EFFECT OF ACIDOSIS, OXIDATIVE STRESS, AND GLUTAMATE TOXICITY ON THE SURVIVAL OF MATURE AND IMMATURE CULTURED CEREBELLAR GRANULE CELLS

Stelmashuk E.V., Belyaeva E.A., Isaev N.K.

Neurochemical Journal 1 (2007) 66-69.

The effects of various pathogenic factors on the viability of cultured immature and mature granule cells of rat cerebellum that developed during brain hypoxia and reoxygenation were compared. These factors included acidosis, oxidative stress, and glutamate toxicity. Incubation of both mature (seven-nine daysof culturing) and immature (three-four days of culturing) cerebellar granule cells for 24 hours under the conditions of external acidosis or oxidative stress was shown to induce neuronal death in these cultures. Immature neurons were significantly more sensitive to these factors than mature ones. Immature neurons were also more sensitive to the staurosporine apoptosis inductor. However, glutamate treatment (100 mu M) resulted in neuronal death only in the mature cultures. The results demonstrated that immature neurons were more sensitive to damaging factors not connected with glutamate toxicity than the mature neurons.

NEW METHOD OF IN VITRO CULTURING OF PIGMENT RETINAL EPITHELIUM IN THE STRUCTURE OF THE POSTERIOR EYE SECTOR OF ADULT RAT

Grigoryan E.N., Novikova Y.P., Kilina O.V., Philippov P.P.

Bulletin of Experimental Biology and Medicine 144 (2007) 618-625.

We propose a new method of organotypic roller 3D-culturing of the posterior sector of the eye. The method allows maintaining tissue viability in vitro for 14 days (which considerably surpasses the capacities of stationary culturing) and studying of the behavior of pigment retinal epithelial cells and choriocapillary membrane. Using this method we demonstrated phenotypic transformation, migration, and proliferation of pigment retinal epithelial cells under conditions of roller organotypic culture. In the absence of the retina, these cells exhibit properties of scavenger cells (phagocytes) both within and outside the layer. Under conditions of roller culturing in vitro, cells of the pigment retinal epithelium undergo changes similar to those observed in various retinal pathologies in vivo, including age-associated changes. Here we discuss the possibility of using the proposed method for evaluation of the effect of various factors added to the culture medium on the pigment epithelium, for modeling of processes developing in damaged pigment epithelium or under conditions of various pathologies, and for the study of regeneration responses in cells of pigment retinal epithelium in adult vertebrates.

OPTIMAL COUPLING OF SUBANTENNAS AS A STRATEGY FOR EFFICIENT FUNCTIONING OF THE LIGHT-HARVESTING ANTENNAS IN PHOTOSYNTHESIZING ORGANISMS: MODEL COMPUTATIONS

Zobova A.V., Fetisova Z.G.

Doklady Biochemistry and Biophysics 416 (2007) 281-284.

QUANTUM YIELD OF CHARGE SEPARATION AND FLUORESCENCE IN PHOTOSYSTEM II OF GREEN PLANTS

Shuvalov V.A., Dolgova T.A.

Doklady Biochemistry and Biophysics 416 (2007) 268-270.

DETECTION OF TYPE 2 HERPES SIMPLEX VIRUS IN CELLS OF SPERMATOGENIC EPITHELIUM IN INFECTED TESTES OF GUINEA PIGS

Gribencha S.V., Bragina E.E., Abdumalikov R.A., Bocharova E.N., Kurilo L.F.

Bulletin of Experimental Biology and Medicine 144 (2007) 73-76.

We developed a model of herpetic orchitis in guinea pigs. Intratesticular inoculation of type 2 herpes simplex virus suspension results in infection of the testicular spermatocytes and spermatides. The possibility of viral infection dissemination from infected into intact testis is proven.

INFLORESCENCE AND EARLY FLOWER DEVELOPMENT IN LOTEAE (LEGUMINOSAE) IN A PHYLOGENETIC AND TAXONOMIC CONTEXT

Sokoloff D.D., Degtjareva G.V., Endress P.K., Remizowa M.V., Samigullin T.H., Valiejo-Roman C.M.

International Journal of Plant Sciences 168 (2007) 801-833.

Molecular phylogeny shows that the temperate legume tribe Loteae is close to the mostly tropical Robinieae and monogeneric Sesbanieae, but comparative morphological studies of these groups are limited. Unusual patterns of inflorescence symmetry and calyx development have been described in some Loteae, but taxon sampling was low. We studied these features with scanning electron microscopy in 25 species of Loteae plus in three Robinia species. Phylogenetic trees of Loteae based on nuclear ribosomal ITS sequences and 77 morphological characters are constructed. Our data show that whorled flower arrangement is a synapomorphy of Loteae; joint initiation of the two adaxial sepals is a synapomorphy of a clade containing Hippocrepis, Scorpiurus, and Coronilla; floral buds bent backward early in development are a synapomorphy of Coronilla; bilateral umbel symmetry and the presence of a single whorl of flowers are probably primitive within Loteae. Inflorescences of Robinia show no special similarities with those of Loteae. Developmental data support homologies between sterile bracts in all Loteae. Even if the sterile bract is situated at the top of the peduncle, it is morphologically the first leaf on the peduncle. Monosymmetric umbels of Loteae (including the model legume Lotus japonicus) could be useful for investigating genetic control of symmetry in structures of hierarchic levels higher than flowers.

EFFICIENT TRANSLATION INITIATION DIRECTED BY THE 900-NUCLEOTIDE-LONG AND GC-RICH 5 ' UNTRANSLATED REGION OF THE HUMAN RETROTRANSPOSON LINE-1 MRNA IS STRICTLY CAP DEPENDENT RATHER THAN INTERNAL RIBOSOME ENTRY SITE MEDIATED

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

Molecular and Cellular Biology 27 (2007) 4685-4697.

Retrotransposon L1 is a mobile genetic element of the LINE family that is extremely widespread in the mammalian genome. It encodes a dicistronic mRNA, which is exceptionally rare among eukaryotic cellular mRNAs. The extremely long and GC-rich L1 5' untranslated region (5'UTR) directs synthesis of numerous copies of RNA-binding protein ORF1p per mRNA. One could suggest that the 5'UTR of L1 mRNA contained a powerful internal ribosome entry site (IRES) element. Using transfection of cultured cells with the polyadenylated monocistronic (L1 5'UTR-Fluc) or bicistronic (Rluc-L1 5'UTR-Fluc) RNA constructs, capped or uncapped, it has been firmly established that the 5'UTR of L1 does not contain an IRES. Uncapping reduces the initiation activity of the L1 5'UTR to that of background. Moreover, the translation is inhibited by upstream AUG codons in the 5'UTR. Nevertheless, this cap-dependent initiation activity of the L1 5'UTR was unexpectedly high and resembles that of the beta-actin 5'UTR (84 nucleotides long). Strikingly, the deletion of up to 80% of the nucleotide sequence of the L1 5'UTR, with most of its stem loops, does not significantly change its translation initiation efficiency. These data can modify current ideas on mechanisms used by 40S ribosomal subunits to cope with complex 5'UTRs and call into question the conception that every long GC-rich 5'UTR working with a high efficiency has to contain an IRES. Our data also demonstrate that the ORF2 translation initiation is not directed by internal initiation, either. It is very inefficient and presumably based on a reinitiation event.

CHANGES IN THE SHAPE OF PHOTODYNAMICALLY DAMAGED TETRAHYMENA PYRIFORMIS CELLS

Brailovskaya I.V., Kudryavtseva T.A., Larionov V.N., Prikhod'ko E.A., Mokhova E.N.

Doklady Biochemistry and Biophysics 413 (2007) 72-75.

WHEAT ENDONUCLEASE WEN1 DEPENDENT ON S-ADENOSYL-L-METHIONINE AND SENSITIVE TO DNA METHYLATION STATUS

Fedoreyeva L.I., Sobolev D.E., Vanyushin B.F.

Epigenetics 2 (2007) 50-53.

Ca2+-, Mg2+-dependent wheat endonuclease WEN1 with molecular mass of about 27 kDa was isolated from coleoptyles. Methylated DNA of l phage grown on E. coli dam(+), dcm(+) cells was hydrolyzed by WEN1 more effectively than DNA of phage grown on dam(-), dcm(-) cells. Two pH activity maxima (pH 6.5-7.5 and 9.0-10.5) were observed when double-stranded DNA was hydrolyzed. WEN1 is stable at elevated temperatures (65 degrees C) and in wide range of pH values. WEN1 is activated by S-adenosyl-L-methionine, S-adenosyl-L-homocysteine and S-isobutyladenosine. It is a first case to show that higher eukaryote endonuclease discriminates between DNA of various methylation status and is modulated by S-AdoMet and its analogs.

IS MONOMERIC ALPHA-SYNUCLEIN ABLE TO FORM ION CHANNELS?

Zakharov S.D., Dutseva E., Hulleman J.D., Antonenko Y.N., Rochet J.C., Cramer W.A.

Biophysical Journal (2007) 396A-397A.

COMPARATIVE STUDY OF TOPOGENESIS OF CYTOCHROME P450SCC (CYP11A1) AND ITS HYBRIDS WITH ADRENODOXIN EXPRESSED IN ESCHERICHIA COLI CELLS

Vinogradova A.A., Luzikov V.N., Novikova L.A.

Biochemistry-Moscow 72 (2007) 208-214.

Hybrid proteins consisting of the mature form of cytochrome P450scc (mP) and adrenodoxin (Ad), attached to either the NH2-or COOH-terminus (Ad-mP and mP-Ad, respectively), were expressed in E. coli. Spectral and catalytic properties of P450scc were studied using the membrane fraction of E. coli cells. It has been shown that the Ad amino acid sequence attached to the termini of the P450scc-domain neither affects the insertion of a hybrid protein into the cytoplasmic membrane nor influences its heme binding ability. The results suggest that Ad attached to the NH2-terminus does not markedly affect the folding of the P450scc-domain, but cholesterol hydroxylase/lyase activity of the Ad-mP hybrid was found to be much lower than that of the native P450scc enzyme. The modification of the COOH-terminus does not alter the specific P450scc activity, but results in a dramatic increase in the amount of hybrid protein with incorrectly folded P450scc domain.

CLOCKWISE OR ANTICLOCKWISE? TURNING THE CENTRIOLE TRIPLETS IN THE RIGHT DIRECTION!

Uzbekov R., Prigent C.

Febs Letters 581 (2007) 1251-1254.

Centrosomes are small cytoplasmic macromolecular assemblies composed from two major components, centrioles and pericentriolar material, each with its own complex architecture. This organelle is of interest because it plays a role in a number of fundamental cellular processes and defects in these processes have recently been correlated with variety of human disease. Increasingly, what is known about the structure of this organelle has been overshadowed by the increasing wealth of information on its biochemistry. In this short review, we highlight some of the common centriole structural errors found in the literature and define a set of rules that define centriole structure. .

KINETIC AND MUTATIONAL ANALYSES OF THE MAJOR CYTOSOLIC EXOPOLYPHOSPHATASE FROM SACCHAROMYCES CEREVISIAE

Tammenkoski M., Moiseev V.M., Lahti M., Ugochukwu E., Brondijk T.H.C., White S.A., Lahti R., Baykov A.A.

Journal of Biological Chemistry 282 (2007) 9302-9311.

Yeast exopolyphosphatase (scPPX) processively splits off the terminal phosphate group from linear polyphosphates longer than pyrophosphate. scPPX belongs to the DHH phosphoesterase superfamily and is evolutionarily close to the well characterized family II pyrophosphatase (PPase). Here, we used steady-state kinetic and binding measurements to elucidate the metal cofactor requirement for scPPX catalysis over the pH range 4.2-9.5. A single tight binding site for Mg2+ (K-d of 24 mu m) was detected by equilibrium dialysis. Steady-state kinetic analysis of tripolyphosphate hydrolysis revealed a second site that binds Mg2+ in the millimolar range and modulates substrate binding. This step requires two protonated and two deprotonated. enzyme groups with pK(a) values of 5.0-5.3 and 7.6-8.2, respectively. The catalytic step requiring two deprotonated groups (pK, of 4.6 and 5.6) is modulated by ionization of a third group (pK, of 8.7). Conservative mutations of Asp(127), His(148), His(149) (conserved in scPPX and PPase), and Asn(35) (His in PPase) reduced activity by a factor of 600-5000. N35H and D127E substitutions reduced the Mg2+ affinity of the tight binding site by 25-60-fold. Contrary to expectations, the N35H variant was unable to hydrolyze pyrophosphate, but markedly altered metal cofactor specificity, displaying higher catalytic activity with Co2+ bound to the weak binding site versus the Me2+-or Mn2+-bound enzyme. These results provide an initial step toward understanding the dynamics of scPPX catalysis and reveal significant functional differences between structurally similar scPPX and familly II PPase.

PREPARATION OF FUNCTIONALLY ACTIVE RECOMBINANT HUMAN INTERLEUKIN-6

Spiridonova V.A., Lygina A.S., Anohina M.M., Tupitsyn N.N.

Biochemistry-Moscow 72 (2007) 424-429.

The gene of human interleukin-6 (hIL-6) with an additional 20 amino acids on the N-end, including six histidine residues, was cloned into the expression plasmid pET28b(+). The conditions were elaborated for preparing highly active protein both using denaturing agents and without them. Application of a dialysis cascade allowed us to prepare a functionally active hIL-6 of 90-95% purity with the yield of 3 mg from liter of the cell culture. The highest activity was detected by ELISA in the preparation obtained without denaturing agents. The functional activity of hIL-6 was studied by flow cytofluorimetry. Addition of hIL-6 to the cells of immortal lines of human multiple myeloma resulted in dimerization of the gp130 receptor molecule.

INTERACTION OF SULFONATED METALLOPHTHALOCYANINES WITH BILAYER LIPID MEMBRANES: PHOTOCHEMICAL ACTIVITY VERSUS ADSORPTION ON THE MEMBRANE SURFACE

Sokolenko E.A., Pashkovskaya A.A., Kotova E.A., Sokolov V.S., Antonenko Y.N.

Biophysical Journal (2007) 239A-239A.

BINDING OF SUBSTRATE AT THE EFFECTOR SITE OF PYROPHOSPHATASE INCREASES THE RATE OF ITS HYDROLYSIS AT THE ACTIVE SITE

Sitnik T.S., Avaeva S.M.

Biochemistry-Moscow 72 (2007) 68-76.

It is shown that in addition to the active site, each subunit of Escherichia coli inorganic pyrophosphatase (E-PPase) contains an extra binding site for the substrate magnesium pyrophosphate or its non-hydrolyzable analog magnesium methylenediphosphonate. The occupancy of the extra site stimulates the substrate conversion. Binding affinity of this site decreased or disappeared upon the conversion of E-PPase into a trimeric form or introduction of point mutations. However, when the slowly hydrolyzed substrate, lanthanum pyrophosphate (LaPPi), is used, the extra site was revealed in all enzyme forms of E-PPase and of Y-PPase (Saccharomyces cerevisiae PPase), resulting in about 100-fold activation of hydrolysis. A hypothesis on the localization of the extra site and the mechanism of its effect in E-PPase is presented.

BRANCHED CHAIN KETO-ACIDS EXERT BIPHASIC EFFECTS ON ALPHA-KETOGLUTARATE-STIMULATED RESPIRATION IN INTACT RAT LIVER MITOCHONDRIA

Shestopalov A.I., Kristal B.S.

Neurochemical Research 32 (2007) 947-951.

Pathophysiological concentrations of branched chain keto-acids (BCKAs), such as those that occur in maple syrup urine disease, inhibit oxygen consumption in liver homogenates and brain slices and the enzymatic activity of alpha-ketoglutarate- and pyruvate dehydrogenase complexes. Consistent with previous work, studies in isolated rat liver mitochondria indicate that three BCKAs, alpha-ketoisocaproate (KIC), alpha-keto-beta-methylvalerate (KMV) and alpha-ketoisovalerate (KIV), preferentially inhibited State 3 respiration supported by alpha-ketoglutarate relative to succinate or glutamate/malate (KIC, > 100-fold; KMV, > 10-fold; KIV, > 4-fold). KIC was also the most potent inhibitor (K-i,K-app 13 +/- 2 mu M). Surprisingly, sub-inhibitory concentrations of KIC and KMV can markedly stimulate State 3 respiration of mitochondria utilizing alpha-ketoglutarate and glutamate/malate, but not succinate. The data suggest that physiological concentrations of the BCKAs may modulate mitochondrial respiration.

NUCLEOTIDE-INDUCED AND ACTIN-INDUCED STRUCTURAL CHANGES IN SH1-SH2-MODIFIED MYOSIN SUBFRAGMENT 1

Shakirova L., Mikhailova V., Siletskaya E., Timofeev V.P., Levitsky D.I.

Journal of Muscle Research and Cell Motility 28 (2007) 67-78.

We compared the structural properties of myosin subfragment 1 (S1) modified at both reactive SH-groups, SH1 (Cys707) and SH2 (Cys697), with the properties of unmodified S1 and SH1-modified S1. It is shown using differential scanning calorimetry (DSC) that SH1 modification has no noticeable influence on the changes in S1 thermal unfolding induced by the formation of S1 ternary complexes with ADP and P-i analogs (V-i, AlF4-, and BeF (x) ). These changes, however, normally expressed in a significant increase of S1 thermal stability, are almost fully prevented by modification of both SH1 and SH2. In contrast, SH2 modification had no effect on the changes induced by the formation of the ternary complexes S1-ADP-V-i, S1-ADP-AlF4-, and S1-ADP-BeF (x) in EPR spectra of S1 spin-labeled at SH1 group. Interaction of S1 with F-actin substantially increased the thermal stability of S1; a similar effect was observed by DSC with both SH1- and SH1-SH2-modified S1. Overall, our results demonstrate that modification of both reactive SH-groups on S1 has no influence on the actin-induced changes of S1 and on the local nucleotide-induced conformational changes in the SH1 group region, but strongly prevents the global nucleotide-induced structural changes in the entire S1 molecule. The results suggest that modification of SH1 and SH2 impairs the spread of nucleotide-induced conformational changes from the ATPase site throughout the structure of the entire S1 molecule, thus disturbing a coupling between the motor and regulatory domains in the myosin head.

RIBOSOMAL RNA GUANINE-(N2)-METHYLTRANSFERASES AND THEIR TARGETS

Sergiev P.V., Bogdanov A.A., Dontsova O.A.

Nucleic Acids Research 35 (2007) 2295-2301.

Five nearly universal methylated guanine-(N2) residues are present in bacterial rRNA in the ribosome. To date four out of five ribosomal RNA guanine-(N2)-methyltransferases are described. RsmC(YjjT) methylates G1207 of the 16S rRNA. RlmG(YgjO) and RlmL(YcbY) are responsible for the 23S rRNA m(2)G1835 and m(2)G2445 formation, correspondingly. RsmD(YhhF) is necessary for methylation of G966 residue of 16S rRNA. Structure of Escherichia coli RsmD(YhhF) methyltransferase and the structure of the Methanococcus jannaschii RsmC ortholog were determined. All ribosomal guanine-(N2)-methyltransferases have similar AdoMet-binding sites. In relation to the ribosomal substrate recognition, two enzymes that recognize assembled subunits are relatively small single domain proteins and two enzymes that recognize naked rRNA are larger proteins containing separate methyltransferase- and RNA-binding domains. The model for recognition of specific target nucleotide is proposed. The hypothetical role of the m(2)G residues in rRNA is discussed.

PRIMARY EVENTS IN CYANOBACTERIAL PHOTOSYSTEM I COMPLEXES STUDIED USING FEMTOSECOND SELECTIVE EXCITATION OF ANTENNA AND REACTION CENTER CHLOROPHYLLS

Semenov A., Shelaev I., Gostev F., Nadtochenko V., Mamedov M., Gopta O., Shuvalov V., Sarkisov O.

Photosynthesis Research 91 (2007) S262.

REVERSIBLE INHIBITION OF ESCHERICHIA COLI INORGANIC PYROPHOSPHATASE BY FLUORIDE: TRAPPED CATALYTIC INTERMEDIATES IN CRYO-CRYSTALLOGRAPHIC STUDIES

Samygina V.R., Moiseev V.M., Rodina E.V., Vorobyeva N.N., Porov A.N., Kurilova S.A., Nazarova T.I., Avaeva S.M., Bartunik H.D.

Journal of Molecular Biology 366 (2007) 1305-1317.

Here, we describe high-resolution X-ray structures of Escherichia coli inorganic pyrophosphatase (E-PPase) complexed with the substrate, magnesium, or manganese pyrophosphate. The structures correspond to steps in the catalytic synthesis of enzyme-bound pyrophosphate (PPi) in the presence of fluoride as an inhibitor of hydrolysis. The catalytic reaction intermediates were trapped applying a new method that we developed for initiating hydrolytic activity in the E-PPase crystal. X-ray structures were obtained for three consecutive states of the enzyme in the course of hydrolysis. Comparative analysis of these structures showed that the Mn2+-supported hydrolysis of the phosphoanhydride bond is followed by a fast release of the leaving phosphate from the P1 site. The electrophilic phosphate P2 is trapped in the "down" conformation. Its movement into the "up" position most likely represents the rate-limiting step of Mn2+-supported hydrolysis. We further determined the crystal structure of the Arg43Gln mutant variant of E-PPase complexed with one phosphate and four Mn ions. .

ATP AS EFFECTOR OF INORGANIC PYROPHOSPHATASE OF ESCHERICHIA COLI. THE ROLE OF RESIDUE LYS112 IN BINDING EFFECTORS

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

Biochemistry-Moscow 72 (2007) 100-108.

It has been shown that PPi, methylenediphosphonate, and ATP act as effectors of Escherichia coli inorganic pyrophosphatase (E-PPase), and that they compete for binding at the allosteric regulatory site. On the basis of chemical modification and computer modeling of a structure of the enzyme-ATP complex, a number of amino acid residues presumably involved in binding effectors has been revealed. Mutant variants Lys112Gln, Lys112Gln/Lys148Gln, and Lys112Gln/Lys115Ala of E-PPase have been obtained, as well as a modified variant of wild type E-PPase ((Ad)wt PPase) with a derivative of ATP chemically attached to the amino group of Lys146. Kinetic properties of these variants have been investigated and compared to the earlier described variants Lys115Ala, Arg43Gln, and Lys148Gln. Analysis of the data confirms the proposed location of an effector binding site in a cluster of positively charged amino acid residues including the side chains of Arg43, Lys146 (subunit A), Lys112, and Lys115 (subunit B). Lys112 is supposed to play a key role in forming contacts with the phosphate groups of the three studied effectors.

ATP AS EFFECTOR OF INORGANIC PYROPHOSPHATASE OF ESCHERICHIA COLI. IDENTIFICATION OF THE BINDING SITE FOR ATP

Rodina E.V., Vorobyeva N.N., Kurilova S.A., Belenikin M.S., Fedorova N.V., Nazarova T.I.

Biochemistry-Moscow 72 (2007) 93-99.

The interaction of Escherichia coli inorganic pyrophosphatase (E-PPase) with effector ATP has been studied. The E-PPase has been chemically modified with the dialdehyde derivative of ATP. It has been established that in the experiment only one molecule of effector ATP is bound to each subunit of the hexameric enzyme. Tryptic digestion of the adenylated protein followed by isolation of a modified peptide by HPLC and its mass-spectrometric identification has showed that it is an amino group of Lys146 that undergoes modification. Molecular docking of ATP to E-PPase indicates that the binding site for effector ATP is located in a cluster of positively charged amino acid residues proposed earlier on the basis of site-directed mutagenesis to participate in binding of effector pyrophosphate. Molecular docking also reveals several other amino acid residues probably involved in the interaction with effectors.

CRITICAL ROLE OF ELECTROSTATIC INTERACTIONS OF AMINO ACIDS AT THE CYTOPLASMIC REGION OF HELICES 3 AND 6 IN RHODOPSIN CONFORMATIONAL PROPERTIES AND ACTIVATION

Ramon E., Cordomi A., Bosch L., Zernii E.Y., Senin I.I., Manyosa J., Philippov P.P., Perez J.J., Garriga P.

Journal of Biological Chemistry 282 (2007) 14272-14282.

The cytoplasmic sides of transmembrane helices 3 and 6 of G-protein-coupled receptors are connected by a network of ionic interactions that play an important role in maintaining its inactive conformation. To investigate the role of such a network in rhodopsin structure and function, we have constructed single mutants at position 134 in helix 3 and at positions 247 and 251 in helix 6, as well as combinations of these to obtain double mutants involving the two helices. These mutants have been expressed in COS-1 cells, immunopurified using the rho-1D4 antibody, and studied by UV-visible spectrophotometry. Most of the single mutations did not affect chromophore formation, but double mutants, especially those involving the T251K mutant, resulted in low yield of protein and impaired 11-cisretinal binding. Single mutants E134Q, E247Q, and E247A showed the ability to activate transducin in the dark, and E134Q and E247A enhanced activation upon illumination, with regard to wild-type rhodopsin. Mutations E247A and T251A (in E134Q/E247A and E134Q/T251A double mutants) resulted in enhanced activation compared with the single E134Q mutant in the dark. A role for Thr(251) in this network is proposed for the first time in rhodopsin. As a result of these mutations, alterations in the hydrogen bond interactions between the amino acid side chains at the cytoplasmic region of transmembrane helices 3 and 6 have been observed using molecular dynamics simulations. Our combined experimental and modeling results provide new insights into the details of the structural determinants of the conformational change ensuing photoactivation of rhodopsin.

INHIBITION OF HIV-1 INTEGRASE BY MODIFIED OLIGONUCLEOTIDES: OPTIMIZATION OF THE INHIBITOR STRUCTURE

Prikazchikova T.A., Volkov E.M., Zubin E.M., Romanova E.A., Gottikh M.B.

Molecular Biology 41 (2007) 118-125.

Integration of human immunodeficiency virus type 1 DNA into the infected cell genome is one of the key steps of the viral replication cycle. Therefore, viral integrase is of interest as a target for new antiviral drugs. Conjugates of 11-mer single-stranded oligonucleotides with hydrophobic molecules were shown to be efficient integrase inhibitors, inducing dissociation of the integrase-viral DNA complex. The dependence of the conjugate inhibitory activity on the oligonucleotide length and structure as well as on the structure of hydrophobic molecules was studied. Conjugates with eosin and oleic acid proved to be the most active. Conjugates of these molecules with 2'-O-methyl-oligonucleotide inhibited integrase at concentrations 50-100 nM but did not influence some other DNA-binding enzymes.

KINETIC ANALYSIS OF THE INTERACTION OF GUANINE NUCLEOTIDES WITH EUKARYOTIC TRANSLATION INITIATION FACTOR EIF5B

Pisareva V.P., Hellen C.U.T., Pestova T.V.

Biochemistry 46 (2007) 2622-2629.

Eukaryotic translation initiation factor eIF5B is a ribosome-dependent GTPase that is responsible for the final step in initiation, which involves the displacement of initiation factors from the 40S ribosomal subunit in initiation complexes and its joining with the 60S subunit. Hydrolysis of eIF5B-bound GTP is not required for its function in subunit joining but is necessary for the subsequent release of eIF5B from assembled 80S ribosomes. Here we investigated the kinetics of guanine nucleotide binding to eIF5B by a fluorescent stopped-flow technique using fluorescent mant derivatives of GTP and GDP and of the GTP analogues GTP gamma S and GMPPNP. The affinity of eIF5B for mant-GTP (K-d similar to 14-18 mu M) was approximately 7-fold less than for mant-GDP (K-d similar to 2.3 mu M), and both guanine nucleotides dissociated rapidly from eIF5B (k(-1)(mant-GTP) similar to 22-28 s(-1),k(-1)(mant-GDP) similar to 10-14 s(-1)). These properties of eIF5B suggest a rapid spontaneous GTP/GDP exchange on eIF5B and are therefore consistent with it having no requirement for a special guanine nucleotide exchange factor. The affinity of eIF5B for mant-GTP gamma S was about 2 times lower (K-d similar to 6.9 mu M) and for mant-GMPPNP 1.5 times higher (K-d similar to 25.7 mu M) than for mant-GTP, indicating that eIF5B tolerates modifications of the triphosphate moiety well.

MOLECULAR PHYLOGENY OF GASTROTRICHA ON THE BASIS OF A COMPARISON OF THE 18S RRNA GENES: REJECTION OF THE HYPOTHESIS OF A RELATIONSHIP BETWEEN GASTROTRICHA AND NEMATODA

Petrov N.B., Pegova A.N., Manylov O.G., Vladychenskaya N.S., Mugue N.S., Aleshin V.V.

Molecular Biology 41 (2007) 445-452.

Gastrotricha are the small meiobenthic acoelomate worms whose phylogenetic relationships between themselves and other invertebrates remain unclear, despite all attempts to clarify them on the basis of both morphological and molecular analyses. The complete sequences of the I SS rRNA genes (8 new and 7 known) were analyzed in 15 Gastrotricha species to test different hypotheses on the phylogeny of this taxon and to determine the reasons for the contradictions in earlier results. The data were analyzed using both maximum likelihood and Bayes an methods. Based on the results, it was assumed that gastrotrichs form a monophyletic group within the Spiralia clade, which also includes Gnathostomulida, Plathelminthes, Syndermata (Rotifera + Acanthocephala), Nemertea, and Lophotrochozoa. Statistical tests rejected a phylogenetic hypotheses considering Gastrotricha to be closely related to Nematoda and other Ecdysozoa or placing them at the base of the Bilateria tree, close to Acoela or Nemertodermatida. Among gastrotrichs, species belonging to the orders Chaetonotida and Macrodasyida form two well-supported clades. The analysis confirmed monophyly of the families Chaetonotidae and Xenotrichulidae from the order Chaetonida, as well as the families Turbanellidae and Thaumastodermatidae from the order Macrodasyida. Lepidodasyidae is a polyphyletic family, because the genus Mesodasys forms a sister group for Turbanellidae; genus Cephalodasys forms a separate branch at the base of Macrodasyida; and Lepidodasys groups with Neodasys between Thaumastodermatidae and Turbanellidae. To confirm these conclusions and to get an authentic view of the phylogeny of Gastrotricha, it is necessary to study more Gastrotricha species and to analyze some other genes.

THE STUDY OF AMORPHOUS AGGREGATION OF TOBACCO MOSAIC VIRUS COAT PROTEIN BY DYNAMIC LIGHT SCATTERING

Panyukov Y., Yudin I., Drachev V., Dobrov E., Kurganov B.

Biophysical Chemistry 127 (2007) 9-18.

The kinetics of heat-induced and cetyltrimethylammonium bromide induced amorphous aggregation of tobacco mosaic virus coat protein in Na+/Na+ phosphate buffer, pH 8.0, have been studied using dynamic light scattering. In the case of thermal aggregation (52 degrees C) the character of the dependence of the hydrodynamic radius (R-h) on time indicates that at certain instant the population of aggregates is split into two components. The size of the aggregates of one kind remains practically constant in time, whereas the size of aggregates of other kind increases monotonously in time reaching the values characteristic of aggregates prone to precipitation (R-h = 900-1500 nm). The construction of the light scattering intensity versus R-h plots shows that the large aggregates (the start aggregates) exist in the system at the instant the initial increase in the light scattering intensity is observed. For thermal aggregation the R-h value for the start aggregates is independent of the protein concentration and equal to 21.6 nm. In the case of the surfactant-induced aggregation (at 25 degrees C) no splitting of the aggregates into two components is observed and the size of the start aggregates turns out to be much larger (107 nm) than on the thermal aggregation. The dependence of R-h on time for both heat-induced aggregation and surfactant-induced aggregation after a lapse of time follows the power law indicating that the aggregation process proceeds in the kinetic regime of diffusion-limited cluster-cluster aggregation. Fractal dimension is close to 1.8. The molecular chaperone alpha-crystallin does not affect the kinetics of tobacco mosaic virus coat protein thennal aggregation. .

INFLUENCE OF DONOR SUBSTRATE ON KINETIC PARAMETERS OF THIAMINE DIPHOSPHATE BINDING TO TRANSKETOLASE

Ospanov R.V., Kochetov G.A., Kurganov B.I.

Biochemistry-Moscow 72 (2007) 84-92.

The two-step mechanism of interaction of thiamine diphosphate (ThDP) with transketolase (TK) has been studied: TK + ThDP <-> TK center dot center dot center dot ThDP <-> TK*-ThDP. The scheme involves the formation of inactive intermediate complex TK center dot center dot center dot ThDP followed by its transformation into catalytically active holoenzyme, TK*-ThDP. The dissociation and kinetic constants for individual stages of this process have been determined. The values of forward and backward rate constants change in the presence of the donor substrate hydroxypruvate. This finally leads to an increase in the overall affinity of the coenzyme to TK.

KEY ROLE OF THE INTERNAL 5'-UTR SEGMENT IN THE TRANSCRIPTION ACTIVITY OF THE HUMAN L1 RETROTRANSPOSON

Olovnikov I.A., Adyanova Z.V., Galimov E.R., Andreev D.E., Terenin I.M., Ivanov D.S., Prassolov V.S., Dmitriev S.E.

Molecular Biology 41 (2007) 453-458.

The long 5'-untranslated region (5'-UTR) of the human L1 retrotransposon contains a unique internal promoter. allowing new L1 copies to be less dependent on the integration site at the transcriptional level. The mechanism of action of this promoter still remains unclear; however, some early studies have build up an opinion that the first 5'-UTR segment of 100-150 nt (known as the minimal promoter) is most crucial for the functioning of the full-length promoter. This study shows that the activity of the minimal promoter is rather low in comparison with the activity of the full-length 5'-UTR. Instead, 5'-UTR internal segment 390-662, containing numerous binding sites for various transcription factors, is indispensable for effective L1 transcription and can be considered as a transcriptional enhancer. Deletion of this segment dramatically reduces the level of transcription irrespective of the cell type, whereas deletion of the first 100 nt decreases the transcription level only by a factor of 1.5-2. Thus, the L1 regulatory region remains to be structurally similar to that of well-studied invertebrate LINEs. It is also possible that the internal 5'-UTR segment of L1 contains an alternative promoter, driving synthesis of 5'-truncated L1 mRNA.

ANALYSIS OF SPECTRAL CONJUGATION OF NONUNIFORM SUBANTENNAE IN THE LIGHT-HARVESTING SUPERANTENNA OF OSCILLOCHLORIDACEAE PHOTOSYNTHETIC GREEN BACTERIA

Novikov A.A., Taisova A.S., Fetisova Z.G.

Biofizika 52 (2007) 63-68.

The study is concerned with the problem of optimal spectral coupling of subantennal as a strategy of effective functioning of light-harvesting antennal of photosynthesizing organisms. A theoretical analysis of the optimality of spectral coupling of currently known spectrally inhomogeneous subantennal (B750, B805-860) in the superantenna of green bacteria Oscillochloris trichoides (from the new family Oscillochloridacea discovered by Russian researchers in 2000), performed in the study, showed that the spectral composition of subantennal is functionally nonoptimal. This made it possible to predict the occurrence of an additional subantenna (B-X) with an intermediate energy position (750 nm < X < 805 nm) for the optimization of energy transfer along this superantenna by the functional criterion.

ONCOIMMUNOLOGY: SOME FUNDAMENTAL PROBLEMS OF CANCER IMMUNOTHERAPY

Nedospasov S.A., Kuprash D.V.

Molecular Biology 41 (2007) 316-328.

The review briefly discusses several central problems of modern oncoimmunology. The controversies surrounding the concept of immunological surveillance, as well as the problem of immunological tolerance to tumors, are considered. The discovery of tumor antigens is a great advance towards the identification of possible therapeutic targets. However, antigen-specific vaccinations against cancer have, so far, a very limited use, mainly for prevention of virus-associated cancers, which is essentially based on the antiviral immune response. On the other hand, antibodies to cancer antigens are widely used in cancer diagnosis, and there are remarkable examples of their therapeutic applications. The future opportunities in both theoretical and applied oncoimmunology will directly depend on further advances in basic science.

A KINETIC MODEL OF FUNCTIONING AND REGULATION OF ESCHERICHIA COLI ISOCITRATE DEHYDROGENASE

Mogilevskaya E.A., Lebedeva G.V., Goryanin I.I., Dentin O.V.

Biofizika 52 (2007) 47-56.

A Rapid Equilibrium Random Bi Ter mechanism of formation of two dead-end complexes was proposed to describe the experimental data on the functioning of E.coli isocitrate dehydrogenase (IDH). A kinetic model for the enzyme functioning was constructed, which assumes that it is regulated through reversible phosphorylation by its kinase/phosphatase, which in turn is regulated by IDH substrates and central metabolites such as pyruvate (Pyr), 3-phosphoglycerate (3-PG), and AMP. It was shown using the model that increasing the concentration of these effectors results in an increase of the active part of IDH, thus leading to an increase in the Krebs cycle flux. We predict that the ratio of the phosphorylated and free forms of IDH (IDHP/IDH) is more sensitive to AMP, NADPH, and isocitrate concentrations than to Pyr and 3-PG. The model allows a realistic prediction of changes in the IDHP/IDH ratio, which would occur under changes of biosynthetic and energetic loading of the E.coli cell.

.

THE EFFECT OF MUTATIONS IN ALPHA-TROPOMYOSIN (E40K AND E54K) THAT CAUSE FAMILIAL DILATED CARDIOMYOPATHY ON THE REGULATORY MECHANISM OF CARDIAC MUSCLE THIN FILAMENTS

Mirza M., Robinson P., Kremneva E., Copeland O., Nikolaeva O., Watkins H., Levitsky D., Redwood C., EL-Mezgueldi M., Marston S.

Journal of Biological Chemistry 282 (2007) 13487-13497.

E40K and E54K mutations in alpha-tropomyosin cause inherited dilated cardiomyopathy. Previously we showed, using Ala-Ser alpha-tropomyosin (AS-alpha-Tm) expressed in Escherichia coli, that both mutations decrease Ca2+ sensitivity. E40K also reduces V-max of actin-Tm-activated S-1 ATPase by 18%. We investigated cooperative allosteric regulation by native Tm, AS-alpha-Tm, and the two dilated cardiomyopathy-causing mutants. AS-alpha-Tm has a lower cooperative unit size (6.5) than native-alpha-tropomyosin (10.0). The E40K mutation reduced the size of the cooperative unit to 3.7, whereas E54K increased it to 8.0. For the equilibrium between On and Off states, the K-T value was the same for all actin-Tm species; however, the K-T value of actin-Tm-troponin at pCa 5 was 50% less for AS-alpha-Tm E40K than for AS-alpha-Tm and AS-alpha-Tm E54K. K-b, the "closed" to "blocked" equilibrium constant, was the same for all tropomyosin species. The E40K mutation reduced the affinity of tropomyosin for actin by 1.74-fold, but only when in the On state (in the presence of S-1). In contrast the E54K mutation reduced affinity by 3.5-fold only in the Off state. Differential scanning calorimetry measurements of AS-alpha-Tm showed that domain 3, assigned to the N terminus of tropomyosin, was strongly destabilized by both mutations. Additionally with AS-alpha-Tm E54K, we observed a unique new domain at 55 degrees C accounting for 25% of enthalpy indicating stabilization of part of the tropomyosin. The disease-causing mechanism of the E40K mutation may be accounted for by destabilization of the On state of the thin filaments; however, the E54K mutation has a more complex effect on tropomyosin structure and function.

TWO DCM MUTATIONS IN CARDIAC TROPOMYOSIN ALTER THIN FILAMENT FUNCTION BY DIFFERENT MECHANISMS

Marston S., Mirza M., Copeland O., Robinson P., Redwood C., Levitsky D., Kremneva E., Nikolaieva O., EL-Mezgueldi M.

Biophysical Journal (2007) 625A-625A.

ATOMIC TRITIUM AS AN INSTRUMENT FOR STUDY OF PROTEIN BEHAVIOR AT THE AIR-WATER INTERFACE

Lukashina E.V., Badun G.A., Chulichkov A.L.

Biomolecular Engineering 24 (2007) 125-129.

Atomic tritium was successfully applied as an instrument for study of protein behavior at the air-water interface. Samples of lysozyme solution in 20 mM phosphate buffer (pH 7.0) with concentration of 2 mg/ml incubated at the room temperature for I h were exposed to bombardment with tritium atoms generated on hot tungsten wire in special vacuum device. This procedure resulted in substitution of hydrogen atoms by radioactive tritium in the thin surface layer of studied preparations. Analysis of experimental data on intramolecular radioactivity distribution in lysozyme and computer simulation of tritium bombardment allowed us to suggest two equally probable opposite orientations of lysozyme molecule in the adsorption layer at the air-water interface. .

METHYLTRANSFERASE THAT MODIFIES GUANINE 966 OF THE 16 S rRNA - FUNCTIONAL IDENTIFICATION AND TERTIARY STRUCTURE

Lesnyak D.V., Osipiuk J., Skarina T., Sergiev P.V., Bogdanov A.A., Edwards A., Savchenko A., Joachimiak A., Dontsova O.A.

Journal of Biological Chemistry 282 (2007) 5880-5887.

N-2-Methylguanine 966 is located in the loop of Escherichia coli 16 S rRNA helix 31, forming a part of the P-site tRNA-binding pocket. We found yhhF to be a gene encoding for m(2)G966 specific 16 S rRNA methyltransferase. Disruption of the yhhF gene by kanamycin resistance marker leads to a loss of modification at G966. The modification could be rescued by expression of recombinant protein from the plasmid carrying the yhhF gene. Moreover, purified m(2)G966 methyltransferase, in the presence of S-adenosylomethionine (AdoMet), is able to methylate 30 S ribosomal subunits that were purified from yhhF knock-out strain in vitro. The methylation is specific for G966 base of the 16 S rRNA. The m(2)G966 methyltransferase was crystallized, and its structure has been determined and refined to 2.05 angstrom. The structure closely resembles RsmC rRNA methyltransferase, specific for m(2)G1207 of the 16 S rRNA. Structural comparisons and analysis of the enzyme active site suggest modes for binding AdoMet and rRNA to m(2)G966 methyltransferase. Based on the experimental data and current nomenclature the protein expressed from the yhhF gene was renamed to RsmD. A model for interaction of RsmD with ribosome has been proposed.

PEPTIDE DERIVATIVES OF TYLOSIN-RELATED MACROLIDES

Korshunova G.A., Sumbatyan N.V., Fedorova N.V., Kumetsova I.V., Shishkina A.V., Bogdanov A.A.

Russian Journal of Bioorganic Chemistry 33 (2007) 218-226.

Approaches to the synthesis of model compounds based on the tylosin-related macrolides desmycosin and O-mycaminosyltylonolide were developed to study the conformation and topography of the nascent peptide chain in the ribosome tunnel using specially designed peptide derivatives of macrolide antibiotics. A method for selective bromoacetylation of desmycosin at the hydroxyl group of mycinose was developed, which involves preliminary acetylation of mycaminose. The reaction of the 4 ''-bromoacetyl derivative of the antibiotic with cesium salts of the dipeptide Boc-Ala-Ala-OH and the hexapeptide MeOTr-Gly-Pro-Gly-ProGly-Pro-OH led to the corresponding peptide derivatives of desmycosin. The protected peptides Boc-Ala-AlaOH, Boc-Ala-Ala-Phe-OH, and Boc-Gly-Pro-Gly-Pro-Gly-Pro-OH were condensed with the C23-hydroxyl group of O-mycaminosyltylonolide.

CAJAL BODIES AND THE NUCLEOLUS ARE REQUIRED FOR A PLANT VIRUS SYSTEMIC INFECTION

Kim S.H., Ryabov E.V., Kalinina N.O., Rakitina D.V., Gillespie T., MacFarlane S., Haupt S., Brown J.W.S., Taliansky M.

Embo Journal 26 (2007) 2169-2179.

The nucleolus and Cajal bodies (CBs) are prominent interacting subnuclear domains involved in a number of crucial aspects of cell function. Certain viruses interact with these compartments but the functions of such interactions are largely uncharacterized. Here, we show that the ability of the groundnut rosette virus open reading frame (ORF) 3 protein to move viral RNA long distances through the phloem strictly depends on its interaction with CBs and the nucleolus. The ORF3 protein targets and reorganizes CBs into multiple CB-like structures and then enters the nucleolus by causing fusion of these structures with the nucleolus. The nucleolar localization of the ORF3 protein is essential for subsequent formation of viral ribonucleoprotein (RNP) particles capable of virus long-distance movement and systemic infection. We provide a model whereby the ORF3 protein utilizes trafficking pathways involving CBs to enter the nucleolus and, along with fibrillarin, exit the nucleus to form viral 'transport-competent' RNP particles in the cytoplasm.

KINETICS OF MORPHOGEN GRADIENT FORMATION

Kicheva A., Pantazis P., Bollenbach T., Kalaidzidis Y., Bittig T., Julicher F., Gonzalez-Gaitan M.

Science 315 (2007) 521-525.

In the developing fly wing, secreted morphogens such as Decapentaplegic (Dpp) and Wingless (Wg) form gradients of concentration providing positional information. Dpp forms a longer-range gradient than Wg. To understand how the range is controlled, we measured the four key kinetic parameters governing morphogen spreading: the production rate, the effective diffusion coefficient, the degradation rate, and the immobile fraction. The four parameters had different values for Dpp versus Wg. In addition, Dynamin-dependent endocytosis was required for spreading of Dpp, but not Wg. Thus, the cellular mechanisms of Dpp and Wingless spreading are different: Dpp spreading requires endocytic, intracellular trafficking.

A SYSTEM FOR HETEROLOGOUS EXPRESSION AND ISOLATION OF ESCHERICHIA COLI RNA POLYMERASE AND ITS COMPONENTS

Khodak Y.A., Koroleva O.N., Drutsa V.L.

Biochemistry-Moscow 72 (2007) 178-187.

A set of plasmid vectors for expression of all major Escherichia coli RNA polymerase subunits as fusion proteins with intein-and chitin-binding domains, allowing protein purification in accordance with IMPACT technology, was constructed. It is demonstrated that the fusion subunits alpha, beta, or beta' in conjunction with the natural subunits alpha, beta, beta', and sigma can participate in RNA polymerase assembly in vivo, providing affinity-based isolation of the enzyme. Functional activity of the enzyme preparations was demonstrated in the experiments on in vitro transcription and promoter complex formation. With the use of IMPACT technology, sigma(70) subunit can be isolated as an individual protein without admixture of RNA polymerase.

EFFECT OF ALPHA-CRYSTALLIN ON THERMAL DENATURATION AND AGGREGATION OF RABBIT MUSCLE GLYCERALDEHYDE-3 PHOSPHATE DEHYDROGENASE

Khanova H.A., Markossian K.A., Kleimenov S.Y., Levitsky D.I., Chebotareva N.A., Golub N.V., Asryants R.A., Muronetz V.I., Saso L., Yudin I.K., Muranov K.O., Ostrovsky M.A., Kurganov B.I.

Biophysical Chemistry 125 (2007) 521-531.

The study of thermal denaturation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the presence of alpha-crystallin by differential scanning calorimetry (DSC) showed that the position of the maximum on the DSC profile (T-max) was shifted toward lower temperatures with increasing alpha-crystallin concentration. The diminishing GAPDH stability in the presence of alpha-crystallin has been explained assuming that beating of GAPDH induces dissociation of the tetrameric form of the enzyme into dimers interacting with alpha-crystallin. The dissociation of the enzyme tetramer was shown by sedimentation velocity at 45 degrees C. Suppression of thermal aggregation of GAPDH by alpha-crystallin was studied by dynamic light scattering under the conditions wherein temperature was elevated at a constant rate. The construction of the light scattering intensity versus the hydrodynamic radius (R-h) plots enabled estimating the hydrodynamic radius of the start aggregates (R-h,R-o)- When aggregation of GAPDH was studied in the presence of alpha-crystallin, the start aggregates of lesser size were observed. .

NEW MUTATIONS IN THE HUMAN P53 GENE A REGULATOR OF THE CELL CYCLE AND CARCINOGENESIS

Kashkin K.N., Khlgatian S.V., Gurova O.V., Kuprash D.V., Nedospasov S.A.

Biochemistry-Moscow 72 (2007) 282-292.

Mutations in the tumor suppressor gene p53 often lead to disarrangement of the cell cycle and of genetic integrity control of cells that may contribute to tumor development. We studied p53 gene Mutations in 26 primary tumors of colorectal cancer patients. Mutations in p53 were found in 17 tumors (65.4%). All point Mutations affected the DNA binding domain of p53 and were localized in exons 4-8 of the gene. Mutant p53 isoforms with altered domain structure and/or with alternative C-terminus arising from frameshift Mutations or abnormal splicing were found in six tumors. Mutations Leu111Gln and Ser127Phe were shown in colorectal cancer for the first time. Isoforms p53-305 with C-4 insertion in codons 300/301 and p53i9* including an additional 44 nucleotides of the 3'-end of intron 9 were discovered for the first time. Mutations of p53 were associated with lymph node metastases and III/IV stage of tumors that are signs Of unfavorable prognosis in colorectal cancer.

CHANGES IN PHYSICOCHEMICAL CHARACTERISTICS OF RABBIT SCLERA AS A RESULT OF REINFORCEMENT TREATMENT

Ignat'eva N.Y., Averkiev S.V., Iomdina E.N., Ivashchenko Z.N., Baratova L.A., Lukashina E.V., Lunin V.V.

Biofizika 52 (2007) 324-331.

It has been shown by biochemical analysis and differential scanning calorimetry that the connective tissue formed around a transplant as a result of sclero-reinforcing interference (capsula) is similar to intact sclera. The main component of newly formed capsules is collagen I whose fibers have a perfect structure and the amount of cross-links sufficient to provide normal thermomechanical properties. A fraction of collagen having thermally labile immature)) cross-links in capsules formed around the transplant impregraned with Panaxal has been detected by differential scanning calorimetry. It was suggested that fibroblasts in tissues of these capsules have a high synthetic activity.

CA2+-DEPENDENT CONFORMATIONAL CHANGES IN THE NEURONAL CA2+-SENSOR RECOVERIN PROBED BY THE FLUORESCENT DYE ALEXA647

Gensch T., Komolov K.E., Senin I.I., Philippov P.P., Koch K.W.

Proteins-Structure Function and Bioinformatics 66 (2007) 492-499.

Recoverin belongs to the superfamily of EF-hand Ca2+-binding proteins and operates as a Ca2+-sensor in vertebrate photoreceptor cells, where it regulates the activity of rhodopsin kinase GRK1 in a Ca2+-dependent manner. Ca2+-dependent conformational changes in recoverin are allosterically controlled by the covalently attached myristoyl group. The amino acid sequence of recoverin harbors a unique cysteine at position 38. The cysteine can be modified by the fluorescent dye Alexa647 using a maleimide-thiol coupling step. Introduction of Alexa647 into recoverin did not disturb the biological function of recoverin, as it can regulate rhodopsin kinase activity like unlabeled recoverin. Performance of the Ca2+-myristoyl switch of labeled recoverin was monitored by Ca2+-dependent association with immobilized lipids using surface plasmon resonance spectroscopy. When the Ca2+-concentration was varied, labeled myristoylated recoverin showed a 37%-change in fluorescence emission and a 34%-change in excitation intensity, emission and excitation maxima shifted by 6 and 18 nm, respectively. In contrast, labeled nonmyristoylated recoverin exhibited only minimal changes. Time-resolved fluorescence measurements showed biexponentiell fluorescence decay, in which the slower time constant of 2 ns was specifically influenced by Ca2+-induced conformational changes. A similar influence on the slower time constant was observed with the recoverin mutant Rec(E85Q) that has a disabled EF-hand 2, but no such influence was detected with the mutant Rec(E121Q) (EF-hand 3 is nonfunctional) that contains the myristoyl group in a clamped position. We conclude from our results that Alexa647 bound to cysteine 38 can monitor the conformational transition in recoverin that is under control of the myristoyl group.

A TRIMETAL SITE AND SUBSTRATE DISTORTION IN A FAMILY II INORGANIC PYROPHOSPHATASE

Fabrichniy I.P., Lehtio L., Tammenkoski M., Zyryanov A.B., Oksanen E., Baykov A.A., Lahti R., Goldman A.

Journal of Biological Chemistry 282 (2007) 1422-1431.

We report the first crystal structures of a family II pyrophosphatase complexed with a substrate analogue, imidodiphosphate (PNP). These provide new insights into the catalytic reaction mechanism of this enzyme family. We were able to capture the substrate complex both by fluoride inhibition and by site-directed mutagenesis providing complementary snapshots of the Michaelis complex. Structures of both the fluoride-inhibited wild type and the H98Q variant of the PNP-Bacillus subtilis pyrophosphatase complex show a unique trinuclear metal center. Each metal ion coordinates a terminal oxygen on the electrophilic phosphate and a lone pair on the putative nucleophile, thus placing it in line with the scissile bond without any coordination by protein. The nucleophile moves further away from the electrophilic phosphorus site, to the opposite side of the trimetal plane, upon binding of substrate. In comparison with earlier product complexes, the side chain of Lys(296) has swung in and so three positively charged side chains, His(98), Lys(205) and Lys(296), now surround the bridging nitrogen in PNP. Finally, one of the active sites in the wild-type structure appears to show evidence of substrate distortion. Binding to the enzyme may thus strain the substrate and thus enhance the catalytic rate.

WHICH STAGE OF THE PROCESS OF APOTRANSKETOLASE INTERACTION WITH THIAMINE DIPHOSPHATE IS AFFECTED BY THE REGULATORY ACTIVITY OF THE DONOR SUBSTRATE?

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

Iubmb Life 59 (2007) 104-109.

The interaction of thiamine diphosphate (ThDP) with transketolase (TK) involves at least two stages: [GRAPHICS] During the first stage, an inactive intermediate complex (TK ... ThDP) is formed, which is then transformed into a catalytically active holoenzyme (TK*-ThDP). The second stage is related to conformational changes of the protein. In the preceding publication (Esakova, O. A., Meshalkina, L. E., Golbik, R., Hubner, G., and Kochetov, G. A. Eur. J. Biochem. 2004, 271, 4189-4194) we reported that the affinity of ThDP for TK considerably increases in the presence of the donor substrate, which may be a mechanism whereby the activity of the enzyme is regulated under the conditions of the coenzyme deficiency. Here, we 14 demonstrate that the substrate affects the stage of the reverse A conformational transition, characterized by the constant k(-1): in the presence of the substrate, its value is decreased several fold, whereas K-d and k(+1) remain unchanged.

EXTRACELLULAR ALKALINE PROTEINASE OF COLLETOTRICHUM GLOESOSPORSIOIDES

Dunasevsky Y.E., Matveeva A.R., Beliakova G.A., Domash V.I., Belozersky M.A.

Biochemistry-Moscow 72 (2007) 345-350.

The main proteinase of the filamentous fungus Colletotrichum gloeosporioides causing anthracnoses and serious problems for production and storage of agricultural products has molecular mass of 57 kD and was purified more than 200-fold to homogeneity with the yield of 5%. Maximal activity of the proteinase is at pH 9.0-10.0, and the enzyme is stable at pH 6.0-11.5 (residual activity not less than 70%). The studied enzyme completely kept its activity to 55 degrees C. with a temperature optimum of 45 degrees C. The purified C. gloeosporioides proteinase is stable at alkaline pH values, but rapidly loses its activity at pH Values lower than 5.0. Addition of bovine serum albumin stabilizes the enzyme Under acidic conditions. Data on inhibitor analysis and substrate specificity of the enzyme allow its classification as a serine proteinase of subtilisin family. It is demonstrated that the extracellular proteinase of C. gloeosporioides specifically effects plant cell wall proteins. It is proposed that the studied proteinase - via hydrolysis of cell wall - provides for penetration of the fungus into the tissues of the host plant.

SUPEREXPRESSION OF TUBERCULOSIS ANTIGENS IN PLANT LEAVES

Dorokhov Y.L., Sheveleva A.A., Frolova O.Y., Komarova T.V., Zvereva A.S., Ivanov P.A., Atabekov J.G.

Tuberculosis 87 (2007) 218-224.

Recent developments in genetic engineering allow the employment of plants as factories for 1/foreign protein production. Thus, tuberculosis (TB) ESAT6 antigen was expressed in different plant systems, but the level of vaccine protein accumulation was extremely low. We describe the technology for superexpression of TB vaccine proteins (Ag85B, ESAT6, and ESAT6:Ag85B fusion) in plant leaves which involves: (i) construction of tobacco mosaic virus-based vectors with the coat protein genes substituted by those for TB antigens; (ii) Agrobacterium-mediated delivery to plant leaf tissues of binary vectors containing the cDNA copy of the vector virus genome; and (iii) replication of virus vectors in plant cells under conditions suppressing the virus-induced gene silencing. This technology enables efficient production of the TB vaccine proteins in plants; in particular, the level of Ag85B antigen accumulation was not less than 800mg/kg of fresh leaves. Expression of TB antigens in plant cells as His(6)-tagged proteins promoted their isolation and purification by Ni-NTA affinity chromatography. Deletion of transmembrane domains from Ag85B caused a dramatic increase in its intracellular stability. We propose that the strategy of TB antigens superproduction in a plant might be used as a basis for the creation of prophylactic and therapeutic vaccine against TB. .

ISOLATION AND SITE-DIRECTED MUTAGENESIS OF DNA METHYLTRANSFERASE SSSI

Darii M.V., Kirsanova O.V., Drutsa V.L., Kochetkov S.N., Gromova E.S.

Molecular Biology 41 (2007) 110-117.

Prokaryotic DNA methyltransferase SssI (M.SssI) methylates cytosines at C5 in CpG sequences. Bacterial strains that produced M.SssI and its mutants as His(6)-tagged proteins were constructed. To verify the role of Ser300 in recognizing the CpG sequence by the enzyme, Ser300 was replaced by Gly or Pro. The substitutions had virtually no effect on DNA binding and methylation by M.SssI apart from a slight decrease in binding in the case of S300P. It was assumed that no contact with DNA is formed by the side chain of Ser300 and the carbonyl oxygen and amide nitrogen of its peptide bonds. In addition, Ala was substituted for highly conserved Val188, presumably involved in stabilization of the flipped-out cytosine during the reaction. The substitution decreased fivefold the dissociation constant of the enzyme-substrate complex and halved the initial rate of DNA methylation. Despite the lack of a considerable effect of V188A, it was assumed that Val188 does form a contact with the target cytosine, but such a contact is formed with Ala in the case of the V188A mutant.

ENZYME-CATALYZED SIDE REACTIONS WITH MOLECULAR OXYGEN MAY CONTRIBUTE TO CELL SIGNALING AND NEURODEGENERATIVE DISEASES

Bunik V.I., Schloss J.V., Pinto J.T., Gibson G.E., Cooper A.J.L.

Neurochemical Research 32 (2007) 871-891.

A link between neurodegeneration and well-characterized enzymatic and non-enzymatic reactions that produce reactive oxygen species (ROS) from O-2 is well established. Several enzymes that contain pyridoxal 5'-phosphate (PLP) or thiamine diphosphate (ThDP) catalyze side reactions (paracatalytic reactions) in the presence of ambient O-2. These side reactions produce oxidants such as hydrogen peroxide [H2O2] or extremely reactive peracids [RC(O)OOH]. We hypothesize that although these enzymes normally produce oxidants at low or undetectable levels, changes in substrate levels or disease-induced structural alterations may enhance interactions with O-2, thereby generating higher levels of reactive oxidants. These oxidants may damage the enzymes producing them, alter nearby macromolecules and/or destroy important metabolites/coenzymes. We propose that paracatalytic reactions with O-2 catalyzed by PLP-dependent decarboxylases and by ThDP-dependent enzymes within the alpha-keto acid dehydrogenase complexes may contribute to normal cellular signaling and to cellular damage in neurodegenerative diseases.

REDOX CONTROL OF FAST LIGAND DISSOCIATION FROM ESCHERICHIA COLI CYTOCHROME BD

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

Biochemical and Biophysical Research Communications 355 (2007) 97-102.

Bacterial bd-type quinol oxidases, such as cytochrome bd from Escherichia coli, contain three hemes, but no copper. In contrast to heme-copper oxidases and similarly to globins, single electron-reduced cytochrome bd forms stable complexes with 02, NO and CO at ferrous heme d. Kinetics of ligand dissociation from heme d(2+) in the single electron- and fully-reduced cytochrome bd from E coli has been investigated by rapid mixing spectrophotometry at 20 degrees C. Data show that (i) O-2 dissociates at 78 s(-1), (ii) NO and CO dissociation is fast as compared to heme-copper oxidases and (iii) dissociation in the single electron-reduced state is hindered as compared to the fully-reduced enzyme. Presumably, rapid ligand dissociation requires reduced heme b(595). As NO, an inhibitor of respiratory oxidases, is involved in the immune response against microbial infection, the rapid dissociation of NO from cytochrome bd may have important bearings on the patho-physiology of enterobacteria. .

PHOTORECEPTOR PROTEINS AS CANCER-RETINA ANTIGENS

Bazhin A.V., Schadendorf D., Willner N., De Smet C., Heinzelmann A., Tikhomirova N.K., Umansky V., Philippov P.P., Eichmuller S.B.

International Journal of Cancer 120 (2007) 1268-1276.

Melanocytes, melanoma and photoreceptor cells are of neuroectodermal origin and have a certain sensitivity to light. In this study, we present evidence for photoreceptor proteins that are responsible for visual transduction and its regulation function as a new class of cancer antigens in melanoma. Visual rhodopsin, transducin, cGMP-phosphodiesterase 6, cGMP-dependent channels, guanylyl cyclase, rhodopsin kinase, recoverin and arrestin are expressed in melanoma and can induce antibody responses in patients. Melanocytes also express mRNA of all photoreceptor genes besides transducin, but were devoid of the corresponding protein, which was tested for rhodopsin, cGMP-phosphodiesterase, guanylyl cyclase and recoverin. Furthermore, we show for the first time that some healthy tissues express mRNA of these genes, but never protein. Expression profiles and autoantibody responses were confirmed in the MT/ret and the HGF(tg)/Ink4a(-/-) transgenic mouse melanoma models. We propose a molecular transition of cancer-retina antigens from mRNA expression in melanocytes to protein expression in melanoma. Our work provides the basis for analyzing regulation of photoreceptor gene expression in normal and malignant cells as well as possible therapeutic tumor targeting using the newly defined class of cancer-retina antigens. .

RECOVERIN AS A CANCER-RETINA ANTIGEN

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

Cancer Immunology Immunotherapy 56 (2007) 110-116.

In photoreceptor cells the Ca2+-binding protein recoverin controls phosphorylation of the visual receptor rhodopsin by inhibiting rhodopsin kinase (GRK-1). It can also serve as a paraneoplastic antigen in the development of retinal degeneration in some patients with cancer. The aberrant expression of recoverin in cancer cells and the presence of autoantibodies against recoverin are essential for the occurrence of cancer-associated retinopathy, which finally results in the apoptosis of photoreceptor cells. Noteworthy in cancer patients, the aberrant recoverin expression and the appearance of autoantibodies against recoverin are more frequent than paraneoplastic syndromes. We suggest the term "cancer-retina antigens" for this kind of proteins like recoverin that are solely expressed in retina and tumor tissues and evoke antibodies and/or T cells in patients with cancer. The rare development of a paraneoplastic syndrome is possibly caused by this immune response and probably depends on further events allowing to overcome the blood-retina barrier and the immune privileged status of the retina. It is still unknown whether aberrantly expressed recoverin could have a specific function in cancer cells, though it is suggested that it can be functionally associated with G-protein-coupled receptor kinases. This paper reviews the present knowledge on paraneoplastic syndromes associated with the aberrant expression of recoverin. A possible application of recoverin as a potential target for immunotherapy of cancer is discussed.

RECRUITMENT OF LIPIDS INTO MICRODOMAINS BY ELECTROSTATIC INTERACTIONS WITH MODEL PEPTIDES

Antonenko Y.N., Pohl P.

Biophysical Journal (2007) 572A-572A .

BAFILOMYCIN A1 IS A POTASSIUM IONOPHORE THAT IMPAIRS MITOCHONDRIAL FUNCTIONS

Teplova V.V., Tonshin A.A., Grigoriev P.A., Saris N.E.L., Salkinoja-Salonen M.S.

Journal of Bioenergetics and Biomembranes 39 (2007) 321-329.

Novel activities of bafilomycin A1, a macrolide antibiotic known as an inhibitor of V-ATPases, were discovered. Bafilomycin A1 induced uptake of potassium ions by energized mitochondria and caused mitochondrial swelling, loss of membrane potential, uncoupling of oxidative phosphorylation, inhibition of the maximal respiration rates, and induced pyridine nucleotide oxidation. The mitochondrial effects provoked by nanomolar concentrations of bafilomycin A1 were connected to its activity as a potent, K+-specific ionophore. The K+ ionophoric activity of bafilomycin A1 was observed also in black lipid membranes, indicating that it was an inherent property of the bafilomycin A1 molecule. It was found that bafilomycin A1 is a K+ carrier but not a channel former. Bafilomycin A1 is the first and currently unique macrolide antibiotic with K+ ionophoric properties. The novel properties of bafilomycin A1 may explain some of the biological effects of this plecomacrolide antibiotic, independent of V-ATPase inhibition.

RECYCLING OF EUKARYOTIC POSTTERMINATION RIBOSOMAL COMPLEXES

Pisarev A.V., Hellen C.U.T., Pestova T.V.

Cell 131 (2007) 286-299.

After translational termination, mRNA and P site deacylated tRNA remain associated with ribosomes in posttermination complexes ( post-TCs), which must therefore be recycled by releasing mRNA and deacylated tRNA and by dissociating ribosomes into subunits. Recycling of bacterial post-TCs requires elongation factor EF-G and a ribosome recycling factor RRF. Eukaryotes do not encode a RRF homolog, and their mechanism of ribosomal recycling is unknown. We investigated eukaryotic recycling using post-TCs assembled on a model mRNA encoding a tetrapeptide followed by a UAA stop codon and report that initiation factors elF3, elF1, elF1A, and elF3j, a loosely associated subunit of eIF3, can promote recycling of eukaryotic post-TCs. elF3 is the principal factor that promotes splitting of posttermination ribosomes into 60S subunits and tRNA- and mRNA bound 40S subunits. Its activity is enhanced by elFs 3j, 1, and 1A. elF1 also mediates release of P site tRNA, whereas eIF3j ensures subsequent dissociation of mRNA.

NEW HIGHLY FLUORESCING BIS-HOECHSTS: PHYSICOCHEMICAL, BIOCHEMICAL AND CYTOLOGICAL STUDIES

Zhuze A.L., Gromyko A.V., Ivanov A.A., Salyanov V.I., Popov K.V., Korolev S.P., Gottikh M.B., Oleinikov V.A., Streltsov S.A.

Journal of Biomolecular Structure & Dynamics 24 (2007) 83.

NONEQUIVALENCE OF TRANSKETOLASE ACTIVE CENTERS WITH RESPECT TO ACCEPTOR SUBSTRATE BINDING

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

Biochemical and Biophysical Research Communications 361 (2007) 1044-1047.

The interaction of transketolase with its acceptor substrate, ribose 5-phosphate, has been studied. The active centers of the enzyme were shown to be functionally nonequivalent with respect to ribose 5-phosphate binding. Under the conditions where only one out of the two active centers of transketolase is functional, their affinities for ribose 5-phosphate are identical. The phenomenon of nonequivalence becomes apparent when the substrate interacts with one of the two active centers. As a consequence of such interaction, the affinity of the second active center for ribose 5-phosphate decreases. .

COMPLETION OF THE MAPPING OF TRANSCRIPTION START SITES FOR THE FIVE-GENE BLOCK SUBGENOMIC RNAS OF BEET YELLOWS CLOSTEROVIRUS AND IDENTIFICATION OF PUTATIVE SUBGENOMIC PROMOTERS

Vitushkina M.V., Rogozin I.B., Jelkmann W., Koonin E.V., Agranovsky A.A.

Virus Research 128 (2007) 153-158.

In the positive-sense RNA genome of Beet yellows Closterovirus (BYV), the 3'-terminal open reading frames (ORFs) 2-8 are expressed as a nested set of subgenomic (sg) RNAs. ORFs 2-6, coding for the structural and movement proteins, form a 'five-gene block' conserved in closteroviruses. We mapped the 5'-end of the ORF 4 sgRNA, which encodes the p64 protein, at adenosine-11169 in the BYV genome. This completes the mapping of the transcription start sites for the five-gene block sgRNAs of BYV. Computer-assisted analysis of the sequences upstream of BYV ORFs 2, 3, 4,5, and 6 revealed two conserved motifs, which might constitute the subgenomic promoter elements. These motifs are conserved in the equivalent positions upstream of three orthologous genes of Citrus tristeza Closterovirus and two orthologous genes of Beet yellow stunt Closterovirus. .

PROTEINASE, AMYLASE, AND PROTEINASE-INHIBITOR ACTIVITIES IN THE GUT OF SIX COCKROACH SPECIES

Vinokurov K., Taranushenko Y., Krishnan N., Sehnal F.

Journal of Insect Physiology 53 (2007) 794-802.

Representative species, two from each of the cockroach families Blattidae, Blattellidae, and Blaberidae, have similar morphology of the digestive tract but differ in the physiology of digestion. The pH of crop and along the midgut varies in different species from 5.9 to 9.0 and the redox parameter from 10.1 to 12.9. Activities of proteinases and amylases in comparable gut regions differ among the species up to 100 times. Proteolytic activity is high in the midgut and moderate in the crop of Blattidae; in the other species, it is very low in the crop and increases to a moderate level in the posterior half of midgut (PM). The level of amylolytic activity is similar in the examined gut compartments of Blattidae and Blattellidae but low in the PM of Blaberidae. Blaberidae are also characterized by a high potential of the salivary glands, crop, and midgut to inhibit subtilisin, trypsin, and chymotrypsin. Inhibition of these proteinases by the extracts of salivary glands and gut is several orders of magnitude lower and often undetectable in the representatives of Blattidae and Blattellidae. .

POSITION OF EUKARYOTIC INITIATION FACTOR EIF5B ON THE 80S RIBOSOME MAPPED BY DIRECTED HYDROXYL RADICAL PROBING

Unbehaun A., Marintchev A., Lomakin I.B., Didenko T., Wagner G., Hellen C.U.T., Pestova T.V.

Embo Journal 26 (2007) 3109-3123.

Eukaryotic translation initiation factor eIF5B is a ribosome-dependent GTPase that mediates displacement of initiation factors from the 40S ribosomal subunit in 48S initiation complexes and joining of 40S and 60S subunits. Here, we determined eIF5B's position on 80S ribosomes by directed hydroxyl radical cleavage. In the resulting model, eIF5B is located in the intersubunit cleft of the 80S ribosome: domain 1 is positioned near the GTPase activating center of the 60S subunit, domain 2 interacts with the 40S subunit (helices 3, 5 and the base of helix 15 of 18S rRNA and ribosomal protein (rp) rpS23), domain 3 is sandwiched between subunits and directly contacts several ribosomal elements including Helix 95 of 28S rRNA and helix 44 of 18S rRNA, domain 4 is near the peptidyl-transferase center and its helical subdomain contacts rpL10E. The cleavage data also indicate that binding of eIF5B might induce conformational changes in both subunits, with ribosomal segments wrapping around the factor. Some of these changes could also occur upon binding of other translational GTPases, and may contribute to factor recognition.

EFFECT OF THE INHIBITORY NEUROTRANSMITTER GLYCINE ON SLOW DESTRUCTIVE PROCESSES IN BRAIN CORTEX SLICES UNDER ANOXIC CONDITIONS

Tonshin A.A., Lobysheva N.V., Yaguzhinsky L.S., Bezgina E.N., Moshkov D.A., Nartsissov Y.R.

Biochemistry-Moscow 72 (2007) 509-517.

Slow destructive processes in brain cortex were studied under deep hypoxia (anoxia). Study of the character and dynamics of DNA destruction showed that apoptosis and necrosis run in parallel under the experimental conditions. These processes typically develop in tens of hours. A similar conclusion was reached from electron microscopic study of the tissue ultrastructure. More detailed study revealed that a relatively rare type of apoptosis not involving cytochrome c release from the intermembrane space of mitochondria and not associated with opening of the mitochondrial nonspecific pore occurs under the experimental conditions. As this is occurring, the process can be slowed by high concentrations of glycine, an inhibitory neurotransmitter. The study of DNA destruction demonstrated that high concentrations of glycine selectively slow apoptosis but have almost no effect on necrosis. Glycine also drastically decreases changes in the tissue ultrastructure, particularly of mitochondria, arising under anoxia. Glycine does not notably influence the mitochondrial oxidative phosphorylation system. Study of impairment of mitochondrial function demonstrated that the oxidative phosphorylation system is not disturbed for 1 h, which is several times longer than the inhibition time of brain function under deep hypoxia. The mitochondrial respiratory system is preserved for a relatively long time (24 h). Malate oxidase activity is deactivated after 48 h. The succinate oxidase fragment of the mitochondrial respiratory chain proved especially resistant; it retains activity under anoxia for more than 72 h. A possible mechanism of the effect of high glycine concentrations is discussed.

PROSTAGLANDIN SYNTHESIS IN RAT BRAIN ASTROCYTES IS UNDER THE CONTROL OF THE N-3 DOCOSAHEXAENOIC ACID, RELEASED BY GROUP VIB CALCIUM-INDEPENDENT PHOSPHOLIPASE A(2)

Strokin M., Sergeeva M., Reiser G.

Journal of Neurochemistry 102 (2007) 1771-1782.

In the current study, we reveal that in astrocytes the VIB Ca2+-independent phospholipase A(2) is the enzyme responsible for the release of docosahexaenoic acid (22:6n-3). After pharmacological inhibition and siRNA silencing of VIB Ca2+-independent phospholipase A(2), docosahexaenoic acid release was strongly suppressed in astrocytes, which were acutely stimulated (30 min) with ATP and glutamate or after prolonged (6 h) stimulation with the endotoxin lipopolysaccharide. Docosahexaenoic acid release proceeds simultaneously with arachidonic acid (20:4n-6) release and prostaglandin liberation from astrocytes. We found that prostaglandin production is negatively controlled by endogenous docosahexaenoic acid, since pharmacological inhibition and siRNA silencing of VIB Ca2+-independent phospholipase A(2) significantly amplified the prostaglandin release by astrocytes stimulated with ATP, glutamate, and lipopolysaccharide. Addition of exogenous docosahexaenoic acid inhibited prostaglandin synthesis, which suggests that the negative control of prostaglandin synthesis observed here is likely due to competitive inhibition of cyclooxygenase-1/2 by free docosahexaenoic acid. Additionally, treatment of astrocytes with docosahexaenoic acid leads to the reduction in cyclooxygenase-1 expression, which also contributes to reduced prostaglandin production observed in lipopolysaccharide-stimulated cells. Thus, we identify a regulatory mechanism important for the brain, in which docosahexaenoic acid released from astrocytes by VIB Ca2+-independent phospholipase A(2) negatively controls prostaglandin production.

RAP80/UIMC1 AS CANCER-ASSOCIATED ANTIGEN: ALTERNATIVE SPLICE VARIANTS AND THEIR IMMUNOGENICITY

Shebzukhov Y.V., Koroleva E.P., Khlgatian S.V., Belousov P.V., Sazykin A.Y., Kadachigova T.S., Pomerantseva E.A., Lagarkova M.A., Nedospasov S.A., Kuprash D.V.

Cancer Letters 255 (2007) 255-262.

We have identified RAP80/UIMC1, the protein highly expressed in testis, as a new cancer-associated antigen. Sera from 5% to 10%, of patients with different types of cancer contain specific antibodies to RAP80/UIMC1. In order to investigate the possible reasons for RAP80/UIMC1 immunogenicity, we characterized its numerous splice isoforms and mapped immunogenic regions of the protein. The majority of RAP80/UIMC1 transcripts was detected both in normal tissues and in colon tumors. There are several RAP80/UIMC1 isoforms that are predominantly expressed in testis, however we did not observe elevated expression of these transcripts in tumors from seropositive patients. We mapped the major immunogenic region of RAP80/UIMC1 to the central part of the protein encoded by exon 9 which is present in a number of ubiquitous splice forms. Thus, based on our data, autoreactivity to RAP80/UfMC1 is related to reasons other than overexpression or tumor-specific splicing. .

ISOLATION OF ACTIVE YEAST TELOMERASE PROTEIN EST3P AND INVESTIGATION OF ITS DIMERIZATION IN VITRO

Sharanov Y.S., Smekalova E.M., Zvereva M.I., Dontsova O.A.

Biochemistry-Moscow 72 (2007) 702-706.

In this study we proposed a method for isolation of Est3p modified with various affinity tags, which is applicable for structural and functional studies, and investigated homo-and heterodimer formation with various recombinant forms of Est3p.

INTERACTION OF POLYELECTROLYTES WITH PROTEINS, 3 INFLUENCE OF COMPLEXING POLYCATIONS ON THE THERMOAGGREGATION OF OLIGOMERIC ENZYMES

Shalova I.N., Naletova I.N., Saso L., Muronetz V.I., Izumrudov V.A.

Macromolecular Bioscience 7 (2007) 929-939.

DECREASE OF DEHYDROGENASE ACTIVITY OF CEREBRAL GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE IN DIFFERENT ANIMAL MODELS OF ALZHEIMER'S DISEASE

Shalova I.N., Cechalova K., Rehakova Z., Dimitrova P., Ognibene E., Caprioli A., Schmalhausen E.V., Muronetz V.I., Saso L.

Biochimica et Biophysica Acta-General Subjects 1770 (2007) 826-832.

Recently, a relationship between glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the beta-amyloid precursor protein (LAPP) in relationship with the pathogenesis of Alzheimer's disease (AD) has been suggested. Therefore, we studied the specific activity of GAPDH in the, different animal models of AD: transgenic mice (Tg2576) and rats treated with beta-amyloid, or thiorphan, or lipopolysaccharides (LPS) and interferon gamma (INF gamma). We observed that GAPDH activity was significantly decreased in the brain samples from TG mice. The injection of beta-amyloid, or thiorphan, an inhibitor of neprilysin involved in beta-amyloid catabolism, in rat brains resulted in a pronounced reduction of the enzyme activity. The infusion of LPS and IFN gamma, which can influence the progression of the AD, significantly reduced the enzyme activity. .

EFFECTS OF LIPOPHILIC DICATIONS ON PLANAR BILAYER PHOSPHOLIPID MEMBRANE AND MITOCHONDRIA

Severina I.I., Vyssokikh M.Y., Pustovidko A.V., Simonyan R.A., Rokitskaya T.I., Skulachev V.P.

Biochimica et Biophysica Acta-Bioenergetics 1767 (2007) 1164-1168.

In this paper, we studied effects of phosphonium dications P2C5 and P2C10 on bilayer planar phospholipid membrane (BLM) and rat liver mitochondria. In line with our previous observations [M.F. Ross, T. Da Ros, F.H. Blaikie, T.A. Prime, C.M. Porteous, I.I. Severina, VP. Skulachev, H.G. Kjaergaard, R.A. Smith, M.P. Murphy, Accumulation of lipophilic dications by mitochondria and cells, Biochem. J. 400 (2006) 199-208], we showed both P2C5 and P2C10 are cationic penetrants for BLM. They generated transmembrane difftision potential (Delta Psi), the compartment with a lower dication concentration positive. However, the Delta Psi values measured proved to be lower that the Nernstian. This fact could be explained by rather low BLM conductance for the cations at their small concentrations and by induction of some BLM damage at their large concentrations. The damage in question consisted in appearance of non-Ohmic current/voltage relationships which increased in time. Such a non-Ohmicity was especially strong at Delta Psi > 100 mV. Addition of penetrating lipophilic anion TPB, which increases the BLM conductance for lipophilic cations, yielded the Nernstian Delta Psi, i.e. 30 mV per ten-fold dication gradient. In the State 4 mitochondria, dications stimulated respiration and lowered Delta Psi. Moreover, they inhibited the State 3 respiration with succinate or glutamate and malate (but not with TMPD and ascorbate) in an uncoupler-sensitive fashion. Effect on the in State 4 mitochondria, similarly to that on BLM, was accounted for by a time-dependent membrane damage. On the other hand, the State 3 effect was most probably due to inhibition of the respiratory chain Complex I and/or Complex III. The damaging and inhibitory activities of lipophilic dications should be taken into account when one considers a possibility to use them as a vehicle to target antioxidants or other compounds to mitochondria. .

CASPASE-RESISTANT VIRD2 PROTEIN PROVIDES ENHANCED GENE DELIVERY AND EXPRESSION IN PLANTS

Reavy B., Bagirova S., Chichkova N.V., Fedoseeva S.V., Kim S.H., Vartapetian A.B., Taliansky M.E.

Plant Cell Reports 26 (2007) 1215-1219.

Agrobacterium tumefaciens VirD2 protein is one of the key elements of Agrobacterium-mediated plant transformation, a process of transfer of T-DNA sequence from the Agrobacterium tumour inducing plasmid into the nucleus of infected plant cells and its integration into the host genome. The VirD2 protein has been shown to be a substrate for a plant caspase-like protease activity (PCLP) in tobacco. We demonstrate here that mutagenesis of the VirD2 protein to prevent cleavage by PCLP increases the efficiency of reporter gene transfer and expression. These results indicate that PCLP cleavage of the Agrobacterium VirD2 protein acts to limit the effectiveness of T-DNA transfer and is a novel resistance mechanism that plants utilise to combat Agrobacterium infection.

ROLE OF CA2+ IN ACTIVATION OF REACTIVE OXYGEN SPECIES PRODUCTION IN POLYMORPHONUCLEAR LEUKOCYTES DURING TUMOUR GROWTH IN RATS

Pustovidko A., Potselueva M., Kochegarov A., Evtodienko Y.

Luminescence 22 (2007) 199-205.

The role of Ca2+ ions in PMA-induced generation of reactive oxygen species (ROS) by polymorphonuclear leukocytes (PMNL) was studied during Zajdela hepatoma growth in the peritoneal cavity of rats. In PMNL from control healthy animals, a manifold Ca2+-induced enhancement of ROS generation and its significant reduction in the presence of Ca2+ binding agent (BAPTAAM) were observed. In contrast, ROS generation by PMNL from tumour-carrying animals dramatically increased in Ca2+ free medium, being practically insensitive to the agents, which can increase or decrease intracellular Ca2+ levels. Free cytosolic Ca2+ ([Ca2+](i)) in control PMNL was found to be relatively low (-250 nmol/L), rising slowly after Ca2+ addition and further to two-fold in the presence of Ca2+ and ionomycin in the incubating medium. Tumour growth in animals was accompanied with a significant [Ca2+], elevation. In Ca2+ free medium, [Ca2+](i) elevation was up to 480 nmol/L in tPMNL with the additions of Ca(2+)and ionomycin as well as EGTA and ionomycin being able to increase [Ca2+](i) to 700-900 nmol/L onward. It was concluded that a higher Ca2+ permeability of the plasma membrane and higher Ca2+ accumulation in intracellular pools of PMNL was developed at the advanced stages of malignant disease. These results indicate the primed state of circulating PMNL and the independence of PMA-induced ROS generation at intra- and extracellular Ca2+ levels at the advanced stages of tumour growth in animals. .

SEQUENCE ANALYSIS AND MOLECULAR CHARACTERIZATION OF LARVAL MIDGUT CDNA TRANSCRIPTS ENCODING PEPTIDASES FROM THE YELLOW MEALWORM, TENEBRIO MOLITOR L

Prabhakar S., Chen M.S., Elpidina E.N., Vinokurov K.S., Smith C.M., Marshall J., Oppert B.

Insect Molecular Biology 16 (2007) 455-468.

Peptidase sequences were analysed in randomly picked clones from cDNA libraries of the anterior or posterior midgut or whole larvae of the yellow mealworm, Tenebrio molitor Linnaeus. Of a total of 1528 sequences, 92 encoded potential peptidases, from which 50 full-length cDNA sequences were obtained, including serine and cysteine proteinases and metallopeptidases. Serine proteinase transcripts were predominant in the posterior midgut, whereas transcripts encoding cysteine and metal lopeptidases were mainly found in the anterior midgut. Alignments with other proteinases indicated that 40% of the serine proteinase sequences were serine proteinase homologues, and the remaining ones were identified as either trypsin, chymotrypsin or other serine proteinases. Cysteine proteinase sequences included cathepsin B- and L-like proteinases, and metal lopeptidase transcripts were similar to carboxypeptidase A. Northern blot analysis of representative sequences demonstrated the differential expression profile of selected transcripts across five developmental stages of Te. molitor. These sequences provide insights into peptidases in coleopteran insects as a basis to study the response of coleopteran larvae to external stimuli and to evaluate regulatory features of the response.

ACCUMULATION OF SLIGHTLY DELETERIOUS MUTATIONS IN MITOCHONDRIAL PROTEIN-CODING GENES OF LARGE VERSUS SMALL MAMMALS

Popadin K., Polishchuk L.V., Mamirova L., Knorre D., Gunbin K.

Proceedings of the National Academy of Sciences of the United States of America 104 (2007) 13390-13395.

After the effective size of a population, N-e,N- declines, some slightly deleterious amino acid replacements which were initially suppressed by purifying selection become effectively neutral and can reach fixation. Here we investigate this phenomenon for a set of all 13 mitochondrial protein-coding genes from 110 mammalian species. By using body mass as a proxy for N., we show that large mammals (i.e., those with low N-e) as compared with small ones (in our sample these are, on average, 369.5 kg and 275 g, respectively) have a 43% higher rate of accumulation of nonsynonymous nucleotide substitutions relative to synonymous substitutions, and an 8-40% higher rate of accumulation of radical amino acid substitutions relative to conservative substitutions, depending on the type of amino acid classification. These higher rates result in a 6% greater amino acid dissimilarity between modern species and their most recent reconstructed ancestors in large versus small mammals. Because nonsynonymous substitutions are likely to be more harmful than synonymous substitutions, and radical amino acid substitutions are likely to be more harmful than conservative ones, our results suggest that large mammals experience less efficient purifying selection than small mammals. Furthermore, because in the course of mammalian evolution body size tends to increase and, consequently, N tends to decline, evolution of mammals toward large body size may involve accumulation of slightly deleterious mutations in mitochondrial protein-coding genes, which may contribute to decline or extinction of large mammals.

MIXING OF EXCITON AND CHARGE-TRANSFER STATES IN PHOTOSYSTEM II REACTION CENTERS: MODELING OF STARK SPECTRA WITH MODIFIED REDFIELD THEORY

Novoderezhkin V.I., Dekker J.P., van Grondelle R.

Biophysical Journal 93 (2007) 1293-1311.

We propose an exciton model for the Photosystem II reaction center ( RC) based on a quantitative simultaneous fit of the absorption, linear dichroism, circular dichroism, steady- state fluorescence, triplet- minus- singlet, and Stark spectra together with the spectra of pheophytin- modified RCs, and so- called RC5 complexes that lack one of the peripheral chlorophylls. In this model, the excited state manifold includes a primary charge- transfer ( CT) state that is supposed to be strongly mixed with the pure exciton states. We generalize the exciton theory of Stark spectra by 1), taking into account the coupling to a CT state ( whose static dipole cannot be treated as a small parameter in contrast to usual excited states); and 2), expressing the line shape functions in terms of the modified Redfield approach ( the same as used for modeling of the linear responses). This allows a consistent modeling of the whole set of experimental data using a unified physical picture. We show that the fluorescence and Stark spectra are sensitive to the assignment of the primary CT state, its energy, and coupling to the excited states. The best fit of the data is obtained supposing that the initial charge separation occurs within the special- pairP(D1)P(D2). Additionally, the scheme with primary electron transfer from the accessory chlorophyll to pheophytin gave a reasonable quantitative fit. We show that the effectiveness of these two pathways is strongly dependent on the realization of the energetic disorder. Supposing a mixed scheme of primary charge separation with a disorder- controlled competition of the two channels, we can explain the coexistence of fast sub- ps and slow ps components of the Phe- anion formation as revealed by different ultrafast spectroscopic techniques.

HIGH-PRESSURE TREATMENT OF POLYTENE CHROMOSOMES IMPROVES STRUCTURAL RESOLUTION

Novikov D.V., Kireev I., Belmont A.S.

Nature Methods 4 (2007) 483-485.

The exceptional cytology provided by polytene chromosomes has made Drosophila melanogaster a premier model for chromosome studies, but full exploitation of polytene cytology is impeded by the difficulty in preparing high-quality chromosome spreads. Here we describe use of high pressure to produce formaldehyde-fixed chromosome spreads, which upon light-microscopy examination reveal structural detail previously observed only in electron microscopy preparations. We demonstrate applications to immunofluorescence and in situ hybridization.

ENZYMES CATALYZING PROTEIN FOLDING AND THEIR CELLULAR FUNCTIONS

Nagradova N.

Current Protein & Peptide Science 8 (2007) 273-282.

In live cells, protein folding often cannot occur spontaneously, but requires the participation of helper proteins - molecular chaperones and foldases. The mechanisms employed by chaperones markedly increase the effectiveness of protein folding, but have no bearing on the rate of this process, whereas foldases actually accelerate protein folding by exerting a direct influence on the rate-limiting steps of the overall reaction. Two types of foldases are known, using different principles of action. Peptidyl-prolyl cis/trans isomerase and protein-disulfide isomerase catalyze the folding of every protein that needs isomerization of prolyl peptide bonds or formation and isomerization of disulfide bonds for proper folding. By contrast, some foldases operating in the periplasm of bacterial cells are specifically designed to help in the folding of substrate proteins whose primary structure does not contain sufficient information for correct folding. In this review, we discuss recent data on the catalytic mechanisms of both types of foldases, focusing specifically on how a catalyst provides the structural information required for the folding of a target protein. Comparative analysis of the mechanisms employed by two different periplasmic foldases is used to substantiate the notion that combinations of a protein which is unable to fold independently and a specific catalyst delivering the necessary steric information are probably designed to achieve some particular biological purposes. The review also covers the problem of participation of peptidyl-prolyl cis/trans isomerase in different cellular functions, highlighting the role of this enzyme in conformational rearrangements of folded native proteins.

STUDY OF THREE-DIMENSIONALLY ORDERED STRUCTURES OF INTACT MITOCHONDRIA BY SMALL-ANGLE NEUTRON SCATTERING

Murugova T.N., Gordeliy V.I., Kuklin A.I., Sollodovnikova I.M., Yaguzhinsky L.S.

Crystallography Reports 52 (2007) 521-524.

The ultrastructure of cristae of intact mitochondria, in which the activity of the oxidative phosphorylation system was retained, was investigated by sinall-angle neutron scattering. Osmotic swelling of organelles was found to cause ultrastructural rearrangements of the inner mitochondrial membrane in rat liver and heart mitochondria. This process was accompanied by the appearance of diffraction peaks indicative of the formation of ordered structures. The lamellar packing of the membranes in heart mitochondria, which is observed under normal conditions, was found to undergo a transition to a nonlamellar hexagonal packing, whose scattering curve shows two peaks with the scattering vectors characterized by a spacing ratio of 1 : root(3) over bar. The electron-microscopy results confirm the possibility of the formation of a hexagonal packing of the inner membrane in heart mitochondria.

PHYSICO-CHEMICAL AND EVOLUTIONARY CONSTRAINTS FOR THE FORMATION AND SELECTION OF FIRST BIOPOLYMERS: TOWARDS THE CONSENSUS PARADIGM OF THE ABIOGENIC ORIGIN OF LIFE

Mulkidjanian A.Y., Galperin M.Y.

Chemistry & Biodiversity 4 (2007) 2003-2015.

During the last two decades, the common school of thought has split into two, so that the problem of the origin of life is tackled in the framework of either the 'replication first' paradigm or the 'metabolism first' scenario. The first paradigm suggests that the life started from the spontaneous emergence of the first, supposedly RNA-based 'replicators' and considers in much detail their further evolution in the so-called 'RNA world'. The alternative hypothesis of 'metabolism first' derives the life from increasingly complex autocatalytic chemical cycles. In this work, we emphasize the role of selection during the prebiological stages of evolution and focus on the constraints that are imposed by physical, chemical, and biological laws. We try to demonstrate that the 'replication first' and 'metabolism first' hypotheses complement, rather than contradict, each other. We suggest that life on Earth has started from a 'metabolism-driven replication'; the suggested scenario might serve as a consensus scheme in the framework of which the molecular details of origin of life can be further elaborated. The key feature of the scenario is the participation of the UV irradiation both as driving and selecting forces during the earlier stages of evolution.

PROTON TRANSLOCATION BY THE CYTOCHROME BC(1) COMPLEXES OF PHOTOTROPHIC BACTERIA: INTRODUCING THE ACTIVATED Q-CYCLE

Mulkidjanian A.Y.

Photochemical & Photobiological Sciences 6 (2007) 19-34.

The cytochrome b(C1) complexes are proton-translocating, dimeric membrane ubiquinol: cytochrome c oxidoreductases that serve as "hubs" in the vast majority of electron transfer chains. After each ubiquinol molecule is oxidized in the catalytic center P at the positively charged membrane side, the two liberated electrons head out, according to the Mitchell's Q-cycle mechanism, to different acceptors. One is taken by the [2Fe-2S] iron-sulfur Rieske protein to be passed further to cytochrome c(1). The other electron goes across the membrane, via the low- and high-potential hemes of cytochrome b, to another ubiquinone-binding site N at the opposite membrane side. It has been assumed that two ubiquinol molecules have to be oxidized by center P to yield first a semiquinone in center N and then to reduce this semiquinone to ubiquinol. This review is focused on the operation of cytochrome b(C1) complexes in phototrophic purple bacteria. Their membranes provide a unique system where the generation of membrane voltage by light-driven, energy-converting enzymes can be traced via spectral shifts of native carotenoids and correlated with the electron and proton transfer reactions. An "activated Q-cycle" is proposed as a novel mechanism that is consistent with the available experimental data on the electron/proton coupling. Under physiological conditions, the dimeric cytochrome b(C1) complex is suggested to be continually primed by prompt oxidation of membrane ubiquinol via center N yielding a bound semiquinone in this center and a reduced, high-potential heme b in the other monomer of the enzyme. Then the oxidation of each ubiquinol molecule in center P is followed by ubiquinol formation in center N, proton translocation and generation of membrane voltage.

IN VIVO AND IN VITRO INVESTIGATION OF TRANSCRIPTIONAL REGULATION BY DntR

Lonneborg R., Smirnova I., Dian C., Leonard G.A., Brzezinski P.

Journal of Molecular Biology 372 (2007) 571-582.

DntR is a bacterial transcription factor that has been isolated from Burkholderia species that are able to degrade the nitro-aromatic compound 2,4-dinitrotoluene. We recently solved the X-ray crystal structure of DntR, which suggested a putative location of an inducer-binding cavity (IBC). In this study, we constructed mutants of DntR in which residues lining the proposed IBC were modified in order to identify the structural elements involved in inducer binding, to modulate the inducer binding specificity, transcriptional activation of the reporter gene gfp induced by the wild-type and mutant DntRs was monitored by analysing whole-cell fluorescence using flow-cytometry after addition of a number of potential inducer compounds. Three of the mutant proteins (F111L; F111V/H169V and **Y110S/ F111V) were purified and the binding constants for several of the potential inducers to these mutants were estimated. Furthermore, crystal structures the F111L and **Y110S/F111V mutant proteins were solved and used to explain changes in the inducer binding specificity at an atomic level. A comparison of the inducing capability in the whole-cell system and binding constants for a number of potential inducers suggests a mechanism where binding of an inducer molecule is not the sole requirement for transcriptional activation. In addition, specific interactions between DntR and the inducer molecule resulting in a conformational change of the protein are needed. .

SOMATIC AND SPERM-SPECIFIC ISOENZYMES OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE: COMPARATIVE ANALYSIS OF PRIMARY STRUCTURES AND FUNCTIONAL FEATURES

Kuravsky M.L., Muronetz V.I.

Biochemistry-Moscow 72 (2007) 744-749.

The elucidation of the interdependence between structural features and functions of somatic and sperm-specific isoenzymes of glyceraldehyde-3-phosphate dehydrogenase (GAPD and GAPDS, respectively) was the goal of comparative analysis of their primary structures. GAPDS was shown to lack the sequence similar to the atypical nuclear export signal motif (NES) of the somatic isoenzyme GAPD. This finding is confirmed by experimental data on the absence of interaction between GAPDS and antibodies 6C5 recognizing the NES motif in the sequence of GAPD. The lack of NES correlates with functional peculiarities of the sperm-specific enzyme that is tightly bound to the fibrous sheath of the sperm flagellum. The sequences of the two isoenzymes were examined for the short motifs that might participate in apoptosis, endocytosis, and DNA repair. Sites of phosphorylation by different protein kinases have been revealed in both isoenzymes, and their characteristic features are discussed. These observations can serve as the basis for subsequent search for new ways of regulating the two isoenzymes.

A MODEL OF RESTRICTION ENDONUCLEASE MVAI IN COMPLEX WITH DNA: A TEMPLATE FOR INTERPRETATION OF EXPERIMENTAL DATA AND A GUIDE FOR SPECIFICITY ENGINEERING

Kosinski J., Kubareva E., Bujnicki J.M.

Proteins-Structure Function and Bioinformatics 68 (2007) 324-336.

R.MvaI is a Type II restriction enzyme (REase), which specifically recognizes the pentanucleotide DNA sequence 5'-CCWGG-3' (W indicates A or T). It belongs to a family of enzymes, which recognize related sequences, including 5'-CCSGG-3'(S indicates G or C) in the case of R.BcnI, or 5'-CCNGG-3' (where N indicates any nucleoside) in the case of R.ScrFI. REases from this family hydrolyze the phosphodiester bond in the DNA between the 2nd and 3rd base in both strands, thereby generating a double strand break with 5'-protruding single nucleotides. So far, no crystal structures of REases with similar cleavage patterns have been solved. Characterization of sequence-structure-function relationships in this family would facilitate understanding of evolution of sequence specificity among REases and could aid in engineering of enzymes with new specificities. However, sequences of R.MvaI or its homologs show no significant similarity to any proteins with known structures, thus precluding straightforward comparative modeling. We used a fold recognition approach to identify a remote relationship between R.MvaI and the structure of DNA repair enzyme MutH, which belongs to the PD-(D/E)XK superfamily together with many other REases. We constructed a homology model of R.MvaI and used it to predict functionally important amino acid residues and the mode of interaction with the DNA. In particular, we predict that only one active site of R.MvaI interacts with the DNA target at a time, and the cleavage of both strands (5'-CCAGG-3' and 5'-CCTGG-3') is achieved by two independent catalytic events. The model is in good agreement with the available experimental data and will serve as a template for further analyses of R.MvaI, R.BcnI, R.ScrFI and other related enzymes.

IN VITRO RECONSTITUTION AND BIOCHEMICAL CHARACTERIZATION OF TRANSLATION INITIATION BY INTERNAL RIBOSOMA ENTRY

Kolupaeva V.G., de Breyne S., Pestova T.V., Hellen C.U.T.

Translation Initiation: Reconstituted Systems and Biophysical Methods 430 (2007) 409-439.

The internal ribosomal entry sites (IRESs) of encephalomyocarditis virus (EMCV) and related viruses promote initiation of translation by a noncanonical end-independent mechanism. To characterize this mechanism at the molecular level, we have developed biochemical approaches to reconstitute the process in vitro from individual purified components of the translation apparatus, developed methods to characterize steps in this process so that the functions of individual proteins can be characterized, and adapted assays such as primer extension inhibition ("toe printing") to monitor accurate assembly on the IRES of ribosomal 48S and 80S complexes. In vitro reconstitution Of 48S complex formation offers an approach for the functional identification of IRES trans-acting factors (ITAFs) that are required for initiation in addition to canonical initiation factors and revealed that despite being related, different EMCV-like IRESs nevertheless have distinct ITAF requirements. Toe printing revealed that a common feature of initiation on EMCV-like IRESs is the stable binding of an eIF4G/eIF4A complex to them near the initiation codon, where it can locally unwind RNA to facilitate ribosomal attachment. The same toe printing assay indicated that binding of ITAFs to these IRESs enhances binding of these two canonical initiation factors. We also describe protocols for chemical and enzymatic footprinting to determine the interactions of trans-acting factors with the IRES at nucleotide resolution and for directed hydroxyl radical probing to determine their orientation on the IRES.

COMPARATIVE PROTEOMICS OF CELL DIVISION MUTANTS AND WILD-TYPE OF SYNECHOCOCCUS SP STRAIN PCC 7942

Koksharova O.A., Klint J., Rasmussen U.

Microbiology-Sgm 153 (2007) 2505-2517.

Bacterial cell division is a highly co-ordinated and fine-tuned process. In the unicellular cyanobacterium Synechococcus sp. strain PCC 7942, inactivating mutations in the ftn2 and ftn6 genes block cell division and result in a phenotype with extensively elongated cells. In order to establish the pleiotropic responses induced and cellular processes affected by blocked cell division, the proteomes of wild-type and the cell division mutants Ftn2 and l of Synechococcus sp. strain PCC 7942 were characterized and compared. By separating soluble extracted proteins on 2D gels, more than 800 protein spots were visualized on each SYPRO Ruby-stained gel. Quantitative differences in protein composition were detected by using the PDQuest software, and comparative analysis revealed that 76 protein spots changed significantly in the cell division mutants. These protein spots were selected for identification using peptide mass fingerprints generated by MALDI-TOF MS. Fifty-three protein spots were successfully identified, representing 44 different proteins. The upregulated proteins include proteins involved in cell division/cell morphogenesis, protein synthesis and processing, oxidative stress response, amino acid metabolism, nucleotide biosynthesis, and glycolysis, as well as unknown proteins. Among the downregulated proteins are those involved in chromosome segregation, protein processing, photosynthesis, redox regulation, carbon dioxide fixation, nucleotide biosynthesis, the biosynthetic pathway to fatty acids, and energy production. Besides eliciting common responses, inactivation of Ftn2 and Ftn6 in the mutants may result in different responses in protein levels between the mutants. Among 18 identified differentially affected protein spots, 75 % (9/12) of the protein spots affected in the Ftn2 mutant were upshifted, whereas in the Ftn6 mutant 70 % (7/10) of the affected protein spots were downshifted. Identification of such differentially expressed proteins provides new targets for future studies that will allow assessment of their physiological roles and significance in cyanobacterial cell division.

INTERACTION OF A PLANT VIRUS-ENCODED PROTEIN WITH THE MAJOR NUCLEOLAR PROTEIN FIBRILLARIN IS REQUIRED FOR SYSTEMIC VIRUS INFECTION

Kim S.H., MacFarlane S., Kalinina N.O., Rakitina D.V., Ryabov E.V., Gillespie T., Haupt S., Brown J.W.S., Taliansky M.

Proceedings of the National Academy of Sciences of the United States of America 104 (2007) 11115-11120.

The nucleolus and specific nucleolar proteins are involved in the life cycles of some plant and animal viruses, but the functions of these proteins and of nucleolar trafficking in virus infections are largely unknown. The ORF3 protein of the plant virus, groundnut rosette virus (an umbravirus), has been shown to cycle through the nucleus, passing through Cajal bodies to the nucleolus and then exiting back into the cytoplasm. This journey is absolutely required for the formation of viral ribonucleoprotein particles (RNPs) that, themselves, are essential for the spread of the virus to noninoculated leaves of the shoot tip. Here, we show that these processes rely on the interaction of the ORF3 protein with fibrillarin, a major nucleolar protein. Silencing of the fibrillarin gene prevents long-distance movement of groundnut rosette virus but does not affect viral replication or cell-to-cell movement. Repressing fibrillarin production also localizes the ORF3 protein to multiple Cajal body-like aggregates that fail to fuse with the nucleolus. Umbraviral ORF3 protein and fibrillarin interact in vitro and, when mixed with umbravirus RNA, form an RNP complex. This complex has a filamentous structure with some regular helical features, resembling the RNP complex formed in vivo during umbravirus infection. The filaments formed in vitro are infectious when inoculated to plants, and their infectivity is resistant to RNase. These results demonstrate previously undescribed functions for fibrillarin as an essential component of translocatable viral RNPs and may have implications for other plant and animal viruses that interact with the nucleolus.

INTRACELLULAR OBJECTS TRACKING

Kalaidzidis Y.

European Journal of Cell Biology 86 (2007) 569-578.

The tracking of intracellular organelles and vesicles is becoming increasingly important for understanding cellular dynamics. Originally, the development of tracking algorithms was mainly pursued in other fields, e.g. aerospace/military/street surveillance. However, most of this algorithm is not directly applicable to live cell microscopy data. Here we describe the algorithms that have been successfully applied to object detection and tracking specifically in vivo and in vitro motility assays. The characteristics advantages and disadvantages of the different approaches are compared. .

DIFFERENTIAL PROTEIN EXPRESSION IN PANCREATIC ISLETS AFTER TREATMENT WITH AN IMIDAZOLINE COMPOUND

Jagerbrink T., Lexander H., Palmberg C., Shafqat J., Sharoyko V., Berggren P.O., Efendic S., Zaitsev S., Jornvall H.

Cellular and Molecular Life Sciences 64 (2007) 1310-1316.

The effects of an imidazoline compound (BL11282) on protein expression in rat pancreatic islets were investigated with a proteomic approach. The compound increases insulin release selectively at high glucose concentrations and is therefore of interest in type 2 diabetes. Whole cell extracts from isolated drug-treated and native pancreatic rat islets were compared after separation by 2-D gel electrophoresis. Differentially expressed proteins were identified by mass spectrometry; 15 proteins were selectively up-regulated and 7 selectively down-regulated in drug-treated islets. Of special interest among the differentially expressed proteins are those involved in protein folding (Hsp60, protein disulfide isomerase, calreticulin), Ca2+ binding (calgizzarin, calcyclin and annexin I) and metabolism or signalling (pyruvate kinase, alpha enolase and protein kinase C inhibitor 1).

CELLULAR MECHANISMS OF BRAIN HYPOGLYCEMIA

Isaev N.K., Stel'mashuk E.V., Zorov D.B.

Biochemistry-Moscow 72 (2007) 471-478.

Data on intracellular processes induced by a low glucose level in nerve tissue are presented. The involvement of glutamate and adenosine receptors, mitochondria, reactive oxygen species (ROS), and calcium ions in the development of hypoglycemia-induced damage of neurons is considered. Hypoglycemia-induced calcium overload of neuronal mitochondria is suggested to be responsible for the increased ROS production by mitochondria.

ACTIVATION OF MECHANISMS OF PHOTOPROTECTION BY DESICCATION AND BY LIGHT: POIKILOHYDRIC PHOTOAUTOTROPHS

Heber U., Azarkovich M., Shuvalov V.

Journal of Experimental Botany 58 (2007) 2745-2759.

Mechanisms of protection against photo-oxidation in selected desiccation-tolerant lichens and mosses have been investigated by measuring loss of light absorption during desiccation and chlorophyll fluorescence as indicators of photoprotection. Apparent absorption (1-7) spectra measured in the reflectance mode revealed stronger absorption of photosynthetic pigments in hydrated than in desiccated organisms, but differences were pronounced only in a cyanolichen, less so in some chlorolichens, and even less in mosses. Since the amplitude of chlorophyll. fluorescence is a product of (1-7) light absorption by chlorophyll and quantum yield of fluorescence and since fluorescence is inversely related to thermal energy dissipation, when chemical fluorescence quenching is negligible, fluorescence measurements were used to measure changes in energy dissipation. Preincubation of the hydrated organisms and desiccation in darkness excluded the contribution of mechanisms of energy dissipation to photoprotection which are dependent on the presence of zeaxanthin or on the light-dependent formation of a quencher of fluorescence within the reaction centre of photosystem II. Fast drying in darkness or in very low light was less effective in decreasing chlorophyll fluorescence than slow drying. Heating the desiccated organisms increased fluorescence by inactivating the mechanism responsible for. uorescence quenching. Glutaraldehyde inhibited. uorescence quenching during desiccation. Prolonged exposure of a desiccated moss or a desiccated lichen to very strong light caused more photo-induced damage after fast drying than after slow drying. The photo-oxidative nature of damage was emphasized by the observation that irreversible loss of. uorescence was larger in air than in a nitrogen atmosphere. It is concluded from these observations that desiccation-induced conformational changes of a chlorophyll protein complex result in the fast radiationless dissipation of absorbed light energy. This mechanism of photoprotection is more effective in preventing photooxidative damage than other mechanisms of energy dissipation which require light for activation such as zeaxanthin-dependent energy dissipation or quencher formation within the reaction centre of photosystem II.

SUPPORT FROM DNA DATA FOR A NARROW SPECIES CONCEPT IN SCHISTIDIUM (GRIMMIACEAE, MUSCI)

Goryunov D.V., Ignatova E.A., Ignatov M.S., Milyutina I.A., Troitsky A.V.

Journal of Bryology 29 (2007) 98-103.

A study of ITS1 of 28 specimens of eight species of Schistidium from well-separated populations in Russia and northwest Europe revealed that there are very big differences between species (up to 16 substitutions and 256 indels), whereas within the species differences in DNA there are very few (none to four substitutions and none to six indels). These results strongly support the narrow species concept in Schistidium. Schistidium papillosum is represented by two quite distinct genotypes and probably needs further splitting.

EVIDENCE FOR THE FORMATION OF START AGGREGATES AS AN INITIAL STAGE OF PROTEIN AGGREGATION

Golub N., Meremyanin A., Markossian K., Eronina T., Chebotareva N., Asryants R., Muronets V., Kurganov B.

Febs Letters 581 (2007) 4223-4227.

The kinetics of thermal aggregation of glycogen phosphorylase b and glyceraldehyde 3-phosphate dehydrogenase from rabbit skeletal muscles were studied using dynamic light scattering. Use of high concentrations of the enzymes (1-3 mg/ml) provided a simultaneous registration of the native enzyme forms and protein aggregates. It was shown that initially registered aggregates (start aggregates) were large-sized particles. The hydrodynamic radius of the start aggregates was about 100 nm. The intermediate states between the native enzyme forms and start aggregates were not detected. The initial increase in the light scattering intensity is connected with accumulation of the start aggregates, the size of the latter remaining unchanged. From a certain moment in time aggregates of higher order, formed as a result of sticking of the start aggregates, make a major contribution to the enhancement of the light scattering intensity. .

OVEREXPRESSION AND REFOLDING OF THIOREDOXIN/TRAIL FUSION FROM INCLUSION BODIES AND FURTHER PURIFICATION OF TRAIL AFTER CLEAVAGE BY ENTEROPEPTIDASE

Gasparian M.E., Ostapchenko V.G., Yagolovich A.V., Tsygannik I.N., Chernyak B.V., Dolgikh D.A., Kirpichnikov M.P.

Biotechnology Letters 29 (2007) 1567-1573.

The human TRAIL gene (encoding residues 114-281) was synthesized by PCR and cloned into plasmid pET-32a. High level expression (1.5 g l(-1)) of thioredoxin/TRAIL fusion was achieved in Escherichia coli strain BL21(DE3), mainly as inclusion bodies. Refolded fusion thioredoxin/TRAIL was cleaved by enteropeptidase and TRAIL was separated from thioredoxin on Ni-NTA agarose. High yield (400 mg l(-1)) of TRAIL without N-terminal methionine and His tag was obtained. Sedimentation coefficient demonstrated that 98% of TRAIL formed trimers. TRAIL formed crystals of space group P3(1) with unit-cell dimensions a = b = 72.5 angstrom, c = 141.5 angstrom. Apoptosis induced in HeLa cells by purified TRAIL was 5-fold enhanced by emetine.

REGULATION OF EXPRESSION OF NA+-TRANSLOCATING NADH : QUINONE OXIDOREDUCTASE GENES IN VIBRIO HARVEYI AND KLEBSIELLA PNEUMONIAE

Fadeeva M.S., Yakovtseva E.A., Belevich G.A., Bertsova Y.V., Bogachev A.V.

Archives of Microbiology 188 (2007) 341-348.

The expression of genes encoding sodium-translocating NADH:quinone oxidoreductase (Na+-NQR) was studied in the marine bacterium Vibrio harveyi and in the enterobacterium Klebsiella pneumoniae. It has been shown that such parameters as NaCl concentration, pH value, and presence of an uncoupler in the growth media do not influence significantly the level of nqr expression. However, nqr expression depends on the growth substrates used by these bacteria. Na+-NQR is highly repressed in V. harveyi during anaerobic growth, and nqr expression is modulated by electron acceptors and values of their redox potentials. The latter effect was shown to be independent of the ArcAB regulatory system.

SENSITIZED PHOTOINACTIVATION OF MINIGRAMICIDIN CHANNELS IN BILAYER LIPID MEMBRANES

Dutseva E.A., Antonenko Y.N., Kotova E.A., Pfeifer J.R., Koert U.

Biochimica et Biophysica Acta-Biomembranes 1768 (2007) 1230-1237.

The method of sensitized photoinactivation based on the photosensitized damage of gramicidin A (gA) molecules was applied here to study ionic channels formed by minigramicidin (the 11-residue analogue of gramicidin A) in a planar bilayer lipid membrane (BLM) of different thickness. Irradiation of BLM with a single flash of visible light in the presence of a photosensitizer (aluminum phthalocyanine or Rose Bengal) generating singlet oxygen provoked a decrease in the minigramicidin-induced electric current across BLM, the kinetics of which had the characteristic time of several seconds, as observed with gA. For gA, there is good correlation between the characteristic time of photoinactivation and the single-channel lifetime. In contrast to the covalent dimer of gA characterized by extremely long single-channel lifetime and the absence of current relaxation upon flash excitation, the covalent head-to-head dinner of minigramicidin displayed the flash-induced current decrease with the kinetics being strongly dependent on the membrane thickness. The current decrease became slower both upon increasing the concentration of the minigramicidin covalent dimer and upon including cholesterol in the membrane composition. These data in combination with the quadratic dependence of the current on the peptide concentration can be rationalized by hypothesizing that the macroscopic current across BLM measured at high concentrations of the peptide is provided by dimers of minigramicidin covalent dimers in the double beta(5-7)-helical conformation having the lifetime of about 0.4 s, while single channels with the lifetime of 0.01 s, observed at a very low peptide concentration, correspond to the single-stranded beta(6.3)-helical conformation. Alternatively the results can be explained by clustering of channels at high concentrations of the minigramicidin covalent dimer. .

SIGNIFICANCE OF THE C-TERMINAL AMINO ACID RESIDUE IN MENGOVIRUS RNA-DEPENDENT RNA POLYMERASE

Dmitrieva T.M., Alexeevski A.V., Shatskaya G.S., Tolskaya E.A., Gmyl A.P., Khitrina E.V., Agol V.I.

Virology 365 (2007) 79-91.

Replication of picomavirus genomes is accomplished by the virally encoded RNA-dependent RNA polymerase (RdRP). Although the primary structure of this enzyme exhibits a high level of conservation, there are several significant differences among different picomavirus genera. In particular, a comparative alignment indicates that the C-terminal sequences of cardiovirus RdRP (known also as 3D(pol)), are 1-amino-acid residue (arginine or tryptophan) longer than that of the enterovirus or rhinovirus enzymes. Here, it is shown that alterations of the last codon of the RdRP-encoding sequence of mengovirus RNA leading to deletion of the C-terminal Trp460 or its replacement by Ala or Phe dramatically impaired viral RNA replication and, in the former case, resulted in a quasi -infectious phenotype (i.e., the mutant RNA might generate a low yield of pseudorevertants acquiring a Tyr residue in place of the deleted Trp460). The replacement of Trp460 by His or Tyr did not appreciably alter the viral growth potential. Homology modeling of three-dimensional structure of mengovirus RdRP suggested that Trp460 may be involved in interaction between the thumb and palm domains of the enzyme. Specifically, Trp460 of the thumb may forrn a hydrogen bond with Thr-219 and hydrophobically interact with Va1216 of the palm. The proposed interactions were consistent with the results of in vivo SELEX experiment, which demonstrated that infectious virus could contain Ser or Thr at position 219 and hydrophobic Val, Leu, Ile, as well as Arg (whose side chain has a nonpolar part) at position 216. A similar thumb-palm domain interaction may be a general feature of several RdRPs and its possible functional significance is discussed. .

QUANTUM CHEMICAL STUDIES OF THE CATALYTIC MECHANISM OF N-TERMINAL NUCLEOPHILE HYDROLASE

Chilov G.G., Sidorova A.V., Svedas V.K.

Biochemistry-Moscow 72 (2007) 495-500.

Modeling of the catalytic mechanism of penicillin acylase, a member of the N-terminal nucleophile hydrolase superfamily, is for the first time conducted at ab initio quantum chemistry level. The uniqueness of this family of enzymes is that their active site lacks His and Asp (Glu) residues, comprising together with a serine residue the classical catalytic triad. The current investigation confirms that the amino group of the N-terminal serine residue in N-terminal hydrolases is capable of activating its own hydroxyl group. Using the MP2/RHF method with the 6-31+G** basis set, stationary points on the potential energy surface of the considered molecular system were located, corresponding to local minima (complexes of reagents, products, intermediate) and to saddle points (transition states). It turned out that the stage of acyl-serine formation proceeds via two transition states; the first one, which separates reagents from the so-called tetrahedral intermediate, has the highest relative energy (30 kcal/mol). In contrast to recently proposed empiric suggestions, we have found that participation of a bridging water molecule in proton shuttling is not necessary for the catalysis. The quantum chemical calculations showed a crucial role of a specific solvation in decreasing the activation barrier of the reaction by approximately 10 kcal/mol.

DYNAMICS OF ELECTRON TRANSFER FROM HIGH-POTENTIAL CYTOCHROME C TO BACTERIOCHLOROPHYLL DIMER IN PHOTOSYNTHETIC REACTION CENTERS AS PROBED USING LASER-INDUCED TEMPERATURE JUMP

Chamorovsky S.K., Chamorovsky C.S., Knox P.P., Chizhov I.V., Zubov B.V.

European Biophysics Journal with Biophysics Letters 36 (2007) 601-608.

Laser-induced temperature jump experiments were used for testing the rates of thermoinduced conformational transitions of reaction center (RC) complexes in chromatophores of Chromatium minutissimum. The thermoinduced transition of the macromolecular RC complex to a state providing effective electron transport from the multiheme cytochrome c to the photoactive bacteriochlorophyll dimer within the temperature range 220-280 K accounts for tens of seconds with activation energy 0.166 eV/molecule. The rate of the thermoinduced transition in the cytochrome-RC complex was found to be three orders of magnitude slower than the rate of similar thermoinduced transition of the electron transfer reaction from the primary to secondary quinone acceptors studied in the preceding work (Chamorovsky et al. in Eur Biophys J 32:537-543, 2003). Parameters of thermoinduced activation of the electron transfer from the multiheme cytochrome c to the photoactive bacteriochlorophyll dimer are discussed in terms of cytochrome c docking onto the RC.

CORRELATION OF ELECTRON TRANSFER RATE IN PHOTOSYNTHETIC REACTION CENTERS WITH INTRAPROTEIN DIELECTRIC PROPERTIES

Chamorovsky S.K., Cherepanov D.A., Chamorovsky C.S., Semenov A.Y.

Biochimica et Biophysica Acta-Bioenergetics 1767 (2007) 441-448.

A number of the electrogenic reactions in photosystem I, photosystem II, and bacterial reaction centers (RC) were comparatively analyzed, and the variation of the dielectric permittivity (epsilon) in the vicinity of electron carriers along the membrane normal was calculated. The value of e was minimal at the core of the complexes and gradually increased towards the periphery. We found that the rate of electron transfer (ET) correlated with the value of the dielectric permittivity: the fastest primary ET reactions occur in the low-polarity core of the complexes within the picosecond time range, whereas slower secondary reactions take place at the high-polarity periphery of the complexes within micro- to millisecond time range. The observed correlation was quantitatively interpreted in the framework of the Marcus theory. We calculated the reorganization energy of ET carriers using their van der Waals volumes and experimentally determined epsilon values. The electronic coupling was calculated by the empirical Moser-Dutton rule for the distance-dependent electron tunneling rate in nonadiabatic ET reactions. We concluded that the local dielectric permittivity inferred from the electrometric measurements could be quantitatively used to estimate the rate constant of ET reactions in membrane proteins with resolved atomic structure with the accuracy of less than one order of magnitude. .

MICELLAR PEROXIDASE-CATALYZED SYNTHESIS OF CHIRAL POLYANILINE

Caramyshev A.V., Lobachov V.M., Selivanov D.V., Sheval E.V., Vorobiev A.K., Katasova O.N., Polyakov V.Y., Makarov A.A., Sakharov I.Y.

Biomacromolecules 8 (2007) 2549-2555.

Micellar peroxidase-catalyzed synthesis of chiral polyaniline (PANI) in the presence of dodecylbenzenesulfonic acid (DBSA) was developed. The effect of DBSA concentration on the catalytic efficiency of horseradish and palm tree peroxidases was examined. Favorable conditions for the enzymatic synthesis of chiral PANI, determined by a multiple factors design, demonstrated that the PANIs with the highest chirality were produced in the presence of low concentrations of optically active camphorsulphonic acid (CSA). Unexpectedly, the chiral PANI was also synthesized in the absence of CSA in feed. The favorable conditions for the enzymatic production of chiral and conducting PANIs were shown to be different. The morphology of the chiral PANI particles was examined by transmission and scanning electron microscopies.

MICROTUBULES' INTERACTION WITH CELL CORTEX IS REQUIRED FOR THEIR RADIAL ORGANIZATION, BUT NOT FOR CENTROSOME POSITIONING

Brodsky I.B., Burakov A.V., Nadezhdina E.S.

Cell Motility and the Cytoskeleton 64 (2007) 407-417.

Microtubules in interphase mammalian cells usually form a radial array with minus-ends concentrated in the central region and plus-ends placed at the periphery. This is accepted as correct, that two factors determinate the radial organization of microtubules - the centrosome, which nucleate and anchor the microtubules minus-ends, and the interaction of microtubules with cortical dynein, which positions centrosome in the cell center. However, it looks as if there are additional factors, affecting the radial structure of microtubule system. We show here that in aged Vero cytoplasts (17 h after enucleation) microtubule system lost radial organization and became chaotic. To clear up the reasons of that, we studied centrosome activity, its position in the cytoplasts and microtubule dynamics. We found that centrosome in aged cytoplasts was still active and placed in the central region of the cytoplasm, while after total disruption of the microtubules it was displaced from the center. Microtubules in aged cytoplasts were not stabilized, but they lost their ability to stop to grow near cell cortex and continued to grow reaching it. Aged cytoplast lamellae was partially depleted with dynactin though Golgi remained compact indicating dynein activity. We conclude that microtubule stoppage at cell cortex is mediated by some (protein) factors, and these factors influence radial structure of microtubule system. It seems that the key role in centrosome positioning is played by dynein complexes anchored everywhere in the cytoplasm rather than anchored in cell cortex. Cell Motil. Cytoskeleton 64:407-417, 2007. .

REDOX-DEPENDENT SODIUM BINDING BY THE Na+-TRANSLOCATING NADH: QUINONE OXIDOREDUCTASE FROM VIBRIO HARVEYI

Bogachev A.V., Bertsova Y.V., Aitio O., Permi P., Verkhovsky M.I.

Biochemistry 46 (2007) 10186-10191.

Relaxation characteristics of the Na-23 nuclei magnetization were used to determine the sodium-binding properties of the Na+-translocating NADH:quinone oxidoreductase from Vibrio harveyi (NQR). The dissociation constant of Na+ for the oxidized enzyme was found to be 24 mM and for the reduced enzyme about 30 mu M. Such large (3 orders in magnitude) redox dependence of the NQR affinity to sodium ions shows that the molecular machinery was designed to use the drop in redox energy for creating an electrochemical sodium gradient. Redox titration of NQR monitored by changes in line width of the Na-23 NMR signal at 2 mM Na+ showed that the enzyme affinity to sodium ions follows the Nernst law for a one-electron carrier with E-m about -300 mV (vs SHE). The data indicate that energy conservation by NQR involves a mechanism modulating ion affinity by the redox state of an enzyme redox cofactor.

DISCOVERY OF THE TRUE PEROXY INTERMEDIATE IN THE CATALYTIC CYCLE OF TERMINAL OXIDASES BY REAL-TIME MEASUREMENT

Belevich I., Borisov V.B., Verkhovsky M.I.

Journal of Biological Chemistry 282 (2007) 28514-28519.

The sequence of the catalytic intermediates in the reaction of cytochrome bd terminal oxidases from Escherichia coli and Azotobacter vinelandii with oxygen was monitored in real time by absorption spectroscopy and electrometry. The initial binding of O-2 to the fully reduced enzyme is followed by the fast (5 mu s) conversion of the oxy complex to a novel, previously unresolved intermediate. In this transition, low spin heme b(558) remains reduced while high spin heme b(595) is oxidized with formation of a new heme d-oxygen species with an absorption maximum at 635 nm. Reduction of O-2 by two electrons is sufficient to produce ( hydro) peroxide bound to ferric heme d. In this case, the O-O bond is left intact and the newly detected intermediate must be a peroxy complex of heme d (Fe-d(3+)-O-O-( H)) corresponding to compound 0 in peroxidases. The alternative scenario where the O-O bond is broken as in the P-M intermediate of heme-copper oxidases and compound I of peroxidases is not very likely, because it would require oxidation of a nearby amino acid residue or the porphyrin ring that is energetically unfavorable in the presence of the reduced heme b558 in the proximity of the catalytic center. The formation of the peroxy intermediate is not coupled to membrane potential generation, indicating that hemes d and b595 are located at the same depth of the membrane dielectric. The lifetime of the new intermediate is 47 mu s; it decays into oxoferryl species due to oxidation of low spin heme b558 that is linked to significant charge translocation across the membrane.

POTATO VIRUS X: STRUCTURE, DISASSEMBLY AND RECONSTITUTION

Atabekov J., Dobrov E., Karpova O., Rodionova N.

Molecular Plant Pathology 8 (2007) 667-675.

This paper summarizes some structural characteristics of Potato virus X(PVX), the flexuous filamentous plant potexvirus. A model of PVX coat protein (CP) tertiary structure in the virion proposed on the basis of tritium planigraphy combined with predictions of the protein tertiary structure is described. A possible role of glycosylation and phosphorylation in the CP structure and function is discussed. Two forms of PVX virion disassembly are discussed: (i) the virion co-translational disassembly after PVX CP in situ phosphorylation and (ii) disassembly of PVX triggered by different factors after linear destabilization of the virion by binding of the PVX-coded movement protein (TGBp1) to one end of the polar CP-helix. Special emphasis was placed on a translational activation of encapsidated PVX RNA and rapid disassembly of TGBp1-PVX complexes into free RNA and CP. The results of experiments on the PVX CP repolymerization and PVX reconstitution are considered. In particular, the products assembled from PVX RNA, CP and TGBp1 were examined. Single-tailed particles were found with a helical, head-like structure consisting of helically arranged CP subunits located at the 5'-tail of RNA; the TGBp1 was bound to the end of the head. Translatable 'RNA-CP-TGBp1' complexes may represent the transport form of the PVX infection.

DIFFERENTIAL FACTOR REQUIREMENT TO ASSEMBLE TRANSLATION INITIATION COMPLEXES AT THE ALTERNATIVE START CODONS OF FOOT- AND-MOUTH DISEASE VIRUS RNA

Andreev D.E., Fernandez-Miragall O., Ramajo J., Dmitriev S.E., Terenin I.M., Martinez-Salas E., Shatsky I.N.

RNA-A Publication of the RNA Society 13 (2007) 1366-1374.

The foot-and-mouth disease virus (FMDV) RNA contains two in-frame AUG codons separated by 84 nt that direct translation initiation of the viral polyprotein. The mechanism of initiation at the IRES-proximal AUG codon (AUG1) has been previously analyzed, whereas no data on factor requirements for AUG2 have been reported. Here, using the method of 48S translation initiation complex reconstitution, we show that elF1 is indispensable in forming the 48S initiation complex at AUG2. In contrast, it reduces the assembly of this complex at AUG1. Stabilization of a stem - loop between the initiation triplets induces a small decrease in the toeprint intensity at AUG2, accompanied by an increase in the AUG1/AUG2 ratio as well as a moderate reduction of protein synthesis initiated at AUG2 in transfected cells. PTB and ITAF45 exerted an additive positive effect on the 48S complex at AUG2, although a substantial reconstitution on both AUGs occurs on omission of either of these proteins. Relative to the beta-globin mRNA, the 48S complex formation at AUG1 and AUG2 is slow and occurs with the same kinetics as revealed by the "kinetic'' toeprint assay. Mutation of AUG1 to AUA does not abrogate protein synthesis in transfected cells, and has no effect on the rate of the 48S complex formation at AUG2. We conclude that the AUG2 initiation region is selected independently of 48S complex formation at the upstream AUG1. The kinetic toeprint assay also shows that cap- dependent assembly of the 48S complex in vitro occurs faster than the FMDV IRES-mediated complex assembly.

CRYOGENIC FUEL TARGETS FOR INERTIAL FUSION: OPTIMIZATION OF FABRICATION AND DELIVERY CONDITIONS

Aleksandrova I.V., Koresheva E.R., Osipov I.E., Bazdenkov S.V., Belolipetskiy A.A., Chtcherbakov V.I., Tirnasheva T.P., Tonshin A.A., Yaguzinskiy L.S., Dorogotovtsev V.M., Akunets A.A.

Journal of Russian Laser Research 28 (2007) 207-235.

The paper addresses the main problems associated with the fabrication of cryogenic fuel targets and their delivery to the irradiation zone of an inertial confinement fusion (ICF) facility. Optimal solutions of these problems have been developed at the Lebede, Physical Institute for the case of free-standing direct-irradiation cryogenic targets. In contrast to the traditional technology, the approach proposed and demonstrated is shown to conform to all requirements of the current ICF program: (a) quality of fuel layering, (b)stability of layering, and (c) minimization of tritium inventory. This technology is the basis for a designed device of introducing cryogenic targets into the chamber of an ISKRA-6 high-power laser facility being developed in Russia.

HYPERHOMOCYSTEINEMIA AND LIPID PEROXIDATION IN STABLE CORONARY HEART DISEASE

Belaya O.L., Fedorova N.V., Fomina I.G.

Cardiovascular Therapy and Prevention 6 (2007) 41-46.

Aim. To compare homocysteine, lipids, and lipid peroxidation (LP) products' levels in plasma of coronary heart disease (CHD) patients with stable angina and dyslipidemia, and relatively healthy individuals. Material and methods. The Study included 30 CHD patients (Functional Class, FC, II-III angina) and 15 relatively, healthy Volunteers. Plasma levels of lipids, LP products (malone dialdehyde, MDA), homocysteine (by highly effective liquid chromatography method), as well as coagulation and fibrinolysis parameters were Measured. Results. In 86,7% of CHD patients with II-III FC angina, homocysteine and MDA levels were significantly higher than in healthy controls, and in 30916 these levels were higher than standard norm. Plasma homocysteine levels correlated with LP activity in CHD patients (r=0,59). Methionine load test helped to diagnose latent hyperhomocysteinemia. Conclusion. CHD patients with II-III FC angina, hypercholesterolemia and hyperlipoperoxidemia had significantly higher homocysteine levels, compared to relatively healthy individuals (p < 0,001).

STUDY OF THE NEW HIV-1 INTEGRASE INHIBITORS MECHANISM OF ACTION USING MODIFIED SUBSTRATES

Agapkina Y., Korolev S., Aleksandrov D., Romanova E., Shevelev S., Poroikov V., Gottikh M.

Febs Journal 274 (2007) 239-239.

REGULATION OF THE NQR-OPERONS EXPRESSION IN VIBRIO HARVEYI AND KLEBSIELLA PNEUMONIAE

Fadeeva M.S., Yakovtseva E.A., Bertsova Y.V., Bogachev A.V.

Febs Journal 274 (2007) 226-226.

GSK-3 INHIBITOR, LI+ AND INSULIN PREVENT DYSFUNCTION OF MITOCHONDRIA AND CELL DEATH IN RAT KIDNEY AFTER ISCHEMIA/REOXYGENATION(I/R)

Vasileva A.K., Plotnikov E.Y., Kazachenko A.V., Kirpatovsky V.I., Zorov D.B.

Febs Journal 274 (2007) 205-205.

MITOCHONDRIAL FRAGMENTATION INDUCED BY UV AND ISCHEMIA. ROLE OF GLYCOGEN SYNTHASE KINASE-3 (GSK-3)

Plotnikov E.Y., Vasileva A.K., Shashkova A.A., Zorov D.B.

Febs Journal 274 (2007) 205-205.

PRO- AND ANTIOXIDANT PROPERTIES OF MITOCHONDRIA-TARGETED ANTIOXIDANT MITOQ

Izyumov D.S., Mostovenko E.V., Korotetskaya M.V., Chernyak B.V.

Febs Journal 274 (2007) 145-145.

METHYLATION OF GCGG SITES OF THE PATATIN PROMOTER IS ORGAN-SPECIFIC AND INVERSELY CORRELATES WITH ITS ACTIVITY

Romanov G.A., Naumkina E.M., Ashapkin V.V., Vanyushin B.F.

Doklady Biochemistry and Biophysics 417 (2007) 327-330.

EXCITATION DYNAMICS IN STRONGLY BOUNDED ASSOCIATES OF B800-850 AND B800-830 COMPLEXES FROM PHOTOSYNTHETIC PURPLE BACTERIUM THIORHODOSPIRA SIBIRICA

Razjivin A.P., Stepanenko I.A., Kozlovsky V.S., Kompanets V.O., Chekalin S.V., Makhneva Z.K., Moskalenko A.A., Tikhonov A.V., Popov V.O., Tikhonova T.V.

Biologicheskie Membrany 24 (2007) 457-464.

Strongly bounded associates of B800-850 (LH2) and B800-830 (LH3) complexes from photosynthetic purple bacterium Thiorhodospira sibirica were investigated. It was shown that associates contain 6-8 complexes (LH2:LH3 approximate to 1: 1). Absorption spectra of the monomer LH2 and the monomer LH3 complexes were calculated. Excitation of B800 absorption band of associates results in: i) intracomplex excitation energy transfer from B800 to B820 or B850 with time constant of about 500 fs; ii) intercomplex excitation energy transfer from B820 band of LH3 complex to B850 band of LH2 complex with time constant of about 2.5 ps; iii) excitation deactivation in B850 band of LH2 complex with time constant of about 800 ps. Signal polarization at long-wavelength side of associates absorption spectrum near 900 nm was negative (-0.1). The interaction of LH3 and LH2 complexes in associates is, to some extent, analogous to the interaction of LH2 and LH1 complexes in chromatophores. Time constant of excitation energy transfer between LH3 and LH2 complexes in associates may be regarded as a minimal time constant for energy transfer between the peripheral and core antenna complexes.

EFFECTS OF DMSO AND CHOLESTEROL-BINDING PEPTIDES ON PHAGOCYTIC ACTIVITY OF CULTURED MACROPHAGES IC-21

Dunina-Barkovskaya A.Y., Vishniakova K.S., Cheshev D.A., Chekanov N.N., Bujurina I.M.

Biologicheskie Membrany 24 (2007) 451-456.

One of the earliest events of phagocytosis is the formation of lipid domains enriched with cholesterol and sphingomyelin in plasma membrane of a phagocyte. In these domain:;, there cluster phagocytic receptors and other integral proteins involved in the phagocytic signal cascade. Earlier we found that phagocytic receptor Fc gamma R and some ionic channels possess in their transmembrane region an amino-acid sequence analogous to the cholesterol-binding consensus of the peripheral-type benzodiazepine receptor (PBR). To check if this sequence is of functional significance, in this work we studied the effects of two synthetic peptides - a fragment of the PBR cholesterol-binding consensus, VLNYYVW, and its variation, LLVYPW, on the phagocytic activity of cultured macrophages IC-21. Phagocytosis was assessed by fluorescent microscopy, using 2-mu m non-opsonized fluorescent latex beads. Peptides were dissolved in DMSO, which turned to affect phagocytic activity by itself, in the presence of 0.5-1.3% DMSO the number of beads per cell was 20-30% lower than in the absence of DMSO. Peptide VLNYYVW (5-100 mu g/ml) augmented the inhibitory action of DMSO, while LLVYPW (20100 mu g/ml) exerted a differing effect. Our data suggest that the peptides studied may indeed interfere with the interactions between integral proteins and membrane cholesterol and thus affect the phagocytic process. The action of DMSO on phagocytosis found here may also be related to the disturbance in cholesterol-protein interactions. We propose that cholesterol-binding peptides can be used for the design of immunomodulating drugs.

GAP JUNCTION PROTEINS EVOLUTION

Panchin Y.V.

Neuron Glia Biology 2 (2007) S24-S25.

A BIOCHEMICAL APPROACH TO THE PROBLEM OF AGING: "MEGAPROJECT" ON MEMBRANE-PENETRATING IONS. THE FIRST RESULTS AND PROSPECTS

Skulachev V.P.

Biochemistry-Moscow 72 (2007) 1385-1396.

Antioxidants specifically addressed to mitochondria have been studied for their ability to decelerate aging of organisms. For this purpose, a project has been established with participation of several research groups from Belozersky Institute of Physico-Chemical Biology and some other Russian research institutes as well as two groups from the USA and Sweden, with support by the "Mitotechnology" company founded by "RAInKo" company (O. V. Deripaska and Moscow State University). This paper summarizes the first results of the project and estimates its prospects. Within the framework of the project, antioxidants of a new type (SkQ) were synthesized comprising plastoquinone (an antioxidant moiety), a penetrating cation, and decane or pentane linker. Using planar bilayer phospholipid membranes, we selected SkQ derivatives with the highest penetrating ability, namely plastoquinonyl-decyl-triphenylphosphonium (SkQ1), plastoquinonyl-decylrhodamine 19 (SkQR1), and methylplastoquinonyl-decyl-triphenylphosphonium (SkQ3). Anti-and prooxidant properties of these substances and also of ubiquinone and ubiquinonyl-decyl-triphenylphosphonium (MitoQ) were tested on isolated mitochondria. Micromolar concentrations of cationic quinones are found to be very strong prooxidants, but in lower (submicromolar) concentrations they display antioxidant activity. The antioxidant activity decreases in the series SkQ1 = SkQR1 > SkQ3 > MitoQ, so the window between the anti-and prooxidant effects is smallest for MitoQ. SkQ1 is rapidly reduced by complexes I and II of the mitochondrial respiratory chain, i.e. it is a rechargeable antioxidant. Extremely low concentrations of SkQ1 and SkQR1 completely arrest the H2O2-induced apoptosis in human fibroblasts and HeLa cells (for SkQ1 C (1/2) = 1.10(-9) stop M) Higher concentrations of SkQ are required to block necrosis initiated by reactive oxygen species (ROS). In mice, SkQ1 decelerates the development of three types of accelerated aging (progeria) and also of normal aging, and this effect is especially demonstrative at early stages of aging. The same pattern is shown in invertebrates (drosophila and daphnia). In mammals, the effect of SkQs on aging is accompanied by inhibition of development of such age-related diseases as osteoporosis, involution of thymus, cataract, retinopathy, etc. SkQ1 manifests a strong therapeutic action on some already developed retinopathies, in particular, congenital retinal dysplasia. With drops containing 250 nM skQ1, vision is recovered in 50 of 66 animals who became blind because of retinopathy. SkQ1-containing drops instilled in the early stage of the disease prevent the loss of sight in rabbits with experimental uveitis and restore vision to animals that had already become blind. A favorable effect is also achieved in experimental glaucoma in rabbits. Moreover, the pretreatment of rats with 0.2 nmol SkQ1 per kg body weight significantly decreases the H2O2-induced arrhythmia of the isolated heart. SkQ1 strongly reduces the damaged area in myocardial infarction or stroke and prevents the death of animals from kidney infarction. In p53(-/-) stop mice, SkQ1 decreases the ROS level in the spleen cells and inhibits appearance of lymphomas which are the main cause of death of such animals. Thus, it seems reasonable to perform clinical testing of SkQ preparations as promising drugs for treatment of age-related and some other severe diseases of human and animals.

CONTRIBUTION OF GENOSYSTEMATICS TO CURRENT CONCEPTS OF PHYLOGENY AND CLASSIFICATION OF BRYOPHYTES

Troitsky A.V., Ignatov M.S., Bobrova V.K., Milyutina I.A.

Biochemistry-Moscow 72 (2007) 1368-1376.

This paper is a survey of the current state of molecular studies on bryophyte phylogeny. Molecular data have greatly contributed to developing a phylogeny and classification of bryophytes. The previous traditional systems of classification based on morphological data are being significantly revised. New data of the authors are presented on phylogeny of Hypnales pleurocarpous mosses inferred from nucleotide sequence data of the nuclear DNA internal transcribed spacers ITS1-2 and the trnL-F region of the chloroplast genome.

Genetic structure of the Salvelinus genus chars from reservoirs of the Kuril Islands

Shubina E.A., Ponomareva E.V., Gritsenko O.F.

Biochemistry-Moscow 72 (2007) 1331-1348.

Genetic resemblance of chars Salvelinus alpinus krasheninnikovi (Salvelinus malma krasheninnikovi) of 35 samples collected in five Kuril Islands-Shumshu, Paramushir, Onekotan, Iturup, and Kunashir-has been studied by the PCR-RAPD method. In the limits of each island, both resident isolates and anadromous forms give strictly supported clusters distinct from samples from the other islands. The samples from five islands form three superclusters: the first from Kunashir and Iturup Islands, the second from Paramushir and Onekotan Islands, and the third from Shumshu Island. The possible reasons for genetic similarity of resident and anadromous forms of Dolly Varden chars inhabiting reservoirs of a definite island are considered (the founder effect, homing, limited migration).

PHYLOGENY OF FLOWERING PLANTS BY THE CHLOROPLAST GENOME SEQUENCES: IN SEARCH OF A "LUCKY GENE"

Logacheva M.D., Penin A.A., Samigullin T.H., Vallejo-Roman C.M., Antonov A.S.

Biochemistry-Moscow 72 (2007) 1324-1330.

One of the most complicated remaining problems of molecular-phylogenetic analysis is choosing an appropriate genome region. In an ideal case, such a region should have two specific properties: (i) results of analysis using this region should be similar to the results of multigene analysis using the maximal number of regions; (ii) this region should be arranged compactly and be significantly shorter than the multigene set. The second condition is necessary to facilitate sequencing and extension of taxons under analysis, the number of which is also crucial for molecular phylogenetic analysis. Such regions have been revealed for some groups of animals and have been designated as "lucky genes". We have carried out a computational experiment on analysis of 41 complete chloroplast genomes of flowering plants aimed at searching for a "lucky gene" for reconstruction of their phylogeny. It is shown that the phylogenetic tree inferred from a combination of translated nucleotide sequences of genes encoding subunits of plastid RNA polymerase is closest to the tree constructed using all protein coding sites of the chloroplast genome. The only node for which a contradiction is observed is unstable according to the different type analyses. For all the other genes or their combinations, the coincidence is significantly worse. The RNA polymerase genes are compactly arranged in the genome and are fourfold shorter than the total length of protein coding genes used for phylogenetic analysis. The combination of all necessary features makes this group of genes main candidates for the role of "lucky gene" in studying phylogeny of flowering plants.

DO WE NEED MANY GENES FOR PHYLOGENETIC INFERENCE?

Aleshin V.V., Konstantinova A.V., Mikhailov K.V., Nikitin M.A., Petrov N.B.

Biochemistry-Moscow 72 (2007) 1313-1323.

Fifty-six nuclear protein coding genes from Taxonomically Broad EST Database and other databases were selected for phylogenomic-based examination of alternative phylogenetic hypotheses concerning intergroup relationship between multicellular animals (Metazoa) and other representatives of Opisthokonta. The results of this work support sister group relationship between Metazoa and Choanoflagellata. Both of these groups form the taxon Holozoa along with the monophyletic Ichthyosporea or Mesomycetozoea (a group that includes Amoebidium parasiticum, Sphaeroforma arctica, and Capsaspora owczarzaki). These phylogenetic hypotheses receive high statistical support both when utilizing whole alignment and when only 5000 randomly selected alignment positions are used. The presented results suggest subdivision of Fungi into Eumycota and lower fungi, Chytridiomycota. The latter form a monophyletic group that comprises Chytridiales + Spizellomycetales + Blastocladiales (Batrachochytrium, Spizellomyces, Allomyces, Blastocladiella), contrary to the earlier reports based on the analysis of 18S rRNA and a limited set of protein coding genes. The phylogenetic distribution of genes coding for a ubiquitin-fused ribosomal protein S30 implies at least three independent cases of gene fusion: in the ancestors of Holozoa, in heterotrophic Heterokonta (Oomycetes and Blastocystis), and in the ancestors of Cryptophyta and Glaucophyta. Ubiquitin-like sequences fused with ribosomal protein S30 outside of Holozoa are not FUBI orthologs. Two independent events of FUBI replacement by the ubiquitin sequence were detected in the lineage of C. owczarzaki and in the monophyletic group of nematode worms Tylenchomorpha + Cephalobidae. Bursaphelenchus xylophilus (Aphelenchoidoidea) retains a state typical of the rest of the Metazoa. The data emphasize the fact that the reliability of phylogenetic reconstructions depends on the number of analyzed genes to a lesser extent than on our ability to recognize reconstruction artifacts.

A VIEW OF AN ELEMENTAL NATURALIST AT THE DNA WORLD (BASE COMPOSITION, SEQUENCES, METHYLATION)

Vanyushin B.F.

Biochemistry-Moscow 72 (2007) 1289-1298.

The pioneering data on base composition and pyrimidine sequences in DNA of pro- and eukaryotes are considered , and their significance for the origin of genosystematics is discussed. The modern views on specificity and functional role of enzymatic DNA methylation in eukaryotes are described. DNA methylation controls all genetic functions and is a mechanism of cellular differentiation and gene silencing. A model of regulation of DNA replication by methylation is suggested . Adenine DNA methylation in higher eukaryotes ( higher plants) was first observed, and it was established that one and the same gene can be methylated at both cytosine and adenine moieties. Thus, there are at least two different and seemingly interdependent DNA methylation systems present in eukaryotic cells. The first eukaryotic adenine DNA-methyl- transferase is isolated from wheat seedlings and described: the enzyme methylates DNA with formation of N-6-methylade- nine in the sequence TGATCA -> TGm(6)ATCA. It is found that higher plants have endonucleases that are dependent on S- adenosyl-L-methionine (SAM) and sensitive to DNA methylation status. Therefore, as in bacteria, plants seem to have a restriction-modification (R-M) system. A system of conjugated up- and down-regulation of SAM-dependent endonucleases by SAM modulations is found in plants. Revelation of an essential role of DNA methylation in regulation of genetic processes is a fundament of materialization of epigenetics and epigenomics.

CHIRAL HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY ANALYSIS OF ALPHA-AMINO ACID MIXTURES USING A NOVEL SH REAGENT-N-R-MANDELYL-L-CYSTEINE AND TRADITIONAL ENANTIOMERIC THIOLS FOR PRECOLUMN DERIVATIZATION

Chernobrovkin M.G., Shapovalova E.N., Guranda D.T., Kudryavtsev P.A., Svedas V.K., Shpigun O.A.

Journal of Chromatography A 1175 (2007) 89-95.

Several chiral thiols, i.e. traditionally used enantionterically pure SH reagents and novel N-R-mandelyl-L-cysteine (R-NMC) containing additional chiral center, have been applied as co-reagents in precolumn derivatization with o-phthalaldehyde for enantiomeric HPLC analysis of individual a-amino acids and their mixtures. The R-NMC-derived isoindoles as well as adducts with other thiols have a characteristic absorption maximum at 340 nm, and are highly fluorescent allowing detection of 10 mu g/l of an amino acid. Investigated 19 amino acids were analyzed separately and in a mixture by a gradient HPLC after precolumn derivatization. The chromatographic behavior of formed isoindoles substantially differs for each of the thiols used for modification. In contrast to traditional enantiomeric thiols application of diastereomeric R-NMC provides higher resolution for a-amino acid enantiomers, with L,D-elution order (except for Arg). Combined use of R-NMC and other thiol enlarges the possibilities of this method, allowing accurate chiral analysis of complex amino acid mixtures. .

HELICAL ALPHA-SYNUCLEIN FORMS HIGHLY CONDUCTIVE ION CHANNELS

Zakharov S.D., Hulleman J.D., Dutseva E.A., Antonenko Y.N., Rochet J.C., Cramer W.A.

Biochemistry 46 (2007) 14369-14379.

alpha-Synuclein (alpha S) is a cytosolic protein involved in the etiology of Parkinson's disease (PD). Disordered in an aqueous environment, alpha S develops a highly helical conformation when bound to membranes having a negatively charged surface and a large curvature. It exhibits a membrane-permeabilizing activity that has been attributed to oligomeric protofibrillar forms. In this study, monomeric wild-type alpha S and two mutants associated with familial PD, E46K and A53T, formed ion channels with well-defined conductance states in membranes containing 25-50% anionic lipid and 50% phosphatidylethanolamine (PE) in the presence of a trans-negative potential. Another familial mutant, A30P, known to have a lower membrane affinity, did not form ion channels. Ca2+ prevented channel formation when added to membranes before alpha S and decreased channel conductance when added to preformed channels. In contrast to the monomer, membrane permeabilization by oligomeric alpha S was not characterized by formation of discrete channels, a requirement for PE lipid, or a membrane potential. Channel activity, alpha-helical contents thermal stability of membrane-bound alpha S determined by far-UV CD, and lateral mobility of alpha S bound to planar membranes measured by fluorescence correlation spectroscopy were correlated. It was inferred that discrete ion channels with well-defined conductance states were formed in the presence of a membrane potential by one or several molecules of monomeric alpha S in an alpha-helical conformation and that such channels may have a role in the normal function and/or pathophysiology of the protein.

DNA SEQUENCE-SPECIFIC LIGANDS: XIII. NEW DIMERIC HOECHST 33258 MOLECULES, INHIBITORS OF HIV-1 INTEGRASE IN VITRO

Gromyko A.V., Salyanov V.I., Strel'tsov S.A., Oleinikov V.A., Korolev S.P., Gottikh M.B., Zhuze A.L.

Russian Journal of Bioorganic Chemistry 33 (2007) 569-578.

Dimeric Hoechst 33258 molecules [dimeric bisbenzimidazoles (DBBIs)] that, upon binding, occupy one turn of the B form of DNA in the narrow groove were constructed by computer simulation. Three fluorescent DBBIs were synthesized; they consist of two bisbenzimidazole units tail-to-tail linked to phenolic hydroxy groups via penta-or heptamethylene or tri(ethylene glycol) spacers and have terminal positively charged N,N-dimethylaminopropyl carboxamide groups in the molecule. The absorption spectra of the DBBIs in the presence of different DNA concentrations showed a hypochromic effect and a small shift of the absorption band to longer wavelengths, which indicated the formation of a complex with DNA. The presence of an isobestic point in the spectrum indicates the formation of one type of DBBI-DNA complexes. The interaction of DBBIs with DNA was studied by CD using a cholesteric liquid-crystalline dispersion (CLD) of DNA. The appearance of a positive band in the absorption region of ligand chromophores in the CD spectrum of the DNA CLD indicates the formation of a DBBI-DNA complex in which ligand chromophores are arranged at an angle close to 54 degrees relative to the helix axis of DNA, which suggests the localization of the DBBI in the narrow groove of DNA. All the DBBIs were found to be in vitro inhibitors of HIV-1 DNA integrase in the 3'-processing reaction, and, of the three DBBIs, two dimers inhibit HIV-1 integrase even in submicromolar concentrations.

ARACHIDONIC ACID SIGNALING IS OF IMIDAZOLINE-INDUCED K-ATP INVOLVED IN THE MECHANISM CHANNEL-INDEPENDENT STIMULATION OF INSULIN SECRETION

Sharoyko V.V., Zaitseva I.I., Leibiger B., Efendic S., Berggren P.O., Zaitsev S.V.

Cellular and Molecular Life Sciences 64 (2007) 2985-2993.

The mechanism by which the novel, pure glucose-dependent insulinotropic, imidazoline derivative BL11282 promotes insulin secretion in pancreatic islets has been investigated. The roles of K-ATP channels, Q2-adrenoreceptors, the 11-receptor-phosphatidylcholine-specific phospholipase (PC-PLC) pathway and arachidonic acid signaling in BL11282 potentiation of insulin secretion in pancreatic islets were studied. Using SUR1((-/-)) deficient mice, the previous notion that the insulinotropic activity of BLI1282 is not related to its interaction with K-ATP channels was confirmed. Insulinotropic activity of 131-11282 was not related to its effect on alpha(2)-adrenoreceptors, I-1-imidazoline receptors or PC-PLC. BL11282 significantly increased [H-1]arachidonic acid production. This effect was abolished in the presence of the iPLA2 inhibitor, bromoenol lactone. The data suggest that potentiation of glucose-induced insulin release by BL11282, which is independent of concomitant changes in cytoplasmic free Ca2+ concentration, involves release of arachidonic acid by iPLA(2) and its metabolism to epoxyeicosatrienoic acids through the cytochrome P-450 pathway.

COOPERATIVE INTERACTION OF HIGH-POTENTIAL HEMES IN THE CYTOCHROME SUBUNIT OF THE PHOTOSYNTHETIC REACTION CENTER OF BACTERIUM ECTOTHIORHODOSPIRA SHAPOSHNIKOVII

Pottosin I.I., Chamorovsky C.S., Chamorovsky S.K.

Biochemistry-Moscow 72 (2007) 1254-1260.

Cooperative interaction of the high-potential hemes (C-h) in the cytochrome subunit of the photosynthesizing bacterium Ectothiorhodospira shaposhnikovii was studied by comparing redox titration curves of the hemes under the conditions of pulse photoactivation inducing single turnover of electron-transport chain and steady-state photoactivation, as well as by analysis of the kinetics of laser-induced oxidation of cytochromes by reaction center (RC). A mathematical model of the processes of electron transfer in cytochrome-containing RC was considered. Theoretical analysis revealed that the reduction of one heme C-h, facilitated the reduction of the other heme, which was equivalent to a 60 mV positive shift of the midpoint potential. In addition, reduction of the second heme Ch caused a three- to four-fold acceleration of the electron transfer from the cytochrome subunit to RC.

THE CENTROSOME KEEPS NUCLEATING MICROTUBULES BUT LOOSES THE ABILITY TO ANCHOR THEM AFTER THE INHIBITION OF DYNEIN-DYNACTIN COMPLEX

Zhapparova O.N., Burakov A.V., Nadezhdina E.S.

Biochemistry-Moscow 72 (2007) 1233-1240.

We inhibited dynein in cells either by the expression of coiled coil-1 (CC1) fragment of dynactin p150Glued subunit or by the microinjection of CC1 protein synthesized in Escherichia coli. CC1 impeded the aggregation of pigment granules in fish melanophores and caused the dispersion of Golgi in Vero and HeLa cells. These data demonstrated the inhibiting effect of CC1 on dynein. In cultured cells, CC1 expression caused the disruption of microtubule array, while the nucleation of new microtubules remained unaltered. This was proved both with in vivo microtubule recovery after nocodazole treatment and with in vitro microtubule polymerization on centrosomes, when the number of nucleated microtubules marginally reduced after the incubation with CC1 Moreover, the inhibiting anti-dynein 74.1 antibodies caused the same effect. Thus we have shown that though dynein is not important for microtubule nucleation, it is essential for the radial organization of microtubules presumably being involved in microtubule anchoring on the centrosome.

PREPARATION OF 2 '-HYDRAZINO OLIGONUCLEOTIDES AND THEIR REACTION WITH ALDEHYDES AND 1,3-DIKETONES

Zatsepin T.S., Oretskaya T.S., Gait M.J., Stetsenko D.A.

Nucleosides Nucleotides & Nucleic Acids 26 (2007) 795-798.

Oligodeoxyribonucleotides that contain a hydrazino nucleoside, 2 '-O-(2-hydrazinoethyl)uridine were prepared and shown to react with aldehydes or 1,3-diketones with the formation of hydrazones or pyrazoles, respectively. The method may be applicable for the preparation of oligonucleotide-peptide conjugates.

A CONVENIENT SOLID-PHASE METHOD FOR THE SYNTHESIS OF NOVEL OLIGONUCLEOTIDE-FOLATE CONJUGATES

Kazanova E.V., Zubin E.M., Kachalova A.V., Volkov E.M., Oretskaya T.S., Stetsenko D.A., Gottikh M.B.

Nucleosides Nucleotides & Nucleic Acids 26 (2007) 1273-1276.

We describe the Preparation of two batches of a polymer support for the incorporation of folic acid into oligonucleotides. The method permits the regioselective attachment of a target nucleic acid sequence through its 3'-end to either the alpha- or gamma-carboxyl group of L-glutamic acid, respectively. The supports have been tested in solid-phase synthesis of oligonucleotide-folate conjugates for cell delivery studies.

PHYLOGENY OF THE GENUS LOPHOZIA (DUMORT.) DUMORT. S. STR. INFERRED FROM NUCLEAR AND CHLOROPLAST SEQUENCES ITS1-2 AND TRNL-F

Vilnet A.A., Milyutina I.A., Konstantinova N.A., Ignatov M.S., Troitsky A.V.

Russian Journal of Genetics 43 (2007) 1306-1313.

Maximum parsimony and maximum likelihood phylogenetic trees were constructed for 21 taxa of Lophozia s. str. and the related genera, Schistochilopsis (5 species), Protolophozia elongata, and Obtusifolium obtusum based on combined nuclear ITS1-2 and chloroplast trnL-F DNA sequences. The trees were characterized by similar topology. It was demonstrated that the genus Lophozia s. str. was monophyletic, excluding L. sudetica, which deserved isolation into a distinct cryptic genus. The species distribution among the clades disagreed with the sections distinguished based on anatomical and morphological data. The relationships within the genus Schistochilopsis were consistent with the sectioning of the genus, based on morphological characters. Analysis of molecular data provided more precise definition of the systematic position of a number of taxa. A low level of genetic divergence of geographically distant forms was demonstrated.

MULTISTATE CONFORMATIONAL MODEL OF A SINGLE LH2 COMPLEX: QUANTITATIVE PICTURE OF TIME-DEPENDENT SPECTRAL FLUCTUATIONS

Novoderezhkin V.I., Rutkauskas D., van Grondelle R.

Chemical Physics 341 (2007) 45-56.

The fluorescence (FL) spectrum of single LH2 complexes fluctuates on a time-scale of seconds in wavelength, spectral shape and intensity [D. Rutkauskas, V. Novoderezhkin, R.J. Cogdell, R. van Grondelle, Biophys. J. 88 (2005) 422]. Here we propose a model capable to explain the statistics of these time-dependent FL-fluctuations. In this model this evolution of the spectra is described in terms of slow conformational motion of the pigment-protein complex inducing random shifts of the site energies. These shifts manifest themselves as inhomogeneous broadening of the bulk spectra and can be directly observed as spectral fluctuations in single molecule experiments. Our model assumes a finite number of conformational sub-states for each pigment of the complex and allows a calculation of the conformational dynamics by introducing phenomenological transfer rates between these sub-states. The simplest version of the model with two conformations for each pigment in enough to explain the fast small-size (+/- 10 nm) fluctuations within the 860-880 nm spectral region. Larger spectral jumps that are observed in the experiment (10-20 nm jumps to the blue and 10-40 nm to the red) can only be reproduced by including additional conformational states. The simplest model includes one more pair of states (producing big red and big blue shifts of the site energies). This four-state model enables us to reproduce quantitatively (i) the distribution of the FL peak positions, (ii) the changes of the width and asymmetry of the FL profiles as a function of the FL peak position, and (iii) distribution of the amplitudes and times of spectral jumps as a function of the initial FL peak position. We classify and describe the dynamic patterns that appear most often in the measured FL traces and in the calculated dynamics. We demonstrate a similarity between the measured and modelled patterns and show how these main types of the dynamics are related to specific changes in the conformational state of the 18 pigments of the LH2-B850 antenna. .

TIME-RESOLVED SINGLE-TURNOVER OF BA(3) OXIDASE FROM THERMUS THERMOPHILUS

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

Biochimica et Biophysica Acta-Bioenergetics 1767 (2007) 1383-1392.

The kinetics of the oxidation of fully-reduced ba(3) cytochrome c oxidase from Thermus thermophilus by oxygen were followed by time-resolved optical spectroscopy and electrometry. Four catalytic intermediates were resolved during this reaction. The chemical nature and the spectral properties of three intermediates (compounds A, P and O) reproduce the general features of aa(3)-type oxidases. However the F intermediate in ba(3) oxidase has a spectrum identical to the P state. This indicates that the proton taken up during the P -> F transition does not reside in the binuclear site but is rather transferred to the covalently cross-linked tyrosine near that site. The total charge translocation associated with the F -> O transition in ba(3) oxidase is close to that observed during the F -> O transition in the aa(3) oxiclases. However, the P-R -> F transition is characterized by significantly lower charge translocation, which probably reflects the overall lower measured pumping efficiency during multiple turnovers. .

NPIDB: A DATABASE OF NUCLEIC ACIDS-PROTEIN INTERACTIONS

Spirin S., Titov M., Karyagina A., Alexeevski A.

Bioinformatics 23 (2007) 3247-3248.

The database NPIDB (Nucleic AcidsProtein Interaction DataBase) contains information derived from structures of DNAprotein and RNAprotein complexes extracted from PDB (1834 complexes in July 2007). It is organized as a collection of files in PDB format and is equipped with a web-interface and a set of tools for extracting biologically meaningful characteristics of complexes. The content of the database is weekly updated.

SYNERGISTIC ACTION OF THE EXTRACT FROM MYCELIAL FUNGUS PLEUROTES OSTREATUS AND SOME CYTOSTATIC DRUGS ON PROLIFERATION AND APOPTOSIS OF TRANSFORMED CELLS

Polyakov V.Y., Kir'yanov G., Gerasimenia V.P., Orlov A.E., Lazareva E.A., Murashova M., Chentsov Y.S.

Biologicheskie Membrany 24 (2007) 379-388.

Effects produced by the P. ostreatus mycelium extract alone and in combination with doxorubicine and cyclophosphamide were studied on HeLa and myeloid leukosis cell lines. To assess cell viability, mitotic and apoptotic indexes were calculated; cell membrane status was evaluated using Trypan blue staining. Results show that mycelium extract alone acts as a weak inducer of apoptosis; it causes abnormal chromosome segregation and formation of chromosome bridges during mitosis. A combined application of the extract together with cyclophosphamide reveals an apparent synergism of the two agents, manifested as a prominent increase of cytotoxicity and a rise of apototic index. Results are discussed in relation to the hypothesis of possible intracellular targets for the extract.

A CBS DOMAIN-CONTAINING PYROPHOSPHATASE OF MOORELLA THERMOACETICA IS REGULATED BY ADENINE NUCLEOTIDES

Jamsen J., Tuominen H., Salminen A., Belogurov G.A., Magretova N.N., Baykov A.A., Lahti R.

Biochemical Journal 408 (2007) 327-333.

CBS (cystathionine P-synthase) domains are found in proteins from all kingdoms of life, and point mutations in these domains are responsible for a variety of hereditary diseases in humans; however, the functions of CBS domains are not well understood. In the present study, we cloned, expressed in Escherichia coli, and characterized a family 11 PPase (inorganic pyrophosphatase) from Moorella thermoacetica (mtCBS-PPase) that has a pair of tandem 60-amino-acid CBS domains within its N-terminal domain. Because mtCBS-PPase is a dimer and requires transition metal ions (Co2+ or Mn2+) for activity, it resembles common family II PPases, which lack CBS domains. The mtCBS-PPase, however, has lower activity than common family II PPases, is potently inhibited by ADP and AMP, and is activated up to 1.6-fold by ATP Inhibition by AMP is competitive, whereas inhibition by ADP and activation by ATP are both of mixed types. The nucleotides are effective at nanomolar (ADP) or micromolar concentrations (AMP and ATP) and appear to compete for the same site on the enzyme. The nucleotide-binding affinities are thus 100-10000-fold higher than for other CBS-domain-containing proteins. Interestingly, genes. encoding CBS-PPase occur most frequently in bacteria that have a membrane-bound H+-translocating PPase with a comparable PPi-hydrolysing activity. Our results suggest that soluble nucleotide-regulated PPases act as amplifiers of metabolism in bacteria by enhancing or suppressing ATP production and biosynthetic reactions at high and low [ATP]/([AMP] + [ADP]) ratios respectively.

INTERACTION WITH KEAP1 DOES NOT LEAD TO UBIQUITINATION AND DEGRADATION OF PROTHYMOSIN ALPHA

Melnikov S.V., Evstafieva A.G., Vartapetian A.B.

Molecular Biology 41 (2007) 790-796.

Prothymosin alpha (ProT alpha) is highly conserved among vertebrates and has many biological functions, including an enforcement of cell antioxidant defense. This function is due to ProT alpha interaction with Keap1, a repressor of the Nrf2 transcription factor, activating the expression of several antioxidant protein genes. Keap1 exports Nrf2 from the cell nucleus into the cytoplasm and, acting as an adaptor protein for ubiquitin ligase, facilitates ubiquitination of Nrf2 and its subsequent degradation by the 26S proteasome. ProT alpha and Nrf2 compete for Keap1 binding. ProT alpha is capable of displacing Nrf2 from its complex with Keap1, thereby increasing the expression of the Nrf2 target genes. It was found that ProT alpha remained stable upon its interaction with Keap1. In contrast to Nrf2, ProT alpha escaped Keap1-dependent ubiquitination, proteasomal degradation, and export from the nucleus. Moreover, ProT alpha ubiquitination was not detected even when Keap1 and ubiquitin were overproduced. Hence, activation of Nrf2-dependent transcription by ProT alpha was assumed to result from the increase in free Nrf2 rather than from an increase in total Nrf2.

THE MITOCHONDRION AS JANUS BIFRONS

Zorov D.B., Isaev N.K., Plotnikov E.Y., Zorova L.D., Stelmashook E.V., Vasileva A.K., Arkhangelskaya A.A., Khrjapenkova T.G.

Biochemistry-Moscow 72 (2007) 1115-1126.

The signaling function of mitochondria is considered with a special emphasis on their role in the regulation of redox status of the cell, possibly determining a number of pathologies including cancer and aging. The review summarizes the transport role of mitochondria in energy supply to all cellular compartments (mitochondria as an electric cable in the cell), the role of mitochondria in plastic metabolism of the cell including synthesis of heme, steroids, iron-sulfur clusters, and reactive oxygen and nitrogen species. Mitochondria also play an important role in the Ca2+-signaling and the regulation of apoptotic cell death. Knowledge of mechanisms responsible for apoptotic cell death is important for the strategy for prevention of unwanted degradation of postmitotic cells such as cardiomyocytes and neurons.

PEROXIDASE ACTIVITY OF MITOCHONDRIAL CYTOCHROME C OXIDASE

Vygodina T.V., Konstantinov A.A.

Biochemistry-Moscow 72 (2007) 1056-1064.

Mitochondrial cytochrome c oxidase is able to oxidize various aromatic compounds like o-dianisidine, benzidine and its derivatives (diaminobenzidine, etc.), p-phenylenediamine, as well as amidopyrine, melatonin, and some other pharmacologically and physiologically active substances via the peroxidase, but not the oxidase mechanism. Although specific peroxidase activity of cytochrome c oxidase is low compared with classical peroxidases, its activity may be of physiological or pathophysiological significance due to the presence of rather high concentrations of this enzyme in all tissues, as well as specific localization of the enzyme in the mitochondrial membrane favoring accumulation of hydrophobic aromatic substances.

MET23LYS MUTATION IN SUBUNIT GAMMA OF FOF1-ATP SYNTHASE FROM RHODOBACTER CAPSULATUS IMPAIRS THE ACTIVATION OF ATP HYDROLYSIS BY PROTONMOTIVE FORCE

Feniouk B.A., Rebeechi A., Giovannini D., Anefors S., Mulkidjanian A.Y., Junge W., Tunina P., Melandri A.

Biochimica et Biophysica Acta-Bioenergetics 1767 (2007) 1319-1330.

H+-F0F1-ATP synthase couples proton flow through its membrane portion, F-0, to the synthesis of ATP in its headpiece, F-1. Upon reversal of the reaction the enzyme functions as a proton pumping ATPase. Even in the simplest bacterial enzyme the ATPase activity is regulated by several mechanisms, involving inhibition by MgADP, conformational transitions of the epsilon subunit, and activation by protonmotive force. Here we report that the Met23Lys mutation in the gamma subunit of the Rhodobacter capsulatus ATP synthase significantly impaired the activation of ATP hydrolysis by protonmotive force. The impairment in the mutant was due to faster enzyme deactivation that was particularly evident at low ATP/ADP ratio. We suggest that the electrostatic interaction of the introduced gamma Lys23 with the DELSEED region of subunit stabilized the ADP-inhibited state of the enzyme by hindering the rotation of subunit gamma rotation which is necessary for the activation. .

PARAQUAT POTENTIATES GLUTAMATE TOXICITY IN IMMATURE CULTURES OF CEREBELLAR GRANULE NEURONS

Stelmashook E.V., Isaev N.K., Zorov D.B.

Toxicology Letters 174 (2007) 82-88.

Toxic concentrations of paraquat (0.2 mM, 24 h) caused death of both mature and immature cerebellar granule neurons (CGNs), which could be prevented by blockers of ionotropic glutamate receptors, or by removal of glutamine from cultural medium. Glutamate (Glu, 0.05-1 mM, 24 h) was highly toxic for mature CGNs while young CGNs were insensitive to the toxic effect of Glu. Measurements of the relative intracellular calcium ion concentration showed that the Glu-induced [Ca2+](i) rise in mature neurons was two times higher than that in young neurons. Subtoxic concentrations of paraquat did not affect the Glu-induced [Ca2+](i) rise in neurons, but lowered the CGNs survival only in immature cultures. These data provide evidence that oxidative stress induced by paraquat is a strong factor modulating the glutamate-induced damage to immature CGNs. .

PHOTODYNAMIC ACTIVITY AND BINDING OF SULFONATED METALLOPHTHALOCYANINES TO PHOSPHOLIPID MEMBRANES: CONTRIBUTION OF METAL-PHOSPHATE COORDINATION

Pashkovskaya A.A., Sokolenko E.A., Sokolov V.S., Kotova E.A., Antonenko Y.N.

Biochimica et Biophysica Acta-Biomembranes 1768 (2007) 2459-2465.

Photosensitized efficacy of tetrasulfonated phthalocyanines of zinc, aluminum and nickel (ZnPcS4,, AlPcS4 and NiPcS4, respectively) as studied by gramicidin channel (gA) photoinactivation was compared with adsorption of the dyes on the surface of a bilayer lipid membrane as measured by the inner field compensation method. The adsorption of the negatively charged phthalocyanines on diphytanoylphosphatidylcholine (DPhPC) membranes led to formation of a negative boundary potential difference between the membrane/water interfaces. Good correlation was shown between the photodynamic activity and the membrane binding of the three metallophthalocyanines. ZnPcS4 appeared to be the most potent of these photosensitizers, while NiPcS4 was completely ineffective. All of these phthalocyanines displayed no binding and negligible gA photoinactivation with membranes formed of glycerol monooleate (GMO), whereas Rose Bengal exhibited significant binding and photodynamic efficacy with GMO membranes. Gramicidin photoinactivation in the presence of AlPcS4, being insensitive to the ionic strength of the bathing solution, was inhibited by fluoride and attenuated by phosphate ions. A blue shift of the fluorescence peak position of ZnPcS4 dissolved in ethanol was elicited by phosphate, similarly to fluoride, which was indicative of the coordination interaction of these ions with the central metal atom of the phthalocyanine macrocycle. This interaction was enhanced in the medium modeling the water-membrane interface. The results obtained imply that binding of tetrasulfonated metallophthalocyanines to phospholipid membranes is determined primarily by metal-phosphate coordination. .

ION CHANNELS OF VARIOUS TYPES INDUCED IN LIPID MEMBRANES BY GRAMICIDIN A DERIVATIVES CARRYING A CATIONIC SEQUENCE AT THEIR C-TERMINI

Stoilova T.B., Dutseva E.A., Pashkovskaya A.A., Sychev S.V., Koval'chuk S.V., Sobko A.A., Egorova N.S., Kotova E.A., Antonenko Y.N., Surovoi A.Y., Ivanov V.T.

Russian Journal of Bioorganic Chemistry 33 (2007) 474-481.

The channel-forming activity of gramicidin A derivatives carrying positively charged amino acid sequences at their C-termini was studied on planar bilayer lipid membranes and liposomes. We showed previously (FEBS Lett., 2005, vol. 579, pp. 5247-5252) that, at low concentrations, these peptides form classical cation-selective pores typical of gramicidin A, whereas, at high concentrations, they form large nonselective pores. The ability of the peptides to form nonselective pores, which was determined by the efflux of carboxyfluorescein, an organic dye, from liposomes, decreased substantially as the length of the gramicidin fragment in the series of cationic analogues was truncated. CD spectra showed that large pores are formed by peptides having both beta(6.3) single-stranded and beta(5.6) double-stranded helical conformations of the gramicidin fragment, with the C-terminal cationic sequence being extended. The dimerization of the peptides by the oxidation of the terminal cysteine promoted the formation of nonselective pores. It was shown that nonselective pores are not formed in membranes of erythrocytes, which may indicate a dependence of the channel-forming ability on the membrane type. The results may be of interest for the directed synthesis of peptides with antibacterial activity.

CENTRIOLE DUPLICATION IN PE (SPEV) CELLS STARTS BEFORE THE ONSET OF THE DNA REPLICATION

Uzbekov R.E.

Biologicheskie Membrany 24 (2007) 292-298.

Centrosome includes two centrioles and serves as a structural basis of mitotic spindle pole. Duplication of centrosome and doubling of chromosome quantity during DNA replication are two principal events of cell cycle in the course of preparation for cell division. In the present work, cells of the porcine embryonic kidney cell line PE (SPEV) were individually monitored after mitosis, and the procentriole appearance was detected by electron microscopy as soon as 5-6 h after mitosis. This period was 1-2 h shorter than minimal duration of G(1)-phase in this cell line. Ultrastructural analysis of centrosomes in the cells with known "cell cycle age" in combination with autoradiography study of the same cells using H-3-thimidine directly confirmed that duplication of centrioles started before the cells entered S-phase of cell cycle, i.e. preceded DNA replication.

CYTOCHROME BD FROM AZOTOBACTER VINELANDII: EVIDENCE FOR HIGH-AFFINITY OXYGEN BINDING

Belevich I., Borisov V.B., Bloch D.A., Konstantinov A.A., Verkhovsky M.I.

Biochemistry 46 (2007) 11177-11184.

Cytochrome bd from Azotobacter vinelandii is a respiratory quinol oxidase that is highly efficient in reducing intracellular oxygen concentration, thus enabling nitrogen fixation under ambient aerobic conditions. Equilibrium measurements Of 02 binding to ferrous heme d in the one-electron-reduced form of the A. vinelandii enzyme give K-d(O2)= 0.5 mu M, close to the value for the Escherichia coli cytochrome bd (ca. 0.3 mu M); thus, both enzymes have similar, high affinity for oxygen. The reaction of the A. vinelandii cytochrome bd in the one-electron-reduced and fully reduced states with 02 is extremely fast approaching the diffusion-controlled limit in water. In the fully reduced state, the rate Of 02 binding depends linearly on the oxygen concentration consistently with a simple, single-step process. In contrast, in the one-electron-reduced state the rate of oxygen binding is hyperbolic, implying a more complex binding pattern. Two possible explanations for the saturation kinetics are considered: (A) There is a spectroscopically silent prebinding of oxygen to an unidentified low-affinity saturatable site followed by the oxygen transfer to heme d. (B) Oxygen binding to heme d requires an "activated" state of the enzyme in which an oxygen channel connecting heme d to the bulk is open. This channel is permanently open in the fully reduced enzyme (hence no saturation behavior) but flickers between the open and closed states in the one-electron-reduced enzyme.

MODIFIED MODEL OF THE STRUCTURE OF THE POTATO VIRUS X COAT PROTEIN

Dobrov E.N., Nemykh M.A., Lukashina E.V., Baratova L.A., Drachev V.A., Efimov A.V.

Molecular Biology 41 (2007) 638-641.

A modified model was proposed for the tertiary structure of the coat protein (CP) molecules in potato virus X (PVX) virions, similar to the original model of 2001 describing the structure of CP of potato virus A, a member of another group of filamentous viruses. According to the new model, CP comprises two main structural domains, namely, a bundle of alpha-helices, located near the long axis of the virion, and the socalled RNP fold (or abCd fold), located in the vicinity of its surface. The model made it possible to suggest a possible mechanism of the PVX virion structural rearrangement (remodeling) resulting from translational activation of virions by the TGB1 movement protein according to Atabekov and colleagues.

COMPARATIVE STRUCTURAL STABILITY OF SUBUNITS OF THE POTATO VIRUS X COAT PROTEIN IN SOLUTION AND IN VIRUS PARTICLES

Nemykh M.A., Novikov V.K., Arutyunyan A.M., Kalmykov P.V., Drachev V.A., Dobrov E.N.

Molecular Biology 41 (2007) 630-637.

Several optical methods and differential scanning calorimetry were used to study the structure and stability of free coat protein (CP) molecules and CP molecules in the virion of the potato virus X (PVX), a filamentous plant virus. All criteria suggest that PVX CP (hereinafter, CP) subunits in solution at room temperature display a certain preserved tertiary structure; however, this structure is very unstable and already denatures at 35 degrees C. Very low concentrations of sodium dodecylsulfate or cetyltrimethylammonium bromide also disrupt the CP tertiary structure, three-five molecules of these detergents per one protein molecule being sufficient. However, the secondary structure of CP molecules does not change under the same conditions. Once included into the virion, CP subunits become considerably more stable towards increased temperature and detergents. This combination of a highly labile tertiary structure and a fairly stable secondary structure of free CP can be a structural basis for the recently discovered ability of PVX CP to assume two distinct functional states within the virion.

GENE SILENCING IN PLANTS

Dorokhov Y.L.

Molecular Biology 41 (2007) 519-530.

The review considers the cytoplasmic silencing of viral RNAs by short RNAs and the silencing of endogenous mRNAs by specific short double-stranded microRNAs. The role of some cell factors such as Dicer, Argonaute, RNA-dependent RNA polymerase, RNA polymerase IV, and pectin methylesterase is described in detail. The role of viral proteins and nucleic acids in silencing suppression and possible biotechnological applications of this mechanism are discussed.

FUNCTIONALISATION OF CALCIUM PHOSPHATE NANOPARTICLES BY OLIGONUCLEOTIDES AND THEIR APPLICATION FOR GENE SILENCING

Sokolova V., Kovtun A., Prymak O., Meyer-Zaika W., Kubareva E.A., Romanova E.A., Oretskaya T.S., Heumann R., Epple M.

Journal of Materials Chemistry 17 (2007) 721-727.

In molecular biology, the production of proteins can be effectively inhibited by introducing specific oligonucleotides into a living cell (gene silencing or antisense strategy; important for gene therapy). Calcium phosphate nanoparticles can serve as carriers for biomolecules in such therapeutic applications due to their high biocompatibility and biodegradability. Stable colloids were prepared by coating the inorganic nanoparticles with single- and double-stranded oligonucleotides. The dispersions were analysed by dynamic light scattering, zeta potential measurements, transmission electron microscopy, and scanning electron microscopy. Particles with a diameter of about 100 nm were obtained under optimized conditions. The efficiency of such nanoparticles to specifically inhibit protein synthesis was tested on HeLa-EGFP cells whose green fluorescence was turned off by the coated nanoparticles (gene silencing with siRNA). If siRNA was incorporated into the calcium phosphate particle and thereby protected from intracellular degradation, the transfection efficiency was significantly increased. The dispersions were stable and could be stored at 4 degrees C without loss of activity for several weeks, making them available as biochemical reagents.

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

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

Biopolymers 88 (2007) 589-589.

ANTIOXIDANT AND PROOXIDANT EFFECTS OF QUERCETIN ON GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE

Schmalhausen E.V., Zhlobek E.B., Shalova I.N., Firuzi O., Saso L., Muronetz V.I.

Food and Chemical Toxicology 45 (2007) 1988-1993.

Anti- and prooxidant properties of quercetin under different conditions were investigated using glyceraldehyde-3-phosphate dehydrogenase, a glycolytic enzyme containing essential cysteine residues. Quercetin was shown to produce hydrogen peroxide in aqueous solutions at pH 7.5, this resulting in the oxidation of the cysteine residues of the enzyme. Quercetin significantly increased oxidation of GAPDH observed in the presence of ferrous ions, particularly when FeSO4 was added to the solution containing GAPDH and quercetin. The results suggest the formation of hydroxyl radical in the case of the addition of FeSO4 to a quercetin solution. At the same time, quercetin protects GAPDH from oxidation in the presence of ascorbate and Fe3+. In the absence of metals, quercetin protects SH-groups of GAPDH from oxidation by the superoxide anion generated by the system containing xanthine/xanthine oxidase. .

PHOTOSYNTHETIC ELECTRON TRANSPORT IN THE CYANOBACTERIUM SYNECHOCYSTIS SP PCC 6803: HIGH-FIELD W-BAND AND X-BAND EPR STUDY OF ELECTRON FLOW THROUGH PHOTOSYSTEM I

Savitsky A., Trubitsin B.V., Mobius K., Semenov A.Y., Tikhonov A.N.

Applied Magnetic Resonance 31 (2007) 221-236.

In this work, by using the respective advantages of W- and X-band electron paramagnetic resonance (EPR) spectroscopy techniques to investigate electron transport processes, we have studied the light-induced redox transients of the primary electron donor P-700, and the secondary acceptor A, in photosystem (PS) I complexes of intact cyanobacterial cells Synechocystis sp. PCC 6803. We found that the kinetic behavior of the cation radical P-700(center dot+). generated by illumination with continuous light, and the EPR intensity of the radical pair P-700(center dot+), A(1)(center dot-), generated upon laser pulse illumination strongly depend on the illumination prehistory (either the sample was frozen in the dark or during illumination). Both these processes were sensitive to the presence of electron transport inhibitors which block electron flow between the two photosystems. In line with our X-band EPR data on the kinetics of light-induced redox transients of P-700, our high-field W-band EPR study of the radical-pair state P(700)(center dot+)A(1)(center dot-) shows that photosynthetic electron flow through the PS I reaction center is controlled both on the donor and on the acceptor side of PS.

ASSEMBLY AND ANALYSIS OF EUKARYOTIC TRANSLATION INITIATION COMPLEXES

Pisarev A.V., Unbehaun A., Hellen C.U.T., Pestova T.V.

Translation Initiation: Reconstituted Systems and Biophysical Methods 430 (2007) 147-177.

The canonical initiation process is the most complex aspect of translation in eukaryotes. It involves the coordinated interactions of at least 11 eukaryotic initiation factors, 40S and 60S ribosomal subunits, mRNA, and aminoacylated initiator tRNA (Met-tRNA(i)(Met)), as well as binding and hydrolysis of GTP and ATR I The factor requirements for many individual steps in this process, including scanning, initiation codon recognition, and ribosomal subunit joining, have until recently been obscure. We established the factor requirements for these steps by reconstituting the initiation process in vitro from individual purified components of the translation apparatus and developed approaches to explain the mechanism of individual steps and the roles of individual factors and to characterize the structure of initiation complexes. Here we describe protocols for the purification of native initiation factors and for expression and purification of active recombinant forms of all single subunit initiation factors, for the reconstitution of the initiation process, and for determination of the position of ribosomal complexes on mRNA by primer extension inhibition ("toe printing"). We also describe protocols for site-directed ultraviolet (UV) cross-linking to determine the interactions of individual nucleotides in mRNA with components of the initiation complex and for directed hydroxyl radical probing to determine the position of initiation factors on the ribosome.

CHANGES IN CHROMOSOME POSITIONING MAY CONTRIBUTE TO THE DEVELOPMENT OF DISEASES RELATED TO X-CHROMOSOME ANEUPLOIDY

Petrova N.V., Yakilitenko I.I., Alexeevski A.V., Verbovoy V.A., Razin S.V., Iarovaia O.V.

Journal of Cellular Physiology 213 (2007) 278-283.

The radial positions of the centromeric regions of chromosomes I and X were determined in normal male fibroblasts (XY) and in fibroblasts from a patient with a rare case of XXXXY polysomy. The centromeric regions and presumably the whole territories of active X chromosomes were demonstrated to occupy similar, although not identical, positions in XY and XXXXY cells. The centromeres of inactive X chromosomes (Barr bodies) were located closer to the nuclear periphery as compared with the centromeres of active X chromosomes. In addition, it was established that the nuclear radial position of gene-rich chromosome I was changed in XXXXY cells as compared to normal XY cells. The data are discussed in the context of the hypothesis postulating that changes in nuclear positioning of chromosomal territories induced by the presence of extra copies of individual chromosomes may contribute to the development of diseases related to different polysomies. .

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

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

Biopolymers 88 (2007) 624-624.

REGULATION OF STRESS-INDUCED INTRACELLULAR SORTING AND CHAPERONE FUNCTION OF HSP27 (HSPB1) IN MAMMALIAN CELLS

Bryantsev A.L., Kurchashova S.Y., Golyshev S.A., Polyakov V.Y., Wunderink H.F., Kanon B., Budagova K.R., Kabakov A.E., Kampinga H.H.

Biochemical Journal 407 (2007) 407-417.

In vitro, small Hsps (heat-shock proteins) have been shown to have chaperone function capable of keeping unfolded proteins in a form competent for Hsp70-dependent refolding. However, this has never been confirmed in living mammalian cells. In the present study, we show that Hsp27 (HspB1) translocates into the nucleus upon heat shock, where it forms granules that co-localize with IGCs (interchromatin granule clusters). Although heat-induced changes in the oligomerization status of Hsp27 correlate with its phosphorylation and nuclear translocation, Hsp27 phosphorylation alone is not sufficient for effective nuclear translocation of HspB1. Using firefly luciferase as a heat-sensitive reporter protein, we demonstrate that HspB 1 expression in HspB1-deficient fibroblasts enhances protein refolding after heat shock. The positive effect of HspB 1 on refolding is completely diminished by overexpression of Bag-1 (Bcl-2-associated athanogene), the negative regulator of Hsp70, consistent with the idea of HspB 1 being the substrate holder for Hsp70. Although HspB1 and luciferase both accumulate in nuclear granules after heat shock, our results suggest that this is not related to the refolding activity of HspB1. Rather, granular accumulation may reflect a situation of failed refolding where the substrate is stored for subsequent degradation. Consistently, we found 20S proteasomes concentrated in nuclear granules of HspB1 after heat shock. We conclude that HspB1 contributes to an increased chaperone capacity of cells by binding unfolded proteins that are hereby kept competent for refolding by Hsp70 or that are sorted to nuclear granules if such refolding fails.

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

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

Biopolymers 88 (2007) 624-624.