I. Structure and functions of biological macromolecules

and macromolecular complexes. Biocalalysis.

MODULATION OF PENTOSE PHOSPHATE PATHWAY DURING CELL CYCLE PROGRESSION IN HUMAN COLON ADENOCARCINOMA CELL LINE HT29.

P. Vizan, G. Alcarraz-Vizan, S. Diaz-Moralli, O. N. Solovjeva, W. M. Frederiks, M. Cascante.

International Journal of Cancer 124 (12):2789-2796, 2009.

Cell cycle regulation is dependent on multiple cellular and molecular events. Cell proliferation requires metabolic sources for the duplication of DNA and cell size. However, nucleotide reservoirs are not sufficient to support cell duplication and, therefore, bio-synthetic pathways should be upregulated during cell cycle. Here, we reveal that glucose-6-phosphate dehydrogenase (G6PDH) and transketolase (TKT), the 2 key enzymes of oxidative and nonoxidative branches of the pentose phosphate pathway respectively, which is necessary for nucleotide synthesis, are enhanced during cell cycle progression of the human colon cancer cell line HT29. These enhanced enzyme activities coincide with an increased ratio of pentose monophosphate to hexose monophosphate pool during late G1 and S phase, suggesting a potential role for pentose phosphates in proliferating signaling. Isotopomeric analysis distribution of nucleotide ribose synthesized from 1,2-(I3)- C-2-glucose confirms the activation of the PPP during late G1 and S phase and reveals specific upregulation of the oxidative branch. Our data sustain the idea of a critical oxidative and nonoxidative balance in cancer cells, which is consistent with a late G1 metabolic check point. The distinctive modulation of these enzymes during cell cycle progression may represent a new strategy to inhibit proliferation in anticancer treatments.

ANTICHAPERONE ACTIVITY OF CYCLODEXTRIN DERIVATIVES.

O. I. Maloletkina, K. A. Markosyan, R. A. Asriyants, V. N. Orlov, B. I. Kurganov.

Doklady Biochemistry and Biophysics 427 (1):199-201, 2009.

NEW FUNCTION OF THE AMINO GROUP OF THIAMINE DIPHOSPHATE IN THIAMINE CATALYSIS.

L. E. Meshalkina, G. A. Kochetov, G. Hubner, K. Tittmann, R. Golbik.

Biochemistry-Moscow 74 (3):293-300, 2009.

In this work, we investigated the rate of formation of the central intermediate of the transketolase reaction with thiamine diphosphate (ThDP) or 4'-methylamino-ThDP as cofactors and its stability using stopped-flow spectroscopy and circular dichroism (CD) spectroscopy. The intermediates of the transketolase reaction were analyzed by NMR spectroscopy. The kinetic stability of the intermediate was shown to be dependent on the state of the amino group of the coenzyme. The rates of the intermediate formation were the same in the case of the native and methylated ThDP, but the rates of the protonation or oxidation of the complex in the ferricyanide reaction were significantly higher in the complex with methylated ThDP. A new negative band was detected in the CD spectrum of the complex transketolase-4'-methylamino-ThDP corresponding to the protonated dihydroxyethyl-4'-methylamino-ThDP released from the active sites of the enzyme. These data suggest that transketolase in the complex with the NH2-methylated ThDP exhibits dihydroxyethyl-4'-methylamino-ThDP-synthase activity. Thus, the 4'-amino group of the coenzyme provides kinetic stability of the central intermediate of the transketolase reaction, dihydroxyethyl-ThDP.

THE STUDY OF TWO ALKALIPHILIC THERMOPHILE BACTERIA OF THE Anoxybacillus GENUS AS PRODUCERS OF EXTRACELLULAR PROTEINASE.

E. V. Lavrenteva, A. P. Shagzhina, O. B. Babasanova, Y. E. Dunaevsky, Z. B. Namsaraev, D. D. Barkhutova.

Applied Biochemistry and Microbiology 45 (5):484-488, 2009.

Two strains of alkaliphilic thermophile bacteria of the genus Anoxybacillus from hydrothermal vents of Lake Baikal were detected and characterized. It was demonstrated that proteinases secreted by these bacteria had wide substrate specificity, hydrolyzed proteins and n-nitroanilide substrates, and showed maximal activity at pyroglutamyl-alanine-alanine-leucine n-nitroanilide hydrolysis. We determined maximal activity of the proteinases at alkaline pH values (10.0-10.5), the enzymes were thermostable and were characterized by a wide thermal optimum (55-70⁰C). The results of inhibitor analysis and substrate specifity examination of extracellular enzymes demonstrated their belonging to the subtilisin-like serine proteinases.

SHORT SYNTHETIC POLYELECTROLYTES DESTABILIZE PROTEINS MOST EFFICIENTLY.

S. V. Stogov, V. I. Muronetz, V. A. Izumrudov.

Doklady Biochemistry and Biophysics 427 (1):187-190, 2009.

INTERACTION OF POLYANIONS WITH ELECTRONEUTRAL LIPOSOMES IN A SLIGHTLY ACIDIC MEDIUM.

A. K. Berkovich, V. N. Orlov, N. S. Melik-Nubarov.

Polymer Science Series A 51 (6):648-657, 2009.

In this study, the interaction between poly(styrene sulfonic acid), polyacrylic acid, poly(meth-acrylic acid), poly(L-glutamic acid), poly(vinyl sulfate), and ternary copolymer of styrene with maleic anhydride and methacrylic acid (3: 2: 1), as well as DNA with lipid vesicles composed of zwitterion (electroneutral) lipid phosphatidylcholine, has been investigated. The methods of centrifuge ultrafiltration and dynamic light scattering reveal that, at pH 4.2, all polyacids under study are effectively adsorbed on the phospholipid membrane. The polymer-membrane complex is stabilized by hydrogen bonds and hydrophobic interactions in addition to electrostatic bonds. Even though, to a greater or lesser extent, all polyacids are capable of undergoing adsorption on the membrane in a slightly acidic medium, their effect on the membrane permeability is substantially different and is correlated with the ability of a polymer to form multiple interactions with phospholipid molecules. Poly(acrylic acid), poly(methacrylic acid), poly(styrene sulfonic acid), and the ternary copolymer of styrene with maleic anhydride and methacrylic acid can produce the membrane pores that are permeable to low-molecular-mass compounds. At the same time, poly(L-glutamic acid), poly(vinyl sulfate), and DNA exert no effect on the membrane permeability, although they are sorbed on the membrane surface.

PHOSPHONO ANALOGUES OF 2-OXOGLUTARATE PROTECT CEREBELLAR GRANULE NEURONS UPON GLUTAMATE EXCITOTOXICITY.

V. I. Bunik, M. S. Kabysheva, E. I. Klimuk, T. P. Storozhevykh, V. G. Pinelis.

Natural Compounds and Their Role in Apoptotic Cell Signaling Pathways 1171:521-529, 2009.

Glutamate excitotoxicity is an important contributor to neuronal loss. Glutamate-induced Ca²⁺ deregulation and accompanying mitochondrial depolarization are closely associated with the onset of apoptotic and necrotic neuronal death. We investigated the role in these phenomena of 2-oxoglutarate dehydrogenase (OGDH), the enzyme participating in mitochondrial degradation of glutamate. To achieve this goal, we used specific effectors of cellular OGDH, succinyl phosphonate and its phosphonoethyl ether. Preincubation of cerebellar granule neurons with these phosphono analogues of 2-oxoglutarate was shown to protect the cells from glutamate-induced Ca²⁺ deregulation and irreversible mitochondrial depolarization, followed simultaneously by fluorescence of fura-2FF and rhodamine 123, respectively. The protection was characterized by delay in onset and decreased propagation of Ca²⁺ deregulation and by reversibility of the associated mitochondrial depolarization. Compared to its phosphonoethyl ether, succinyl phosphonate exhibited both higher affinity to OGDH in vitro and better protection from Ca²⁺ deregulation in situ, supporting the assumption that neuroprotection by phosphonates involves their interaction with cellular OGDH. Preincubation of cerebellar granule neurons with succinyl phosphonate decreased neuronal death after excitotoxic action of glutamate. Thus, specific inhibitors of OGDH alleviate glutamate-induced calcium deregulation, mitochondrial depolarization, and neuronal death.

METABOLIC CONTROL EXERTED BY THE 2-OXOGLUTARATE DEHYDROGENASE REACTION:

A CROSS-KINGDOM COMPARISON OF THE CROSSROAD BETWEEN ENERGY PRODUCTION AND NITROGEN ASSIMILATION.

V. I. Bunik and A. R. Fernie.

Biochemical Journal 422:405-421, 2009.

Mechanism-based inhibitors and both forward and reverse genetics have proved to be essential tools in revealing roles for specific enzymatic processes in cellular function. Here, we review experimental studies aimed at assessing the impact of OG (2-oxoglutarate) oxidative decarboxylation oil basic cellular activities in a number of biological systems. After summarizing the catalytic and regulatory properties of the OGDHC (OG dehydrogenase complex), we describe the evidence that has been accrued on its cellular role. We demonstrate an essential role of this enzyme in metabolic control in a wide range of organisms. Targeting this enzyme in different cells and tissues, mainly by its specific inhibitors, effects changes in a number of basic functions, such as mitochondrial potential, tissue respiration, ROS (reactive oxygen species) production, nitrogen metabolism, glutamate signalling and survival, Supporting the notion that the evolutionary conserved reaction of OG degradation is required for metabolic adaptation. In particular, regulation of OGDHC under stress conditions may be essential to overcome glutamate excitotoxicity in neurons or affect the wound response in plants. Thus, apart from its role in producing energy, the flux through OGDHC significantly affects nitrogen' assimilation and amino acid metabolism, whereas the side reactions of OGDHC, such as ROS production and the carboligase reaction, have biological functions in signalling and glyoxylate utilization. Our current view on the role of OGDHC reaction in various processes within complex biological systems allows us a far greater fundamental understanding of metabolic regulation and also opens up new opportunities for us to address both biotechnological and medical challenges.

PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR (PPAR)-γ POSITIVELY CONTROLS AND PPAR Α NEGATIVELY CONTROLS CYCLOOXYGENASE-2 EXPRESSION IN RAT BRAIN ASTROCYTES THROUGH a CONVERGENCE ON PPAR β/δ via MUTUAL CONTROL OF PPAR EXPRESSION LEVELS.

S. Aleshin, S. Grabeklis, T. Hanck, M. Sergeeva, G. Reiser.

Molecular Pharmacology 76 (2):414-424, 2009.

Peroxisome proliferator-activated receptor ( PPAR) transcription factors are pharmaceutical drug targets for treating diabetes, atherosclerosis, and inflammatory degenerative diseases. The possible mechanism of interaction between the three PPAR isotypes (α, β/δ, and γ) is not yet clear. However, this is important both for understanding transcription factor regulation and for the development of new drugs. The present study was designed to compare the effects of combinations of synthetic agonists of PPAR α [2-[4-[2-[4-cyclohexylbutyl cyclohexylcarbamoyl)amino]ethyl]phenyl] sulfanyl-2-methylpropanoic acid (GW7647)], PPAR β/δ [4-(3-(2-propyl-3-hydroxy-4-acetyl)phenoxy) propyloxyphenoxy acetic acid, (L-165041)], and PPAR γ (rosiglitazone, ciglitazone) on inflammatory gene regulation in rat primary astrocytes. We measured cyclooxygenase-2 (COX-2) expression and prostaglandin E 2 synthesis in lipopolysaccharide (LPS)-stimulated cells. PPAR α, PPAR β/δ, and PPAR γ knockdown models served to delineate the contribution of each PPAR isotype. Thiazolidinediones enhanced the LPS-induced COX-2 expression via PPAR γ-dependent pathway, whereas L-165041 and GW7647 had no influence. However, the addition of L-165041 potentiated the effect of PPAR γ activation through PPAR β/δ-dependent mechanism. On the contrary, PPAR α activation (GW7647) suppressed the effect of the combined L-165041/rosiglitazone application. The mechanism of the interplay arising from combined applications of PPAR agonists involves changes in PPAR expression levels. A PPAR β/δ overexpression model confirmed that PPAR β/δ expression level is the point at which PPAR γ and PPAR α pathways converge in control of COX-2 gene expression. Thus, we discovered that in primary astrocytes, PPAR γ has a positive influence and PPAR γ has a negative influence on PPAR β/δ expression and activity. A positive/negative-feedback loop is formed by PPAR β/δ-dependent increase in PPAR α expression level. These findings elucidate a novel principle of regulation in the signaling by synthetic PPAR agonists that involves modulating the interaction between PPAR α, -β/δ, and -γ isoforms on the level of their expression.

STRUCTURAL AND KINETIC FEATURES OF FAMILY I INORGANIC PYROPHOSPHATASE

FROM Vibrio cholerae.

E. V. Rodina, V. R. Samygina, N. N. Vorobyeva, T. S. Sitnik, S. A. Kurilova, T. I. Nazarova.

Biochemistry-Moscow 74 (7):734-742, 2009.

In this paper, kinetic properties of a soluble inorganic pyrophosphatase of family I from Vibrio cholerae (V-PPase), intestinal pathogen and causative agent of human cholera, are characterized in detail, and the crystal structure of a metal-free enzyme is reported. Hydrolytic activity of V-PPase has been studied as a function of pH, concentration of metal cofactors (Mg²⁺ or Mn²⁺), and ionic strength. It has been found that, despite the high conservation of amino acid sequences for the known bacterial PPases of family I, V-PPase differs from the other enzymes of the same family in a number of parameters. Dissociation constants of V-PPase complexed with Mg²⁺ or Mn²⁺ were essentially the same as for Escherichia coli PPase (E-PPase). However, the pH optimum of MgPPi hydrolysis by V-PPase was shifted to more alkaline pH due to higher values of the pK_a of ionizable groups for both the free enzyme and the enzyme-substrate complex. The stability of a hexameric form of V-PPase has been studied as a function of pH. The corresponding pK_a of a group that controls the stability of the hexamer at pH below 6 (pK_a = 4.4) was significantly lower than in the other hexameric PPases. The crystal structure reported here is analyzed and compared with the structure of E-PPase. The location of amino acid residues that differ in V-PPase and E-PPase is discussed. Since V-PPase has been found to retain its hydrolytic activity in high ionic strength media, the observed structural and kinetic features are analyzed in view of the possible osmoadaptation of this protein.

STRUCTURAL REARRANGEMENTS IN TRANSFER-MESSENGER RNA IN THE PROCESS OF TRANS-TRANSLATION - PROTEIN SYNTHESIS QUALITY CONTROL SYSTEM IN BACTERIA.

O. Dontsova, O. Shpanchenko, A. Bogdanov, E. Bugaeva, A. Golovin, L. Isaksson.

FEBS Journal 276:38, 2009.

COOPERATIVE BINDING OF SUBSTRATES TO TRANSKETOLASE FROM Saccharomyces cerevisiae.

I. A. Sevostyanova, V. A. Selivanov, V. A. Yurshev, O. N. Solovjeva, S. V. Zabrodskaya, G. A. Kochetov.

Biochemistry-Moscow 74 (7):789-792, 2009.

Catalytic activity of two active sites of transketolase and their affinity towards the substrates (xylulose-5-phosphate and ribose-5-phosphate) has been studied in the presence of Ca²⁺ and Mg²⁺. In the presence of Ca²⁺, the active sites exhibit negative cooperativity in binding both xylulose-5-phosphate (donor substrate) and ribose-5-phosphate (acceptor substrate) and positive cooperativity in the catalytic transformation of the substrates. In the presence of Mg²⁺, nonequivalence of the active sites is not observed.

GROEL-MEDIATED DE NOVO FORMATION OF Β-STRUCTURED AGGREGATES OF TWO OVINE PRION PROTEIN ALLELIC VARIANTS PRP-VRQ AND PRP-ARR.

G. Kisselev, I. Naletova, Y. Shchutskaya, T. Haertle, V. Muronetz.

FEBS Journal 276:145, 2009.

YELLOW MEALWORM Tenebrio molitor AS A SOURCE OF SPECIFIC PEPTIDASES FOR TREATMENT OF CELIAC DISEASE.

E. Elpidina, I. Goptar, I. Filippova.

FEBS Journal 276:307-308, 2009.

STRUCTURAL STUDYING OF CONFORMATIONAL INTERPLAY WITHIN TWO-MODULE THROMBIN BINDING DNA APTAMERS.

A. Kopylov, A. Golovin, R. Reshetnikov, T. Turchaninov, A. Yuminova, V. Spiridonova, A. Arutyunyan.

FEBS Journal 276:319-320, 2009.

Tribolium castaneum LARVAL GUT TRANSCRIPTOME AND PROTEOME: A RESOURCE FOR THE STUDY OF THE COLEOPTERAN GUT.

K. Morris, M. D. Lorenzen, Y. Hiromasa, J. M. Tomich, C. Oppert, E. N. Elpidina, K. Vinokurov, J. L. Jurat-Fuentes, J. Fabrick, B. Oppert.

Journal of Proteome Research 8 (8):3889-3898, 2009.

Tribolium castaneum is an important agricultural pest and an advanced genetic model for coleopteran insects. We have taken advantage of the recently acquired T. castaneum genome to identify T. castaneum genes and proteins in one of the more critical environmental inter-faces of the insect, the larval alimentary tract. Genetic transcripts isolated from the T. castaneum larval gut were labeled and hybridized to a custom array containing oligonucleotides from predicted genes in the T. castaneum genome. Through a ranking procedure based on relative labeling intensity, we found that approximately 17.6% of the genes represented in the array were predicted to be highly expressed in gut tissue. Several genes were selected to compare relative expression levels in larval gut, head, or carcass tissues using quantitative real-time PCR, and expression levels were, with few exceptions, consistent with the gut rankings. In parallel with the microarrays, proteins extracted from the T. castaneum larval gut were subjected to proteomic analysis. Two-dimensional electrophoretic analysis combined with MALDI-TOF resulted in the identification of 37 of 88 selected protein samples. As an alternative strategy, one-dimensional electrophoretic separation of T. castaneum larval gut proteins followed by two-dimensional nano-HPLC and ESI-MS/MS resulted in the identification of 98 proteins. A comparison of the proteomic studies indicated that 16 proteins were commonly identified in both, whereas 80 proteins from the proteomic analyses corresponded to genes with gut rankings indicative of high expression in the microarray analysis. These data serve as a resource of T. castaneum transcripts and proteins in the larval gut and provide the basis for comparative transcriptomic and proteomic studies related to the gut of coleopteran insects.

DIGESTIVE CYSTEINE CATHEPSINS AGAINST CELIAC DISEASE.

T. Semashko, S. Popletaeva, I. Filippova, E. Elpidina.

FEBS Journal 276:388, 2009.

USE OF BUCKWHEAT SEED PROTEASE INHIBITOR GENE FOR IMPROVEMENT OF TOBACCO AND POTATO PLANT RESISTANCE TO BIOTIC STRESS.

N. V. Khadeeva, E. Z. Kochieva, M. Y. Tcherednitchenko, E. Y. Yakovleva, K. V. Sydoruk, V. G. Bogush, Y. E. Dunaevsky, M. A. Belozersky.

Biochemistry-Moscow 74 (3):260-267, 2009.

The possibility to use agrobacterial transformation of leaf discs to produce resistance to bacterial infections in tobacco and potato plants by introduction of a single gene encoding the serine proteinase inhibitor BWI-1a (ISP) from buckwheat seeds is shown. All studied PCR-positive transgenic plants exhibited antibacterial activity in biotests. It was shown that the presence of just a single gene of serine proteinase inhibitor provides sufficient protection at least against two bacterial phytopathogens, Pseudomonas syringae pv. tomato and Clavibacter michiganensis sbsp. michiganensis. The biotest including tobacco plant infection by the white wings butterfly in the green house has also demonstrated the existence of protective effect in transgenic tobacco plants. Significant genotypic variations in the protection efficiency were found between members of different genera of the same family (potato and tobacco) as well as between different lines of the same species. Northern blot analysis of four transgenic potato lines and three tobacco lines transformed by a vector plasmid containing the ISP gene of serine proteinases BWI-1a from buckwheat seeds has shown the presence of the expected size mRNA transcript.

SYNTHETIC REGULATORS OF THE 2-OXOGLUTARATE OXIDATIVE DECARBOXYLATION ALLEVIATE THE GLUTAMATE EXCITOTOXICITY IN CEREBELLAR GRANULE NEURONS.

M. S. Kabysheva, T. P. Storozhevykh, V. G. Pinelis, V. I. Bunik.

Biochemical Pharmacology 77 (9):1531-1540, 2009.

Impairment of the 2-oxoglutarate oxidative decarboxylation by the 2-oxoglutarate dehydrogenase complex (OGDHC) is associated with the glutamate accumulation, ROS production and neuropathologies. We hypothesized that correct function of OGDHC under metabolic stress is essential to overcome the glutamate excitotoxic action on neurons. We show that synthetic phosphono analogs of 2-oxoglutarate, succinyl phosphonate and its phosphono ethyl ester, improve the catalysis by brain OGDHC through inhibiting the side reaction of irreversible inactivation of its first component, 2-oxoglutarate dehydrogenase. Under the Substrate and cofactor saturation, the component and complex undergo the inactivation during catalysis with the apparent rate constant 0.2 min^-1. The inactivation rate is reduced by 90% and 60% in the presence of 50 mu M succinyl phosphonate and its phosphono ethyl ester, correspondingly. In cultured cerebellar granule neurons exposed to excitotoxic glutamate, the phosphonates (100 mu M) protect from the irreversible impairment of mitochondrial function and delayed calcium deregulation. The deregulation amplitude is decreased by succinyl phosphonate and its phosphono ethyl ester by 50% and 30%, correspondingly. Thus, succinyl phosphonate is more potent than its phosphono ethyl ester in protecting both the isolated brain OGDHC from inactivation and cultured neurons from the glutamate-induced calcium deregulation. The correlation of the relative efficiency of the phosphonates in vitro and in situ indicates that their cellular effects are due to targeting OGDHC, which is in accord with independent studies. We conclude that the compounds preserving the 2-oxoglutarate dehydrogenase activity are of neuroprotective value upon metabolic disbalance induced by glutamate excess.

CHAPERONE-LIKE ACTIVITIES OF DIFFERENT MOLECULAR FORMS OF β-CASEIN. IMPORTANCE OF POLARITY OF N-TERMINAL HYDROPHILIC DOMAIN.

R. Yousefi, Y. Y. Shchutskaya, J. Zimny, J. C. Gaudin, A. A. Moosavi-Movahedi, V. I. Muronetz, Y. F. Zuev, J. M. Chobert, T. Haertle.

Biopolymers 91 (8):623-632, 2009.

As a member of intrinsically unstructured protein family, β-casein (β-CN) contains relatively high amount of prolyl residues, adopts noncompact and flexible structure and exhibits chaperone-like activity in vitro. Like many chaperones, native β-CN does not contain cysteinyl residues and exhibits strong tendencies for self-association. The chaperone-like activities of three recombinant β-CNs wild type (WT) β-CN, C4 β-CN (with cysteinyl residue in position 4) and C208 β-CN (with cysteinyl residue in position 208), expressed and purified from E. coli, which, consequently, lack the phosphorylated residues, were examined and compared with that Of native β-CN using insulin and alcohol dehydrogenase as target/substrate proteins. The dimers (β-CND) of C4-β-CN and C208 β-CN were also studied and their chaperone-like activities were compared with those of their monomeric forms. Lacking phosphorylation, WT β-CN, C208 β-CN, C4 β-CN and C4 β-CND exhibited significantly lower chaperone-like activities than native β-CN. Dimerization of C208 β-CN with two distal hydrophilic domains considerably improved its chaperone-like activity in comparison with its monomeric form. The obtained results demonstrate the significant role played by the polar contributions of phosphorylated residues and N-terminal hydrophilic domain as important functional elements in enhancing the chaperone-like activity of native β-CN.

THE YFIC GENE OF E-COLI ENCODES AN ADENINE-N6 METHYLTRANSFERASE THAT SPECIFICALLY MODIFIES A37 OF tRNA(1)(VAL)(CMO(5)UAC).

A. Y. Golovina, P. V. Sergiev, A. V. Golovin, M. V. Serebryakova, I. Demina, V. M. Govorun, O. A. Dontsova.

RNA-A Publication of the RNA Society 15 (6):1134-1141, 2009.

Transfer RNA is highly modified. Nucleotide 37 of the anticodon loop is represented by various modified nucleotides. In Escherichia coli, the valine-specific tRNA (cmo(5) UAC) contains a unique modification, N-6-methyladenosine, at position 37; however, the enzyme responsible for this modification is unknown. Here we demonstrate that the yfiC gene of E. coli encodes an enzyme responsible for the methylation of A37 in tRNA(1)(Val). Inactivation of yfiC gene abolishes m(6)A formation in tRNA(1)(Val), while expression of the yfiC gene from a plasmid restores the modification. Additionally, unmodified tRNA(1)(Val) can be methylated by recombinant YfiC protein in vitro. Although the methylation of m(6)A in tRNA(1)(Val) by YfiC has little influence on the cell growth under standard conditions, the yfiC gene confers a growth advantage under conditions of osmotic and oxidative stress.

INFLUENCE OF ACETYLTRANSFERASE ACTIVITY ON THE PROGNOSIS AND THE COURSE OF CORONARY ARTERY DISEASE IN PATIENTS AFTER CORONARY STENTING.

S. P. Semitko, I. A. Kuznecova, D. T. Guranda, D. G. Iosseliani.

American Journal of Cardiology 103 (9A):AS17, 2009.

NITRIC OXIDE-INDUCED MEMBRANE TUBULOVESICULAR EXTENSIONS (CYTONEMES) OF HUMAN NEUTROPHILS CATCH AND HOLD SALMONELLA ENTERICA SEROVAR TYPHIMURIUM AT A DISTANCE FROM THE CELL SURFACE.

S. I. Galkina, J. M. Romanova, V. I. Stadnichuk, J. G. Molotkovsky, G. F. Sud'ina, T. Klein.

Fems Immunology and Medical Microbiology 56 (2):162-171, 2009.

Nitric oxide (NO) plays an important role in host defense against bacterial infections such as salmonellosis. NO and 4-bromophenacyl bromide (BPB) induce the formation of long tubulovesicular extensions (TVE, cytonemes, membrane tethers) from human neutrophils. These TVE serve as cellular sensory and adhesive organelles. In the present study, we demonstrated that in the presence of the NO donor, diethylamine NONOate or BPB human neutrophils bound and aggregated Salmonella enterica serovar Typhimurium bacteria extracellularly by TVE. In contrast, inhibition of NO-synthase activity by N-omega-nitro-l-arginine methyl ester stimulated neutrophil phagocytosis (ingestion) of bacteria. Neutrophil TVE consisted of membrane-covered cytoplasm as was shown by the fluorescent cytoplasmic dye 2',7'-bis(2carboxyethyl)-5,(6)-carboxyfluorescein, and the fluorescent lipid, BODIPY-labeled sulfatide. Disruption and shedding of TVE were accompanied by the appearance of specific invaginations (porosomes) on neutrophil cell bodies. These invaginations corresponded to the variations in diameter of TVE (160-240 nm). We hypothesized that TVE represented protrusions of neutrophil exocytotic trafficking through special structures on the neutrophil surface. In conclusion, we propose a novel mechanism by which NO-induced TVE formation enables neutrophils to bind and aggregate bacteria at a distance.

PHOSPHOLIPIDS INFLUENCE THE AGGREGATION OF RECOMBINANT OVINE PRIONS FROM RAPID EXTENSIVE AGGREGATION TO AMYLOIDOGENIC CONVERSION.

K. Tsiroulnikov, Y. Shchutskaya, V. Muronetz, J. M. Chobert, T. Haertle.

Biochimica et Biophysica Acta-Proteins and Proteomics 1794 (3):506-511, 2009.

The transformation of prion protein (PrP) into its insoluble amyloid form correlates with neurodegeneration in transmissible spongiform encephalopathies. PrP is connected to the neuronal membrane by a covalently-linked glycosylphosphatidylinositol (GPI) anchor. The current study demonstrates that phosphatidylinositol and phosphatidylethanolamine in low concentrations (0.5-50 mu M) stimulate rapid unlimited aggregation of PrP. At a higher concentration (500 mu M), lipid particles prevent the formation of large PrP aggregates and induce an increase in the β-sheet structure content of PrP protein. Thus, the liberation of PrP from the membrane and its direct interaction with its own GPI moiety, as well as with membrane lipids. can promote the formation of aggregated structures of PrP. The phospholipids studied are also able to upregulate the aggregation of oligomeric PrP forms (12-mers and 36-mers), the neurotoxicity of which has been reported recently. Low phosphatidylinositol concentrations induce these oligomers to form aggregates of smaller size when compared with aggregates formed directly from monomers. The inhibition of extensive aggregation observed at a high concentration of phosphatidylinositol (500 mu M) results in both the formation of amyloids from PrP monomers and the interaction of protein molecules with lipid micelles. Thus, phospholipids are not only involved in the aggregation of prion monomers and their amyloidogenic conversion, but also regulate the aggregative status of prion oligomers already formed. Consequently, depending on their micellar status, phospholipids can either promote amyloidogenic conversion and conserve neurotoxic oligomeric forms (lipid micelles) or mediate the formation of large-size amorphous aggregates (non-micellar phospholipids).

SMALL HEAT SHOCK PROTEIN HSP27 PROTECTS MYOSIN S1 FROM HEAT-INDUCED AGGREGATION, BUT NOT FROM THERMAL DENATURATION AND ATPASE INACTIVATION (VOL 582, PG 1407, 2008).

D. I. Markov, A. V. Pivovarova, I. S. Chernik, N. B. Gusev, D. I. Levitsky.

FEBS Letters 583 (5):949, 2009.

DIGESTIVE PROTEOLYSIS ORGANIZATION IN TWO CLOSELY RELATED TENEBRIONID BEETLES: RED FLOUR BEETLE (Tribolium castaneum) AND CONFUSED FLOUR BEETLE (Tribolium confusum).

K. S. Vinokurov, E. N. Elpidina, D. P. Zhuzhikov, B. Oppert, D. Kodrik, F. Sehnal.

Archives of Insect Biochemistry and Physiology 70 (4):254-279, 2009.

The spectra of Tribolium castaneum and T. confusum larval digestive peptidases were characterized with respect to the spatial organization of protein digestion in the midgut. The pH of midgut contents in both species increased from 5.6-6.0 in the anterior to 7.0-7.5 in the posterior midgut. However, the pH optimum of the total proteolytic activity of the gut extract for either insect was pH 4.1. Approximately 80% of the total proteolytic activity was in the anterior and 20% in the posterior midgut of either insect when evaluated in buffers simulating the pH and reducing conditions characteristics for each midgut section. The general peptidase activity of gut extracts from either insect in pH 5.6 buffer was mostly due to cysteine peptidases. In the weakly alkaline conditions of the posterior midgut, the serine endopeptidases, 8 and 10 for T.castaneum, and 7 and 9 for T. confusum, repectively. Serine peptidases included trypsin-, chymotrypsin-, and elastase-like enzymes, the latter being for the first time reported in Tenebrionid insects. These data support a complex system of protein digestion in the Tribolium midgut with the fundamental role of cysteine peptidases.

QUANTITATIVE CHARACTERISTIC OF THE CATALYTIC PROPERTIES AND MICROSTRUCTURE

OF CROSS-LINKED ENZYME AGGREGATES OF PENICILLIN ACYLASE.

N. A. Pchelintsev, M. I. Youshko, V. K. Svedas.

Journal of Molecular Catalysis B-Enzymatic 56 (4):202-207, 2009.

The microstructure and the catalytic properties of cross-linked enzyme aggregates (CLEA) of penicillin acylase (PA) obtained under different conditions were investigated. The period of time left between the enzyme precipitation and the cross-linking step was found to influence the structural organization of the resulting enzyme preparation. Confocal fluorescent microscopy of the so-called "fresh" and "mature" CLEAs PA allowed to estimate the "characteristic" diameter of CLEA PA particles, which appeared to be about 1.6 mu m, and revealed that the "mature" type was composed of relatively big particles as compared to the "fresh" type. Complementary kinetic studies showed that the "mature" CLEA PA were more effective in both hydrolytic and synthetic reactions. It was suggested that the aggregate size might regulate the extent of covalent modification of PA and thereby influence the catalytic properties of CLEA.

HALF-OF-THE-SITES REACTIVITY OF TRANSKETOLASE FROM Saccharomyces cerevisiae.

I. Sevostyanova, O. Solovjeva, V. Selivanov, G. Kochetov.

Biochemical and Biophysical Research Communications 379 (4):851-854, 2009.

Cleavage by yeast transketolase of the donor substrate, D-xylulose 5-phosphate, in the absence of the acceptor substrate was studied using stopped-flow spectrophotometry. One mole of the substrate was shown to be cleaved in the prestationary phase, leading to the formation of one mole of the reaction product per mole enzyme, which has two active centers. This observation indicates that only one out of the two active centers functions (i.e., binds and cleaves the substrate) at a time. Such half-of-the-sites reactivity of transketolase conforms well with our understanding, proposed previously, that the active centers of the enzyme operate in sequence (in phase opposition): the cleavage of a ketose within one center (first phase of the transketolase reaction) is paralleled by its formation in the other center (glycolaldehyde residue is condensed with the acceptor substrate, and the second stage of the transketolase reaction is thereby completed) [M.V. Kovina, G.A. Kochetov, FEBS Lett. 440 (1998) 81-84].

COLORIMETRIC ASSAY OF CHITOSAN IN PRESENCE OF PROTEINS AND POLYELECTROLYTES

BY USING O-PHTHALALDEHYDE.

N. I. Larionova, D. K. Zubaerova, D. T. Guranda, M. A. Pechyonkin, N. G. Balabushevich.

Carbohydrate Polymers 75 (4):724-727, 2009.

A novel approach of colorimetric quantification of chitosan based on the derivatization reaction of its primary amino groups with o-phthalaldehyde and a thiol - N-acetyl-L-cysteine has been developed. The reaction of equal volumes of sample solution and the reagent solution was allowed to proceed for 1 h, and then the absorbance values were measured at 340 nm against the reference solution. The procedure conditions have been optimized for chitosan assay in the presence of polyanionic electrolyte dextran sulphate (pH 8.9, the reagent solution: 4.0 mM o-phthalaldehyde. 2.6 mM N-acetyl-L-cysteine, 0.25 M NaCl). The method has proven to be convenient and reliable for quantitative determination of either the concentrations of chitosans of various molecular weights or their degree of deacetylation. The different reactivity of chitosans and proteins can be used in order to determine chitosan in presence of the protein. This approach ensured accurate assay within the chitosan concentrations ranging from 0.01 to 0.15 mg/ml and could be applied for quantitative analysis of chitosan in protein-loaded microparticles.

α-KETOGLUTARATE DEHYDROGENASE CONTRIBUTES TO PRODUCTION OF REACTIVE OXYGEN SPECIES IN GLUTAMATE-STIMULATED HIPPOCAMPAL NEURONS in situ.

G. Zundorf, S. Kahlert, V. I. Bunik, G. Reiser.

Neuroscience 158 (2):610-616, 2009.

The α-ketoglutarate dehydrogenase complex (KGDHC) which catalyzes the conversion of a-ketoglutarate to succinyl-CoA and NADH in mitochondria, is known to generate O₂· - in vitro. To find out if KGDHC contributes to neuronal reactive oxygen species (ROS) increase in situ, we investigated whether the specific inhibitors of cellular KGDHC, succinyl phosphonate (SP) and the SP triethyl ester (TESP), might affect the glutamate-induced ROS production in cultured hippocampal neurons from rats. The concentration-dependent decrease in the mitochondrial potential of the glutamate-overstimulated neurons in the presence of SP or TESP indicated that under the conditions inducing neuronal ROS generation, the inhibitors are delivered to mitochondria, and their subsequent inhibition of KGDHC decreases the mitochondrial potential. The production of O₂·- was detected by reaction with hydroethidine. The distribution of the resulting fluorescence of DNA-ethidium coincided with that of the mitochondrial marker Mitotracker, pointing to the mitochondrial origin of the hydroethidine-detected ROS in response to glutamate (100 mu M). At 200 mu M, both TESP and SP administered together with glutamate, inhibited the glutamate-induced ROS production by about 20%, with the inhibition increasing to 44% at 500 mu M TESP. The decrease in neuronal ROS by specific inhibitors of KGDHC demonstrates that KGDHC is a source of ROS in cultured neurons responding to glutamate. However, increasing the concentration of the strongest KGDHC inhibitor SP to 500 mu M even increased the ROS production compared with glutamate alone, presumably due to secondary effects arising upon the strong KGDHC inhibition. Our work extends the current understanding of the glutamate-induced ROS generation in neurons, shedding light on the pathological mechanisms of the KGDHC involvement in glutamate neurotoxicity. In conclusion, potent KGDHC inhibitors are promising diagnostic tools for in situ study of neurodegenerative mechanisms.

IMPACT OF 7,8-DIHYDRO-8-OXOGUANINE ON METHYLATION OF THE CPG SITE BY DNMT3A.

D. V. Maltseva, A. A. Baykov, A. Jeltsch, E. S. Gromova.

Biochemistry 48 (6):1361-1368, 2009.

7,8-Dihydro-8-oxoguanine (8-oxoG) is a ubiquitous oxidative DNA lesion resulting from injury to DNA via reactive oxygen species. 8-oxoG lesions may play a role in the formation of aberrant DNA methylation patterns during carcinogenesis. In this study, we assessed the effects of 8-oxoG on methylation and complex formation of nine 30-mer oligodeoxynucleotide duplexes by the catalytic domain of murine Dnmt3a DNA methyltransferase (Dnmt3a-CD). The effects of 8-oxoG on the methylation rate of hemimethylated duplexes varied from a 25-fold decrease to a 1.8-fold increase, depending on the position of the lesion relative to the Dnmt3a-CD recognition site (CpG) and target cytosine (C). The most significant effect was observed when 8-oxoG replaced guanine within the recognition site immediately downstream of the target cytosine. Fluorescence polarization experiments with fluorescein-labeled duplexes revealed that two molecules of Dnmt3a-CD bind per duplex, generating sigmoid binding curves. Duplexes exhibiting the highest apparent binding cooperativity formed the least stable 1:2 complexes with Dnmt3a-CD and were methylated at the lowest rate. Kinetic analyses disclosed the formation of very stable nonproductive enzyme-substrate complexes with hemimethylated duplexes that act as suicide substrates of Dnmt3a-CD. The presence of 8-oxoG within the CpG site downstream of the target cytosine markedly diminished productive versus nonproductive binding. We propose that 8-oxoG located adjacent to the target cytosine interferes with methylation by weakening the affinity of DNA for Dnmt3a-CD, thereby favoring a nonproductive binding mode.

ROLE OF 2-OXOGLUTARATE DEHYDROGENASE IN BRAIN PATHOLOGIES INVOLVING GLUTAMATE NEUROTOXICITY.

A. Graf, M. Kabysheva, E. Klimuk, L. Trofimova, T. Dunaeva, G. Zundorf, S. Kahlert, G. Reiser, T. Storozhevykh, V. Pinelis, N. Sokolova, V. Bunik.

Journal of Molecular Catalysis B-Enzymatic 61 (1-2):80-87, 2009.

Decreased activity of the mitochondrial thiamin-dependent 2-oxoglutarate dehydrogenase complex (OGDHC) is associated with a number of inborn and acquired neuropathologies. We hypothesized that perturbation in flux through the complex influences brain development and function, in particular, because the OGDHC reaction is linked to the synthesis/degradation of neurotransmitters glutamate and GABA. Developmental impact of this metabolic knot was studied by characterizing the brain OGDHC activity in offspring of rats exposed to acute hypobaric hypoxia at a critical organogenesis period of pregnancy. In this model, we detected the hypoxia-induced changes in the brain OGDHC activity and in certain physiologic and morphometric parameters. The changes were mostly abrogated by application of specific effector of cellular OGDHC, the phosphonate analog of 2-oxoglutarate (succinyl phosphonate), shortly before hypoxia. The glutamate excitotoxicity known to greatly contribute to hypoxic damage was alleviated by succinyl phosphonate in situ. That is, the delayed calcium deregulation, mitochondrial depolarization and reactive oxygen species (ROS) production became less pronounced in cultivated neurons loaded with succinyl phosphonate. In vitro, succinyl phosphonate protected OGDHC from the catalysis-induced inactivation. Thus, the protective effects of the phosphonate upon hypoxic insult in vivo may result from the preservation of mitochondrial function and Ca²⁺ homeostasis due to the phosphonate inhibition of both the OGDHC-dependent ROS production and associated OGDHC inactivation. As a result, we showed for the first time that the hypoxia- and glutamate-induced cerebral damage is linked to the function of OGDHC. introducing the phosphonate analogs of 2-oxoglutarate as promising diagnostic tools to reveal the role of OGDHC in brain function and development.

INHIBITION OF THROMBIN ACTIVITY WITH DNA-APTAMERS.

A. B. Dobrovolsky, E. V. Titaeva, S. G. Khaspekova, V. A. Spiridonova, A. M. Kopylov, A. V. Mazurov.

Bulletin of Experimental Biology and Medicine 148 (1):33-36, 2009.

The effects of two DNA aptamers (oligonucleotides) 15TBA and 31TBA (15- and 31-mer thrombin-binding aptamers, respectively) on thrombin activity were studied. Both aptamers added to human plasma dose-dependently increased thrombin time (fibrin formation upon exposure to exogenous thrombin), prothrombin time (clotting activation by the extrinsic pathway), and activated partial thromboplastin time (clotting activation by the intrinsic pathway). At the same time, these aptamers did not modify amidolytic activity of thrombin evaluated by cleavage of synthetic chromogenic substrate. Aptamers also inhibited thrombin-induced human platelet aggregation. The inhibitory effects of 31TBA manifested at lower concentrations than those of 15TBA in all tests. These data indicate that the studied antithrombin DNA aptamers effectively suppress its two key reactions, fibrin formation and stimulation of platelet aggregation, without modifying active center of the thrombin molecule.

STRUCTURAL FEATURES OF THE tmRNA-RIBOSOME INTERACTION.

E. Y. Bugaeva, S. Surkov, A. V. Golovin, L. G. Ofverstedt, U. Skoglund, L. A. Isaksson, A. A. Bogdanov, O.V. Shpanchenko, O. A. Dontsova.

RNA-A Publication of the RNA Society 15 (12):2312-2320, 2009.

Trans-translation is a process which switches the synthesis of a polypeptide chain encoded by a nonstop messenger RNA to the mRNA-like domain of a transfer-messenger RNA (tmRNA). It is used in bacterial cells for rescuing the ribosomes arrested during translation of damaged mRNA and directing this mRNA and the product polypeptide for degradation. The molecular basis of this process is not well understood. Earlier, we developed an approach that allowed isolation of tmRNA-ribosomal complexes arrested at a desired step of tmRNA passage through the ribosome. We have here exploited it to examine the tmRNA structure using chemical probing and cryo-electron microscopy tomography. Computer modeling has been used to develop a model for spatial organization of the tmRNA inside the ribosome at different stages of trans-translation.

STRUCTURAL FEATURES OF THE tmRNA-RIBOSOME INTERACTION.

E.Y. Bugaeva, S. Surkov, A.V. Golovin, L.G. Ofverstedt, U. Skoglund, L.A. Isaksson, A.A. Bogdanov, O.V. Shpanchenko, O.A. Dontsova.

RNA (Cold Spring Harbor) 15 (12):2312-2320, 2009.

Three 'triple gene block' proteins known as TGBp1, TGBp2 and TGBp3 are required for cell-to-cell movement of plant viruses belonging to a number of genera including Hordeivirus. Hordeiviral TGBp1 interacts with viral genomic RNAs to form ribonucleoprotein (RNP) complexes competent for translocation between cells through plasmodesmata and over long distances via the phloem. Binding of hordeivirus TGBp1 to RNA involves two protein regions, the C-terminal NTIPase/ helicase domain and the N-terminal extension region. This study demonstrated that the extension region of hordeivirus TGBp1 consists of two structurally and functionally distinct domains called the N-terminal domain (NTD) and the internal domain (ID). In agreement with secondary structure predictions, analysis of circular dichroism spectra of the isolated NTD and ID demonstrated that the NTD represents a natively unfolded protein domain, whereas the ID has a pronounced secondary structure. Both the NTD and ID were able to bind ssRNA non-specifically. However, whilst the NTD interacted with ssRNA non-cooperatively, the ID bound ssRNA in a cooperative manner. Additionally, both domains bound dsRNA. The NTD and ID formed low-molecular-mass oligomers, whereas the ID also gave rise to high-molecular-mass complexes. The isolated ID was able to interact with both the NTD and the C-terminal NTPase/helicase domain in solution. These data demonstrate that the hordeivirus TGBp1 has three RNA-binding domains and that interaction between these structural units can provide a basis for remodelling of viral RNP complexes at different steps of cell-to-cell and long-distance transport of virus infection.

HALOGENATED PYRUVATE DERIVATIVES AS SUBSTRATES OF TRANSKETOLASE FROM Saccharomyces cerevisiae.

O. A. Esakova, L. E. Meshalkina, G. A. Kochetov, R. Golbik.

Biochemistry-Moscow 74 (11):1234-1238, 2009.

Pyruvate derivatives halogenated at C3 were shown to be donor substrates in the transketolase reaction. No drastic differences between the derivatives were observed in the value of the catalytic constant, whereas the Michaelis constant increased in the following order: Br-pyruvate < Cl-pyruvate < Cl-2-pyruvate < F-pyruvate < Br-2-pyruvate. The presence of the halogenated pyruvate derivatives increased the affinity of apotransketolase for the coenzyme; of note, the extent of this effect was equal with both of the active centers of the enzyme. In contrast, the presence of any other substrate known to date, including hydroxypyruvate (i.e. pyruvate hydroxylated at C3), induced nonequivalence of the active centers in that they differed in the extent to which the affinity for the coenzyme increased. Consequently, the β-hydroxyl of dihydroxyethylthiamine diphosphate (an intermediate of the transketolase reaction) played an important role in the phenomenon of non-equivalence of the active centers associated with the coenzyme binding. The fundamental possibility was demonstrated of using halogenated pyruvate derivatives as donors of the halogen-hydroxyethyl group in organic synthesis of halogenated carbohydrates involving transketolase.

ENGINEERING OF CASEINS AND MODULATION OF THEIR STRUCTURES AND INTERACTIONS.

J. C. Gaudin, A. Le Parc, B. Castrec, M. H. Ropers, Y. Choiset, J. Shchutskaya, R. Yousefi, V. I. Muronetz, Y. Zuev, J. M. Chobert, T. Haertle.

Biotechnology Advances 27 (6):1124-1131, 2009.

β-Casein (β-CN) is a milk protein widely used in food industries because of its mild emulsifying properties due to its amphiphilicity. However. the elements determining its micellization behavior in solution and interfacial behavior at the air-water interface are not well known In order to Study flow the forced dimerisation influences functional properties of β-CN, recombinant wild-type β-CN was produced and distal cysteinylated forms of recombinant β-CN were engineered We show that 1) cysteinylated β-CN formed mainly dimers bridged by disulfide bonds, 2) the process of dimerization adds to the micellization process with temperature and is poorly reversible, 3) covalent disulfide linkage forms at the air-water interface at a lower temperature than in bulk. In conclusion, the location of the cysteinylation in the C-terminus or N-terminus or both is of importance for the properties of β-CN

MOLECULAR MECHANISMS OF TRANSFORMATION OF SKQ MITOTROPIC QUINONES AND THE SEARCH FOR NEW APPROACHES TO CREATION OF SELECTIVE FREE RADICAL TRAPS.

S. A. Eremeyev, V. I. Kargin, K. A. Motovilov, V. N. Tashlitsky, V. Y. Markov, G. A. Korshunova, N. V. Sumbatyan, M. Y. Vyssokikh, L. Yaguzhinsky.

Biochemistry-Moscow 74 (10):1114-1124, 2009.

Features of the mechanism of action of positively charged benzoquinone derivatives (SkQ), which are the analogs of coenzyme Q (I), plastoquinone (II), and tocopherol (III), are discussed. It is usually considered that the main target of these compounds is mitochondria, where they accumulate due to the positive charge of the molecule. In the present work, it is shown with model systems that the reduced forms of compounds (I-III) under certain conditions can transform into electrically neutral cyclic zwitterions, which theoretically can escape from the matrix of energized mitochondria against the concentration gradient. A weak uncoupling effect of molecules I-III has been found on mitochondria. Its existence is in agreement with the abovementioned transformation of positively charged hydroquinones of type Ia-IIIa into electrically neutral molecules. The data obtained with model systems suggest that the target of SkQ hydroquinones as free radical traps may be not only mitochondria but also biochemical systems of the cytoplasm. Due to the presence of a large number of reactive oxygen species (ROS)-dependent signal systems in a cell, the functioning of cytoplasmic systems might be disturbed under the action of antioxidants. The problem of selective effect of antioxidants is discussed in detail in the present work, and a functional diagram of selective decrease of the "background level" of ROS based on differences in the intensity of background and "signal" ROS fluxes is considered.

ROLE OF THERMOINDUCED DISSOCIATION IN INTERACTION BETWEEN α-CRYSTALLIN AS AN OLIGOMERIC CHAPERONE AND GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE AS AN OLIGOMERIC PROTEIN SUBSTRATE.

N. A. Chebotareva, B. I. Kurganov, K. O. Muranov, R. A. Asryants, M. A. Ostrovsky.

Doklady Biochemistry and Biophysics 428 (1):245-248, 2009.

STERIC AND ELECTRONIC PROPERTIES OF THE COFACTOR'S AMINO GROUP CONTROL THE LIFETIME OF THE CENTRAL CARBANION/ENAMINE INTERMEDIATE IN TRANSKETOLASE.

L. E. Meshalkina, G. A. Kochetov, G. Hubner, K. Tittmann, R. Golbik.

Journal of Molecular Catalysis B-Enzymatic 61 (1-2):67-72, 2009.

Transketolase (TK), a thiamin diphosphate (ThDP) dependent enzyme, catalyzes the reversible transfer of a two-carbon unit from keto- to aldo-substrates. Dihydroxyethylthiamin diphosphate (DHEThDP), formed as a result of cleavage of the donor substrate, serves as an intermediate of the TK reaction. TK from the yeast Saccharomyces cerevisiae is unique among thiamin enzymes displaying enzymatic activity after reconstitution with a methylated analogue of the native cofactor, 4'-methylamino-ThDP. The reconstitution of the apoenzyme with both ThDP and the methylated analogue can be analyzed by near UV circular dichroism. It was demonstrated that in the native holoenzyme and in the complex of TK with 4'-methylamino-ThDP the formation of the dihydroxyethyl-based carbanion/enamine took place with comparable rate constants, whereas the protonation of the reactive species was much faster in the complex with the analogue. The enzymatic activity of the enzyme reconstituted with 4'-methylamino-ThDP was 10fold higher in the ferricyanide assay. We suggest that a methylation of the 4'-amino group of ThDP impairs the resonance stabilization of the carbanion/enanline intermediate both sterically and electronically, thus allowing either a faster protonation or oxidation reaction by ferricyanide. The formation of the optically active DHE-4'-methylamino-ThDP was monitored by near UV circular dichroism spectra and corroborated by H-1 NMR analysis. The protonated form of the intermediate DHE-4'-methylamino-ThDP was released from the active sites of TK and accumulated in the medium on preparative scale.

II. Structure and functions of biological membranes. Bioenergetics. Photosynthesis.

ANTIBACTERIAL EFFECTS OF SILVER IONS ON GROWTH OF GRAM-NEGATIVE BACTERIA AND BIOFILM FORMATION.

M. A. Radzig, O. A. Koksharova, I. A. Khmel'.

Molecular Genetics Microbiology and Virology 24 (4):194-199, 2009.

The average minimal inhibiting concentrations (MICs) of AgNO3 for the gram-negative bacteria Escherichia coli K12, Serratia proteamaculans 94, and Serratia liquefaciens MG1 were found to be 0.075-0.3 mu g/ml and, for Pseudomonas aeruginosa PAO1 and P. chlororaphis 449, this concentration was 0.15-0.3 mu g/ml. The formation of a biofilm by Escherichia coli AB1157 and S. proteamaculans 94 was completely inhibited by 0.3 mu g/ml of AgNO3 and, in the case of Pseudomonas aeruginosa PAO1, biofilm formation was inhibited by 0.6 mu g/ml of AgNO3. Mutations in E. coli genes that encode global regulators of gene expression, such as sigma S and sigma N subunits of RNA polymerase, the catabolite repression protein CRP, and the Lon protease, had no marked effect on the sensitivity of cells to silver. Wild-type E. coli strains and strains deficient in excision repair (uvrA and uvrB), SOS repair, and recombination (recA, lexA, recBC, and recF mutants) did not differ in their sensitivity to silver. This suggests that the sensitivity of the bacteria to the silver was not associated with DNA lesions that could be repaired by these repair and recombination systems. E. coli mutant strains deficient in OmpF or OmpC porins were three to four times more resistant to silver ions than the wild-type strain. Experiments with the pME6863 plasmid, which bears the gene of N-acyl-homoserine lactonase AiiA, demonstrated that the quorum sensing (QS) regulation did not participate in controlling the sensitivity of S. proteamaculans 94 and P. chlororaphis 449 to silver. The same conclusion was drawn from a comparison of AgNO3 MICs of the S. liquefaciens wild-type strain and a mutant strain deficient in QS.

NEW DATA ON BIOCHEMICAL MECHANISM OF PROGRAMMED SENESCENCE OF ORGANISMS AND ANTIOXIDANT DEFENSE OF MITOCHONDRIA.

V. P. Skulachev.

Biochemistry-Moscow 74 (12):1400-1403, 2009.

Much evidence has recently been reported suggesting that reactive oxygen species (ROS) produced in mitochondria play a crucial role in the programmed senescence of organisms. In particular, it has been shown that antioxidants addressed to mitochondria slow down the appearance of symptoms of senescence and development of senile diseases and increase the median lifespan of various organisms from fungi to mammals. At the biochemical level, the mechanism of action of such rechargeable antioxidants as plastoquinonyldecyltriphenyl phosphonium (SkQ1) includes, in particular, prevention of oxidation of mitochondrial cardiolipin by ROS. The hormone melatonin also exhibits a number of such effects, and decrease in its level with age could explain the weakening of antioxidant protection upon aging. According to Moosmann et al., there exists a natural mechanism of antioxidant protection that, like SkQ1, is localized in the internal mitochondrial membrane and is rechargeable. It involves methionine residues in the surface regions of proteins encoded by mitochondrial DNA. It appears that in organisms with high respiratory metabolism the genetic code in the mitochondrial system of protein biosynthesis has changed. In these organisms (including some yeasts, insects, crustaceans, and vertebrates), the AUA codon codes for methionine rather than isoleucine, as in the case of synthesis of proteins encoded either in the nucleus or in mitochondria of organisms with lower rates of metabolism (other yeast species, sponges, and echinoderms). Methionine quenches ROS, being converted to methionine sulfoxide, which is re-reduced to the initial methionine by NADPH.

ELECTROSTATIC BINDING OF SUBSTITUTED METAL PHTHALOCYANINES TO ENTEROBACTERIAL CELLS: ITS ROLE IN PHOTODYNAMIC INACTIVATION.

M. G. Strakhovskaya, Y. N. Antonenko, A. A. Pashkovskaya, E. A. Kotova, V. Kireev, V. G. Zhukhovitsky, N. A. Kuznetsova, O. A. Yuzhakova, V. M. Negrimovsky, A. B. Rubin.

Biochemistry-Moscow 74 (12):1305-1314, 2009.

The effect of ionic substituents in zinc and aluminum phthalocyanine molecules and of membrane surface charge on the interaction of dyes with artificial membranes and enterobacterial cells, as well as on photosensitization efficiency was studied. It has been shown that increasing the number of positively charged substituents enhances the extent of phthalocyanine binding to Escherichia coli cells. This, along with the high quantum yield of singlet oxygen generation, determines efficient photodynamic inactivation of Gram-negative bacteria by zinc and aluminum octacationic phthalocyanines. The effect of Ca²⁺ and Mg²⁺ cations and pH on photodynamic inactivation of enterobacteria in the presence of octacationic zinc phthalocyanine has been studied. It has been shown that effects resulting in lowering negative charge on outer membrane protect bacteria against photoinactivation, which confirms the crucial role in this process of the electrostatic interaction of the photosensitizer with the cell wall. Electrostatic nature of binding is consistent with mainly electrostatic character of dye interactions with artificial membranes of different composition. Lower sensitivity of Proteus mirabilis to photodynamic inactivation, compared to that of E. coli and Salmonella enteritidis, due to low affinity of the cationic dye to the cells of this species, was found.

PRIMARY PROCESSES OF CHARGE SEPARATION IN REACTION CENTERS OF YM210L/FM197Y AND YM210L MUTANTS OF Rhodobacter sphaeroides.

A. G. Yakovlev, L. G. Vasilieva, A. Y. Shkuropatov, V. A. Shuvalov.

Biochemistry-Moscow 74 (11):1203-1210, 2009.

Difference femtosecond absorption spectroscopy with 20-fsec temporal resolution was applied to study a primary stage of charge separation and transfer processes in reaction centers of YM210L and YM210L/FM197Y site-directed mutants of the purple bacterium Rhodobacter sphaeroides at 90 K. Photoexcitation was tuned to the absorption band of the primary electron donor P at 880 nm. Coherent oscillations in the kinetics of stimulated emission of P* excited state at 940 nm and of anion absorption of monomeric bacteriochlorophyll B_A^- at 1020 nm were monitored. The absence of tyrosine YM210 in RCs of both mutants leads to strong slowing of the primary reaction P* à P⁺B_A^- and to the absence of stabilization of separated charges in the state P⁺B_A^-. Mutation FM197Y increases effective mass of an acetyl group of pyrrole ring I in the bacteriochlorophyll molecule P-B of the double mutant YM210L/FM197Y by a hydrogen bond with OH-TyrM197 group that leads to a decrease in the frequency of coherent nuclear motions from 150 cm^-1 in the single mutant YM210L to similar to 100 cm^-1 in the double mutant. Oscillations with 100-150 cm^-1 frequencies in the dynamics of the P* stimulated emission and in the kinetics of the reversible formation of P⁺B_A^- state of both mutants reflect a motion of the P-B molecule relatively to P-A in the area of mutual overlapping of their pyrrole rings I. In the double mutant YM210L/FM197Y the oscillations in the P* emission band and the B_A^- absorption band are conserved within a shorter time similar to 0.5 psec (1.5 psec in the YM210L mutant), which may be a consequence of an increase in the number of nuclei forming a wave packet by adding a supplementary mass to the dimer P.

MODELING OF ATP-ADP STEADY-STATE EXCHANGE RATE MEDIATED BY THE ADENINE NUCLEOTIDE TRANSLOCASE IN ISOLATED MITOCHONDRIA.

E. Metelkin, O. Demin, Z. Kovacs, C. Chinopoulos.

FEBS Journal 276 (23):6942-6955, 2009.

A computational model for the ATP-ADP steady-state exchange rate mediated by adenine nucleotide translocase (ANT) versus mitochondrial membrane potential dependence in isolated rat liver mitochondria is presented. The model represents the system of three ordinary differential equations, and the basic components included are ANT, F-0/F-1-ATPase, and the phosphate carrier. The model reproduces quantitatively the relationship between mitochondrial membrane potential and the ATP-ADP steady-state exchange rate mediated by the ANT operating in the forward mode, with the assumption that the phosphate carrier functions under rapid equilibrium. Furthermore, the model can simulate the kinetics of experimentally measured data on mitochondrial membrane potential titrated by an uncoupler. Verified predictions imply that the ADP influx rate is highly dependent on the mitochondrial membrane potential, and in the 0-100 mV range it is close to zero, owing to extremely low matrix ATP values. In addition to providing theoretical values of free matrix ATP and ADP, the model explains the diminished ADP-ATP exchange rate in the presence of nigericin, a condition in which there is hyperpolarization of the inner mitochondrial membrane at the expense of the mitochondrial ∆ pH gradient; the latter parameter influences matrix inorganic phosphate and ATP concentrations in a manner also described.

QUANTUM EFFICIENCY OF THE MINOR CHANNEL FOR EXCITATION MIGRATION FROM B800 TO B875 BACTERIOCHLOROPHYLL FRACTIONS IN PURPLE BACTERIA.

A. Y. Borisov and A. P. Gaydukova.

Biologicheskie Membrany 26 (5):370-378, 2009.

Excitation migration in light-harvesting bacteriochlorophyll complexes LH1 and LH2 of purple bacteria has been studied in many experimental and theoretical works. According to the widely accepted notion, it proceeds along the declining stairs, B800* à B850* à B875* à P870*, where symbol * stands for excitations in the corresponding BChl fraction. In this paper we demonstrate the presence of one more way for a direct excitation delivery from B800 to B875, thus bypassing the main route. The comparison modeling enables the estimation of the mean portion of excitation that uses this minor migration way. In some real cases it may reach 9-9.5%. The values of the critical distances for excitation migration from B800 to B850 and from B800 to B875, as well as their values for arbitrary spectral shifts in BChl molecules, are determined.

GLUCOSE DEPRIVATION POTENTIATES TOXICITY OF OUABAIN AND GLUTAMATE IN CORTICAL NEURONS CULTURED FOR DIFFERENT TIME PERIODS.

E. R. Lozier, A. I. Dzhanibekova, E. V. Stel'mashuk, A. V. Graf, D. B. Zorov, N. A. Sokolova, N. K. Isaev.

Neurochemical Journal 3 (3):202-206, 2009.

The addition of glutamate (Glu) to culture medium for 24 hours induced the dose-dependent death of rat cortical neurons cultured for 9-10 days and did not affect neurons cultured for 4-5 days; this suggests that the later neurons are neurochemically immature. In mature cultures, glucose deprivation (GD) enhanced the toxic effect of low Glu concentrations by 15% and did not influence the toxicity of high concentrations of this neuromediator. In immature cultures, GD potentiated the Glu effect independent of the concentration of this neuromediator. Inhibition of Na⁺/K⁺-ATPase induced the death of some of the neurons. In the presence of a normal level of glucose, ouabain decreased the viability of mature and immature neurons to 67 ± 4 % and 79 ± 5%, respectively, and its presence during GD diminished viability to 28 ± 4 % and 56 ± 3%, respectively. The toxicity of ouabain was substantially attenuated when ionotropic glutamate receptors were blocked by MK-801. GD alone caused no significant increase in the death of these cells, even after a 3-hour incubation. Thus, GD strongly increases the susceptibility of neurons to the toxicity mediated by the activation of the NMDA subtype of ionotropic Glu receptors, even in the case of neurochemically immature neurons.

CHITOSAN-INDUCED PROGRAMMED CELL DEATH IN PLANTS.

L. A. Vasil'ev, E. V. Dzyubinskaya, R. A. Zinovkin, D. B. Kiselevsky, N. V. Lobysheva, V. D. Samuilov.

Biochemistry-Moscow 74 (9):1035-1043, 2009.

Chitosan, CN-, or H₂O₂ caused the death of epidermal cells (EC) in the epidermis of pea leaves that was detected by monitoring the destruction of cell nuclei; chitosan induced chromatin condensation and marginalization followed by the destruction of EC nuclei and subsequent internucleosomal DNA fragmentation. Chitosan did not affect stoma guard cells (GC). Anaerobic conditions prevented the chitosan-induced destruction of EC nuclei. The antioxidants nitroblue tetrazolium or mannitol suppressed the effects of chitosan, H₂O₂, or chitosan + H₂O₂ on EC. H₂O₂ formation in EC and GC mitochondria that was determined from 2',7'-dichlorofluorescein fluorescence was inhibited by CN- and the protonophoric uncoupler carbonyl cyanide m-chlorophenylhydrazone but was stimulated by these agents in GC chloroplasts. The alternative oxidase inhibitors propyl gallate and salicylhydroxamate prevented chitosan- but not CN--induced destruction of EC nuclei; the plasma membrane NADPH oxidase inhibitors diphenylene iodonium and quinacrine abolished chitosan- but not CN--induced destruction of EC nuclei. The mitochondrial protein synthesis inhibitor lincomycin removed the destructive effect of chitosan or H₂O₂ on EC nuclei. The effect of cycloheximide, an inhibitor of protein synthesis in the cytoplasm, was insignificant; however, it was enhanced if cycloheximide was added in combination with lincomycin. The autophagy inhibitor 3-methyladenine removed the chitosan effect but exerted no influence on the effect of H₂O₂ as an inducer of EC death. The internucleosome DNA fragmentation in conjunction with the data on the 3-methyladenine effect provides evidence that chitosan induces programmed cell death that follows a combined scenario including apoptosis and autophagy. Based on the results of an inhibitor assay, chitosan-induced EC death involves reactive oxygen species generated by the NADPH oxidase of the plasma membrane.

INTERACTION OF TETRASUBSTITUTED CATIONIC ALUMINUM PHTHALOCYANINE WITH ARTIFICIAL AND NATURAL MEMBRANES.

A. A. Pashkovskaya, I. V. Perevoshchikova, V. E. Maizlish, G. P. Shaposhnikov, E. A. Kotova, Y. N. Antonenko.

Biochemistry-Moscow 74 (9):1021-1026, 2009.

A study of the properties of water-soluble tetrasubstituted cationic aluminum phthalocyanine (AlPcN4) revealed efficient binding of this photosensitizer to phospholipid membranes as compared with tetrasulfonated aluminum and zinc phthalocyanine complexes. This also manifested itself in enhanced photodynamic activity of AlPcN4 as measured by the photosensitized damage of gramicidin channels in a planar bilayer lipid membrane. The largest difference in the photodynamic activity of cationic and anionic phthalocyanines was observed in a membrane containing negatively charged lipids, thereby pointing to significant contribution of electrostatic interactions to the binding of photosensitizers to a membrane. Fluoride anions suppressed the photodynamic activity and binding to membrane of both tetraanionic and tetracationic aluminum phthalocyanines, which supports our hypothesis that interaction of charged metallophthalocyanines with phospholipid membranes is mostly determined by coordination of the central metal atom with the phosphate group of lipid.

INVESTIGATION OF THE LOW-AFFINITY OXIDATION SITE FOR EXOGENOUS ELECTRON DONORS

IN THE Mn-DEPLETED PHOTOSYSTEM II COMPLEXES.

V. N. Kurashov, E. R. Lovyagina, D. Y. Shkolnikov, M. K. Solntsev, M. D. Mamedov, B. K. Semin.

Biochimica et Biophysica Acta-Bioenergetics 1787 (12):1492-1498, 2009.

In the manganese-depleted photosystem II (PSII[-Mn]) preparations, oxidation of exogenous electron donors is carried out through the high-affinity (HA) and the low-affinity (LA) sites. This paper investigates the LA oxidation site in the PSII(-Mn) preparations where the HA, Mn-binding site was blocked with ferric cations [[11] B.K. Semin, M.L Ghirardi, M. Seibert, Blocking of electron donation by Mn(II) to Y-z(center dot) following incubation of Mn-depleted photosystem II membranes with Fe(II) in the light, Biochemistry 41 (2002) 5854-5864.]. In blocked (PSII[-Mn,+Fe]) preparations electron donation by Mn(II) cations to Y-z(center dot) was not detected at Mn(II) concentration 10 mu M (corresponds to K-m for Mn(II) oxidation at the HA site), but detected at Mn concentration 100 mu M (corresponds to K-m for the LA site) by fluorescence measurements. Comparison of pH-dependencies of electron donation by Mn(II) through the HA and the LA sites revealed the similar pK_a equal to 6.8. Comparison of K_m for diphenylcarbazide (DPC) oxidation at the LA site and K_d for A_T thermoluminescence band suppression by DPC in PSII(-Mn,+Fe) samples suggests that there is relationship between the LA site and A_T band formation. The role of D1-His190 as an oxidant of exogenous electron donors at the LA site is discussed. in contrast to electrogenic electron transfer from Mn(II) at the HA site to Y-z(center dot), photovoltage due to Mn(II) oxidation in iron-blocked PSII(-Mn) core particles was not detected.

ON THE ORIGIN OF LIFE IN THE ZINC WORLD. 2. VALIDATION OF THE HYPOTHESIS ON THE PHOTOSYNTHESIZING ZINC SULFIDE EDIFICES AS CRADLES OF LIFE ON EARTH.

A. Y. Mulkidjanian and M. Y. Galperin.

Biology Direct 4, 2009.

Background: The accompanying article (A.Y. Mulkidjanian, Biology Direct 4: 26) puts forward a detailed hypothesis on the role of zinc sulfide (ZnS) in the origin of life on Earth. The hypothesis suggests that life emerged within compartmentalized, photosynthesizing ZnS formations of hydrothermal origin (the Zn world), assembled in sub-aerial settings on the surface of the primeval Earth. Results: If life started within photosynthesizing ZnS compartments, it should have been able to evolve under the conditions of elevated levels of Zn²⁺ ions, byproducts of the ZnS-mediated photosynthesis. Therefore, the Zn world hypothesis leads to a set of testable predictions regarding the specific roles of Zn²⁺ ions in modern organisms, particularly in RNA and protein structures related to the procession of RNA and the "evolutionarily old" cellular functions. We checked these predictions using publicly available data and obtained evidence suggesting that the development of the primeval life forms up to the stage of the Last Universal Common Ancestor proceeded in zinc-rich settings. Testing of the hypothesis has revealed the possible supportive role of manganese sulfide in the primeval photosynthesis. In addition, we demonstrate the explanatory power of the Zn world concept by elucidating several points that so far remained without acceptable rationalization. In particular, this concept implies a new scenario for the separation of Bacteria and Archaea and the origin of Eukarya. Conclusion: The ability of the Zn world hypothesis to generate non-trivial veritable predictions and explain previously obscure items gives credence to its key postulate that the development of the first life forms started within zinc-rich formations of hydrothermal origin and was driven by solar UV irradiation. This concept implies that the geochemical conditions conducive to the origin of life may have persisted only as long as the atmospheric CO2 pressure remained above ca. 10 bar. This work envisions the first Earth biotopes as photosynthesizing and habitable areas of porous ZnS and MnS precipitates around primeval hot springs. Further work will be needed to provide details on the life within these communities and to elucidate the primordial (bio) chemical reactions. Reviewers: This article was reviewed by Arcady Mushegian, Eugene Koonin, and Patrick Forterre. For the full reviews, please go to the Reviewers' reports section.

ON THE ORIGIN OF LIFE IN THE ZINC WORLD: I. PHOTOSYNTHESIZING, POROUS EDIFICES BUILT OF HYDROTHERMALLY PRECIPITATED ZINC SULFIDE AS CRADLES OF LIFE ON EARTH.

A. Y. Mulkidjanian

Biology Direct 4, 2009.

Background: The complexity of the problem of the origin of life has spawned a large number of possible evolutionary scenarios. Their number, however, can be dramatically reduced by the simultaneous consideration of various bioenergetic, physical, and geological constraints. Results: This work puts forward an evolutionary scenario that satisfies the known constraints by proposing that life on Earth emerged, powered by UV-rich solar radiation, at photosynthetically active porous edifices made of precipitated zinc sulfide (ZnS) similar to those found around modern deep-sea hydrothermal vents. Under the high pressure of the primeval, carbon dioxide-dominated atmosphere ZnS could precipitate at the surface of the first continents, within reach of solar light. It is suggested that the ZnS surfaces (1) used the solar radiation to drive carbon dioxide reduction, yielding the building blocks for the first biopolymers, (2) served as templates for the synthesis of longer biopolymers from simpler building blocks, and (3) prevented the first biopolymers from photo-dissociation, by absorbing from them the excess radiation. In addition, the UV light may have favoured the selective enrichment of photostable, RNA-like polymers. Falsification tests of this hypothesis are described in the accompanying article (A.Y. Mulkidjanian, M.Y. Galperin, Biology Direct 2009, 4: 27). Conclusion: The suggested "Zn world" scenario identifies the geological conditions under which photosynthesizing ZnS edifices of hydrothermal origin could emerge and persist on primordial Earth, includes a mechanism of the transient storage and utilization of solar light for the production of diverse organic compounds, and identifies the driving forces and selective factors that could have promoted the transition from the first simple, photostable polymers to more complex living organisms.

PREPARATION AND CHARACTERIZATION OF MOUSE EMBRYONIC FIBROBLASTS WITH K72W MUTATION IN SOMATIC CYTOCHROME C GENE.

I. A. Mufazalov, D. N. Penkov, B. V. Chernyak, O. Y. Pletjushkina, M. Y. Vyssokikh, R. V. Chertkova, M. P. Kirpichnikov, D. A. Dolgikh, A. A. Kruglov, D. V. Kuprash, V. P. Skulachev, S. A. Nedospasov.

Molecular Biology 43 (4):596-603, 2009.

Mouse embryonic fibroblasts (MEF) with point mutation in somatic cytochrome c gene were generated and characterized. It was shown that the substitution of lysine for tryptophan in position 72 (K72W) decreased the proapoptotic functions of cytochrome c in response to staurosporin treatment without disrupting its respiratory functions. The presence of this mutation did not affect the pattern of cytochrome c gene expression or its localization inside the cell. These cell lines therefore represent an interesting model for the study of apoptotic signaling and physiological functions of cytochrome c.

EFFECT OF NITROFURANS AND NO GENERATORS ON BIOFILM FORMATION BY

Pseudomonas aeruginosa PAO1 AND Burkholderia cenocepacia 370.

J. Zaitseva, V. Granik, A. Belik, O. Koksharova, I. Khmel.

Research in Microbiology 160 (5):353-357, 2009.

Antibacterial drugs in the nitrofuran series, such as nitrofurazone, furazidin, nitrofurantoin and nifuroxazide, as well as the nitric oxide generators sodium nitroprusside and isosorbide mononitrate in concentrations that do not suppress bacterial growth, were shown to increase the capacity of pathogenic bacteria Pseudomonas aeruginosa PAO1 and Burkholderia cenocepacia 370 to form biofilms. At 25-100 mu g/ml, nitrofurans 2-2.5-fold enhanced biofilm formation of P. aeruginosa PAO1, and NO donors 3-6-fold. For B. cenocepacia 370, the enhancement was 2-5-fold (nitrofurans) and 4.5-fold (sodium nitroprusside), respectively.

FEMTOSECOND PHASE OF CHARGE SEPARATION IN REACTION CENTERS OF Chloroflexus aurantiacus.

A. G. Yakovlev, T. A. Shkuropatova, L. G. Vasilieva, A. Y. Shkuropatov, V. A. Shuvalov.

Biochemistry-Moscow 74 (8):846-854, 2009.

Difference absorption spectroscopy with temporal resolution of similar to 20 fsec was used to study the primary phase of charge separation in isolated reaction centers (RCs) of Chloroflexus aurantiacus at 90 K. An ensemble of difference (light-minus-dark) absorption spectra in the 730-795 nm region measured at -0.1 to 4 psec delays relative to the excitation pulse was analyzed. Comparison with analogous data for RCs of HM182L mutant of Rhodobacter sphaeroides having the same pigment composition identified the 785 nm absorption band as the band of bacteriopheophytin I_B in the B-branch. By study the bleaching of this absorption band due to formation of I_B^-, it was found that a coherent electron transfer from P* to the B-branch occurs with a very small delay of 10-20 fsec after excitation of dimer bacteriochlorophyll P. Only at 120 fsec delay electron transfer from P* to the A-branch occurs with the formation of bacteriochlorophyll anion B_A^- absorption band at 1028 nm and the appearance of P* stimulated emission at 940 nm, as also occurs in native RCs of Rb. sphaeroides. It is concluded that a nuclear wave packet motion on the potential energy surface of P* after a 20-fsec light pulse excitation leads to the coherent formation of the P⁺B^- and P⁺B_A^- states.

HISTIDINE 440 CONTROLS THE OPENING OF COLICIN E1 CHANNELS IN A LIPID-DEPENDENT MANNER.

A. A. Sobko, T. I. Rokitskaya, E. A. Kotova.

Biochimica et Biophysica Acta-Biomembranes 1788 (9):1962-1966, 2009.

The in vitro activity of many pore-forming toxins, in particular, the rate of increase in the membrane conductance induced by the channel-forming domain (P178) of colicin E1 is maximum at an acidic pH. However, after P178 binding at acidic conditions, a subsequent pH shift from 4 to 6 on both sides of the planar bilayer lipid membrane caused a large increase in the trans-membrane current which was solely due to an increase in the number of open channels. This effect required the presence of anionic lipid. Replacing the His440 residue of P178 by alanine eliminated the pH-shift effect thereby showing that it is associated with deprotonation of this histidine residue. It was concluded that alkalinization-induced weakening of the electrostatic interactions between colicin and the membrane surface facilitates conformational changes required for the transition of membrane-bound colicin molecules to an active channel state.

TWO-PHOTON EXCITATION SPECTROSCOPY OF CAROTENOID-CONTAINING AND CAROTENOID-DEPLETED LH2 COMPLEXES FROM PURPLE BACTERIA.

I. Stepanenko, V. Kompanetz, Z. Makhneva, S. Chekalin, A. Moskalenko, A. Razjivin.

Journal of Physical Chemistry B 113 (34):11720-11723, 2009.

We applied two-photon fluorescence excitation spectroscopy to LH2 complex from purple bacteria Allochromatium minutissimum and Rhodobacter sphaeroides. Bacteriochlorophyll fluorescence was measured under two-photon excitation of the samples within the 1200-1500 rim region. Spectra were obtained for both carotenoid-containing and -depleted complexes of each bacterium to allow their direct comparison. The depletion of carotenoids did not alter the two-photon excitation spectra of either bacteria. The spectra featured a wide excitation band around 1350 nm (2 x 675 nm, 14 800 cm^-1) which strongly resembled two-photon fluorescence excitation spectra of similar complexes published by other authors. We consider obtained experimental data to be evidence of direct two-photon excitation of bacteriochlorophyll excitonic states in this spectral region.

REACTION OF NITRIC OXIDE WITH THE OXIDIZED DI-HEME AND HEME-COPPER OXYGEN-REDUCING CENTERS OF TERMINAL OXIDASES: DIFFERENT REACTION PATHWAYS AND END-PRODUCTS.

V. B. Borisov, E. Forte, A. Giuffre, A. Konstantinov, P. Sarti.

Journal of Inorganic Biochemistry 103 (8):1185-1187, 2009.

Inhibition of terminal oxidases by nitric oxide (NO) has been extensively investigated as it plays a role in regulation of cellular respiration and pathophysiology. Cytochrome bd is a tri-heme (b₅₅₈, b₅₉₅, d) bacterial oxidase containing no copper that couples electron transfer from quinol to O-2 (to produce H₂O) with generation of a transmembrane protonmotive force. In this work, we investigated by stopped-flow absorption spectroscopy the reaction of NO with Escherichia coli cytochrome bd in the fully oxidized (O) state. We show that under anaerobic conditions, the O state of the enzyme binds NO at heme d with second-order rate constant k_on = 1.5 ± 0.2 x 10² M^-1 s^-1, yielding a nitrosyl adduct (d³⁺-NO or d²⁺-NO⁺) with characteristic optical features (an absorption increase at 639 nm and a red shift of the Soret band). The reaction mechanism is remarkably different from that of O cytochrome c oxidase in which the heme-copper binuclear center reacts with NO approximately three orders of magnitude faster, forming nitrite. The data allow us to conclude that in the reaction of NO with terminal oxidases in the O state, CUB is indispensable for rapid oxidation of NO into nitrite.

HEME/HEME REDOX INTERACTION AND RESOLUTION OF INDIVIDUAL OPTICAL ABSORPTION SPECTRA OF THE HEMES IN CYTOCHROME bd FROM Escherichia coli.

D. A. Bloch, V. B. Borisov, T. Mogi, M. I. Verkhovsky.

Biochimica et Biophysica Acta 1787 (10): 1246-1253, 2009.

Cytochrome bd is a terminal component of the respiratory chain of Escherichia coli catalyzing reduction of molecular oxygen to water. it contains three hemes, b₅₅₈, b₅₉₅, and d. The detailed spectro-electrochemical redox titration and numerical modeling of the data reveal significant redox interaction between the low-spin heme b₅₅₈ and high-spin heme b₅₉₅, whereas the interaction between heme d and either hemes b appears to be rather weak. However, the presence of heme d itself decreases much larger interaction between the two hemes b. Fitting the titration data with a model where redox interaction between the hemes is explicitly included makes it possible to extract individual absorption spectra of all hemes. The α- and β-band reduced-minus-oxidized difference spectra agree with the data published earlier ([22] J.G. Koland, MJ. Miller, R.B. Gennis, Potentiometric analysis of the purified cytochrome d terminal oxidase complex from Escherichia coli, Biochemistry 23 (1984) 1051-1056., and [23] R.M. Lorence, J.G. Koland, R.B. Gennis, Coulometric and spectroscopic analysis of the purified cytochrome d complex of Escherichia coli: evidence for the identification of "cytochrome a₁" as cytochrome b₅₉₅, Biochemistry 25 (1986) 2314-2321.). The Soret band spectra show λ_max = 429.5 nm, λ_min approximate to 413 nm (heme b₅₅₈), λ_max = 439 nm, λ_min approximate to 400 ± 1 nm (heme b₅₉₅), and λ_max = 430 nm, λ_min = 405 nm (heme d). The spectral contribution of heme d to the complex Soret band is much smaller than those of either hemes b; the Soret/α (∆ A(430):∆ A(629)) ratio for heme d is 1.6.

MYOGLOBIN CAUSES OXIDATIVE STRESS, INCREASE OF NO PRODUCTION AND DYSFUNCTION OF KIDNEY'S MITOCHONDRIA.

E. Y. Plotnikov, A. A. Chupyrkina, I. B. Pevzner, N. K. Isaev, D. B. Zorov.

Biochimica et Biophysica Acta-Molecular Basis of Disease 1792 (8):796-803, 2009.

Rhabdomyolysis or crush syndrome is a pathology caused by muscle injury resulting in acute renal failure. The latest data give strong evidence that this syndrome caused by accumulation of muscle breakdown products in the blood stream is associated with oxidative stress with primary role of mitochondria. In order to evaluate the significance of oxidative stress under rhabdomyolysis we explored the direct effect of myoglobin on renal tubules and isolated kidney mitochondria while measuring mitochondrial respiratory control, production of reactive oxygen and nitrogen species and lipid peroxidation. In parallel, we evaluated mitochondrial damage under myoglobinurea in vivo. An increase of lipid peroxidation products in kidney mitochondria and release of cytochrome c was detected on the first day of myoglobinuria. In mitochondria incubated with myoglobin we detected respiratory control drop, uncoupling of oxidative phosphorylation, an increase of lipid peroxidation products and stimulated NO synthesis. Mitochondrial pore inhibitor, cyclosporine A, mitochondria-targeted antioxidant (SkQ1) and deferoxamine (Fe-chelator and ferrylmyoglobin reducer) abrogated these events. Similar effects (oxidative stress and mitochondrial dysfunction) were revealed when myoglobin was added to isolated renal tubules. Thus, rhabdomyolysis can be considered as oxidative stress-mediated pathology with mitochondria to be the primary target and possibly the source of reactive oxygen and nitrogen species. We speculate that rhabdomyolysis-induced kidney damage involves direct interaction of myoglobin with mitochondria possibly resulting in iron ions release from myoglobin's heme, which promotes the peroxidation of mitochondrial membranes. Usage of mitochondrial permeability transition blockers, Fe-chelators or mitochondria-targeted antioxidants, may bring salvage from this pathology.

Zn²⁺ ADDED FROM P-SIDE BLOCK WITH HIGH AFFINITY CYTOCHROME C OXIDASE IN PHOSPHOLIPID VESICLES OR IN INTACT MITOCHONDRIA.

W. Zakirzianova, T. Vygodina, A. Konstantinov.

FEBS Journal 276:354, 2009.

CASPASE-INDEPENDENT NMDA RECEPTOR TRIGGERED NEURONAL CELL DEATH COULD BE PREVENTED BY MITOCHONDRIA PRESERVATION BY GLYCINE.

N. Lobysheva, A. Selin, A. Tonshin, L. Yaguzhinsky, Y. Nartsissov.

FEBS Journal 276:324-325, 2009.

THE STUDY OF SUCCINATE DEHYDROGENASE AND BC1 COMPLEX INTERACTION IN INTACT LIVER MITOCHONDRIA OXIDIZING ARTIFICIAL SUBSTRATE DUROQUINOL.

K. Motovilov, G. Kolesova, N. Sumbatyan, L. Yaguzhinsky.

FEBS Journal 276:213, 2009.

AMIODARONE INHIBITS MULTIPLE DRUG RESISTANCE IN YEAST Saccharomyces cerevisiae.

D. A. Knorre, T. N. Krivonosova, O. V. Markova, F. F. Severin.

Archives of microbiology 191 (8):675-679, 2009.

Amiodarone is a widely used antiarrhythmic drug. There is also evidence that amiodarone decreases multidrug resistance in human cell lines. In this paper, we have shown that amiodarone has similar effect on yeast, Saccharomyces cerevisiae, decreasing multiple drug resistance. Amiodarone stimulates the accumulation of ethidium bromide by inhibiting its efflux from the cells. The effect of amiodarone is much stronger on wild-type cells compared to the mutant with inactivated ABC-transporters. Interestingly, the action of amiodarone is additive with the one of chloroquine, a known inhibitor of ABC-transporters. We speculate that these findings could help in the development of antifungal drug mixes.

MITOCHONDRIA-TARGETED RECHARGEABLE ANTIOXIDANT SKQ1 PREVENTS SENESCENCE

IN MAMMALS.

V. Skulachev.

FEBS Journal 276:35-36, 2009.

FUNCTIONS OF MITOCHONDRIA: FROM INTRACELLULAR POWER STATIONS TO MEDIATORS OF A SENESCENCE PROGRAM.

V. P. Skulachev.

Cellular and Molecular Life Sciences 66 (11-12):1785-1793, 2009.

In 1950 s I started in science by showing that non-phosphorylating respiration is critical for survival of an animal at low temperature. Later, in the 1960 s and 1970 s, I took part in verification of Mitchell's chemiosmotic hypothesis postulating that (i) mitochondria transform energy of respiration to electricity and (ii) uncoupling of respiration represents discharge of this electricity by H+ cycling. Fifteen years ago I turned to a specific kind of mitochondrial respiration which produces O₂·, and I came to the conclusion that it plays an ominous role, killing mitochondria, cells, or even organisms. My present task is a "megaproject" with an ambitious goal of minimizing the damaging effect of O₂·and stopping senescence.

SYNTHESIS AND CHARACTERIZATION OF DE NOVO DESIGNED PEPTIDES MODELLING THE BINDING SITES OF [4FE-4S] CLUSTERS IN PHOTOSYSTEM I.

M. L. Antonkine, M. S. Koay, B. Epel, C. Breitenstein, O. Gopta, W. Gartner, E. Bill, W. Lubitz.

Biochimica et Biophysica Acta-Bioenergetics 1787 (8):995-1008, 2009.

Photosystem I (PS I) converts the energy of light into chemical energy via transmembrane charge separation. The terminal electron transfer cofactors in PS I are three low-potential [4Fe-4S] clusters named F-X, F-A and F-B, the last two are bound by the PsaC subunit. We have modelled the F-A and F-B binding sites by preparing two apo-peptides (maquettes), sixteen amino acids each. These model peptides incorporate the consensus [4Fe-4S] binding motif along with amino acids from the immediate environment of the iron-sulfur clusters F-A and F-B. The [4Fe-4S] clusters were successfully incorporated into these model peptides, as shown by optical absorbance, EPR and Mossbauer spectroscopies. The oxidation-reduction potential of the iron-sulfur cluster in the F-A-maquette is - 0.44 ± 0.03 V and in the F-B-maquette is - 0.47 ± 0.03 V. Both are close to that of F-A and F-B in PS I and are considerably more negative than that observed for other [4Fe-4S] model systems described earlier (Gibney, B. R., Mulholland, S. E., Rabanal, F., and Dutton, P. L Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 15041-15046). Our optical data show that both maquettes can irreversibly bind to PS I complexes, where PsaC-bound F-A and F-B were removed, and possibly participate in the light-induced electron transfer reaction in PS I.

EFFECT OF TRANSITORY GLUCOSE DEPRIVATION ON MITOCHONDRIAL STRUCTURE AND FUNCTIONS IN CULTURED CEREBELLAR GRANULE NEURONS.

E. V. Stelmashook, N. K. Isaev, E. Y. Plotnikov, R. E. Uzbekov, I. B. Alieva, B. Arbeille, D. B. Zorov.

Neuroscience Letters 461 (2):140-144, 2009.

We found that 60-min glucose deprivation leads to progressive decrease in the mitochondrial membrane potential and increase in [Ca²⁺](i) in cultured cerebellar granule neurons. The latter effect was fully reversible. returning to the basal level 60 min after restoration of normal glucose level in the incubation medium, whereas mitochondrial membrane potential remained at 10.0 ± 1.8% below the initial value. Electron microscopy indicated that glucose deprivation induced appearance of mitochondria with local lightening of the matrix and destruction of cristae. This mitochondrial conformation was preserved during the restoration phase after glucose level in the cultivation medium returned to the normal level. Neuronal death within a 24-h period after 60-min glucose deprivation was relatively small, being 14.0 ± 4.4%.

REDOX PROPERTIES OF THE PROSTHETIC GROUPS OF Na⁺-TRANSLOCATING NADH:QUINONE OXIDOREDUCTASE. 2. STUDY OF THE ENZYME BY OPTICAL SPECTROSCOPY.

A. V. Bogachev, D. A. Bloch, Y. V. Bertsova, M. I. Verkhovsky.

Biochemistry 48 (27):6299-6304, 2009.

Redox titration of the electronic spectra of the prosthetic groups of the Na⁺-translocating NADH: quinone oxidoreductase (Na⁺-NQR) from Vibrio harveyi at different pH values showed five redox transitions corresponding to the four flavin cofactors of the enzyme and one additional transition reflecting oxidoreduction of the [2Fe-2S] cluster. The pH dependence of he measured midpoint redox potentials showed that the two-electron reduction of the FAD located in the NqrF subunit was Coupled with the uptake of only one H+. The one-electron reduction of neutral semiquinone of riboflavin and the formation of anion flavosemiquinone from the oxidized FMN bound to the NqrB subunit were not coupled to any proton uptake. The two sequential one-electron reductions of the FMN residue bound to the NqrC subunit showed pH-independent formation of anion radical in the first step and the formation of fully reduced flavin coupled to the uptake of one H+ in the second step. All four flavins stayed in the anionic form in the fully reduced enzyme. None of the six redox transitions in Na⁺-NQR showed dependence of its midpoint redox potential on the concentration of sodium ions. A model of the sequence of electron transfer steps In the enzyme is suggested.

REDOX PROPERTIES OF THE PROSTHETIC GROUPS OF Na⁺-TRANSLOCATING NADH:QUINONE OXIDOREDUCTASE. 1. ELECTRON PARAMAGNETIC RESONANCE STUDY OF THE ENZYME.

A. V. Bogachev, L. V. Kulik, D. A. Bloch, Y. V. Bertsova, M. S. Fadeeva, M. I. Verkhovsky.

Biochemistry 48 (27):6291-6298, 2009.

Redox properties of all EPR-detectable prosthetic groups of Na⁺-translocating NADH:quinone oxidoreductase (Na⁺-NQR) from Vibrio harveyi were studied at pH 7.5 using cryo-EPR spectroelectrochemistry. Titration shows five redox transitions. One with E_m = -275 mV belongs to the reduction of the [2Fe-2S] cluster, and the four others reflect redox transitions of flavin cofactors. Two transitions (E_m (1) = -190 mV and E_m (2) = -275 mV) originate from the formation of FMN anion radical, covalently bound to the NqrC Subunit, and its subsequent reduction. The remaining two transitions arise from the two other flavin cofactors. A high potential (E_m = -10 mV) transition corresponds to the reduction of riboflavin neutral radical, which is stable at rather high redox potentials. An E_m = -130 mV transition reflects the formation of FMN anion radical from a flavin covalently bound to the NqrB Subunit, which stays as a radical down to very low potentials. Taking into account the EPR-silent, two-electron transition of noncovalently bound FAD located in the NqrF subunit, there are four flavins in Na⁺-NQR all together. Defined by dipole-dipole magnetic interaction measurements, the interspin distance between the [2Fe-2S](+) cluster and the NqrB subunit-bound FMN anion radical is found to be 22.5 ± 1.5 angstrom, which means that for the functional electron transfer between these two centers another cofactor, most likely FMN bound to the NqrC subunit, should be located.

THE SEARCH FOR AN OPTIMAL ORIENTATIONAL ORDERING OF Q_y TRANSITION DIPOLES

OF SUBANTENNA MOLECULES IN THE SUPERANTENNA OF PHOTOSYNTHETIC GREEN BACTERIA:

MODEL CALCULATIONS.

A. V. Zobova, A. G. Yakovlev, A. S. Taisova, Z. G. Fetisova.

Molecular Biology 43 (3):426-443, 2009.

This work continues the series of our studies on the basic principles in the organization of natural light-harvesting antennae, which we theoretically predicted for the optimal model light-harvesting systems, initiated by our concept of the rigorous optimization of photosynthetic apparatus structure by functional criterion. This approach allowed us to determine several main principles in the organization of the natural systems. This work deals with the problem of the structure optimization of heterogeneous superantennae of photosynthetic green bacteria, consisting of several uniform subantennae, which raises the problem of their optimal coordination. Here we used mathematical modeling of the functioning of these natural superantennae to consider a possible optimization of this process via optimizing the mutual spatial orientation of Q_y transition dipole vectors of the light-harvesting molecules in neighboring subantennae. This allowed us to determine possible modes for optimal orientational ordering of Q_y transition dipoles of subantenna molecules in the model of green bacterium Chloroflexus aurantiacus superantenna. The calculations have demonstrated that the optimal mutual spatial orientation of the of Q_y dipoles in subantenna pigments leads to a stable minimization of the energy transfer time within superantennae and, consequently, a decrease in the energy losses, thereby ensuring a high efficiency and stability of the overall superantenna function.

RECONSTITUTION OF RAB- AND SNARE-DEPENDENT MEMBRANE FUSION BY SYNTHETIC ENDOSOMES.

T. Zobova, M. Miaczynska, U. Coskun, B. Lommer, A. Runge, D. Drechsel, Y. Kalaidzidis, M. Zerial.

Nature 459 (7250):1091-1U77, 2009.

Rab GTPases and SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are evolutionarily conserved essential components of the eukaryotic intracellular transport system. Although pairing of cognate SNAREs is sufficient to fuse membranes in vitro, a complete reconstitution of the Rab-SNARE machinery has never been achieved. Here we report the reconstitution of the early endosomal canine Rab5 GTPase, its key regulators and effectors together with SNAREs into proteoliposomes using a set of 17 recombinant human proteins. These vesicles behave like minimal 'synthetic' endosomes, fusing with purified early endosomes or with each other in vitro. Membrane fusion measured by content-mixing and morphological assays requires the cooperativity between Rab5 effectors and cognate SNAREs which, together, form a more efficient 'core machinery' than SNAREs alone. In reconstituting a fusion mechanism dependent on both a Rab GTPase and SNAREs, our work shows that the two machineries act coordinately to increase the specificity and efficiency of the membrane tethering and fusion process.

SAFRANINE O AS A FLUORESCENT PROBE FOR MITOCHONDRIAL MEMBRANE POTENTIAL STUDIED ON THE SINGLE PARTICLE LEVEL AND IN SUSPENSION.

I. V. Perevoshchikova, A. I. Sorochkina, D. B. Zorov, Y. N. Antonenko.

Biochemistry-Moscow 74 (6):663-671, 2009.

The permeant cationic dye safranine O is often used to measure mitochondrial membrane potential due to the dependence of both its absorption and fluorescence on mitochondrial energization, which causes its oligomerization inside mitochondria. In the present study we have used fluorescent correlation spectroscopy (FCS) to record the fluorescence changes on a micro level, i.e. under conditions permitting resolution of contributions from single particles (molecules of the dye and stained mitochondria). We have shown that the decrease in fluorescence signal from a suspension of energized mitochondria stained with a high safranine concentration (10 mu M) is explained by the decrease in dye concentration in the medium in parallel with the accumulation of the dye inside the mitochondria, which results in fluorescence quenching. With 1 mu M safranine O, the fluorescence rise after energization is caused by the accumulation of the dye up to a level not sufficient for full fluorescence quenching and also by the higher intensity of mitochondrial fluorescence on immersion of the dye in the hydrophobic milieu. Besides the estimation of the inner mitochondrial membrane potential, this approach also assesses the concentration of fluorescent particles. The non-monotonic dependence of the FCS parameter 1/G(τ à 0) on the concentration of mitochondrial protein suggests heterogeneity of the system with respect to fluorescence of particles. An important advantage of the described method is its high sensitivity, which allows measurements with low concentrations and quantities of mitochondrial protein in samples (less than 10 mu g).

BACTERIORHODOPSINS CONTAINING MODIFIED CHROMOPHORES: A STUDY ON THE WILD TYPE AND D96N MUTANT OF Halobacterium salinarum.

L. V. Khitrina, E. V. Mironova, A. A. Khodorov.

Biologicheskie Membrany 26 (3):194-200, 2009.

Modification of the chromophore in bacteriorhodopsin (BR) from ET1001 and D96N strains of Halobacterium salinarum was carried out. Purple membranes were decolored by means of light-dependent hydroxylaminolysis. The all- trans-isomers of retinal and its 3,4-didehydro-, 4-keto- and phenyl analogs were reconstituted into apomembranes. Absorption maxima of the homonymic pigments in both strains were similar. The kinetics of the M-intermediates in the mode of a single turnover of the photocycle induced by a short light flash (532 nm, 15 ns) was compared. For the investigated bacteriorhodopsin analogs the efficiency of the M-intermediate formation did not exhibit any reliable dependence on the point mutation. Both for ET1001 and for D96N strains the M-relaxation of the 4-ketoBR was distinctly biphasic, with the slow phase comprising about 10-15% of the signal amplitude. Replacement of the ionone ring by phenyl caused a weak deceleration of the M relaxation (similar to 1.5-fold decrease in t(1/2)). Independence of the photocycle deceleration of the point mutation and chromophore modification was shown for all BR analogs studied.

TWO-PHOTON EXCITATION SPECTRUM OF FLUORESCENCE OF THE LIGHT-HARVESTING

COMPLEX B800-850 FROM Allochromatium minutissimum WITHIN 1200-1500 (600-750) NM

SPECTRAL RANGE IS NOT CAROTENOID MEDIATED.

I. A. Stepanenko, V. O. Kompanets, S. V. Chekalin, Z. K. Makhneva, A. A. Moskalenko, R. Y. Pishchainikov, A. P. Razjivin.

Biologicheskie Membrany 26 (3):180-187, 2009.

Two types of peripheral light-harvesting complexes LH2 (B800-850) from photosynthetic purple bacterium Allochromatium minutissimum were studied. First type containing carotenoids was prepared from wild type cells. The other one was obtained from carotenoid-depleted cells grown with diphenylamine. We have shown that under laser femtosecond excitation within absorption wavelength range of 1200-1500 nm the two-photon excitation of LH2 complexes takes place. This excitation is a result of fluorescence of bacteriochlorophyll (BChl) spectral form B850 (BChl molecules of circular aggregate with strong exaction interaction in 850 urn spectral domain). LH2 fluorescence excitation spectra under two-photon excitation are the same for carotenoid-containing and carotenoidless preparations. In both cases the broad band with peak near 1350 (675) nm (FWHM similar to 240 (120) nm) was found. It is concluded that the broad band with peak near 1350 (675) nm in two-photon excitation spectra of LH2 complexes from Allochromatium minutissimum can not be interpreted as two-photon excitation band of the optically forbidden S₀ à S₁ transition of carotenoids (rhodopin). Possible nature of this band is discussed.

MORPHOLOGICAL CHANGES IN THE KIDNEYS OF RATS WITH POSTISCHEMIC ACUTE RENAL FAILURE AFTER INTRARENAL ADMINISTRATION OF FETAL MESENCHYMAL STEM CELLS FROM HUMAN BONE MARROW.

Y. V. Kudryavtsev, V. I. Kirpatovskii, E. Y. Plotnikov, A. V. Kazachenko, M. V. Marei, T. G. Khryapenkova, D. B. Zorov, G. T. Sukhikh.

Bulletin of Experimental Biology and Medicine 147 (1):113-119, 2009.

Chronic experiments on outbred albino rats were performed to compare the dynamics of histological signs for postischemic renal injury (90-min thermal ischemia) after intraparenchymal injection of cultured fetal MSC from human bone marrow. Functional indexes of the ischemic kidney were predetermined. In the early period after ischemia (day 4), administration of human bone marrow MSC was followed by the increase in blood flow in the microcirculatory bed and decrease in the degree of alteration in renal tubules. An increase in the area of zones with histological signs for normal function of tubules was accompanied by the improvement of biochemical indexes for renal function. In the delayed period, a protective effect of cell therapy was manifested in the prevention of death of renal tubules. Mild calcification of the necrotic tubular epithelium served as a marker of this process. Human bone marrow MSC were labeled with the fluorescent probe Calcein. These cells migrated from the site of injection, spread in the interstitium, and retained viability for 7 days. During this period, some cells were incorporated into the lumen of renal tubules.

GENERATION OF NEW TRAIL MUTANTS DR5-A AND DR5-B WITH IMPROVED SELECTIVITY TO DEATH RECEPTOR 5.

M. E. Gasparian, B. V. Chernyak, D. A. Dolgikh, A. V. Yagolovich, E. N. Popova, A. M. Sycheva, S. A. Moshkovskii, M.P. Kirpichnikov.

Apoptosis 14 (6):778-787, 2009.

TRAIL (tumor necrosis factor (TNF) related apoptosis-inducing ligand) has been introduced as an extrinsic pathway inducer of apoptosis that does not have the toxicities of Fas and TNF. However, the therapeutic potential of TRAIL is limited because of many primary tumor cells are resistant to TRAIL. Despite intensive investigations, little is known in regards to the mechanisms underlying TRAIL selectivity and efficiency. A major reason likely lies in the complexity of the interaction of TRAIL with its five receptors, of which only two DR4 and DR5 are death receptors. Binding of TRAIL with decoy receptors DcR1 and DcR2 or soluble receptor osteoprotegerin (OPG) fail to induce apoptosis. Here we describe design and expression in Escherichia coli of DR5-selective TRAIL variants DR5-A and DR5-B. The measurements of dissociation constants of these mutants with all five receptors show that they practically do not interact with DR4 and DcR1 and have highly reduced affinity to DcR2 and OPG receptors. These mutants are more effective than wild type TRAIL in induction of apoptosis in different cancer cell lines. In combination with the drugs targeted to cytoskeleton (taxol, cytochalasin D) the mutants of TRAIL induced apoptosis in resistant Hela cells overexpressing Bcl-2. The novel highly selective and effective DR5-A and DR5-B TRAIL variants will be useful in studies on the role of different receptors in TRAIL-induced apoptosis in sensitive and resistant cell lines.

CO-EVOLUTION OF PRIMORDIAL MEMBRANES AND MEMBRANE PROTEINS.

A. Y. Mulkidjanian, M. Y. Galperin, E. V. Koonin.

Trends in Biochemical Sciences 34 (4):206-215, 2009.

Studies of the past several decades have provided major insights into the structural organization of biological membranes and mechanisms of many membrane molecular machines. However, the origin(s) of the membrane(s) and membrane proteins remains enigmatic. We discuss different concepts of the origin and early evolution of membranes with a focus on the evolution of the (im)permeability to charged molecules such as proteins, nucleic acids and small ions. Reconstruction of the evolution of F-type and A/V-type membrane ATPases (ATP synthases), which are either proton- or sodium-dependent, might help us to understand not only the origin of membrane bioenergetics but also of membranes themselves. We argue that evolution of biological membranes occurred as a process of co-evolution of lipid bilayers, membrane proteins and membrane bioenergetics.

AN ATTEMPT TO PREVENT SENESCENCE: A MITOCHONDRIAL APPROACH.

V. P. Skulachev, V. N. Anisimov, Y. N. Antonenko, L. E. Bakeeva, B. V. Chernyak, V. P. Erichev, O. F. Filenko, N. I. Kalinina, V. I. Kapelko, N. G. Kolosova, B. P. Kopnin, G. A. Korshunova, M. R. Lichinitser, L. A. Obukhova, E. G. Pasyukova, O. I. Pisarenko, V. A. Roginsky, E. K. Ruuge, I. I. Senin, I. I. Severina, M. V. Skulachev, I. M. Spivak, V. N. Tashlitsky, V. A. Tkachuk, M. Y. Vyssokikh, L. S. Yaguzhinsky, D. B. Zorov.

Biochimica et Biophysica Acta-Bioenergetics 1787 (5):437-461, 2009.

Antioxidants specifically addressed to mitochondria have been studied to determine if they can decelerate senescence of organisms. For this purpose, a project has been established with participation of several research groups from Russia and some other countries. This paper summarizes the first results of the project. A new type of compounds (SkQs) comprising plastoquinone (an antioxidant moiety), a penetrating cation, and a decane or pentane linker has been synthesized. Using planar bilayer phospholipid membrane (BLM), we selected SkQ derivatives with the highest permeability, namely plastoquinonyl-decyl-triphenylphosphonium (SkQ1), plastoquinonyl-decyl-rhodamine 19 (SkQR1), and methylplastoquinonyldecyltriphenyl-phosphonium (SkQ3). Anti- and prooxidant properties of these substances and also of ubiquinonyl-decyl-triphenylphosphonium (MitoQ) were tested in aqueous solution, detergent micelles, liposomes, BLM, isolated mitochondria, and cell cultures. In mitochondria, micromolar cationic quinone derivatives were found to be prooxidants, but at lower (sub-micro molar) concentrations they displayed antioxidant activity that decreases in the series SkQ1 = SkQR1 > SkQ3 > MitoQ. SkQ1 was reduced by mitochondrial respiratory chain, i.e. it is a rechargeable antioxidant. Nanomolar SkQ1 specifically prevented oxidation of mitochondrial cardiolipin. In cell cultures, SkQR1, a fluorescent SkQ derivative, stained only one type of organelles, namely mitochondria. Extremely low concentrations of SkQ1 or SkQR1 arrested H₂O₂-induced apoptosis in human fibroblasts and HeLa cells. Higher concentrations of SkQ are required to block necrosis initiated by reactive oxygen species (ROS). In the fungus Podospora anserina, the crustacean Ceriodaphnia affinis, Drosophila, and mice, SkQ1 prolonged lifespan, being especially effective at early and middle stages of aging. In mammals, the effect of SkQs on aging was accompanied by inhibition of development of such age-related diseases and traits as cataract, retinopathy, glaucoma, balding, canities, osteoporosis, involution of the thymus, hypothermia, torpor, peroxidation of lipids and proteins, etc. SkQ1 manifested a strong therapeutic action on some already pronounced retinopathies, in particular, congenital retinal dysplasia. With drops containing 250 nM SkQ1, vision was restored to 67 of 89 animals (dogs, cats, and horses) that became blind because of a retinopathy. Instillation of SkQ1-containing drops prevented the loss of sight in rabbits with experimental uveitis and restored vision to animals that had already become blind. A favorable effect of the same drops was also achieved in experimental glaucoma in rabbits. Moreover, the SkQ1 pretreatment of rats significantly decreased the H₂O₂ or ischemia-induced arrhythmia of the isolated heart. SkQs strongly reduced the damaged area in myocardial infarction or stroke and prevented the death of animals from kidney ischemia. In p53(-/-) mice, 5 nmol/kgxday SkQ1 decreased the ROS level in the spleen and inhibited appearance of lymphomas to the same degree as million-fold higher concentration of conventional antioxidant NAC. Thus, SkQs look promising as potential tools for treatment of senescence and age-related diseases.

SELF-OSCILLATING WATER LUMINESCENCE INDUCED BY LASER IRRADIATION.

V. I. Bruskov, S. V. Gudkov, S. F. Chalkin, E. G. Smirnova, L. S. Yaguzhinskii.

Doklady Biochemistry and Biophysics 425 (1):114-116, 2009.

ASSEMBLY OF A CHIMERIC RESPIRATORY CHAIN FROM BOVINE HEART SUBMITOCHONDRIAL PARTICLES AND CYTOCHROME BD TERMINAL OXIDASE OF Escherichia coli.

E. V. Gavrikova, V. G. Grivennikova, V. B. Borisov, G. Cecchini, A. D. Vinogradov.

FEBS Letters 583 (8):1287-1291, 2009.

Cytochrome bd is a terminal quinol oxidase in Escherichia coli. Mitochondrial respiration is inhibited at cytochrome bc1 (complex III) by myxothiazol. Mixing purified cytochrome bd oxidase with myxothiazol-inhibited bovine heart submitochondrial particles (SMP) restores up to 50% of the original rotenone-sensitive NADH oxidase and succinate oxidase activities in the absence of exogenous ubiquinone analogs. Complex III bypassed respiration and is saturated at amounts of added cytochrome bd similar to that of other natural respiratory components in SMP. The cytochrome bd tightly binds to the mitochondrial membrane and operates as an intrinsic component of the chimeric respiratory chain.

COUPLED DIFFUSION OF PERIPHERALLY BOUND PEPTIDES ALONG THE OUTER AND INNER MEMBRANE LEAFLETS.

A. Horner, Y. N. Antonenko, P. Pohl.

Biophysical Journal 96 (7):2689-2695, 2009.

Transmembrane signaling implies that peripheral protein binding to one leaflet be detected by the opposite leaflet. Therefore, protein recruitment into preexisting cholesterol and sphingolipid rich platforms may be required. However, no clear molecular picture has evolved about how these rafts in both leaflets are connected. By using planar lipid bilayers, we show that the peripheral binding of a charged molecule (poly-lysine, PLL) is detected at the other side of the bilayer without involvement of raft lipids. The diffusion coefficient, D-P, of PLL differed by a factor of root 2 when PLL absorbed to one or to both leaflets of planar membranes. Fluorescence correlation spectroscopy showed that the changes of the lipid diffusion coefficient, D-M, were even more pronounced. Although D-M remained larger than D-P on PLL binding to the first membrane leaflet, D-M dropped to D-P on PLL binding to both leaflets, which indicated that the lipids sandwiched between two PLL molecules had formed a nanodomain. Due to its small area of similar to 20 nm(2) membrane electrostriction or leaflet interaction at bilayer midplane can only make a small contribution to interleaflet coupling. The tendency of the system to maximize the area where the membrane is free to undulate seems to be more important. As a spot with increased bending stiffness, the PILL bound patch in one leaflet attracts a stiffening additive on the other leaflet. That is to say, instead of suppressing undulations in two spots, two opposing PLL molecules migrate along a membrane at matching positions and suppress these undulations in a single spot. The gain in undulation energy is larger than the energy required for the alignment of two small PLL domains in opposite leafs and their coordinated diffusion. We propose that this type of mechanical interaction between two membrane separated ligands generally contributes to transmembrane signaling.

TIME-RESOLVED O-H à E-H TRANSITION OF THE ABERRANT BA(3) OXIDASE

FROM Thermus thermophilus.

S. A. Siletsky, I. Belevich, M. Wikstrom, T. Soulimane, M. I. Verkhovsky.

Biochimica et Biophysica Acta-Bioenergetics 1787 (3):201-205, 2009.

The kinetics of single-electron injection into the oxidized nonrelaxed state (O-H à E-H transition) of the aberrant ba₃ cytochrome oxidase from Thermus thermophilus, noted for its lowered efficiency of proton pumping, was investigated by time-resolved optical spectroscopy. Two main phases of intraprotein electron transfer were resolved. The first component (τ similar to 17 mu s) reflects oxidation of Cu-A and reduction of the heme groups (low-spin heme b and high-spin heme a₃ in a ratio close to 50:50). The subsequent component (τ similar to 420 mu s) includes reoxidation of both hemes by Cu-B. This is in significant contrast to the O-H à E-H transition of the aa₃-type cytochrome oxidase from Paracoccus denitrificans, where the fastest phase is exclusively due to transient reduction of the low-spin heme a, without electron equilibration with the binuclear center. On the other hand, the one-electron reduction of the relaxed O state in ba₃ oxidase was similar to that in aa₃ oxidase and only included rapid electron transfer from Cu-A to the low-spin heme b. This indicates a functional difference between the relaxed O and the pulsed O-H forms also in the ba₃ oxidase from T thermophilus.

POSITIVELY CHARGED GRAMICIDIN A BASED PEPTIDES FORM TWO TYPES OF MEMBRANE CHANNELS.

S. I. Kovalchuk, E. A. Kotova, T. B. Stoilova, S. V. Sychev, N. S. Egorova, A. Y. Surovoy, Y. N. Antonenko, V. T. Ivanov.

Peptides for Youth 611:565-566, 2009.

PROTEIN AGGREGATION AND NEURODEGENERATION: CLUES FROM A YEAST MODEL OF HUNTINGTON'S DISEASE.

N. Bocharova, R. Chave-Cox, S. Sokolov, D. Knorre, F. Severin.

Biochemistry-Moscow 74 (2):231-234, 2009.

A number of neurodegenerative diseases are accompanied by the appearance of intracellular protein aggregates. Huntington's disease (HD) is caused by a mutation in a gene encoding huntingtin. The mutation causes the expansion of the polyglutamine (polyQ) domain and consequently polyQ-containing aggregates accumulate and neurons in the striatum die. The role of the aggregates is still not clear: they may be the cause of cytotoxicity or a manifestation of the cellular attempt to remove the misfolded proteins. There is accumulating evidence that the main cause of HD is the interaction of the mutated huntingtin with other polyQ-containing proteins and molecular chaperones and most studies based on a yeast model of HD support this point of view. Data obtained using yeasts suggest pathological consequences of polyQ-proteasomal interaction: proteasomal overload by polyQs may interfere with functions of the cell cycle-regulating proteins.

MULTIPLE OBJECTS TRACKING IN FLUORESCENCE MICROSCOPY.

Y. Kalaidzidis.

Journal of Mathematical Biology 58 (1-2):57-80, 2009.

Many processes in cell biology are connected to the movement of compact entities: intracellular vesicles and even single molecules. The tracking of individual objects is important for understanding cellular dynamics. Here we describe the tracking algorithms which have been developed in the non-biological fields and successfully applied to object detection and tracking in biological applications. The characteristics features of the different algorithms are compared.

PRIMARY STEPS OF THE Na⁺-TRANSLOCATING NADH: UBIQUINONE OXIDOREDUCTASE CATALYTIC CYCLE RESOLVED BY THE ULTRAFAST FREEZE-QUENCH APPROACH.

V. Bogachev, N. P. Belevich, Y. V. Bertsova, M. I. Verkhovsky.

Journal of Biological Chemistry 284 (9):5533-5538, 2009.

The Na⁺-translocating NADH: ubiquinone oxidoreductase (Na⁺ - NQR) is a component of respiratory chain of various bacteria, and it generates a redox-driven transmembrane electrochemical Na⁺ potential. Primary steps of the catalytic cycle of Na⁺ - NQR from Vibrio harveyi were followed by the ultrafast freeze-quench approach in combination with conventional stopped-flow technique. The obtained sequence of events includes NADH binding (similar to 1.5 x 10⁷ M^-1 s^-1), hydride ion transfer from NADH to FAD (similar to 3.5 x 10³ s^-1), and partial electron separation and formation of equivalent fractions of reduced 2Fe-2S cluster and neutral semiquinone of FAD (similar to 0.97 x 10³ s^-1). In the last step, a quasi-equilibrium is approached between the two states of FAD: two-electron reduced (50%) and one-electron reduced (the other 50%) species. The latter, neutral semiquinone of FAD, shares the second electron with the 2Fe-2S center. The transient midpoint redox potentials for the cofactors obtained during the fast kinetics measurements are very different from ones achieved during equilibrium redox titration and show that the functional states of the enzyme realized during its turning over cannot be modeled by the equilibrium approach.

ELECTROGENIC REACTIONS ON THE DONOR SIDE OF MN-DEPLETED PHOTOSYSTEM II CORE PARTICLES IN THE PRESENCE OF MNCL2 AND SYNTHETIC TRINUCLEAR MN-COMPLEXES.

V. N. Kurashov, S. I. Allakhverdiev, S. K. Zharmukhamedov, T. Nagata, V. V. Klimov, A. Y. Semenov, M. D. Mamedov.

Photochemical & Photobiological Sciences 8 (2):162-166, 2009.

An electrometric technique was used to investigate the generation of a photovoltage ∆Ψ by Mn-depleted spinach photosystem II (PS II) core particles incorporated into liposomes. In the presence of MnCl2, the fast kinetically unresolvable phase of ∆Ψ generation, related to electron transfer between the redox-active tyrosine Y-Z and the primary plastoquinone acceptor Q_A was followed by an additional electrogenic phase (τ similar to 20 mu s, similar to 5% of the phase attributed to Y_Z^oxQ_A^-). The latter phase was ascribed to the transfer of an electron from the Mn, bound to the Mn-binding site of the PS II reaction center to the Y_Z^ox. An additional electrogenicity observed upon addition of synthetic trinuclear Mn complex-1 has a τ similar to 50 ms (similar to 4% of the Y_Z^oxQ_A) and τ similar to 160 ms (similar to 25%). The fast electrogenic component could be ascribed to reduction of Y_Z^ox by Mn, delivered to the Mn-binding site in Mn-depleted samples after the release of the tripod ligands from the complex-1 while the slow electrogenic phase to the electron transfer from the Mn-containing complex-1 attached to the protein-water boundary to the oxidized Mn at the protein-embedded Mn-binding site.

KINETIC MODELLING OF NSAID ACTION ON COX-1: FOCUS ON in vitro/in vivo ASPECTS AND DRUG COMBINATIONS.

A. Goitsov, A. Maryashkin, M. Swat, Y. Kosinsky, I. Humphery-Smith, O. Demin, I. Goryanin, G. Lebedeva.

European Journal of Pharmaceutical Sciences 36 (1):122-136, 2009.

The detailed kinetic model of Prostaglandin H Synthase-1 (COX-1) was developed to in silico test and predict inhibition effects of nonsteroidal anti-inflammatory drugs (NSAIDs) on target. The model takes into account key features of the complex catalytic mechanism of cyclooxygenase-1, converting arachidonic acid to prostaglandin PGH(2), and includes the description of the enzyme interaction with various types of NSAIDs (reversible/irreversible, non-selective and selective to COX-1/COX-2). Two different versions of the model were designed to simulate the inhibition of COX-1 by NSAIDs in two most popular experimental settings-in vitro studies with purified enzyme, and the experiments with platelets. The developed models were applied to calculate the dose-dependence of aspirin and celecoxib action on COX-1 in vitro and in vivo conditions. The mechanism of the enhancement of aspirin efficiency in platelet as compared to its action on purified COX-1 was elucidated. The dose-dependence of celecoxib simulated with the use of the "in vivo" version of the model predicted potentially strong inhibitory effect of celecoxib on thromboxan production in platelets. Simulation of the combined effect of two NSAIDs, aspirin and celecoxib, on COX-1 allowed us to reveal the mechanism underlying the suppression of aspirin-mediated COX-1 inhibition by celecoxib. We discuss our modelling results in the context of the ongoing debates on the potential cardio-vascular risks associated with co-administration of various types of NSAIDs.

OPTIMIZATION OF ANTIMICROBIAL DRUG GRAMICIDIN S DOSING REGIME USING BIOSIMULATIONS.

S. Smirnov, A. Belashov, O. Demin.

European Journal of Pharmaceutical Sciences 36 (1):105-109, 2009.

In this paper we have developed a model of antimicrobial effect of gramicidin S. This model has allowed us to predict the dependence of antimicrobial effect of the drug applied as oral melting tablets on dosage, time of resorption and minimal inhibitory concentration (MIC) of the drug characterizing its ability to kill different bacteria. The model has been employed to optimize dosing regime of gramicidin S containing drug Grammidin. Efficacy of the drug has been studied for the diverse gram-positive and gram-negative bacteria with different MIC. The number of bacteria located in the oral cavity and killed by one-pass administration of the drug (resolution of one tablet) has been calculated under condition of various dosing regimes. Based on the simulation results it has been found that (1) twofold prolongation of prescribed resorption time (from 30 to 60 min) of the tablet comprising standard dosage of 3 mg of gramicidin S results in 1.5-fold increase in efficacy, (2) 1.5-fold decrease in gramicidin S dosage (from 3 to 2 mg per administration) under condition of holding prescribed resorption time (30 min) does not lead. to any considerable decrease in the efficacy of the drug.

THE Na⁺-TRANSLOCATING NADH:UBIQUINONE OXIDOREDUCTASE OF Azotobacter vinelandii NEGATIVELY REGULATES ALGINATE SYNTHESIS.

C. Nunez, A. V. Bogachev, G. Guzman, I. Tello, J. Guzman, G. Espin.

Microbiology-Sgm 155:249-256, 2009.

Azotobacter vinelandii is a nitrogen-fixing soil bacterium that produces the exopolysaccharide alginate. In this report we describe the isolation and characterization of A. vinelandii strain GG4, which carries an nqrE::Tn5 mutation resulting in alginate overproduction. The nqrE gene encodes a subunit of the Na⁺-translocating NADH:ubiquinone oxidoreductase (Na⁺-NQR). As expected, Na⁺-NQR activity was abolished in mutant GG4. When this strain was complemented with the nqrEF genes this activity was restored and alginate production was reduced to wild-type levels. Na⁺-NQR may be the main sodium pump of A. vinelandii under the conditions tested (similar to 2 mM Na⁺) since no Na⁺/H+-antiporter activity was detected. Collectively our results indicate that in A. vinelandii the lack of Na⁺-NQR activity caused the absence of a transmembrane Na⁺ gradient and an increase in alginate production.

III. Cell structure and function. Intracellular interactions. Molecular mechanisms of cell differentiation, immunity and oncogenesis. Virus-cell interaction.

SUBCELLULAR LOCALIZATION OF THE NEW PLANT PROTEIN 4/1 AND ANALYSIS OF HETEROLOGOUS PROTEIN-PROTEIN INTERACTIONS INDICATE ITS ABILITY FOR NUCLEAR-CYTOPLASMIC TRANSPORT.

E. A. Minina, T. N. Erokhina, S. K. Garushyants, A. G. Solovyev, S. Y. Morozov.

Doklady Biochemistry and Biophysics 429 (1):296-300, 2009.

INTERACTION OF AFOBAZOLE WITH σ₁-RECEPTORS.

S. B. Seredenin, T. A. Antipova, M. V. Voronin, S. Y. Kurchashova, A. N. Kuimov.

Bulletin of Experimental Biology and Medicine 148 (1):42-44, 2009.

In vitro radioligand assay revealed interaction of afobazole with σ₁-receptors (K_i=5.9 x 10^-6 M). Translocation of σ₁-receptors from the endoplasmic reticulum to the outer membrane was demonstrated by confocal microscopy. Experiments were performed on the model of HT-22 immortalized hippocampal cells after incubation with afobazole in a concentration of 10^-8 M.

TRITIUM PLANIGRAPHY STUDY OF STRUCTURAL ALTERATIONS IN THE COAT PROTEIN OF POTATO VIRUS X INDUCED BY BINDING OF ITS TRIPLE GENE BLOCK 1 PROTEIN TO VIRIONS.

E. Lukashina, G. Badun, N. Fedorova, A. Ksenofontov, M. Nemykh, M. Serebryakova, A. Mukhamedzhanova, O. Karpova, N. Rodionova, L. Baratova, E. Dobrov.

FEBS Journal 276 (23):7006-7015, 2009.

Alterations in Potato virus X (PVX) coat protein structure after binding of the protein, encoded by the first gene of PVX triple gene block (triple gene block 1 protein, TGBp1), to the virions were studied using tritium planigraphy. Previously, it has been shown that TGBp1 molecules interact with the PVX particle end, containing the 5'-terminus of PVX RNA, and that this interaction results in a strong decrease in virion stability and its transformation to a translationally active state. In this work, it has been shown that the interaction of TGBp1 with PVX virions leads to an increase of similar to 50% in tritium label incorporation into the 176-198 segment of the 236-residue-long PVX coat protein subunit, with some decrease in label incorporation into the N-terminal coat protein region. According to the new 'sandwich' variant of our recently proposed model of the three-dimensional structure of the intravirus PVX coat protein, the 176-198 segment is assigned to the β-sheet region located at the subunit surface, presumably participating in coat protein interactions with the intravirus RNA and/or in protein-protein interactions, whereas the N-terminal coat protein region corresponds to the other part of the same β-sheet. For the remaining segments of the PVX coat protein subunit, no significant difference between tritium incorporation into untreated and TGBp1-treated PVX was observed. A detailed description of the 'sandwich' version of the intravirus PVX coat protein model is presented.

THE in situ STRUCTURAL CHARACTERIZATION OF THE INFLUENZA A VIRUS MATRIX M1 PROTEIN WITHIN A VIRION.

A. V. Shishkov, E. N. Bogacheva, A. A. Dolgov, A. L. Chulichkov, D. G. Knyazev, N. V. Fedorova, A. L. Ksenofontov, L. V. Kordyukova, E. V. Lukashina, V. M. Mirsky, L. A. Baratova.

Protein and Peptide Letters 16 (11):1407-1413, 2009.

The first attempt has been made to suggest a model of influenza A virus matrix M1 protein spatial structure and molecule orientation within a virion on the basis of tritium planigraphy data and theoretical prediction results. Limited in situ proteolysis of the intact virions with bromelain and surface plasmon resonance spectroscopy study of the M1 protein interaction with lipid coated surfaces were used for independent confirmation of the proposed model.

THERMALLY INDUCED STRUCTURAL CHANGES OF INTRINSICALLY DISORDERED SMALL HEAT SHOCK PROTEIN Hsp22.

A. S. Kazakov, D. I. Markov, N. B. Gusev, D. I. Levitsky.

Biophysical Chemistry 145 (2-3):79-85, 2009.

We applied different methods (differential scanning calorimetry, circular dichroism, Fourier transform infrared spectroscopy, and intrinsic fluorescence) to investigate the thermal-induced changes in the structure of small heat shock protein Hsp22. It has been shown that this protein undergoes thermal-induced unfolding that occurs within a very broad temperature range (from 27⁰C to 80⁰C and above), and this is accompanied by complete disappearance of α-helices, significant decrease in β-sheets content, and by pronounced changes in the intrinsic fluorescence. The results confirm predictions that Hsp22 belongs to the family of intrinsically disordered proteins (IDP) with certain parts of its molecule (presumably, in the α-crystallin domain) retaining folded structure and undergoing reversible thermal unfolding. The results are also discussed in terms of downhill folding scenario.

DOMAIN ORGANIZATION OF THE N-TERMINAL PORTION OF HORDEIVIRUS MOVEMENT PROTEIN TGBPL.

Valentin V. Makarov, Ekaterina N. Rybakova, Alexander V. Efimov, Eugene N. Dobrov, Marina V. Serebryakova, Andrey G. Solovyev, Igor V. Yaminsky, Michael E. Taliansky, Sergey Yu. Morozov, Natalia O. Kalinina.

Journal of General Virology 90 (Part 12):3022-3032, 2009.

Three 'triple gene block' proteins known as TGBp1, TGBp2 and TGBp3 are required for cell-to-cell movement of plant viruses belonging to a number of genera including Hordeivirus. Hordeiviral TGBp1 interacts with viral genomic RNAs to form ribonucleoprotein (RNP) complexes competent for translocation between cells through plasmodesmata and over long distances via the phloem. Binding of hordeivirus TGBp1 to RNA involves two protein regions, the C-terminal NTPase/helicase domain and the N-terminal extension region. This study demonstrated that the extension region of hordeivirus TGBp1 consists of two structurally and functionally distinct domains called the N-terminal domain (NTD) and the internal domain (ID). In agreement with secondary structure predictions, analysis of circular dichroism spectra of the isolated NTD and ID demonstrated that the NTD represents a natively unfolded protein domain, whereas the ID has a pronounced secondary structure. Both the NTD and ID were able to bind ssRNA non-specifically. However, whilst the NTD interacted with ssRNA non-cooperatively, the ID bound ssRNA in a cooperative manner. Additionally, both domains bound dsRNA. The NTD and ID formed low-molecular-mass oligomers, whereas the ID also gave rise to high-molecular-mass complexes. The isolated ID was able to interact with both the NTD and the C-terminal NTPase/helicase domain in solution. These data demonstrate that the hordeivirus TGBp1 has three RNA-binding domains and that interaction between these structural units can provide a basis for remodelling of viral RNP complexes at different steps of cell-to-cell and long-distance transport of virus infection.

COOPERATION BETWEEN PICORNAVIRAL PROTEINS FOR THE OVERCOMING OF CELLULAR PROTECTIVE MECHANISMS.

E. S. Gavryushina.

Zhurnal Obshchei Biologii 70 (3):245-248, 2009.

Picornaviruses can overcome cellular mechanisms of antiviral defense by inhibiting cellular RNA and protein synthesis, nuclear-cytoplasmic transport and secretory pathways. The participation of proteases 2A and 3C and membrane proteins 2B and 3A in these processes is described. Four steps of the viral inhibition of cellular gene expression and the reciprocal supplement between these steps are discussed.

CLONING AND FUNCTIONAL EXPRESSION OF THE FIRST EUKARYOTIC Na⁺-TRYPTOPHAN SYMPORTER, AGNAT6.

E. A. Meleshkevitch, M. Robinson, L. B. Popova, M. M. Miller, W. R. Harvey, D. Y. Boudko.

Journal of Experimental Biology 212 (10):1559-1567, 2009.

The nutrient amino acid transporter (NAT) subfamily of the neurotransmitter sodium symporter family (NSS, also known as the solute carrier family 6, SLC6) represents transport mechanisms with putative synergistic roles in the absorption of essential and conditionally essential neutral amino acids. It includes a large paralogous expansion of insect-specific genes, with seven genes from the genome of the malaria mosquito, Anopheles gambiae. One of the An. gambiae NATs, AgNAT8, was cloned, functionally expressed and characterized in X. laevis oocytes as a cation-coupled symporter of aromatic amino acids, preferably L-phenylalanine, L-tyrosine and L-DOPA. To explore an evolutionary trend of NAT-SLC6 phenotypes, we have cloned and characterized AgNAT6, which represents a counterpart of AgNAT8 descending from a recent gene duplication (53.1% pairwise sequence identity). In contrast to AgNAT8, which preferably mediates the absorption of phenol-branched substrates, AgNAT6 mediates the absorption of indole-branched substrates with highest apparent affinity to tryptophan (K_0.5(Trp)=1.3 mu mol l^-1 vs K_0.5(Phe)=430 mu mol l^-1) and [2 or 1 Na⁺ or K⁺]:[aromatic substrate] stoichiometry. AgNAT6 is highly transcribed in absorptive and secretory regions of the alimentary canal and specific neuronal structures, including the neuropile of ventral ganglia and sensory afferents. The alignment of AgNATs and LeuT(Aa), a bacterial NAT with a resolved 3D structure, reveals three amino acid differences in the substrate-binding pocket that may be responsible for the indole- vs phenol-branch selectivity of AgNAT6 vs AgNAT8. The identification of transporters with a narrow selectivity for essential amino acids suggests that basal expansions in the SLC6 family involved duplication and retention of NATs, improving the absorption and distribution of under-represented essential amino acids and related metabolites. The identified physiological and expression profiles suggest unique roles of AgNAT6 in the active absorption of indole- branched substrates that are used in the synthesis of the neurotransmitter serotonin as well as the key circadian hormone and potent free-radical scavenger melatonin.

ISOLATION OF THE INFLUENZA A HA2 C-TERMINAL SEGMENT BY COMBINATION OF NONIONIC DETERGENTS.

Y. A. Smirnova, N. V. Fedorova, A. L. Ksenofontov, L. V. Kordyukova, M. V. Serebryakova, L. A. Baratova, B. V. Vaskovsky.

Peptides for Youth 611:311-312, 2009.

FEATURES OF MITOCHONDRIAL ENERGETICS IN LIVING UNICELLULAR EUKARYOTE Tetrahymena pyriformis. A MODEL FOR STUDY OF MAMMALIAN INTRACELLULAR ADAPTATION.

E. A. Prikhodko, I. V. Brailovskaya, S. M. Korotkov, E. N. Mokhova.

Biochemistry-Moscow 74 (4):371-376, 2009.

Tetrahymena pyriformis is used in diverse studies as a non-mammalian alternative due to their resemblance in many main metabolic cycles. However, such basic features of mitochondrial energetics as ∆I (electrical potential difference across the inner mitochondrial membrane) or maximal stimulation of respiration by uncouplers with different mechanisms of uncoupling, such as DNP (2,4-dinitrophenol) and FCCP (p-trifluoromethoxycarbonylcyanide phenylhydrazone), have not been studied in living ciliates. Tetrahymena pyriformis GL cells during stationary growth phase after incubation under selected conditions were used in this study. Maximal stimulation of cellular respiration by FCCP was about six-fold, thus the proton motive force was high. The DNP uncoupling effect was significantly lower. This suggests low activity of the ATP/ADP-antiporter, which performs not only exchange of intramitochondrial ATP to extramitochondrial ADP, but also helps in the uncoupling process. It participates by a similar mechanism in electrophoretic transport from matrix to cytosol of ATP(4-) and DNP anion, but not FCCP anion. Thus, in contrast with mammalian mitochondria, T. pyriformis mitochondria cannot rapidly supply the cytosol with ATP; possibly the cells need high intramitochondrial ATP. The difference between DNP and FCCP is hypothetically explained by low ∆I value and/or an increase in concentration of long-chain acyl-CoAs, inhibitors of the ATP/ADP-antiporter. The first suggestion is confirmed by absence of mitochondria with bright fluorescence in T. pyriformis stained with the ∆I-sensitive probe MitoTracker Red. These data suggest that T. pyriformis cells are useful as a model for study of mitochondrial role in adaptation at the intracellular level.

PERICHROMOSOMAL LAYER PROTEINS ASSOCIATE WITH CHROMOSOME SCAFFOLD AND NUCLEAR MATRIX THROUGHOUT THE CELL CYCLE.

E. V. Sheval, O. A. Dudnik, S. S. Abramchuk, V. Y. Polyakov.

Biologicheskie Membrany 26 (2):126-142, 2009.

According to the radial loop model of chromosome organization, a major role in the formation and maintenance of chromosomes is played by the residual structures (the nuclear matrix in interphase nuclei and the chromosome scaffold in metaphase chromosomes). However, in vivo microscopy has recently revealed that the components of these "static" structures are highly mobile and continuously exchanged between specific target sites and the nucleoplasm or cytoplasm. This contradiction between predicted stability and observed dynamics led us to reexamine the principles underlying the association of proteins with residual structures. In the present paper, we have analyzed the association of two perichromosomal layer proteins, pKi-67 and B23, with the residual structures. The results show that these two proteins are associated with residual structures throughout the cell cycle; only those structures change that contain proteins precipitated by 2 M NaCl (nucleoli, perichromosomal layer, prenucleolar bodies, cytoplasm of mitotic cells). Both pKi-67 and B23 remain associated with the nuclear matrix even when they are translocated to nucleoplasmic foci due to inhibitor action or hypotonic treatment. However, in most cases it remains possible to extract a structurally visible protein fraction with 2 M NaCl (protein distributed in nucleoplasm). One may suppose that the protein fraction associated with residual structures includes molecules interacting with their binding sites at the moment of permeabilization, while the free proteins are extracted (i.e., during the interaction with binding sites, these proteins form salt-resistant complexes; however, on diffusion the same proteins are extractable by the high-salt solution). The residual structures may be considered as a "snapshot" of all proteins transiently (or statically) bound to their target sites at the moment of permeabilization.

SERUM-INDUCED INHIBITION OF THE PHAGOCYTIC ACTIVITY OF CULTURED MACROPHAGES IC-21.

M. S. Golovkina, I. V. Skachkov, M. V. Metelev, A. V. Kuzevanov, K. S. Vishniakova, I. I. Kireev,

and A. Y. Dunina-Barkovskaya.

Biologicheskie Membrany 26 (5):379-386, 2009.

Fetal calf serum has been shown to considerably inhibit phagocytosis of non-opsonized 2-mu m fluorescent latex beads by cultured macrophages IC-21. Phagocytic activity was assessed using fluorescent microscopy and specially devised ImageJ plugins. Phagocytosis percent, PP (percentage of the bead-containing cells in the cell population under study), and phagocytosis index, PI (mean number of beads per cell in the bead-containing population), were about 2 times lower in the cells incubated in the presence of 10% serum as compared to the respective parameters for the cells incubated in serum-free medium (55 ± 5% vs. 92 ± 1% and 2.0 ± 0.2 vs. 4.3 ± 0.2 beads/cell). The effect of serum was dose-dependent. Albumin (10 mg/ml) did not mimic the effect of serum, suggesting that fatty acid extraction was not the cause of the scrum-induced inhibition. Serum is a source of exogenous cholesterol, therefore we checked if cholesterol removal could stimulate phagocytosis. Cholesterol-sequestering agent methyl-β-cyclodextrin (m β CD) in concentrations of 5-7 mM indeed caused an increase of the phagocytic activity but at 10 rnM exerted an inhibitory effect in serum-free medium. Connexin channel blocker carbenoxolon (CBX, 250-500 mu M) in most cases inhibited phagocytosis; the presence of serum or m β CD modulated CBX effects. The data indicate an important role of serum in regulation of the macrophage phagocytic activity. Stimulating effect produced by serum removal may partly be accounted for by a decrease of cholesterol concentration, which in turn may alter the functioning of integral proteins involved in the mechanisms of phagocytosis.

SELF-ORGANIZATION OF ENDOPLASMIC RETICULUM AGGREGATES IN CELLS WITH OVEREXPRESSION OF NUCLEOPORIN POM121.

E. G. Volkova, S. Y. Kurchashova, E. V. Sheval, V. Y. Polyakov.

Biologicheskie Membrany 26 (5):401-407, 2009.

The pore complexes are compound protein structures located in the nuclear envelope, where they control the nuclear-cytoplasmic transport, and inside the stacks of endoplasmic reticulum cisterns, annulate lamella. After overexpression of some nucleoporins, a numerous granules are visible in the cytoplasm. According to published data, these granules are the annulate lamella. In the current paper, the structural organization of POM121-containing granules was analyzed using correlative light microscopy and electron microscopy. The ultrastructural study demonstrates that POM121-containing granules are not annulate lamella but aggregates of endoplasmic reticulum membranes. Thus, POM121 overexpressed is not able to induce the annulate lamella formation. The mechanisms of self-organization of non-functional structures (such as the aggregates of endoplasmic reticulum membranes described here) and possible involvement of these mechanisms in the formation of cellular structures are discussed.

RESTORATION OF POTATO VIRUS X COAT PROTEIN CAPACITY FOR ASSEMBLY WITH RNA

AFTER HIS-TAG REMOVAL.

O. Zayakina, M. Arkhipenko, A. Smirnov, N. Rodionova, O. Karpova, J. Atabekov.

Archives of Virology 154 (2):337-341, 2009.

We found that the fusion of hexahistidine (H)(6) tag to the potato virus X (PVX) coat protein (CP) abolished its ability to be assembled with viral RNA into helical nucleoprotein virus-like particles (VLPs). Instead, irregular agglomerates were produced upon incubation of PVX RNA with (H)(6)-tagged PVX CP. A factor Xa recognition site, IEGR, was inserted upstream of the CP coding sequence. Removal of the (H)(6) tag from PVX CP by Xa protease restored its ability to bind RNA and to assemble VLPs. In addition to the canonical IEGR site, the factor Xa protease was found to cleave PVX CP at a second (non-consensus) site, AVTRGR, located close to the C-terminus of PVX CP. The latter cleavage did not affect reassembly of the PVX RNA and CP into VLPs.

NONSPECIFIC ACTIVATION OF TRANSLATION OF ENCAPSIDATED POTEXVIRAL RNA WITH INVOLVEMENT OF POTATO VIRUS X MOVEMENT PROTEIN TGB1.

A. A. Mukhamedzhanova, O. V. Karpova, N. P. Rodionova, I. G. Atabekov.

Doklady Biochemistry and Biophysics 428 (1):239-241, 2009.

ROLE OF THE ZINC-FINGER AND BASIC MOTIFS OF CHRYSANTHEMUM VIRUS B P12 PROTEIN IN NUCLEIC ACID BINDING, PROTEIN LOCALIZATION AND INDUCTION OF A HYPERSENSITIVE RESPONSE UPON EXPRESSION FROM A VIRAL VECTOR.

N. I. Lukhovitskaya, I. V. Ignatovich, E. I. Savenkov, J. Schiemann, S. Y. Morozov, A. G. Solovyev.

Journal of General Virology 90:723-733, 2009.

The genomes of carlaviruses encode cysteine-rich proteins (CRPs) of unknown function. The 12 kDa CRP of chrysanthemum virus B (CVB), p12, has been shown previously to induce a hypersensitive response (HR) when expressed from potato virus X (PVX). This study demonstrated that a p12-induced H was preceded by induction of a number of genes related to pathogenesis, stress and systemic acquired resistance. p12 localized predominantly to the nucleus. Interestingly, it was found that p12 bound both RNA and DNA in vitro, but notably exhibited a preference for DNA in the presence of Zn²⁺ ions. Mutational analysis of the p12 conserved sequence motifs demonstrated that the basic motif is required for p12 translocation to the nucleus, thus representing part of the protein nuclear localization signal, whereas the predicted zinc finger motif is needed for both Zn²⁺-dependent DNA binding and eliciting an HR in PVX-infected leaves. Collectively, these results link, for the first time, nuclear localization of the protein encoded by a cytoplasmically replicating virus and its DNA-binding capacity with HR induction. Furthermore, these data suggest that p12 may mediate induction of the host genes by binding to the plant genomic DNA, and emphasize that CVB p12 is functionally distinct from other known nuclear-localized proteins encoded by the plant positive-stranded RNA viruses.

DYNACTIN SUBUNIT P150GLUED ISOFORMS NOTABLE FOR DIFFERENTIAL INTERACTION WITH MICROTUBULES.

O. N. Zhapparova, S. A. Bryantseva, L. V. Dergunova, N. M. Raevskaya, A. V. Burakov, O. B. Bantysh, N. A. Shanina, E. S. Nadezhdina.

Traffic 10 (11):1635-1646, 2009.

Dynactin is a multiprotein complex that enhances dynein activity. The largest dynactin subunit, p150Glued, interacts with microtubules through its N-terminal region that contains a globular cytoskeleton-associated protein (CAP)-Gly domain and basic microtubule-binding domain of unknown structure. The p150Glued gene has a complicated intron-exon structure, and many splice isoforms of p150Glued protein have been predicted. Here we describe novel natural 150 kDa isoforms: the p150Glued-1A isoform, whose basic domain is composed of 41 amino acids, and p150Glued-1B with a basic domain of 21 aa because of the lack of exons 5-7 in the corresponding messenger RNA (mRNA). According to reverse transcriptase-polymerase chain reaction (RT-PCR) and western blot data, p150Glued-1A is expressed in nerve tissues, in cultured cells and in embryonic tissues, while 1B is expressed ubiquitously. Overexpression of GFP-p150Glued-1A and -1B fusion proteins and immunostaining of cultured cells with 1A-specific antibodies show that the p150Glued-1A isoform is distributed along microtubules, whereas 1B is associated with microtubule plus-ends. The higher affinity of the p150Glued-1A isoform for microtubules is confirmed by a co-pelleting assay. In fibroblast-like cells, the interaction of p150Glued-1A with microtubules is less dependent on EB1/EB3 and CLIP170 proteins, compared with p150Glued-1B. In polarized cells, p150Glued-1A decorates microtubules that face the leading edge of the cell. The pattern of p150Glued-1A and p150Glued-1B interaction with microtubules and their tissue-specific expression patterns suggest that these isoforms might be involved in cell differentiation and proliferation.

HIGH-SENSITIVITY EXPRESS IMMUNOCHROMATOGRAPHIC METHOD FOR DETECTION OF PLANT INFECTION BY TOBACCO MOSAIC VIRUS.

Y. F. Drygin, A. N. Blintsov, A. P. Osipov, V. G. Grigorenko, I. P. Andreeva, A. I. Uskov, Y. A. Varitsev, B. V. Anisimov, V. K. Novikov, J. G. Atabekov.

Biochemistry-Moscow 74 (9):986-993, 2009.

A highly sensitive express immunochromatography method for molecular diagnosis of plant virus infections was elaborated on the example of a model object - tobacco mosaic virus (TMV). The analysis time does not exceed 5 min, and the lower limit of TMV detection in non-clarified leaf extract (2-4 ng/ml) is comparable with the sensitivity of the enzyme-linked immunosorbent assay of the virus. A single measurement requires 0.1-0.2 ml tested solution (extract from 10-20 mg of leaf material). The sensitivity of TMV determination in the leaf tissue extract was increased by more than one order of magnitude using signal enhancement by silver and is 0.1 ng/ml. In this case, analysis time did not exceed 25 min. The simplicity of this method makes it especially convenient in express diagnosis of numerous analyzed specimens. The prototype of a diagnostic kit for serial analyses of plant viral infections both in laboratory and field conditions was elaborated.

THE FINE STRUCTURE OF THE INFLUENZA VIRUS ENVELOPE AND THE CONCEPT OF TRANSMEMBRANE ASYMMETRY OF LATERAL DOMAINS IN BIOMEMBRANES.

V. A. Radyukhin.

Molecular Biology 43 (4):533-542, 2009.

The molecular architectures of enveloped viruses provide a demonstrative example of perfectly arranged macromolecular complexes, which are formed via highly specific interactions of all structural components. Virus morphogenesis is a multistep process that depends on the concerted actions of many viral and cell components, as well as a fitted organization of main viral constituents. The virus envelope is composed of a mixture of lipid raft and nonraft domains. The domains are recruited from the host cell membrane as discrete well-ordered lipid-protein units during virus assembly. The raft-like nature of the influenza virus A envelope was visualized using a novel approach of cold solubilization of detergent-resistant membranes from intact influenza virus A virions with a mixture of NP40 and octyl glucopyranoside, two nonionic detergents drastically differing in their raft-solubilizing activities. The virus envelope is apparently an ensemble of flexibly joint platforms, which are composed of surface glycoproteins (hemagglutinin and neuraminidase), the matrix M1 protein, and lipids. The modern concept of the transmembrane asymmetry of lateral domains in biological membranes was used to explain the solubilization mechanism revealed. Based on the principles of this concept, the M1 protein shell was assumed to provide a structure-forming framework to support asymmetrical rafts in the virus envelope.

IV. Genomics, proteomics. Protein and gene ingeneering. Transgenosis, gene terapy. Molecular medicine.

FROM STRUCTURE AND FUNCTIONS OF STEROIDOGENIC ENZYMES TO NEW TECHNOLOGIES OF GENE ENGINEERING.

L. A. Novikova, Y. V. Faletrov, I. E. Kovaleva, S. Mauersberger, V. N. Luzikov, V. M. Shkumatov.

Biochemistry-Moscow 74 (13):1482-1504, 2009.

This review summarizes data about structural and functional organization of steroidogenic P450-dependent enzymatic systems. Problems of catalysis of steroid substrate transformation, special features of mitochondrial type P450scc topogenesis, and abilities of some microbial electron transport proteins to support P450 activity in vitro and in vivo are considered. Principal steps in the creation and catalytic properties of transgenic strains of Escherichia coli, Saccharomyces cerevisiae, and Yarrowia lipolytica expressing both mammalian steroidogenic P450s and the corresponding electron transport proteins are also described. Achievements and prospects of using such transgenic strains for biotechnological synthesis and pharmacological screening are considered.

PRINCIPLES OF CONTROL OVER FORMATION OF STRUCTURES RESPONSIBLE FOR RESPIRATORY FUNCTIONS OF MITOCHONDRIA.

V. N. Luzikov

Biochemistry-Moscow 74 (13):1443-1456, 2009.

Topogenesis of mitochondrial proteins includes their synthesis in cytosol and mitochondria, their translocation across the outer and inner membranes, sorting to various mitochondrial compartments, and assembly of different protein complexes. These complexes are involved in transport functions, electron transfer through the respiratory chain, generation of transmembrane electrochemical potential, oxidative phosphorylation of ADP into ATP, etc. To perform these functions, a special stringent control is required over formation of submitochondrial structures and the mitochondrion as a whole. Such control is expected to rigorously eliminate not only misfolded proteins but also incorrectly incorporated subunits and is realized in mitochondria by means of numerous proteases with different functions and localizations. In the case of more complicated protein formations, e.g. supercomplexes, the protein quality is assessed by their ability to realize the integral function of the respiratory chain and, thus, ensure the stability of the whole system. Considering supercomplexes of the mitochondrial respiratory chain, the present review clearly demonstrates that this control is realized by means of various (mainly vacuolar) proteases with different functions and localizations. The contemporary experimental data also confirm the author's original idea that the general mechanism of assembly of subcellular structures is based on the "selection by performance criterion" and "stabilization by functioning".

SOME CHARACTERISTICS OF GENETIC CONTROL OF Fagopyrum esculentum FLOWER DEVELOPMENT.

A. A. Penin, A. N. Fesenko, I. N. Fesenko, M. D. Logacheva.

Wulfenia 16:117-127, 2009.

In this work we present a study of the genetic control of flower development in common buckwheat (Fagopyrum esculentum) based on the expression analysis of FesAG, FesLFY and FesAPI - genes orthologous to those controlling flower development in Arabidopsis thaliana - in wild type buckwheat and in mutants fagopyrum apetala, tepal-like bract, green perianth. In the fagopyrum apetala Mutant, characterised by the conversion of tepals into carpelloid organs, the expression of FesAG is increased what is consistent with the Current knowledge based on the studies of model plant species. In another mutant - green perianth - characterised by partial conversion of flower into inflorescence, we revealed an increased expression of FesAPI. The latter is an unexpected result contradicting current models based on the Study of Arabidopsis thaliana. Our data suggest a significant difference between the genetic control of flower development in Fagopyrum and in Arabidopsis.

DETECTION OF DNA DAMAGE: EFFECT OF THYMIDINE GLYCOL RESIDUES ON THE THERMODYNAMIC, SUBSTRATE AND INTERFACIAL ACOUSTIC PROPERTIES OF OLIGONUCLEOTIDE DUPLEXES.

F. Yang, E. Romanova, E. Kubareva, N. Dolinnaya, V. Gajdos, O. Burenina, E. Fedotova, J. S. Ellis, T. Oretskaya, T. Hianik, M. Thompson.

Analyst 134 (1):41-51, 2009.

Thymidine glycol residues in DNA are biologically active oxidative molecular damage sites caused by ionizing radiation and other factors. One or two thymidine glycol residues were incorporated in 19- to 31-mer DNA fragments during automatic oligonucleotide synthesis. These oligonucleotide models were used to estimate the effect of oxidized thymidines on the thermodynamic, substrate and interfacial acoustic properties of DNA. UV-monitoring melting data revealed that modified residues in place of thymidines destabilize the DNA double helix by 8-22⁰C, depending on the number of lesions, the length of oligonucleotide duplexes and their GC-content. The diminished hybridizing capacity of modified oligonucleotides is presumably due to the loss of aromaticity and elevated hydrophilicity of thymine glycol in comparison to the thymine base. According to circular dichroism (CD) data, the modified DNA duplexes retain B-form geometry, and the thymidine glycol residue introduces only local perturbations limited to the lesion site. The rate of DNA hydrolysis by restriction endonucleases R.MvaI, R.Bst2UI, R.MspR9I and R.Bme1390I is significantly decreased as the thymidine glycol is located in the central position of the double-stranded recognition sequences 5'-CC / WGG-3' (W = A, T) or 5'-CC / NGG-3' (N = A, T, G, C) adjacent to the cleavage site. On the other hand, the catalytic properties of enzymes R.Psp6I and R.BstSCI recognizing the similar sequence are not changed dramatically, since their cleavage site is separated from the point of modification by several base-pairs. Data obtained by gel-electrophoretic analysis of radioactive DNA substrates were confirmed by direct spectrophotometric assay developed by the authors. The effect of thymidine glycol was also observed on DNA hybridization at the surface of a thickness-shear mode acoustic wave device. A 1.9-fold decrease in the rate of duplex formation was noted for oligonucleotides carrying one or two thymidine glycol residues in relation to the unmodified analog.

BINDING OF THE IRES OF HEPATITIS C VIRUS RNA TO THE 40S RIBOSOMAL SUBUNIT: ROLE OF P40.

A. A. Malygin, Z. V. Bochkaeva, E. I. Bondarenko, O. A. Kossinova, V. B. Loktev, I. N. Shatsky, G. G. Karpova.

Molecular Biology 43 (6):997-1003, 2009.

Ribosomal protein p40 is a structural component of the eukaryotic 40S ribosomal subunit, is partly homologous to prokaryotic ribosomal protein S2, and has a long eukaryote-specific C-terminal region. The internal ribosome entry site (IRES) of the hepatitis C virus (HCV) RNA was tested for the binding to 40S ribosomal subunits deficient in p40, saturated with recombinant p40, or pretreated with monoclonal antibody (MAB) 4F6 against p40. The apparent association constant of HCV IRES binding to 40S subunits was shown to directly depend on the p40 content in the subunits. MAB 4F6 prevented HCV IRES binding to 40S subunits and blocked translation of IRES-containing RNA in a cell-free translation system. The results implicate p40 in the binding of the HCV IRES to the ribosome and, therefore, in translation initiation on HCV RNA.

NOVEL MIR390-DEPENDENT TRANSACTING siRNA PRECURSORS IN PLANTS REVEALED BY A PCR-BASED EXPERIMENTAL APPROACH AND DATABASE ANALYSIS.

M. S. Krasnikova, I. A. Milyutina, V. K. Bobrova, L. V. Ozerova, A. V. Troitsky, A. G. Solovyev, S. Y. Morozov.

Journal of Biomedicine and Biotechnology, 2009.

TAS loci in plant genomes encode transacting small interfering RNAs (ta-siRNAs) that regulate expression of a number of genes. The function of TAS3 precursor in Arabidopsis thaliana is controlled by two miR390 target sites flanking two ta-siARF sequences targeting mRNAs of ARF transcription factors. Cleavage of the 3'-miR390-site initiates ta-siRNAs biogenesis. Here we describe the new method for identification of plant ta-siRNA precursors based on PCR with oligodeoxyribonucleotide primers mimicking miR390. The method was found to be efficient for dicotiledonous plants, cycads, and mosses. Based on sequences of amplified loci and a database analysis, a novel type of miR390-dependent TAS sequences was identified in dicots. These TAS loci are characterized by a smaller distance between miR390 sites compared to TAS3, a single copy of ta-siARF, and a sequence conservation pattern pointing to the possibility that processing of novel TAS-like locus is initiated by cleavage of the 5'-terminal miR390 target site.

PRODUCTION OF BIOLOGICALLY ACTIVE HUMAN MYELOCYTOKINES IN PLANTS.

A. S. Zvereva, L. E. Petrovskaya, A. V. Rodina, O. Y. Frolova, P. A. Ivanov, L. N. Shingarova, T. V. Komarova, Y. L. Dorokhov, D. A. Dolgikh, M. P. Kirpichnikov, J. G. Atabekov.

Biochemistry-Moscow 74 (11):1187-1194, 2009.

An effective system for expression of human granulocyte and granulocyte macrophage colony-stimulating factors (hG-CSF and hGM-CSF) in Nicotiana benthamiana plants was developed using viral vector based on tobacco mosaic virus infecting cruciferous plants. The genes of target proteins were cloned into the viral vector driven by actin promoter of Arabidopsis thaliana. The expression vectors were delivered into plant cells by agroinjection. Maximal synthesis rate was detected 5 days after injection and was up to 500 and 300 mg per kg of fresh leaves for hG-CSF and hGM-CSF, respectively. The yield of purified hG-CSF and hGM-CSF was 100 and 50 mg/kg of fresh leaves, respectively. Recombinant plant-made hG-CSF and hGM-CSF stimulated proliferation of murine bone marrow and human erythroleucosis TF-1 cells, respectively, at the same rate as the commercial drugs.

[NEW DERIVATIVES OF AZOBENZENE FOR THE DIRECTED MODIFICATION OF PROTEINS].

Le Tkhi Khien, B. Shirling, A. Iu Riazanova, T. S. Zatsepin, E. M. Volkov, E. A. Kubareva, T. I. Velichko, A. Pingoud, T.S. Oretskaia.

Bioorg. Khim. 35 (5):610-617, 2009.

Derivatives of azobenzene which contained a maleimide group in one of the benzene rings (for binding to a protein cysteine residue) and maleimide, hydroxyl, or carboxyl substitutes in another benzene ring were synthesized. The reactivity of these compounds towards a cysteine residue of a protein and their optical properties in a free state and after their attachment to the mutant forms of the SsoII restriction endonuclease were studied.

SUPPRESSOR OF CYTOKINE SIGNALING-1 INHIBITS CASPASE ACTIVATION AND PROTECTS FROM CYTOKINE-INDUCED β CELL DEATH.

I. I. Zaitseva, M. Hultcrantz, V. Sharoyko, M. Flodstrom-Tullberg, S. V. Zaitsev, P. O. Berggren.

Cellular and Molecular Life Sciences 66 (23):3787-3795, 2009.

Pancreatic β cell damage caused by pro-inflammatory cytokines interleukin-1 β (IL-1 β), interferon-γ (IFN γ) and tumor necrosis factor-α (TNF α) is a key event in the pathogenesis of type 1 diabetes. The suppressor of cytokine signaling-1 (SOCS-1) blocks IFN γ -induced signaling and prevents diabetes in the non-obese diabetic mouse. Here, we investigated if SOCS-1 overexpression in primary β cells provides protection from cytokine-induced islet cell dysfunction and death. We demonstrate that SOCS-1 does not prevent increase in NO production and decrease in glucose-stimulated insulin secretion in the presence of IL-1 β, IFN γ, TNF α. However, it decreases the activation of caspase-3, -8 and -9, and thereby, promotes a robust protection from cytokine-induced β cell death. Our data suggest that SOCS-1 overexpression may not be sufficient in preventing all the biological activities of IFN γ in β cells. In summary, we show that interference with IFN γ signal transduction pathways by SOCS-1 inhibits cytokine-stimulated pancreatic β cell death.

Punctastriata glubokoensis SPEC. NOV., A NEW SPECIES OF 'FRAGILARIOID' DIATOM FROM LAKE GLUBOKOE, RUSSIA.

D. M. Williams, D. A. Chudaev, M. A. Gololobova.

Diatom Research 24 (2):479-485, 2009.

A new species of 'fragilarioid' diatom belonging to the genus Punctastriata Williams & Round is described from Lake Glubokoe (Moscow Area, Russia). P. glubokoensis sp, nov. shares particular features of the genus Punctastriata: multiseriate striae, lack of rimo-portulae, presence of a single apical pore field and a single apical depression. It has widely elliptical, slightly heteropolar valves, with rounded apices; lanceolate central sternum and radiate striae. Spines are located on the costae, and the base of each spine bears two small flat projections, oriented along sternum. The epicingulum consists of three elements: a wide, open valvocopula and two narrower copulae. Relationships of P. glubokoensis sp. nov. to other species in Punctastriata are discussed.

STRUCTURAL BASIS FOR HIV-1 DNA INTEGRATION IN THE HUMAN GENOME.

F. Michel, S. Eiler, F. Granger, J. F. Mouscadet, M. Gottikh, A. Nazabal, S. Emiliani, R. Benarous, D. Moras, P. Schultz, M. Ruff.

Retrovirology 6, 2009.

DIFFERENTIAL CONTRIBUTION OF THE M(7)G-CAP TO THE 5' END-DEPENDENT TRANSLATION INITIATION OF MAMMALIAN mRNAs.

D. E. Andreev, S. E. Dmitriev, I. M. Terenin, V. S. Prassolov, W. C. Merrick, I. N. Shatsky.

Nucleic Acids Research 37 (18):6135-6147, 2009.

Many mammalian mRNAs possess long 5' UTRs with numerous stem-loop structures. For some of them, the presence of Internal Ribosome Entry Sites (IRESes) was suggested to explain their significant activity, especially when cap-dependent translation is compromised. To test this hypothesis, we have compared the translation initiation efficiencies of some cellular 5' UTRs reported to have IRES-activity with those lacking IRES-elements in RNA-transfected cells and cell-free systems. Unlike viral IRESes, the tested 5' UTRs with so-called 'cellular IRESes' demonstrate only background activities when placed in the intercistronic position of dicistronic RNAs. In contrast, they are very active in the monocistronic context and the cap is indispensable for their activities. Surprisingly, in cultured cells or cytoplasmic extracts both the level of stimulation with the cap and the overall translation activity do not correlate with the cumulative energy of the secondary structure of the tested 5' UTRs. The cap positive effect is still observed under profound inhibition of translation with eIF4E-BP1 but its magnitude varies for individual 5' UTRs irrespective of the cumulative energy of their secondary structures. Thus, it is not mandatory to invoke the IRES hypothesis, at least for some mRNAs, to explain their preferential translation when eIF4E is partially inactivated.

NEW AZOBENZENE DERIVATIVES FOR DIRECTED MODIFICATION OF PROTEINS.

L. T. Hien, B. Schierling, A. Y. Ryazanova, T. S. Zatsepin, E. M. Volkov, E. A. Kubareva, T. I. Velichko, A. Pingoud, T. S. Oretskaya.

Russian Journal of Bioorganic Chemistry 35 (5):549-555, 2009.

Derivatives of azobenzene which contained a maleimide group in one of the benzene rings (for binding to a protein cysteine residue) and maleimide, hydroxyl, or carboxyl substitutes in another benzene ring were synthesized. The reactivity of these compounds towards a cysteine residue of a protein and their optical properties in a free form and after their attachment to the mutant forms of the SsoII restriction endonuclease were studied.

POPULATION GENETIC STRUCTURE OF WALLEYE POLLOCK Theragra chalcogramma (GADIDAE, PISCES) FROM THE BERING SEA AND SEA OF OKHOTSK.

E. A. Shubina, E. V. Ponomareva, A. I. Glubokov.

Molecular Biology 43 (5):855-866, 2009.

Walleye pollock Theragra chalcogramma Pallas occupies a central place in ecosystems of the North Pacific and is an important target species of fisheries. The species is characterized by daily vertical, spawning, feeding, and wintering migrations and spawning occurring under the sea ice. Since population structure estimation by the tagging with recapture is inefficient in walleye pollock, the pollock resources are difficult to estimate by conventional methods, requiring population genetic studies with molecular markers. The population genetic structure of five spawning aggregations from the Bering Sea was for the first time studied with ten microsatellite loci: Tch5, Tch10, Tch11, Tch12, Tch14, Tch16, Tch17, Tch19, Tch20, and Tch22. A spatially distant sample from the Sea of Okhotsk was used as a reference group. Polymorphism for the markers reached 100%, and heterozygosity of individual loci ranged from 41 to 95% in different populations. It was shown the aggregations of interest are in goodness-to-fit the Hardy-Weinberg equilibrium (HWE) at hole, while the Sea of Okhotsk sample demonstrated a sex bias: the heterozygosity at Tch16 in males was significantly lower than in females. The highest discriminative power was observed for Tch10, Tch20, and Tch22. F-ST genetic distances between populations were typical for marine fishes. A mixed composition was supposed for the sample from the region of the underwater Shirshov Ridge, which serves as a natural partial geographic barrier between the Olyutor-Karagin and Koryak walleye pollock stocks. With the Shirshov sample excluded, F-ST scatter plots and the spatial autocorrelation approach supported isolation by distance for the aggregations. An influence of abiotic factors on the population structure was assumed for walleye pollock of the Bering Sea.

ON THE PHYLOGENETIC POSITION OF INSECTS IN THE Pancrustacea CLADE.

V. V. Aleshin, K. V. Mikhailov, A. V. Konstantinova, M. A. Nikitin, L. Y. Rusin, D. A. Buinova, O. S. Kedrova, N. B. Petrov.

Molecular Biology 43 (5):804-818, 2009.

The current views on the phylogeny of arthropods are at odds with the traditional system, which recognizes four independent arthropod classes: Chelicerata, Crustacea, Myriapoda, and Insecta. There is compelling evidence that insects comprise a monophyletic lineage with Crustacea within a larger clade named Pancrustacea, or Tetraconata. However, which crustacean group is the closest living relative of insects is still an open question. In recent phylogenetic trees constructed on the basis of large gene sequence data insects are placed together with primitive crustaceans, the Branchiopoda. This topology is often suspected to be a result of the long branch attraction artifact. We analyzed concatenated data on 77 ribosomal proteins, elongation factor 1A (EF1A), initiation factor 5A (eIF5A), and several other nuclear and mitochondrial proteins. Analyses of nuclear genes confirm the monophyly of Hexapoda, the clade uniting entognath and ectognath insects. The hypothesis of the monophyly of Hexapoda and Branchiopoda is supported in the majority of analyses. The Maxillopoda, another clade of Entomostraca, occupies a sister position to the Hexapoda + Branchiopoda group. Higher crustaceans, the Malacostraca, in most analyses appear a more basal lineage within the Pancrustacea. We report molecular synapomorphies in low homoplastic regions, which support the clade Hexapoda + Branchiopoda + Maxillopoda and the monophyletic Malacostraca including Phyllocarida. Thus, the common origin of Hexapoda and Branchiopoda and their position within Entomostraca are suggested to represent bona fide phylogenetic relationships rather than computational artifacts.

GENOSYSTEMATICS AND NEW INSIGHT INTO THE PHYLOGENY AND TAXONOMY OF LIVERWORTS.

A. A. Vilnet, N. A. Konstantinova, A. V. Troitsky.

Molecular Biology 43 (5):783-793, 2009.

The current state of molecular studies in liverworts, including original data, was considered. The traditional concepts of the liverwort phylogeny and systematics have greatly changed as a result of recent molecular researches. The phylogenetic inferences from studies of different DNA loci of different species sampling are mainly congruent. The phylogeny and systematics of the suborder Jungermaniineae, one of the largest and taxonomically difficult groups, is discussed on the basis of nucleotide sequence analyses of internal transcribed spacers 1 and 2 (ITS1-2) of nuclear rDNA and chloroplast trnL-F in a representative species sampling.

COMPARATIVE GENOMICS, GENOSYSTEMATICS, AND THE SCIENTIFIC SCHOOL OF A.S. ANTONOV.

V. V. Aleshin

Molecular Biology 43 (5):697-700, 2009.

SUICIDAL FUNCTION OF DNA METHYLATION IN AGE-RELATED GENOME DISINTEGRATION.

A. L. Mazin.

Ageing Research Reviews 8 (4):314-327, 2009.

This article is dedicated to the 60th anniversary of 5-methylcytosine discovery in DNA. Cytosine methylation can affect genetic and epigenetic processes, works as a part of the genome-defense system and has mutagenic activity; however, the biological functions of this enzymatic modification are not well understood. This review will put forward the hypothesis that the host-defense role of DNA methylation in silencing and mutational destroying of retroviruses and other intragenomic parasites was extended during evolution to most host genes that have to be inactivated in differentiated somatic cells, where it acquired a new function in age-related self-destruction of the genome. The proposed model considers DNA methylation as the generator of 5mC > T transitions that induce 40-70% of all spontaneous somatic mutations of the multiple classes at CpG and CpNpG sites and flanking nucleotides in the p53, FIX, hprt, gpt human genes and some transgenes. The accumulation of 5mC-dependent mutations explains: global changes in the structure of the vertebrate genome throughout evolution; the loss of most 5mC from the DNA of various species over their lifespan and the Hayflick limit of normal cells; the polymorphism of methylation sites, including asymmetric mCpNpN sites; cyclical changes of methylation and demethylation in genes. The suicidal function of methylation may be a special genetic mechanism for increasing DNA damage and the programmed genome disintegration responsible for cell apoptosis and organism aging and death.

STRUCTURE AND EVOLUTION OF JUNCTIONS BETWEEN INVERTED REPEAT AND SMALL SINGLE COPY REGIONS OF CHLOROPLAST GENOME IN NON-CORE CARYOPHYLLALES.

M. D. Logacheva, A. A. Penin, C. M. Valiejo-Roman, A. S. Antonov.

Molecular Biology 43 (5):757-765, 2009.

The structure of the junction between inverted repeat (IR) and small single copy (SSC) regions of the chloroplast genome in the representatives of non-core Caryophyllales is investigated in this work. It was found that for two families-Polygonaceae and Plumbaginaceae-the extension of inverted region is characteristic. This extension is due to the duplication of the part of the ycf1 gene that is partly located in the small single copy region in plants with typical structure of IR/SSC junctions. Comparison of the position of IR/SSC junctions in different species of Polygonaceae has shown that their exact position is not correlated with the affinity of these species inferred from molecular and morphological data. Possible mechanisms leading to the change in position of IR/SSC junctions observed in this work are discussed.

UNCONTROLLED PROTEIN OVEREXPRESSION LEADS TO PLANT CELL AUTOPHAGY.

T. Komarova, E. Sheval, Y. Dorokhov.

FEBS Journal 276:206, 2009.

GAP JUNCTIONAL INTERCELLULAR COMMUNICATION IN HUMAN EMBRYONIC STEM CELLS DURING SPONTANEOUS DIFFERENTIATION.

Yu Yu Sharovskaya, M. A. Lagarkova, S. L. Kiselev, L. M. Chailakhyan.

Dokl Biol Sci 427:387-390, 2009.

CONSERVED STRUCTURAL FEATURES OF ETS DOMAIN-DNA COMPLEXES.

A. V. Grishin, A. V. Alexeevsky, S. A. Spirin, A. S. Karyagina.

Molecular Biology 43 (4):612-619, 2009.

ETS proteins are a family of widespread transcription factors that regulate the expression of many animal genes. Structurally, ETS proteins are characterized by a conserved DNA-binding ETS domain, which recognizes DNA sequences containing the trinucleotide GGA. The structural features of ETS domain-DNA complexes were analyzed, and conserved contacts important in terms of interaction stability and specificity were identified. The analysis revealed nine conserved hydrogen bonds with oxygens of DNA backbone phosphates, two bidentate hydrogen bonds with DNA major groove atoms, one conserved hydrophobic cluster located on the protein-DNA interface and important for binding site recognition, and 12 conserved water molecules presumably mediating the ETS domain-DNA interaction. The results are represented in specialized data bank of protein-DNA complexes (NPIDB).

β- AND γ-CYTOPLASMIC ACTINS DISPLAY DISTINCT DISTRIBUTION AND FUNCTIONAL DIVERSITY.

V. Dugina, I. Zwaenepoel, G. Gabbiani, S. Clement, C. Chaponnier.

Journal of Cell Science 122 (16):2980-2988, 2009.

Using newly generated monoclonal antibodies, we have compared the distribution of β- and γ-cytoplasmic actin in fibroblastic and epithelial cells, in which they play crucial roles during various key cellular processes. Whereas β-actin is preferentially localized in stress fibers, circular bundles and at cell-cell contacts, suggesting a role in cell attachment and contraction, γ -actin displays a more versatile organization, according to cell activities. In moving cells, γ-actin is mainly organized as a meshwork in cortical and lamellipodial structures, suggesting a role in cell motility; in stationary cells, γ-actin is also recruited into stress fibers. β-actin-depleted cells become highly spread, display broad protrusions and reduce their stress-fiber content; by contrast, γ-actin-depleted cells acquire a contractile phenotype with thick actin bundles and shrinked lamellar and lamellipodial structures. Moreover, β- and γ-actin depleted fibroblasts exhibit distinct changes in motility compared with their controls, suggesting a specific role for each isoform in cell locomotion. Our results reveal new aspects of β- and γ-actin organization that support their functional diversity.

BYPRODUCT WITH ALTERED FLUORESCENT PROPERTIES IS FORMED DURING STANDARD DEPROTECTION STEP OF HEXACHLOROFLUORESCEIN LABELED OLIGONUCLEOTIDES.

A. N. Chuvilin, M. V. Serebryakova, I. P. Smirnov, G. E. Pozmogova.

Bioconjugate Chemistry 20 (8):1441-1443, 2009.

HEX-labeled oligonucleotides obtained via typical synthetic protocols may contain more than 15% of material With altered spectral characteristics. We discovered hexachlorofluorescein residue transformation unknown earlier for standard DNA ammonolysis step. HEX residue reacts with ammonium hydroxide yielding acridine derivative, which has differed UV-VIS and fluorescent properties compared to HEX. Therefore. for critical bioassays where sensitivity and/or fluorescent signal differentiation (e.g., in quantitative or multiplexed assays) are essential. the careful RP-HPLC purification step is required.

DESIGN AND SYNTHESIS OF 2 '-FUNCTIONALISED OLIGONUCLEOTIDES. THEIR APPLICATION FOR COVALENT TRAPPING THE PROTEIN-DNA COMPLEXES.

N. G. Dolinnaya, E. M. Zubin, E. A. Kubareva, T. S. Zatsepin, T. S. Oretskaya.

Current Organic Chemistry 13 (11):1029-1049, 2009.

This review outlines the design and synthesis of oligonucleotides bearing 2'-modified residues at predetermined positions within the strand. The relative merits of incorporation of reactive carboxyl, carbonyl, iodoacetamide and disulfide-containing groups into oligonucleotides were considered along with solid-phase synthesis of DNA 2'-conjugates. The specific cross-linking of 2'-substituted oligonucleotides to nucleic acid-binding proteins (transcription factor NF-kappa B, restriction-modification enzymes) was shown to be helpful in scanning the protein-DNA interface and studying the conformational dynamics of biopolymer ensembles. The future perspectives of chemically reactive DNA constructs as specific protein decoys are discussed.

METAL ION CHELATE-ASSISTED LIGATION (CHESS LIGA) FOR SNP DETECTION ON MICROARRAYS.

L. T. Hien, T. S. Oretskaya, T. S. Zatsepin.

Bioorganic & Medicinal Chemistry Letters 19 (15):4018-4021, 2009.

We developed a metal ion chelate-assisted ligation for SNP detection by microarray. An oligonucleotide probe was separated into two 9-10-mers bearing iminodiacetic residues at the gap point. Duplex formation with the DNA target was possible only if nickel ions were added, but a nucleotide substitution opposite the gap point prevented duplex formation. Here we demonstrate the application of this approach for SNP detection (A1298C) within the 5,10-methylenetetrahydrofolate reductase gene on a microarray.

THE ORIGIN OF METAZOA: A TRANSITION FROM TEMPORAL TO SPATIAL CELL DIFFERENTIATION.

K. V. Mikhailov, A. V. Konstantinova, M. A. Nikitin, P. V. Troshin, L. Y. Rusin, V. A. Lyubetsky, Y. V. Panchin, A. P. Mylnikov, L. L. Moroz, S. Kumar, V. V. Aleoshin.

Bioessays 31 (7):758-768, 2009.

For over a century, Haeckel's Gastraeal theory remained a dominant theory to explain the origin of multicellular animals. According to this theory, the animal ancestor was a blastula-like colony of uniform cells that gradually evolved cell differentiation. Today, however, genes that typically control metazoan development, cell differentiation, cell-to-cell adhesion, and cell-to-matrix adhesion are found in various unicellular relatives of the Metazoa, which suggests the origin of the genetic programs of cell differentiation and adhesion in the root of the Opisthokonta. Multicellular stages occurring in the complex life cycles of opisthokont protists (mesomycetozoeans and choanoflagellates) never resemble a blastula. Here, we discuss a more realistic scenario of transition to multi:. cellularity through integration of pre-existing transient cell types into the body of an early metazoon, which possessed a complex life cycle with a differentiated sedentary filter-feeding trophic stage and a non-feeding blastula-like larva, the synzoospore. Choanoflagellates are considered as forms with secondarily simplified life cycles.

MECHANISMS OF SOCIAL BEHAVIOR OF THE VERTEBRATE TISSUE CELLS: CULTURAL MODELS.

V. I. Samoilov and J. M. Vasilyev.

Zhurnal Obshchei Biologii 70 (3):239-244, 2009.

Morphogenetic interactions between cells in the organism are largely reproduced in the cell cultures of various types, in particular, in that of the epitheliocytes and fibroblasts. The cultured cells interact by means of receptors and ligands, which activate intracellular signal systems. Three microenvironment components (liquid medium, extracellular matrix, and intercellular contacts) play the role of such ligands. The ligands of liquid medium include hormones, growth factors, apoptosis factors and other molecules. Specific structures strongly tied with the cytoskeleton (with the actin one, first of all) use to appear during contacts with other cells and with the cellular base. Encounter between two fibroblasts or between two epitheliocytes causes local inhibiting of pseudopodial activity (so called contact paralysis). Regeneration of the connective tissue and the epithelial layer, as well as epithelial-mesenchimal transformation, is successfully reproduced in the cell culture. Incomplete epithelial-mesenchimal transformation seems to serve as a base for morphogenesis of various glands and lungs, as well as of angiogenesis.

MECHANISM OF RHODOPSIN KINASE REGULATION BY RECOVERIN.

K. E. Komolov, I. I. Senin, N. A. Kovaleva, M. P. Christoph, V. A. Churumova, I. I. Grigoriev, M. Akhtar, P. P. Philippov, K. W. Koch.

Journal of Neurochemistry 110 (1):72-79, 2009.

Recoverin is suggested to inhibit rhodopsin kinase (GRK1) at high [Ca²⁺] in the dark state of the photoreceptor cell. Decreasing [Ca²⁺] terminates inhibition and facilitates phosphorylation of illuminated rhodopsin (Rh*). When recoverin formed a complex with GRK1, it did not interfere with the phosphorylation of a C-terminal peptide of rhodopsin (S338-A348) by GRK1. Furthermore, while GRK1 competed with transducin on interaction with rhodopsin and thereby suppressed GTPase activity of transducin, recoverin in the complex with GRK1 did not influence this competition. Constructs of GRK1 that encompass its N-terminal, catalytic or C-terminal domains were used in pull-down assays and surface plasmon resonance analysis to monitor interaction. Ca²⁺-recoverin bound to the N-terminus of GRK1, but did not bind to the other constructs. GRK1 interacted with rhodopsin also by its N-terminus in a light-dependent manner. No interaction was observed with the C-terminus. We conclude that inhibition of GRK1 by recoverin is not the result of their direct competition for the same docking site on Rh*, although the interaction sites of GRK1/Rh* and GRK1/recoverin partially overlap. The N-terminus of GRK1 is recognized by Rh* leading to a conformational change which moves the C-terminus of Rh* into the catalytic kinase groove. Ca²⁺-recoverin interacting with the N-terminus of GRK1 prevents this conformational change and thus blocks Rh* phosphorylation by GRK1.

ENDONUCLEASES AND THEIR INVOLVEMENT IN PLANT APOPTOSIS.

N. I. Aleksandrushkina and B. F. Vanyushin.

Russian Journal of Plant Physiology 56 (3):291-305, 2009.

This review considers modern data about the set, nature, specificity of action, and other properties of plant endonucleases involved in various forms of programmed cell death (PCD) in various plant tissues (organs). Apoptosis is an obligatory component of plant development; plant development is impossible without apoptosis. In dependence on the conditions of plant growth, this process can be induced by various biotic and abiotic factors, including stressors. Endonucleases accomplishing apoptotic degradation of nuclear material in the plant cell play one of the main roles in PCD. Plant endonucleases belong to at least two classes: (1) Ca²⁺- and Mg²⁺-dependent and (2) Zn²⁺-dependent nucleases. The set and activities of endonucleases change with plant age and during apoptosis in a tissue-specific manner. Apoptosis is accompanied by the induction of specific endonucleases hydrolyzing DNA in chromatin with the formation firstly of large domains and then internucleosomal DNA fragments; the products produced are of about 140 nucleotides in length with their subsequent degradation to low-molecular-weight oligonucleotides and mononucleotides. About 30 enzymes are involved in apoptotic DNA degradation. Histone H1 modulates endonuclease activity; separate (sub)fractions of this nuclear protein can stimulate or inhibit corresponding plant endonucleases. In the nucleus and cytoplasm of the plant cells, Ca²⁺/Mg²⁺-dependent endonucleases recognizing substrate DNA methylation status were revealed and described for the first time; their action resembles that of bacterial restrictases, which activity is modulated by the donor of methyl groups, S-adenosylmethionine. This indicates that higher eukaryotes (higher plants) might possess the system of restriction-modification to some degree analogous to that of prokaryotes.

MOLECULAR APPRAISAL OF Bunium AND SOME RELATED ARID AND SUBARID GEOPHILIC

Apiaceae-Apioideae TAXA OF THE ANCIENT MEDITERRANEAN.

G. V. Degtjareva, E. V. Kljuykov, T. H. Samigullin, C. M. Valiejo-Roman, M. G. Pimenov.

Botanical Journal of the Linnean Society 160 (2):149-170, 2009.

Bunium is unusual in Apiaceae in having a variable cotyledon number and broad infrageneric dysploidy. To test the monophyly of the genus, phylogenetic relationships among 39 Bunium species were investigated with DNA sequence data from nuclear (nrITS) and plastid (psbA-trnH intergenic spacer) regions. Several other taxa with a similar ecology and geography were also included in the analyses. Our results suggest that Bunium is not monophyletic. Bunium spp. in the eastern part of the study area are more closely related to the Central Asian genera Elaeosticta, Galagania, Hyalolaena, Mogoltavia and Oedibasis than to those in the western part. Our study revealed that molecular, morphological (cotyledon number and width of fruit commissure) and karyological data reveal similar patterns in the taxa studied.

STRUCTURAL BASIS FOR HIV-1 DNA INTEGRATION IN THE HUMAN GENOME, ROLE OF THE LEDGF/P75 COFACTOR.

F. Michel, C. Crucifix, F. Granger, S. Eiler, J. F. Mouscadet, S. Korolev, J. Agapkina, R. Ziganshin, M. Gottikh, A. Nazabal, S. Emiliani, R. Benarous, D. Moras, P. Schultz, M. Ruff.

Embo Journal 28 (7):980-991, 2009.

Integration of the human immunodeficiency virus (HIV-1) cDNA into the human genome is catalysed by integrase. Several studies have shown the importance of the interaction of cellular cofactors with integrase for viral integration and infectivity. In this study, we produced a stable and functional complex between the wild-type full-length integrase (IN) and the cellular cofactor LEDGF/p75 that shows enhanced in vitro integration activity compared with the integrase alone. Mass spectrometry analysis and the fitting of known atomic structures in cryo negatively stain electron microscopy (EM) maps revealed that the functional unit comprises two asymmetric integrase dimers and two LEDGF/p75 molecules. In the presence of DNA, EM revealed the DNA-binding sites and indicated that, in each asymmetric dimer, one integrase molecule performs the catalytic reaction, whereas the other one positions the viral DNA in the active site of the opposite dimer. The positions of the target and viral DNAs for the 30 processing and integration reaction shed light on the integration mechanism, a process with wide implications for the understanding of viral-induced pathologies.

ENDONUCLEASE ACTIVITIES IN THE COLEOPTILE AND THE FIRST LEAF OF DEVELOPING ETIOLATED WHEAT SEEDLINGS.

N. I. Aleksandrushkina, A. V. Seredina, B. F. Vanyushin.

Russian Journal of Plant Physiology 56 (2):154-163, 2009.

DNase activity in coleoptiles and the first leaf apices of winter wheat (Triticum aestivum L., cv. Mironovskaya 808) etiolated seedlings was found to increase significantly during seedling growth, peaking on the eighth day of plant development. The maximum of DNase activity was coincident with apoptotic internucleosomal DNA fragmentation in these organs. Wheat endonucleases are capable of hydrolyzing both singleand double-stranded DNA of various origins. The leaf and coleoptiles were found to exhibit nuclease activities that hydrolyzed the λ phage DNA with N-6-methyladenine and 5-methylcytosine more actively compared to the hydrolysis of similar unmethylated DNAs. Thus, the endonucleases of wheat seedlings are sensitive to the methylation status of their substrate DNAs. The leaves and coleoptiles exhibited both Ca²⁺/Mg²⁺- and Zn²⁺-dependent nuclease activities that underwent differential changes during development and senescence of seedling organs. EDTA at a concentration of 50 mM fully inhibited the total DNase activity. Electrophoretic heterogeneity was observed for DNase activities operating simultaneously in the coleoptile and the first leaf at different stages of seedling development. Proteins exhibiting DNase activity (16-80 kD mol wt) were revealed in the first leaf and the coleoptile; these proteins were mostly nucleases with the pH optimum around 7.0. Some endonucleases (mol wts of 36, 39, and 28 kD) were present in both organs of the seedling. Some other DNases (mol wts of 16, 56, and about 80 kD) were found in the coleoptile; these DNases hydrolyzed DNA in the nucleus at terminal stages of apoptosis. Different suites of DNase activities were revealed in the nucleus and the cytoplasm, the nuclear DNase activities being more diverse than the cytoplasmic ones. Thus, the cellular (organspecific) and subcellular heterogeneity in composition and activities of DNases has been revealed in wheat plants. These DNases undergo specific changes during seedling development, serving at various stages of programmed cell death in seedling tissues.

EFFICIENT CAP-DEPENDENT TRANSLATION OF MAMMALIAN mRNAs WITH LONG AND HIGHLY STRUCTURED 5'-UNTRANSLATED REGIONS in vitro AND in vivo.

S. E. Dmitriev, D. E. Andreev, Z. V. Adyanova, I. M. Terenin, I. N. Shatsky.

Molecular Biology 43 (1):108-113, 2009.

According to the generally accepted scanning model proposed by M. Kozak, the secondary structure of the 5'-untranslated region (5'-UTR) of eukaryotic mRNA can only inhibit the translation initiation by counteracting migration of the 40S ribosomal subunit along the mRNA polynucleotide chain. The existence of efficiently translatable mRNAs with long and highly structured 5'-UTRs is incompatible with the cap-dependent scanning mechanism. Such mRNAs are expected to use alternative ways of translation initiation to be efficiently translated, primarily the mechanism of internal ribosome entry mediated by internal ribosome entry sites (IRESs), special RNA structures that reside in the 5'-UTR. The paper shows that this viewpoint is incorrect and is probably based on experiments with mRNA translation in rabbit reticulocyte lysate. This cell-free system fails to adequately reflect the relative translation efficiencies observed for different mRNAs in vivo. Five structurally similar mRNAs with either short leaders of the β-globin and β-actin mRNAs or long and highly structured 5'-UTRs of the c-myc, LINE-1, and Apaf-1 mRNAs displayed comparable translation activities in transfected cells and an entire cytoplasmic extract of cultivated cells. Translation activity proved to strongly depend on the presence of a cap at

the 5' end.

DETECTION OF ABASIC SITES IN DNA BY ELECTROCHEMICAL, IMMUNOELECTROCHEMICAL AND ACOUSTIC METHODS USING OSO4, 2,2 '-BIPYRIDINE AS A PROBE FOR UNPAIRED THYMINE RESIDUES.

M. Bartosik, V. Gajdos, P. Kostecka, M. Fojta, E. Palecek, E. Volkov, T. Oretskaya, T. Hianik.

Electroanalysis 21 (3-5):295-302, 2009.

We report on comparative analysis of the detection of DNA damage modeled by presence of abasic sites (AP) at defined positions using the chemical modification of the DNA by the complex of osmium tetroxide-2,2-bipyridine (Os, bipy) that selectively binds to unpaired thymine residues in the damaged DNA. AP were detected by electrochemical detection (EC) of the Os, bipy-thymine adducts, by immunoelectrochemical (IE) and by thickness shear mode acoustic methods (TSM). EC method of detection can perfectly distinguish between the number of A P. I E and TSM methods were of comparable sensitivity.

DIVERSITY OF NEURODEGENERATIVE PROCESSES IN THE MODEL

OF BRAIN CORTEX TISSUE ISCHEMIA.

N. V. Lobysheva, A. A. Tonshin, A. A. Selin, L. S. Yaguzhinsky, Y. R. Nartsissov.

Neurochemistry International 54 (5-6):322-329, 2009.

Stroke is known to induce massive cell death in the ischemic brain. Either necrotic or apoptotic types of cell death program were observed in neurons in zone of ischemia. We suggest that spatial heterogeneity of glucose and oxygen distribution plays a crucial role in this phenomenon. In order to elucidate the role of glucose and oxygen in ischemic neurons choice of cell death pathway, conditions corresponding to different areas of insult were reproduced in vitro in the model of surviving brain cortex tissue slices. Three zones were modeled in vitro by varying glucose and oxygen concentration in surviving slices incubation media. Modeled ischemic area I (MIA I) was corresponded to the center of suggested ischemic zone where the levels of glucose and oxygen were considered to be extremely low. MIA II was assigned as intermediate area where oxygen concentration was still very low but glucose was present (this area was also divided into two sub-areas MIA IIa and MIA IIb with physiologically low (5 mM) and normal (10 mM) level of glucose respectively). MIA III was considered as a periphery area where glucose concentration was close to physiological level and high level of ROS production had been induced by reoxygenation after anoxia. Analysis of molecular mechanisms of cell death in MIA I, IIa, IIb and III was carried out. Cell death in MIA I was found to proceed by necrotic manner. Apoptosis characterized by cyt c release, caspase 3 activation and internucleosomal DNA fragmentation was observed in MIA Ill. Cell death in MIA II was accompanied by several (not all) hallmarks of apoptosis. Mechanisms of cell death in MIA IIa and MIA IIb were found to be different. Internucleosomal DNA fragmentation in MIA IIa but not in MIA IIb was sensitive to glycine (5 mM), inhibitor of NMDA receptor MK-801 (10 mu M) and PTP inhibitor cyclosporine A (10 mu M). Activation of caspase 3 was detected in MIA IIb but not in MIA IIa. However cytochrome c release was observed neither in MIA IIa nor in MIA IIb. In MIAs II-III apoptosis was accompanied by uncoupling of oxidative phosphorylation, which was induced by rise of intracellular Ca²⁺ and intensive ROS production. Results obtained in present study allow us to propose existence of at least four molecular pathways of cell death development in brain ischemic zone. The choice of cell death pathway is determined by oxygen and glucose concentration in the particular area of the ischemic zone.

MOLECULAR ENVIRONMENT OF THE IIID SUBDOMAIN OF THE IRES ELEMENT OF HEPATITITS C VIRUS RNA ON THE HUMAN 40S RIBOSOMAL SUBUNIT.

E. S. Babaylova, D. M. Graifer, A. A. Malygin, I. N. Shatsky, I. Shtahl, G. Karpova.

Russian Journal of Bioorganic Chemistry 35 (1):94-102, 2009.

The molecular environment of the key subdomain IIId of the internal ribosome entry site (IRES) element of hepatitis C virus (HCV) RNA in the binary complex with the human 40S ribosomal subunit was studied. To this end, HCV IRES derivatives bearing perfluorophenylazido groups activatable by mild UV at nucleotides G263 or A275 in the subdomain IIId stem were used. They were prepared by the complementary addressed modification of the corresponding RNA transcript with alkylating oligodeoxyribonucleotide derivatives. None of the RNA derivatives were shown to be crosslinked to the 18S rRNA. It was found that the photoreactive groups of the IRES G263 and A275 nucleotides are crosslinked to ribosomal proteins S3a, S14, and S16. For the IRES derivative with the photoreactive group in nucleotide G263, the degree of modification of proteins S14 and S16 was greater than that of S3a, whereas the derivative containing the same photoreactive group in nucleotide A275 was mainly crosslinked to proteins S3a and S14. An analysis of the data led to the conclusion that in the binary complex of HCV IRES elements with the small subunit of the 80S ribosome, its subdomain IIId stem is located on the solvent side of the subunit between the head and the body next to the "beak" near the exit of mRNA from the ribosome.

POSITIONING OF SUBDOMAIN IIID AND APICAL LOOP OF DOMAIN II OF THE HEPATITIS C IRES ON THE HUMAN 40S RIBOSOME.

E. Babaylova, D. Graifer, A. Malygin, J. Stahl, I. Shatsky, G. Karpova.

Nucleic Acids Research 37 (4):1141-1151, 2009.

The 5-untranslated region of the hepatitis C virus (HCV) RNA contains a highly structured motif called IRES (Internal Ribosome Entry Site) responsible for the cap-independent initiation of the viral RNA translation. At first, the IRES binds to the 40S subunit without any initiation factors so that the initiation AUG codon falls into the P site. Here using an original site-directed cross-linking strategy, we identified 40S subunit components neighboring subdomain IIId, which is critical for HCV IRES binding to the subunit, and apical loop of domain II, which was suggested to contact the 40S subunit from data on cryo-electron microscopy of ribosomal complexes containing the HCV IRES. HCV IRES derivatives that bear a photoactivatable group at nucleotide A275 or at G263 in subdomain IIId cross-link to ribosomal proteins S3a, S14 and S16, and HCV IRES derivatized at the C83 in the apex of domain II cross-link to proteins S14 and S16.

H1 HISTONE MODULATES DNA HYDROLYSIS WITH WEN1 AND WEN2 ENDONUCLEASES FROM WHEAT COLEOPTILES.

L. I. Fedoreyeva, T. A. Smirnova, G. Y. Kolomijtseva, B. F. Vanyushin.

Biochemistry-Moscow 74 (2):145-152, 2009.

We show that total H1 histone from wheat seedlings or rat liver enhances hydrolysis of λ phage DNA with plant endonucleases WEN1 and WEN2 isolated from wheat coleoptiles. Optimal DNA/protein weight ratio in the hydrolysis reaction is 1: 1. The action of fractions I and IV (obtained from total wheat H1 histone by electrophoresis) on DNA hydrolysis with WEN1 and WEN2 enzymes depends on the DNA methylation status. Fraction IV of wheat histone H1 stimulates hydrolysis of unmethylated λ phage DNA with WEN1 and WEN2 enzymes. Hydrolysis of methylated λ phage DNA (it contains 5-methylcytosine in Cm(5)CWGG sequences and N-6-methyladenine in Gm(6)ATC sites) with WEN1 is inhibited with fractions I and IV of wheat H1 histone. Fractions II and III of wheat H1 histone do not influence DNA hydrolysis with WEN1 and WEN2. S-Adenosyl-L-methionine (SAM) stimulates activity of these plant enzymes. But in the presence of H1 histone, SAM does not add to the ability of the enzyme to hydrolyze more DNA compared with that induced with H1 histone itself. Therefore, the stimulating effects of SAM and H1 histone on DNA hydrolysis with plant endonucleases may be similar. It could be suggested that SAM and H1 histone can induce more or less analogous allosteric transformations in the structure of the investigated plant endonucleases. Thus, DNA hydrolysis with plant endonucleases is modulated with total H1 histone. H1 histone fractions affect DNA hydrolysis in a different fashion; they enhance or inhibit hydrolysis depending on the DNA methylation status. We suggest that H1 histone changes site specificity of endonucleases or it might be responsible for formation of new or masking of old sites available for these enzymes due to changes in DNA structure induced in a DNA-histone complex.

CANCER-RETINA ANTIGENS CGMP-PHOSPHODIESTERASE 6 AND TRANSDUCIN CONTROL CGMP METABOLISM AND Ca²⁺ HOMEOSTASIS IN MELANOMA CELLS.

V. Tambor, B. Dikov, P. Philippov, D. Schadendorf, S. Eichmuller, A. Bazhin.

Experimental Dermatology 18 (3):308, 2009.

Ssoii-LIKE DNA-METHYLTRANSFERASE ECL18KI: INTERACTION BETWEEN REGULATORY AND METHYLATING FUNCTIONS.

E. A. Fedotova, A. S. Protsenko, M. V. Zakharova, N. V. Lavrova, A. V. Alekseevsky, T. S. Oretskaya, A. S. Karyagina, A. S. Solonin, E. A. Kubareva.

Biochemistry-Moscow 74 (1):85-91, 2009.

The interaction of DNA-methyltransferase Ecl18kI (M.Ecl18kI) with a fragment of promoter region of restriction-modification system SsoII was studied. It is shown that dissociation constants of M.Ecl18kI and M.SsoII complexes with DNA ligand carrying a regulatory site previously characterized for M.SsoII have comparable values. A deletion derivative of M.Ecl18kI, ∆(72-379)Ecl18kI, representing the N-terminal protein region responsible for regulation, was obtained. It is shown that such polypeptide fragment has virtually no interaction with the regulatory site. Therefore, the existence of a region responsible for methylation is necessary for maintaining M.Ecl18kI regulatory function. The properties of methyl-transferase NlaX, which is actually a natural deletion derivative of M.Ecl18kI and M.SsoII lacking the first 70 amino acid residues and not being able to regulate gene expression of the SsoII restriction-modification system, were studied. The ability of mutant forms of M.Ecl18kI incorporating single substitutions in regions responsible for regulation and methylation to interact with both sites of DNA recognition was characterized. The data show a correlation between DNA-binding activity of two M.Ecl18kI regions-regulatory and methylating.

DETECTION OF DNA DAMAGE: EFFECT OF THYMIDINE GLYCOL RESIDUES ON THE THERMODYNAMIC, SUBSTRATE AND INTERFACIAL ACOUSTIC PROPERTIES OF OLIGONUCLEOTIDE DUPLEXES.

F. Yang, E. Romanova, E. Kubareva, N. Dolinnaya, V. Gajdos, O. Burenina, E. Fedotova, J. S. Ellis, T. Oretskaya, T. Hianik, M. Thompson.

Analyst 134 (1):41-51, 2009.

Thymidine glycol residues in DNA are biologically active oxidative molecular damage sites caused by ionizing radiation and other factors. One or two thymidine glycol residues were incorporated in 19- to 31-mer DNA fragments during automatic oligonucleotide synthesis. These oligonucleotide models were used to estimate the effect of oxidized thymidines on the thermodynamic, substrate and interfacial acoustic properties of DNA. UV-monitoring melting data revealed that modified residues in place of thymidines destabilize the DNA double helix by 8-22⁰C, depending on the number of lesions, the length of oligonucleotide duplexes and their GC-content. The diminished hybridizing capacity of modified oligonucleotides is presumably due to the loss of aromaticity and elevated hydrophilicity of thymine glycol in comparison to the thymine base. According to circular dichroism (CD) data, the modified DNA duplexes retain B-form geometry, and the thymidine glycol residue introduces only local perturbations limited to the lesion site. The rate of DNA hydrolysis by restriction endonucleases R.MvaI, R.Bst2UI, R.MspR9I and R.Bme1390I is significantly decreased as the thymidine glycol is located in the central position of the double-stranded recognition sequences 5'-CC down arrow WGG-3' (W = A, T) or 5'-CC down arrow NGG-3' (N = A, T, G, C) adjacent to the cleavage site. On the other hand, the catalytic properties of enzymes R.Psp6I and R. BstSCI recognizing the similar sequence are not changed dramatically, since their cleavage site is separated from the point of modification by several base-pairs. Data obtained by gel-electrophoretic analysis of radioactive DNA substrates were confirmed by direct spectrophotometric assay developed by the authors. The effect of thymidine glycol was also observed on DNA hybridization at the surface of a thickness-shear mode acoustic wave device. A 1.9-fold decrease in the rate of duplex formation was noted for oligonucleotides carrying one or two thymidine glycol residues in relation to the unmodified analog.

CANCER-RETINA ANTIGENS AS POTENTIAL PARANEOPLASTIC ANTIGENS IN MELANOMA-ASSOCIATED RETINOPATHY.

A. V. Bazhin, C. Dalke, N. Willner, O. Abschutz, H. G. H. Wildberger, P. P. Philippov, R. Dummer, J. Graw, M. H. de Angel, D. Schadendorf, V. Umansky, S. B. Eichmuller.

International Journal of Cancer 124 (1):140-149, 2009.

Melanoma-associated retinopathy is a rare paraneoplastic neurological syndrome characterized by retinopathy in melanoma patients. The main photoreceptor proteins have been found to be expressed as cancer-retina antigens in melanoma. Here we present evidence that these can function as paraneoplastic antigens in melanoma-associated retinopathy. Sera and one tumor cell line of such patients were studied and ret-transgenic mice spontaneously developing melanoma were used as a murine model for melanoma-associated retinopathy. Splenocytes and sera were used for adoptive transfer from tumor-bearing or control mice to wildtype mice. Retinopathy seas investigated in mice by funduscopy, electroretinography and eye histology. Expression of photoreceptor proteins and autoantibodies against arrestin and transducin were detected in melanoma-associated retinopathy patients. In tumor-bearing ret-transgenic mice, retinopathy was frequently (13/15) detected by electroretinogram and eye histology. These pathological changes were manifested in degenerations of photoreceptors, bipolar cells and pigment epithelium as well as retinal detachment. Mostly these defects were combined. Cancer-retina antigens were expressed in tumors of these mice, and autoantibodies against arrestin were revealed in some of their sera. Adoptive transfer of splenocytes and sera from tumor-bearing into wildtype mice led to the induction of retinopathy in 4/16 animals. We suggest that melanoma-associated retinopathy can be mediated by Immoral and/or cellular immune responses against a number of cancer-retina antigens which may function as paraneoplastic antigens in melanoma-associated retinopathy.

DETECTION OF DNA DAMAGE: EFFECT OF THYMIDINE GLYCOL RESIDUES ON THE THERMODYNAMIC, SUBSTRATE AND INTERFACIAL ACOUSTIC PROPERTIES OF OLIGONUCLEOTIDE DUPLEXES.

F. Yang, E. Romanova, E. Kubareva, N. Dolinnaya, V. Gajdos, O. Burenina, E. Fedotova, J. S. Ellis, T. Oretskaya, T. Hianik, M. Thompson.

Analyst 134 (1):41-51, 2009.

Thymidine glycol residues in DNA are biologically active oxidative molecular damage sites caused by ionizing radiation and other factors. One or two thymidine glycol residues were incorporated in 19- to 31-mer DNA fragments during automatic oligonucleotide synthesis. These oligonucleotide models were used to estimate the effect of oxidized thymidines on the thermodynamic, substrate and interfacial acoustic properties of DNA. UV-monitoring melting data revealed that modified residues in place of thymidines destabilize the DNA double helix by 8-22⁰C, depending on the number of lesions, the length of oligonucleotide duplexes and their GC-content. The diminished hybridizing capacity of modified oligonucleotides is presumably due to the loss of aromaticity and elevated hydrophilicity of thymine glycol in comparison to the thymine base. According to circular dichroism (CD) data, the modified DNA duplexes retain B-form geometry, and the thymidine glycol residue introduces only local perturbations limited to the lesion site. The rate of DNA hydrolysis by restriction endonucleases R.MvaI, R.Bst2UI, R.MspR9I and R.Bme1390I is significantly decreased as the thymidine glycol is located in the central position of the double-stranded recognition sequences 5'-CC down arrow WGG-3' (W = A, T) or 5'-CC down arrow NGG-3' (N = A, T, G, C) adjacent to the cleavage site. On the other hand, the catalytic properties of enzymes R.Psp6I and R. BstSCI recognizing the similar sequence are not changed dramatically, since their cleavage site is separated from the point of modification by several base-pairs. Data obtained by gel-electrophoretic analysis of radioactive DNA substrates were confirmed by direct spectrophotometric assay developed by the authors. The effect of thymidine glycol was also observed on DNA hybridization at the surface of a thickness-shear mode acoustic wave device. A 1.9-fold decrease in the rate of duplex formation was noted for oligonucleotides carrying one or two thymidine glycol residues in relation to the unmodified analog.

MOLECULAR ENVIRONMENT OF THE IIID SUBDOMAIN OF THE IRES ELEMENT OF HEPATITITS C VIRUS RNA ON THE HUMAN 40S RIBOSOMAL SUBUNIT.

E. S. Babaylova, D. M. Graifer, A. A. Malygin, I. N. Shatsky, I. Shtahl, G. Karpova.

Russian Journal of Bioorganic Chemistry 35 (1):94-102, 2009.

The molecular environment of the key subdomain IIId of the internal ribosome entry site (IRES) element of hepatitis C virus (HCV) RNA in the binary complex with the human 40S ribosomal subunit was studied. To this end, HCV IRES derivatives bearing perfluorophenylazido groups activatable by mild UV at nucleotides G263 or A275 in the subdomain IIId stem were used. They were prepared by the complementary addressed modification of the corresponding RNA transcript with alkylating oligodeoxyribonucleotide derivatives. None of the RNA derivatives were shown to be crosslinked to the 18S rRNA. It was found that the photoreactive groups of the IRES G263 and A275 nucleotides are crosslinked to ribosomal proteins S3a, S14, and S16. For the IRES derivative with the photoreactive group in nucleotide G263, the degree of modification of proteins S14 and S16 was greater than that of S3a, whereas the derivative containing the same photoreactive group in nucleotide A275 was mainly crosslinked to proteins S3a and S14. An analysis of the data led to the conclusion that in the binary complex of HCV IRES elements with the small subunit of the 80S ribosome, its subdomain IIId stem is located on the solvent side of the subunit between the head and the body next to the "beak" near the exit of mRNA from the ribosome.

Ssoii-LIKE DNA-METHYLTRANSFERASE ECL18KI: INTERACTION BETWEEN REGULATORY AND METHYLATING FUNCTIONS.

E. A. Fedotova, A. S. Protsenko, M. V. Zakharova, N. V. Lavrova, A. V. Alekseevsky, T. S. Oretskaya, A. S. Karyagina, A. S. Solonin, E. A. Kubareva.

Biochemistry-Moscow 74 (1):85-91, 2009.

The interaction of DNA-methyltransferase Ecl18kI (M.Ecl18kI) with a fragment of promoter region of restriction-modification system SsoII was studied. It is shown that dissociation constants of M.Ecl18kI and M.SsoII complexes with DNA ligand carrying a regulatory site previously characterized for M.SsoII have comparable values. A deletion derivative of M.Ecl18kI, ∆(72-379)Ecl18kI, representing the N-terminal protein region responsible for regulation, was obtained. It is shown that such polypeptide fragment has virtually no interaction with the regulatory site. Therefore, the existence of a region responsible for methylation is necessary for maintaining M.Ecl18kI regulatory function. The properties of methyl-transferase NlaX, which is actually a natural deletion derivative of M.Ecl18kI and M.SsoII lacking the first 70 amino acid residues and not being able to regulate gene expression of the SsoII restriction-modification system, were studied. The ability of mutant forms of M.Ecl18kI incorporating single substitutions in regions responsible for regulation and methylation to interact with both sites of DNA recognition was characterized. The data show a correlation between DNA-binding activity of two M.Ecl18kI regions-regulatory and methylating.

CANCER-RETINA ANTIGENS AS POTENTIAL PARANEOPLASTIC ANTIGENS IN MELANOMA-ASSOCIATED RETINOPATHY.

A. V. Bazhin, C. Dalke, N. Willner, O. Abschutz, H. G. H. Wildberger, P. P. Philippov, R. Dummer, J. Graw, M. H. de Angel, D. Schadendorf, V. Umansky, S. B. Eichmuller.

International Journal of Cancer 124 (1):140-149, 2009.

Melanoma-associated retinopathy is a rare paraneoplastic neurological syndrome characterized by retinopathy in melanoma patients. The main photoreceptor proteins have been found to be expressed as cancer-retina antigens in melanoma. Here we present evidence that these can function as paraneoplastic antigens in melanoma-associated retinopathy. Sera and one tumor cell line of such patients were studied and ret-transgenic mice spontaneously developing melanoma were used as a murine model for melanoma-associated retinopathy. Splenocytes and sera were used for adoptive transfer from tumor-bearing or control mice to wildtype mice. Retinopathy seas investigated in mice by funduscopy, electroretinography and eye histology. Expression of photoreceptor proteins and autoantibodies against arrestin and transducin were detected in melanoma-associated retinopathy patients. In tumor-bearing ret-transgenic mice, retinopathy was frequently (13/15) detected by electroretinogram and eye histology. These pathological changes were manifested in degenerations of photoreceptors, bipolar cells and pigment epithelium as well as retinal detachment. Mostly these defects were combined. Cancer-retina antigens were expressed in tumors of these mice, and autoantibodies against arrestin were revealed in some of their sera. Adoptive transfer of splenocytes and sera from tumor-bearing into wildtype mice led to the induction of retinopathy in 4/16 animals. We suggest that melanoma-associated retinopathy can be mediated by Immoral and/or cellular immune responses against a number of cancer-retina antigens which may function as paraneoplastic antigens in melanoma-associated retinopathy.

V. Matematical models in biology.

EFFECTOR PROTEINS OF Chlamydiae.

A. S. Karyagina, A. V. Alexeevsky, S. A. Spirin, N. A. Zigangirova, A. L. Gintsburg.

Molecular Biology 43 (6):897-916, 2009.

This review summarizes the recently published data on the molecular mechanisms of Chlamydiae-host cell interaction, first of all, on chlamydial effector proteins. Such proteins, along with type III transport system proteins, which transfer many effector proteins into the host cytoplasm, are attractive targets for drug therapy of chlamydial infections. The majority of the data concerns two species, Chlamydia trachomatis and Chlamydophila pneumoniae. The C. trachomatis protein TARP, which is presynthesized in elementary bodies, plays an essential role in the initial stages of infection. The pathogen proteins that are involved in the next stage, which is the intracellular inclusion traffic to the centrosome, are C. trachomatis CT229 and C. pneumoniae Cpn0585, which interact with cell Rab GTPases. In C. trachomatis, IncA plays a key role in the fusion of chlamydial inclusions, CT847 modulates the life cycle of the host cell, and LDA3 is essential for the acquisition of nutrients. The protease CPAF and the inclusion membrane proteins IncG and CADD are involved in suppressing apoptosis of infected cells. The proteases CPAF and CT441 and the deubiquitinating protein ChlaDub1 help the pathogen to evade the immune response.

POINCARE'S THEOREM FOR THE MODULAR GROUP OF REAL RIEMANN SURFACES.

A. F. Costa and S. M. Natanzon.

Differential Geometry and Its Applications 27 (5):680-690, 2009.

Let Mod(g) denote the modular group of (closed and orientable) surfaces S of genus g. Each element vertical bar h vertical bar is an element of Mod(g) induces a symplectic automorphism H(vertical bar h vertical bar) of H-1(S, Z). Poincare showed that H : Mod(g) à Sp(2g, Z) is an epimorphism. A real Riemann surface is a Riemann surface S together with an anticonformal involution sigma. Let (S, sigma) be a real Riemann surface, Homeo(g)(sigma) be the group of orientation preserving homeomorphisms of S such that h o sigma = sigma o h and Homeo(g.0)(sigma) be the subgroup of Homeo(g)(sigma) consisting of those isotopic to the identity by an isotopy in Homeo(g)(sigma). The group Mod(g)(sigma) = Homeo(g)(sigma) / Homeo(g.0)(sigma) plays the role of the modular group in the theory of real Riemann surfaces. In this work we describe the image by H of Mod(g)(sigma). Such image depends on the topological type of the involution sigma.