolev D.O. 23 Mamedov I.Z. 38, 55 Olenina L.V. 36, 56 Korolkova Y.V. 29, 31 Manni S. 57 Oleynik N.V. 42 Korshun V.А. 60 Manuvera V.A. 55 Orlov D.S. 15 Kostromina M.A. 21, 28 Markina N.M. 37 Orlov D.S. 39 Kostrukova E.S. 27 Markossian K.A. 32 Orlov E.E. 32 Kotel’nikova O.V. 28 Markvicheva E.A. 49, 54 Osmakov D.I. 13 Kotov A.A. 56 Martynov V.I. 58 Ostrovsky M.A. 45, 51 Kovalchuk S.I. 16, 25, 58 Martynova N.Y. 32 Ovchinnikova T.V. 17, 23, 30, 32, 36, Kovalenko E.I. 17, 26, 43, 44 Maslennikova A.V. 46 37, 47, 52 Kovaleva G.V. 35 Maslova M.V. 54 Ovchinnikova М.V. 33 Kozhemyako V.B. 38 Masulis I.S. 51 Ozoline O.N. 51 Kozlov S.A 13, 33 Maximov V.I. 20 Pakhomov A.A. 58 Kozmin Yu.P. 21, 22, 46 McNeish J. 43 Panteleev M.A. 51 Kramorenko N.V. 40 Medvinskaya N.I. 47 Panteleev P.V. 17, 30, 37 Kravchenko O.V. 29 Melnikova D.N. 32 Paramonov A.S. 23 Kudryashova K.S. 31 Melnikova I.A. 57 Parshukova D. 41 Kudryavtseva A.V. 18 Mikhailova А.G. 33 Pavlukov M. 58 Kudryavtseva E.I. 37 Mikhaylina А.О. 33 Pazina T. 37 Kudryshova K.S. 29 Mikoulinskaia G.V. 14, 19, 38, 58 Pereverzev A.P. 37 Kudzhaev A.M. 14 Mikov A.N. 33 Permyakov S.E. 27 Kuklin A. 36 Milaeva I.V. 20 Pertseva M.A. 44 Kulbatskii D.S. 29 Mineev K.S. 57 Pestov N.B. 15, 25, 28, 59 Kulikov E.E. 44 Mirgorodskaya O.A. 21, 46 Petrenko A.G. 53 Kurganov B.I. 32 Mironov A.F. 22, 46 Petrovskaya L.E. 55 Kurilshikov A.M. 52, 56 Miroshnikov A.I. 21 Pleshkan V.V. 60 Kutuzov M.M. 30 Miroshnikov K.A. 34, 44 Pletnev V.Z. 37, 42 Kutyakov S.V. 60 Mirzoev R.R. 44 Pletneva N.V. 37, 42 Kutyshenko V.P. 38 Mishin A. 37 Pogorelyy M.V. 38, 55 Kuzmenkov A.I. 31 Mitiouchkina T.Y. 22 Polyansky N.B. 32 Kuzmichev P.K. 17 Mitroshin I.V. 15 Ponomareva E.V. 47 Kuzmin D.V. 30 Mochalov K.E. 19 Popenko V.I. 31 Kuznetsova N.R. 50 Modyanov N. 34 Popova I.S. 56

62 | Acta naturae | SPECIAL ISSUE № 1 2014 Povarova N.V. 38 Shulepko M.A. 29, 57 Vasin A.V. 46 Privalova A.M. 49 Shvets V.I. 20 Vassilevski A.A. 31 Prokhorov D.A. 38 Sivaev I.B. 22 Vassina E.M. 30 Prokhortchouk E.B. 32 Sizova S.V. 41 Vazina A.A. 27 Prostyakova A.I. 26 Skvortsova Yu.V. 41 Vihoreva G.A. 54 Puchko E.N. 39 Smirnova E.V. 31, 46 Vlassov V.V. 18, 35, 52, 53, 56 Rakitina T.V. 46 Smirnova E.Yu. 41 Vodovozova E.L. 50 Rapoport E.M. 56 Smirnova I.V. 31 Volkova T.D. 47 Razgulyaeva O.A. 28 Smirnova L. 41, 54 Volovetsky A.B. 46 Rechetov P.D. 27 Smirnova M.P. 15 Volpina O.M. 47 Rizvanov A.A. 40 Snezhkina A.V. 18 Volynski P.E. 17 Rogov A.G. 38 Snezhkov E.V. 50 Vorobyeva E.E. 46 Romanov G.A. 28, 31 Soboleva A.V. 42 Voytsitskiy V.E. 18, 52 Rotanova T.V. 14 Sokolova E.A. 59 Vyunova T.V. 46 Rudenko N.V. 39, 40, 54 Solovieva D.O. 19 Wang S. 43 Rukavtsova E.B. 28, 39 Solovyev A.G. 21 Yagudaeva E.Yu. 28 Rumsh L.D. 28, 33 Solovyeva D.O. 47 Yakimenko A.O. 51 Ryabchikova E.I. 18 Sonina A.N. 54 Yakimenko Z.A. 23 Ryazantsev D.Yu. 27, 31 Souslova E.A. 42 Yakovleva M.A. 45, 51 Rybakina E.G. 15 Stakheev A.A. 42 Yakubovskaya R.I. 50 Rychkova M.E. 32 Stepanenko V.N. 21, 32, 42 Yampolsky I.V. 42 Rykova E.Y. 56 Stepanova A.O. 59 Yankelevich I.A. 13 Ryzhov I.M. 56 Stolboushkina E.A. 15 Yerov O. 43 Sachivkina N.P. 39 Streltsova M.A. 26, 43 Young M.J. 43 Salina E. 24 Stremovskiy O.A. 59 Yudintsev A. 50 Sapozhnikov A.M. 17, 44 Streshnev P.P. 60 Yukhnev V.A. 45 Sarkisyan K. 37 Stukacheva E.A. 41 Yunusov A.R. 45 Savvichev A.S. 26 Suchanova E.I. 38 Zabrodskaya Y.A. 46 Schmülling T. 31 Sukaeva A.Z. 59 Zaitsev I.S. 44, 47 Schreiber V. 30 Sukhanova M.V. 30 Zaitsev S.Yu. 20, 22, 40, 44, 47 Semashko T.A. 27 Sukhanova T. 43 Zakharchenko N.S. 39, 47 Semenkov V.F. 17 Sultanov D.Ch. 14 Zaporozhchenko I.A. 18, 52 Semke A. 41 Surina A.M. 30 Zaporozhskaya Y.V. 47 Semushina S.G. 14 Surina E.A. 31 Zaraisky A.G. 32 Sepp E.K. 30 Sverdlov E.D. 18, 43, 50, 60 Zaripov М.М. 18, 52 Sergeev V.G. 45 Svirshchevskaya E.V. 22, 27, 34, 41, Zavriev S.K. 31, 42 Sergeeva L.I. 28 55, 58 Zayara D.A. 48 Serova O.V. 28, 53 Sykilinda N.N. 44 Zaytseva-Zotova D.S. 54 Shakhparonov M.I. 28 Tarantul V.Z. 35 Zdobnova T.A. 59 Shaloiko L.A. 22 Tarasenko I.V. 22 Zelenetsky A.N. 49 Shamova O.V. 15, 37, 39, 45 Tarlachkov S.V. 40, 44, 54 Zharkova M.S. 39 Shamraychuk I.L. 40 Tikhonov D.B. 33 Zhigalova N.A. 32 Shaposhnikov M.N. 40, 47 Tishchenko S.V. 33 Zhigis L.S. 28 Shartukova M.A. 45 Tkatch E.N. 30 Zhukova L.V. 48 Shashkov A.S. 34 Tretyak M.V. 22 Ziganshin R. 58 Shavrina М.S. 19, 58 Trofimov D.Yu. 31 Ziganshin R.H. 16, 25, 30 Shcheglovitova O.N. 50 Trofimova I.B. 23 Zimin А.А. 19, 58 Shcherbinina T.S. 41, 58 Troyanova N.I. 44 Zinchenko A.A. 28 Shender V.O. 16, 30, 58 Tsarkova M.S. 44 Zinchenko G.N. 13 Shenkarev Z.O. 23 Tsoy L.V. 23 Zlobin A.E. 48 Shevchenko K.V. 46 Tyulkina D.V. 60 Zlobina M.A. 49 Shevchenko M.A. 44 Umnyakova E.S. 60 Zubareva A.A. 41 Shevchuk T.V. 40, 44, 54 Unuchek D. 36 Zubov V.P. 22, 26, 41 Shilyagina N.Y. 46 Urban A.S. 17 Zueva V.S. 28 Shingarova L.N. 55 Usmanova D.R. 57 Zvereva I.O. 42 Shlikht A.G. 40 Ustinov A.V. 60 Zvyagilskaya R.A. 38 Shneider M.M. 34, 44 Varfolomeev S.D. 16, 20, 57 Zvyagin I.V. 38, 55 Shtilman M.I. 54 Varlamov V.P. 58 Shubin V.V. 32 Vasiliev V.D. 27

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