M.V. Lomonosov Moscow State University

A.N. Belozersky Institute of Physico-Chemical Biology

ANNUAL REPORT

2006

Probing of HIV-1 Integrase/DNA Interactions Using Novel Analogs of Viral DNA

Agapkina J., Smolov M., Barbe S., Zubin E., Zatsepin T., Deprez E., Le Bret M., Mouscadet J.F., Gottikh M.

Journal of Biological Chemistry 281 (2006) 11530-11540.

The specific activity of the human immunodeficiency virus, type 1 ( HIV-1), integrase on the viral long terminal repeat requires the binding of the enzyme to certain sequences located in the U3 and U5 regions at the ends of viral DNA, but the determinants of this specific DNA-protein recognition are not yet completely understood. We synthesized DNA duplexes mimicking the U5 region and containing either 2'-modified nucleosides or 1,3-propanediol insertions and studied their interactions with HIV-1 integrase, using Mn²⁺ or Mg²⁺ ions as integrase cofactors. These DNA modifications had no strong effect on integrase binding to the substrate analogs but significantly affected 3'-end processing rate. The effects of nucleoside modifications at positions 5, 6, and especially 3 strongly depended on the cationic cofactor used. These effects were much more pronounced in the presence of Mg²⁺ than in the presence of Mn²⁺. Modifications of base pairs 7-9 affected 3'-end processing equally in the presence of both ions. Adenine from the 3rd bp is thought to form at least two hydrogen bonds with integrase that are crucial for specific DNA recognition. The complementary base, thymine, is not important for integrase activity. For other positions, our results suggest that integrase recognizes a fine structure of the sugar-phosphate backbone rather than heterocyclic bases. Integrase interactions with the unprocessed strand at positions 5-8 are more important than interactions with the processed strand for specific substrate recognition. Based on our results, we suggest a model for integrase interaction with the U5 substrate.

Cholesterol and its anionic derivatives inhibit 5-lipoxygenase activation in polymorphonuclear leukocytes and MonoMac6 cells.

Aleksandrov D.A., Zagryagskaya A.N., Pushkareva M.A., Bachschmid M., Peters-Golden M., Werz O., Steinhilber D., Sud'ina G.F.

FEBS J. 273 (2006) 548-557.

Lipoxygenase (5-LO) is the key enzyme in the biosynthesis of leukotrienes (LTs), biological mediators of host defense reactions and of inflammatory diseases. While the role of membrane binding in the regulation of 5-LO activity is well established, the effects of lipids on cellular activity when added to the medium has not been characterized. Here, we show such a novel function of the most abundant sulfated sterol in human blood, cholesterol sulfate (CS), to suppress LT production in human polymorphonuclear leukocytes (PMNL) and Mono Mac6 cells. We synthesized another anionic lipid, cholesterol phosphate, which demonstrated a similar capacity in suppression of LT synthesis in PMNL. Cholesteryl acetate was without effect. Cholesterol increased the effect of CS on 5-LO product synthesis. CS and cholesterol also inhibited arachidonic acid (AA) release from PMNL. Addition of exogenous AA increased the threshold concentration of CS required to inhibit LT synthesis. The effect of cholesterol and its anionic derivatives can arise from remodeling of the cell membrane, which interferes with 5-LO activation. The fact that cellular LT production is regulated by sulfated cholesterol highlights a possible regulatory role of sulfotransferases/sulfatases in 5-LO product synthesis.

Noncommutative Two-Dimensional Topological Field Theories and Hurwitz Numbers for Real Algebraic Curves

Alexeevski A., Natanzon S.

Selecta Mathematica-New Series 12 (2006) 307-377.

It is well known that the classical two-dimensional topological field theories are in one-to-one correspondence with the commutative Frobenius algebras. An important extension of classical two-dimensional topological field theories is provided by open-closed two-dimensional topological field theories. In this paper we extend open-closed two-dimensional topological field theories to nonorientable surfaces. We call them Klein topological field theories (KTFT). We prove that KTFTs bijectively correspond to (in general noncommutative) algebras with certain additional structures, called structure algebras. The semisimple structure algebras are classified. Starting from an arbitrary finite group, we construct a structure algebra and prove that it is semisimple. We define an analog of Hurwitz numbers for real algebraic curves and prove that they are correlators of a KTFT. The structure algebra of this KTFT is the structure algebra of a symmetric group.

In vitro Reconstitution of Eukaryotic Translation Reveals Cooperativity Between Release Factors eRF1 and eRF3

Alkalaeva E.Z., Pisarev A.V., Frolova L.Y., Kisselev L.L., Pestova T.V.

Cell 125 (2006) 1125-1136.

Eukaryotic translation termination is triggered by peptide release factors eRF1 and eRF3. Whereas eRF1 recognizes all three termination codons and induces hydrolysis of peptidyl tRNA, eRF3's function remains obscure. Here, we reconstituted all steps of eukaryotic translation in vitro using purified ribosomal subunits; initiation, elongation, and termination factors; and aminoacyl tRNAs. This allowed us to investigate termination using pretermination complexes assembled on mRNA encoding a tetrapeptide and to propose a model for translation termination that accounts for the cooperative action of eRF1 and eRF3 in ensuring fast release of nascent polypeptide. In this model, binding of eRF1, eRF3, and GTP to pretermination complexes first induces a structural rearrangement that is manifested as a:2 nucleotide forward shift of the toeprint attributed to pretermination complexes that leads to GTP hydrolysis followed by rapid hydrolysis of pepticlyl tRNA. Cooperativity between eRF1 and eRF3 required the eRF3 binding C-terminal domain of eRF1.

Similar Features in Mechanisms of Translation Initiation of MRNAS in Eukaryotic and Prokaryotic Systems

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

Molecular Biology 40 (2006) 694-702.

Using as examples non-canonical features of translation initiation for some bacterial and mammalian mRNAs with unusual 5'- untranslated regions (5'-UTR) or lacking these regions (leaderless mRNAs), the authors of this review discuss similarities in mechanisms of translation initiation on prokaryotic and eukaryotic ribosomes.

A Leaderless mRNA Can Bind to Mammalian 80S Ribosomes and Direct Polypeptide Synthesis in the Absence of Translation Initiation Factors

Andreev D.E., Terenin I.M., Dunaevsky Y.E., Dmitriev S.E., Shatsky I.N.

Molecular and Cellular Biology 26 (2006) 3164-3169.

Translation initiation in eukaryotic cells is known to be a complex multistep process which involves numerous protein factors. Here we demonstrate that leaderless mRNAs with initiator Met-tRNA can bind directly to 80S mammalian ribosomes in the absence of initiation factors and that the complexes thus formed are fully competent for the subsequent steps of polypeptide synthesis. We show that the canonical 48S pathway of eukaryotic translation initiation has no obvious advantage over the 80S pathway of translation initiation on leaderless mRNAs and suggest that, in the presence of competing mRNAs containing a leader, the latter mechanism will be preferred. The direct binding of the leaderless mRNA to the 80S ribosome was precluded when such an rnRNA was supplied with a 5' leader, irrespective of whether it was in a totally single-stranded conformation or was prone to base pairing. The striking similarity between the mechanisms of binding of leaderless mRNAs with mammalian 80S or bacterial 70S ribosornes gives support to the idea that the alternative mode of translation initiation used by leaderless mRNAs represents a relic from early steps in the evolution of the translation apparatus.

Redox-Regulated Ion Channel Activity of a Cysteine-Containing Gramicidin A Analogue

Antonenko Y.N., Stoilova T.B., Kovalchuk S.I., Egorova N.S., Pashkovskaya A.A., Sobko A.A., Kotova E.A., Surovoy A.Y.

Biochimica et Biophysica Acta-Biomembranes 1758 (2006) 493-498.

According to recent data, gramicidin A analogues having positively charged amino acid sequences at the C-termini exhibit two types of channel activity in lipid membranes:classical cation-selective channels and large unselective pores. The induction of unselective pores was shown here to strongly depend on the redox state of the membrane-bathing solution. if the gramicidin analogue contained a cysteine residue in the sequence GSGPKKKRKVC attached to the C-terminus. In particular, the addition of H₂O₂ led to an increase in-the trans membrane current and the loss of cationic selectivity on planar bilayer lipid membranes and an increase in the carboxyfluorescein leakage of liposomes. The effect was observed at high concentration of the peptide while was absent at the single-channel level. It was concluded that oxidation led to possible formation of dimers of the peptide, which promoted the formation of large unselective pores.

Antibodies to Inactive Conformations of Glyceraldehyde-3-Phosphate Dehydrogenase Inactivate the Apo- and Holoforms of the Enzyme

Arutiunova E.I., Pleten A.P., Nagradova N.K., Muronetz V.I.

Biochemistry-Moscow 71 (2006) 685 -691.

Polyclonal antibodies produced after the immunization of a rabbit with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus were used to isolate two types of antibodies interacting with different non-native forms of the antigen. Type I antibodies were purified using Sepharose-bound apo-GAPDH that was treated with glutaraldehyde to stabilize the enzyme in the tetrameric form. Type II antibodies were isolated using immobilized denatured monomers of the enzyme. It was shown that the type I antibodies bound to the native holo- and apoforms of the enzyme with the ratio of one antibody molecule per GAPDH tetramer. While interacting with the native holoenzyme, the type I antibodies induce a time-dependent decrease in its activity by 80-90%. In the case of the apoenzyme, the decrease in the activity constitutes only 25%, this indicating that only one subunit of the tetramer is inactivated. Differential scanning calorimetry experiments showed that the formation of the complex between both forms of the enzyme and the type I antibodies resulted in a shift of the maximum of the thermal capacity curves (T. value) to lower temperatures. The extremely stable holoenzyme was affected to the greatest extent, the shift of the T-m value constituting approximately 20 degrees C. We assume that the formation of the complex between the holo- or apo-GAPDH and the type I antibody results in time-dependent conformational changes in the enzyme molecule. Thus, the antibodies induce the structural rearrangements yielding the conformation that is identical to the structure of the antigen used for the selection of the antibodies (i.e., inactive). The interaction of the antibodies with the apo-GAPDH results in the inactivation of the subunit directly bound to the antibody. Virtually complete inactivation of the holoenzyme by the antibodies is likely due to the transmission of the conformational changes through the intersubunit contacts. The type II antibodies, which were selected using the immunosorbent with unfolded enzyme form, do not affect the activity of native holo- and apo-GAPDH, but prevent the reactivation of the denatured GAPDH, binding the denatured forms of the enzyme.

Thermodynamic Properties of the Redox Centers of

Na⁺-Translocating NADH:Quinone Oxidoreductase

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

Biochemistry 45 (2006) 3421-3428.

Redox titration of all optically detectable prosthetic groups of Na⁺-translocating NADH:quinone oxidoreductase (Na⁺-NQR) at pH 7.5 showed that the functionally active enzyme possesses only three titratable flavin cofactors, one noncovalently bound FAD and two covalently bound FMN residues. All three flavins undergo different redox transitions during the function of the enzyme. The noncovalently bound FAD works as a "classical" two-electron carrier with a midpoint potential (E-m) of -200 mV. Each of the FMN residues is capable of only one-electron reduction:one from neutral flavosemiquinone to fully reduced flavin (E-m = 20 mV) and the other from oxidized flavin to flavosemiquinone anion (E-m = -150 mV). The lacking second half of the redox transitions for the FMNs cannot be reached under our experimental conditions and is most likely not employed in the catalytic cycle. Besides the flavins, a [2Fe-2S] cluster was shown to function in the enzyme as a one-electron carrier with an E-m of -270 mV. The midpoint potentials of all the redox transitions determined in the enzyme were found to be independent of Na⁺ concentration. Even the components that exhibit very strong retardation in the rate of their reduction by NADH at low sodium concentrations experienced no change in the E-m values when the concentration of the coupling ion was changed 1000 times. On the basis of these data, plausible mechanisms for the translocation of transmembrane sodium ions by Na⁺-NQR are discussed.

Nitric Oxide Reacts With the Ferryl-Oxo Catalytic Intermediate of the Cu-b-Lacking Cytochrome bd Terminal Oxidase

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

FEBS Letters 580 (2006) 4823-4826.

Cytochrome bd is a bacterial respiratory oxidase carrying three hemes but no copper. We show that nitric oxide (NO) reacts with the intermediate F of cytochrome bd from Azotobacter vinelandii:(i) with a 1:1 stoichiometry, (ii) rapidly (k = 1.2 +/- 0.1 x 10⁵ M^-1 s^-1 at 20 degrees C), and (iii) yielding the oxidized enzyme with nitrite bound to heme d at the active site. Unexpectedly, the NO reaction mechanism of this catalytic intermediate in the CUB-lacking cytochrome bd appears similar to that of beef heart cytochrome c oxidase, where CUB was proposed to play a key role. .

Mitochondrial Contact Sites:Their Role in Energy Metabolism and Apoptosis

Brdiczka D.G., Zorov D.B., Sheu S.S.

Biochimica et Biophysica Acta-Molecular Basis of Disease 1762 (2006) 148-163.

The energy metabolism of the failing heart is characterised by a 30% decrease of the total adenine nucleotides content and what may be more important by a 60% loss of creatine and creatine phosphate [J.S. Ingwall, R.G. Weiss, Is the failing heart energy starved? On using chemical energy to support cardiac function, Circ. Res. 95 (2004) 35 - 145]. Besides the effect of these changes on the energy supply, failing heart is known to be more vulnerable to Ca²⁺ overload and apoptosis-inducing processes. Recent studies have pointed to the critical role of mitochondrial contact sites in controlling both the mitochondrial energy metabolism and Ca²⁺ homeostasis. This review focuses on the structure and function of protein complexes in mitochondrial contact sites and their regulatory role in the cellular bioenergetics, intra- and extra-mitochondrial Ca²⁺ levels, and release of apoptosis-inducing factors. Firstly, we review the compositions of different contact sites following by the discussion of experimental data obtained with isolated and reconstituted voltage-dependent anion channel-adenine nucleotide translocase complexes and consequences of the complex disassembling. Furthermore, we describe experiments involving the complex-stabilizing conditions in vitro and in intact cells. At the end, we discuss unsolved problems and opportunities for clinical application of the complex-stabilizing factors.

Brain Pyruvate and 2-Oxoglutarate Dehydrogenase Complexes Are Mitochondrial Targets of the CoA Ester of the Refsum Disease Marker Phytanic Acid

Bunik V.I., Raddatz G., Wanders R.J.A., Reiser G.

EBS Letters 580 (2006) 3551-3557.

Pyruvate and 2-oxoglutarate dehydrogenase complexes are strongly inhibited by phytanoyl-CoA (IC50 approximate to 10^-6-10^-7 M). Palmitoyl-CoA is 10-fold less potent. Phytanic or palmitic acids have no inhibitory effect up to 0.3 mM. At the substrate saturation, the acyl-CoA's affect the first and second enzymatic components of the 2-oxoglutarate dehydrogenase complex, while the third component is inhibited only at a low saturation with its substrate dihydrolipoamide. Thus, key regulatory branch points of mitochondrial metabolism are targets of a cellular derivative of phytanic acid. Decreased activity of the complexes might therefore contribute to neurological symptoms upon accumulation of phytanic acid in Refstum disease. .

Purification and Characterization of Windmill Palm Tree (Trachycarpus fortunei) Peroxidase

Caramyshev A.V., Firsova Y.N., Slastya E.A., Tagaev A.A., Potapenko N.V., Lobakova E.S., Pletjushkina O.Y., Sakharov I.Y.

Journal of Agricultural and Food Chemistry 54 (2006) 9888-9894.

High peroxidase activity was demonstrated to be present in the leaf of several species of cold-resistant palms. Histochemical studies of the leaf of windmill palm tree (Trachycarpus fortunei) showed the peroxidase activity to be localized in hypoderma, epidermis, cell walls, and conducting bundles. However, chlorophyll-containing mesophyll cells had no peroxidase at all. The leaf windmill palm tree peroxidase (WPTP) was purified to homogeneity and had a specific activity of 6230 units/mg, RZ = 3.0, a molecular mass of 50 kDa, and an isoelectric point of pI 3.5. The electronic spectrum of WPTP with a Soret band at 403 nm was typical of plant peroxidases. The N-terminal amino acid sequence of WPTP was determined. The substrate specificity of WPTP was distinct from that of other palm peroxidases, and the best substrate for WPTP was 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid). The palm peroxidase showed an unusually high stability at elevated temperatures and high concentrations of guanidine.

Production of Reactive Oxygen Species in Mitochondria of HeLa Cells Under Oxidative Stress

Chernyak B.V., Izyumov D.S., Lyamzaev K.G., Pashkovskaya A.A., Pletjushkina O.Y., Antonenko Y.N., Sakharov D.V., Wirtz K.W.A., Skulachev V.P.

Biochimica et Biophysica Acta-Bioenergetics 1757 (2006) 525-534.

Mitochondria can be a source of reactive oxygen species (ROS) and a target of oxidative damage during oxidative stress. In this connection, the effect of photodynamic treatment (PDT) with Mitotracker Red (MR) as a mitochondria-targeted photosensitizer has been studied in HeLa cells. It is shown that MR produces both singlet oxygen and superoxide anion upon photoactivation and causes photoinactivation of gramicidin channels in a model system (planar lipid bilayer). Mitochondria-targeted antioxidant (MitoQ) inhibits this effect. In living cells, MR-mediated PDT initiates a delayed ("dark") accumulation of ROS, which is accelerated by inhibitors of the respiratory chain (piericidin, rotenone and myxothiazol) and inhibited by MitoQ and diphenyleneiodonium (an inhibitor of flavin enzymes), indicating that flavin of Complex I is involved in the ROS production, PDT causes necrosis that is prevented by MitoQ. Treatment of the cell with hydrogen peroxide causes accumulation of ROS, and the effects of inhibitors and MitoQ are similar to that described for the PDT model. Apoptosis caused by H₂O₂ is augmented by the inhibitors of respiration and suppressed by MitoQ. It is concluded that the initial segments of the respiratory chain can be an important source of ROS, which are targeted to mitochondria, determining the fate of the cell subjected to oxidative stress.

Cholesterol-Binding Sites in Transmembrane Domains of Integral Membrane Proteins Involved in Phagocytosis and/or Capable of Clustering in Lipid Rafts

Cheshev D.A., Chekanov N.N., Dunina-Barkovskaya A.Y.

Biologicheskie Membrany 23 (2006) 69-73.

We searched for cholesterol-binding sites in some integral proteins involved in phagocytosis and/or capable of clustering in lipid rafts. Asa cholesterol-binding pattern we used a consensus amino acid sequence -L/V-(X)(1-5)-Y-(X)(1-5)-R/K-, described for a peripheral-type benzodiazepine receptor (PBR) cholesterol-binding site (Li H., Papadopoulos V. H Endocrinology. 1998. V. 139. P. 4991-4997). Alignment of this site versus amino acid sequences of a phagocytic receptor Fc gamma RI and some ionic channels (IRK channels. ATP-gated cation channels (P2X purinoreceptors), connexins Cx32 and Cx43, MIP26, and some other integral proteins) revealed that most of the proteins studied possessed a relatively conservative hydrophobic amino-acid sequence analogous to the PBR cholesterol-binding site. This sequence, which can be expressed as (L/V)(1-2)-(X)(1-4)-Y/F-(X)(1-3)-W, was always localized in a transmembrane domain of a protein. The presence of such amino-acid sequences may point to the direct interactions of integral proteins with cholesterol in lipid rafts. The actual involvement of the potential cholesterol-binding sites in the protein-cholesterol interactions and cellular functions can be experimentally verified by mutations affecting the sterical configuration of this site.

Thymosin Beta(4) Induces a Conformational Change in Actin Monomers

Dedova I.V., Nikolaeva O.P., Safer D., De la Cruz E.M., dos Remedios C.G.

Biophysical Journal 90 (2006) 985 -992.

Using fluorescence resonance energy transfer spectroscopy we demonstrate that thymosin beta(4) (t beta(4)) binding induces spatial rearrangements within the small domain (subdomains 1 and 2) of actin monomers in solution. T beta(4) binding increases the distance between probes attached to Gln-41 and Cys-374 of actin by 2 angstrom and decreases the distance between the purine base of bound ATP (epsilon ATP) and Lys-61 by 1.9 angstrom, whereas the distance between Cys-374 and Lys-61 is minimally affected. Distance determinations are consistent with t beta(4) binding being coupled to a rotation of subdomain 2. By differential scanning calorimetry, tb4 binding increases the cooperativity of ATP- actin monomer denaturation, consistent with conformational rearrangements in the t beta(4)-actin complex. Changes in fluorescence resonance energy transfer are accompanied by marked reduction in solvent accessibility of the probe at Gln-41, suggesting it forms part of the binding interface. T beta(4) and cofilin compete for actin binding. T beta(4) concentrations that dissociate cofilin from actin do not dissociate the cofilin-DNase I-actin ternary complex, consistent with the DNase binding loop contributing to high-affnity t beta(4)-binding. Our results favor a model where thymosin binding changes the average orientation of actin subdomain 2. The t beta(4)-induced conformational change presumably accounts for the reduced rate of amide hydrogen exchange from actin monomers and may contribute to nucleotide-dependent, high affinity binding.

Phylogeny of the Genus Lotus (Leguminosae, Loteae):Evidence From NrITS Sequences and Morphology

Degtjareva G.V., Kramina T.E., Sokoloff D.D., Samigullin T.H., Valiejo-Roman C.M., Antonov A.S.

Canadian Journal of Botany-Revue Canadienne de Botanique 84 (2006) 813-830.

Lotus (120-130 species) is the largest genus of the tribe Loteae. The taxonomy of Lotus is complicated, and a comprehensive taxonomic revision of the genus is needed. We have conducted phylogenetic analyses of Lotus based on nrITS data alone and combined with data on 46 morphological characters. Eighty-one ingroup nrITS accessions representing 71 Lotus species are studied; among them 47 accessions representing 40 species are new. Representatives of all other genera of the tribe Loteae are included in the outgroup (for three genera, nrITS sequences are published for the first time). Forty-two of 71 ingroup species were not included in previous morphological phylogenetic studies. The most important conclusions of the present study are (1) addition of morphological data to the nrITS matrix produces a better resolved phylogeny of Lotus; (2) previous findings that Dorycnium and Tetragonolobus cannot be separated from Lotus at the generic level are well supported; (3) Lotusacreticus should be placed in section Pedrosia rather than in section Lotea; (4) a broad treatment of section Ononidium is unnatural and the section should possibly not be recognized at all; (5) section Heinekenia is paraphyletic; (6) a section Lotus should include Lotusaconimbricensis; then the section is monophyletic; (7) a basic chromosome number of xa= 6 is an important synapomorphy for the expanded section Lotus; (8) the segregation of Lotusaschimperi and allies into section Chamaelotus is well supported; (9) there is an apparent functional correlation between stylodium and keel evolution in Lotus.

Red Fluorescent Protein DsRed:Parametrization of Its Chromophore As an Amino Acid Residue for Computer Modeling in the OPLS-AA Force Field

Dmitrienko D.V., Vrzheshch E.P., Drutsa V.L., Vrzheshch P.V.

Biochemistry-Moscow 71 (2006) 1133-1152.

Topology of the neutral form of the DsRed fluorescent protein chromophore as a residue of [(4-cis)-2-[(1-cis)-4-amino-4-oxobutanimidoyl]-4-(4-hydroxybenzylidene)- 5-oxo-4,5-dihydro-1H-imidazol-1-yl]acetic acid was calculated with OPLS-AA force field. Use of this topology and molecular dynamics simulation allows calculating the parameters of proteins that contain such residue in their polypeptide chains. The chromophore parameters were obtained by ab initio (RHF/6-31G**) quantum chemical calculations applying density functional theory (B3LYP). Using this chromophore, we have calculated the molecular dynamics trajectory of tetrameric fluorescent protein DsRed in solution at 300 K (4 nsec). Correctness of the chromophore parametrization was revealed by comparison of quantitative characteristics of the chromophore structure obtained from the molecular dynamic simulations of DsRed protein with the quantitative characteristics of the chromophore based on the crystallographic X-ray data of fluorescent protein DsRed (PDB ID:1ZGO, 1G7K, and 1GGX), and also with the quantitative characteristics of the chromophore obtained by quantum chemical calculations. Inclusion of the neutral form of DsRed protein chromophore topology into the OPLS-AA force field yielded the extended force field OPLS-AA/DsRed. This force field can be used for molecular dynamics calculations of proteins containing the DsRed chromophore. The parameter set presented in this study can be applied for similar extension in any other force fields.

Adequate System for Investigation of the Human Retrotransposon L1 MRNA Translation Initiation in vitro

Dmitriev S.E., Bykova N.V., Andreev D.E., Terenin I.M.

Molecular Biology 40 (2006) 25-30.

Retrotransposon L1 codes for a unique dicistronic mRNA which serves both a transposition intermediate and a template for the synthesis of two proteins of this mobile element. According to preliminary data, the translation initiation of both cistrons of L1 occurs by non-canonical mechanisms. When translating the L1 mRNA in rabbit reticulocyte lysate (RRL), a standard system routinely used by many researchers to study mechanisms of translation initiation in eukaryotes, we observed along with expected products a number of polypeptides resulted from aberrant initiation at internal AUG codons. Such products are absent on translation of L1 mRNA in vivo. Addition to the system of a cytoplasmic extract from HeLa cells resulted in disappearance of these abberant products whereas the efficiency of translation of the first cistron remained unchanged. The level of translation of the second cistron became significantly lower. This also made the picture closer to that observed in vivo. These and other experiments allowed us to clearly demonstrate that the new combined cell-free system is much more adequate to study mechanisms of translation initiation than a regular RRL.

An Internal Ribosome Entry Site Located Upstream of the Crucifer-Infecting Tobamovirus Coat Protein (CP) Gene Can Be Used for CP Synthesis in vivo

Dorokhov Y.L., Ivanov P.A., Komarova T.V., Skulachev M.V., Atabekov J.G.

Journal of General Virology 87 (2006) 2693-2697.

It was previously shown that, unlike the type member of the genus Tobamovirus (TMV U1), a crucifer-infecting tobamovirus (crTMV) contains a 148 nt internal ribosome entry site (IRES)(CP,148)(CR) upstream of the coat protein (CP) gene. Here, viral vectors with substitutions in the stem-loop (SL) region of CP subgenomic promoters (TMV U1-CP-GFP/SL-mut and crTMV-CP-GFP/SL-mut) were constructed and the levels of CP synthesis in agroinoculation experiments were compared. No CP-GFP (green fluorescent protein) synthesis was detected in Nicotiana benthamiana leaves inoculated with TMV U1-CP-GFP/SL-mut, whereas a small amount of CP-GFP synthesis was obtained in crTMV-CP-GFP/SL-mut-injected leaves. Northern blots proved that both promoters were inactive. It could be hypothesized that IRES-mediated early production of the CP by crTMV is needed for realization of its crucifer-infecting capacity.

Role of the Leader Sequence in Tobacco Pectin Methylesterase Secretion

Dorokhov Y.L., Skurat E.V., Frolova O.Y., Gasanova T.V., Ivanov P.A., Ravin N.V., Skryabin K.G., Makinen K.M., Klimyuk V.I., Gleba Y.Y., Atabekov J.G.

FEBS Letters 580 (2006) 3329-3334.

We report that unprocessed tobacco pectin methylesterase (PME) contains N-terminal pro-sequence including the transmembrane (TM) domain and spacer segment preceding the mature PME. The mature portion of PME was replaced by green fluorescent protein (GFP) gene and various deletion mutants of pro-sequence fused to GFP were cloned into binary vectors and agroinjected in Nicotiana benthamiana leaves. The PME pro-sequence delivered GFP to the cell wall (CW). We showed that a transient binding of PME TM domain to endoplasmic reticulum membranes occurs upon its transport to CW. The CW targeting was abolished by various deletions in the TM domain, i.e., anchor domain was essential for secretion of GFP to CW. By contrast, even entire deletion of the spacer segment had no influence on GFP targeting. .

A Novel Function for a Ubiquitous Plant Enzyme Pectin Methylesterase:The Enhancer of RNA Silencing

Dorokhov Y.L., Frolova O.Y., Skurat E.V., Ivanov P.A., Gasanova T.V., Sheveleva A.A., Ravin N.V., Makinen K.M., Klimyuk V.I., Skryabin K.G., Gleba Y.Y., Atabekov J.G.

FEBS Letters 580 (2006) 3872-3878.

Co-agroinjection of Nicotiana henthamiana leaves with the pectin methylesterase (proPME) gene and the TMV:GFP vector resulted in a stimulation of virus-induced RNA silencing (inhibition of GFP production, virus RNA degradation, stimulation of siRNAs production). Conversely, coexpression of TMV:GFP with either antisense PME construct or with enzymatically inactive proPME restored synthesis of viral RNA. Furthermore, expression of proPME enhanced the GFP transgene-induced gene silencing accompanied by relocation of the DCL1 protein from nucleus to the cytoplasm and activation of siRNAs and miRNAs production. It was hypothesized that DCL1 relocated to the cytoplasm may use as substrates both miRNA precursor and viral RNA. The capacity for enhancing the RNA silencing is a novel function for the polyfunctional PME. .

Extracellular Proteinases of Filamentous Fungi As Potential Markers of Phytopathogenesis

Dunaevskii Y.E., Gruban T.N., Belyakova G.A., Belozerskii M.A.

Microbiology 75 (2006) 649-652.

The presence of proteins in the culture liquid of filamentous fungi under study was found to induce the secretion of proteinases. The inhibitory analysis of the major extracellular proteinases of the saprotrophic fungus Trichoderma harzianum and the phytopathogenic fungus Alternaria alternata showed that they both belong to the group of serine proteinases. The substrate specificity of these proteinases and their sensitivity to inhibitors suggest that the enzyme of T harzianum is a subtilisin-like proteinase and the enzyme of A. alternata is a trypsin-like proteinase. This difference between the proteinases may reflect the physiological difference between their producers (saprotroph and phytopathogen).

Degradation of Proteinaceous Substrates by Xylotrophic Basidiomycetes

Dunaevsky Y.E., Zhang D., Matveeva A.R., Belyakova G.A., Belozersky M.A.

Microbiology 75 (2006) 35-39.

The ability of various xylotrophs to produce extracellular proteolytic enzymes has been studied, with emphasis on medium-related factors regulating their secretion. Direct measurement of proteolytic activity in the culture liquid and postelectrophoresis determination of protease activity in polyacrylamide gel copolymerized with gelatin demonstrated that the secreted enzymes are quantitatively and qualitatively diverse. Activity levels of extracellular proteolytic enzymes strongly depend on pH and contents of protein and carbohydrate in the medium. All secreted proteases notably differed in molecular weight (of 51 kDa or higher and in excess of 95 kDa) and belonged mostly to two classes of proteolytic enzymes (serine proteases and metalloproteinases).

Expression of Pannexin Family of Proteins in the Retina

Dvoriantchikova G., Ivanov D., Panchin Y., Shestopalov V.I.

FEBS Letters 580 (2006) 2178-2182.

Expression of the Panx1 and Panx2 members of the pannexin family of gap junction proteins was studied in the retina by in situ hybridization and qRT-PCR. Both pannexins showed robust expression across the retina with predominant accumulation in the retinal ganglion cells (RGCs). In concordance, immuno-histochemical analysis showed accumulation of the Panx1 protein in RGCs, amacrine, horizontal cells and their processes. Two Panx1 isoforms were detected:a ubiquitously expressed 58 kDa protein, and a 43 kDa isoform that specifically accumulated in the retina and brain. Our results indicated that Panx1 and Panx2 are abundantly expressed in the retina, and may therefore contribute to the electrical and metabolic coupling, or to signaling between retinal neurons via the secondary messengers. .

Death of Stoma Guard Cells in Leaf Epidermis Under Disturbance of Energy Provision

Dzyubinskaya E.V., Kiselevsky D.B., Lobysheva N.V., Shestak A.A., Samuilov V.D.

Biochemistry-Moscow 71 (2006) 1120-1127.

Cyanide is an apoptosis inducer in stoma guard cells from pea leaf epidermis. Unlike CN-, the uncoupler of oxidative and photosynthetic phosphorylation carbonyl cyanide m-chlorophenylhydrazone (CCCP), the combination of CCCP, 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU), benzylhydroxamate (BH), myxothiazol, antimycin A, and a glycolysis inhibitor 2-deoxyglucose (DG) did not induce destruction of guard cell nuclei for 20 h of incubation of epidermal peels in the light. DCMU prevented the effect of CN- as a programmed cell death (PCD) inducer. CCCP, the combination of DCMU and CCCP, or the combination of DCMU, CCCP, BH, myxothiazol, antimycin A, and DG supplemented by CN- caused destruction of cell nuclei; the number of the cells lacking nuclei in this case was higher than with CN- alone. DG and CCCP caused cell destruction after longer incubation of the isolated epidermis - after 2 days and to a greater degree after 4 days. The effect of DG and CCCP was reduced by illumination. Cell destruction during long-term incubation was prevented by the combination of DG and CCCP. From data of electron microscopy, DCMU and dinitrophenyl ester of iodonitrothymol (DNP-INT) prevented apoptotic changes of the nuclear ultrastructure induced by CN-. The suppression of the destruction of the guard cell nuclei under combined action of DG and CCCP was apparently caused by switching of cell death from PCD to necrosis. Thus, the type of cell death - via apoptosis or necrosis - is controlled by the level of energy provision.

Programmed Cell Death in Plants:Effect of Protein Synthesis Inhibitors and Structural Changes in Pea Guard Cells

Dzyubinskaya E.V., Kiselevsky D.B., Bakeeva L.E., Samuilov V.D.

Biochemistry-Moscow 71 (2006) 395 -405.

Pea leafepidermis incubated with cyanide displayed ultrastructural changes in guard cells that are typical of apoptosis. Cycloheximide, an inhibitor of cytoplasmic protein synthesis, and lincomycin, an inhibitor of protein synthesis in chloroplasts and mitochondria, produced different effects on the dynamics of programmed death of guard cells. According to light microscopy data, cycloheximide reinforced and lincomycin suppressed the CN--induced destruction of cell nuclei. Lincomycin lowered the effect of cycloheximide in the light and prevented it in the dark. According to electron microscopy data, the most pronounced effects of cycloheximide in the presence of cyanide were autophagy and a lack of apoptotic condensation of nuclear chromatin, the prevention of chloroplast envelope rupturing and its invagination inside the stroma, and the appearance of particular compartments with granular inclusions in mitochondria. Lincomycin inhibited the CN--induced ultrastructural changes in guard cell nuclei. The data show that programmed death Of guard cells may have a combined scenario involving both apoptosis and autophagy and may depend on the action of both cytoplasm synthesized and chloroplast and mitochondrion synthesized proteins.

On the Nature of Obligate Intracellular Symbiosis of Rickettsiae - Rickettsia prowazekii Cells Import Mitochondrial Porin

Emelyanov V.V. and Vyssokikh M.Y.

Biochemistry-Moscow 71 (2006) 730 -735.

Mitochondrial porin was identified in Rickettsia prowazekii by Western blot analysis of whole cells and membrane fractions with monoclonal antibody against porin VDAC I of animal mitochondria. Using the BLAST server, no protein sequences homologous to mitochondrial porin were found among the rickettsial genomes. Rickettsiae also do not contain their own porin. The protein imported by rickettsiae is weakly extracted by nonionic detergents and, like porin in mitochondria, is insensitive to proteinase K in whole cells. Immunocytochemical analysis showed that it localizes to the outer membrane of the bacterial cells. These data support an earlier suggestion about import by rickettsiae of indispensable proteins from cytoplasm of the host cell as a molecular basis of obligate intracellular parasitism. They are also consistent with the hypothesis invoking a transfer of genes specifying surface proteins from the last common ancestor of rickettsiae and mitochondria to the host genome, and preservation by rickettsiae of the primitive ability to import these proteins.

Metabolic Regulation of Neutrophil Spreading, Membrane Tubulovesicular Extensions (Cytonemes) Formation and Intracellular PH Upon Adhesion to Fibronectin

Galkina S.I., Sud'ina G.F., Klein T.

Experimental Cell Research 312 (2006) 2568-2579.

Circulating leukocytes have a round cell shape and roll along vessel walls. However, metabolic disorders can lead them to adhere to the endothelium and spread (flatten). We studied the metabolic regulation of adhesion, spreading and intracellular pH (pHi) of neutrophils (polymorphonuclear leukocytes) upon adhesion to fibronectin-coated substrata. Resting neutrophils adhered and spread on fibronectin. An increase in pHi accompanied neutrophil spreading. Inhibition of oxidative phosphorylation or inhibition of P- and F-type ATPases affected neither neutrophil spreading nor pHi. Inhibition of glucose metabolism or V-ATPase impaired neutrophil spreading, blocked the increase in the pHi and induced extrusion of membrane tubulovesicular extensions (cytonemes), anchoring cells to substrata. Omission of extracellular Na⁺ and inhibition of chloride channels caused a similar effect. We propose that these tubulovesicular extensions represent protrusions of exocytotic trafficking, supplying the plasma membrane of neutrophils with ion exchange mechanisms and additional membrane for spreading. Glucose metabolism and V-type ATPase could affect fusion of exocytotic trafficking with the plasma membrane, thus controlling neutrophil adhesive state and pHi. Cl- efflux through chloride channels and Na⁺ influx seem to be involved in the regulation of the V-ATPase by carrying out charge compensation for the proton-pumping activity and through V-ATPase in regulation of neutrophil spreading and pHi.

Mapping Contacts of the S12-S7 Intercistronic Region of Str Operon MRNA With Ribosomal Protein S7 of E. coli

Golovin A., Spiridonova V., Kopylov A.

FEBS Letters 580 (2006) 5858-5862.

In E. coli, S7 initiates 30S ribosome assembly by binding to 16S rRNA. It also regulates translation of the S12 and S7 cistrons of the 'streptomycin' operon transcript by binding to the S12-S7 intercistronic region. Here, we describe the contacts of N-terminally His(6)-tagged S7 with this region as mapped by UV-induced cross-linking. The cross-links are located at U(-34), U(-35), quite distant from the start codons of the two cistrons. In order to explain the mechanism of translational repression of S12-S7, we consider a possible conformational rearrangement of the intercistronic RNA structure induced by S7 binding. .

Mutational Analysis of the Catalytic Domain of the Murine Dnmt3a DNA-(Cytosine C5)-Methyltransferase

Gowher H., Loutchanwoot P., Vorobjeva G., Handa V., Jurkowska R.Z., Jurkowski T.P., Jeltsch A.

Journal of Molecular Biology 357 (2006) 928-941.

On the basis of amino acid sequence alignments and structural data of related enzymes, we have performed a mutational analysis of 14 amino acid residues in the catalytic domain of the murine Dnmt3a DNA-(cytosine C5)-methyltransferase. The target residues are located within the ten conserved amino acid sequence motifs characteristic for cytosine-C5 methyltransferases and in the putative DNA recognition domain of the enzyme (TRD). Mutant proteins were purified and tested for their catalytic properties and their abilities to bind DNA and AdoMet. We prepared a structural model of Dnmt3a. to interpret our results. We demonstrate that Phe50 (motif I) and Glu74 (motif II) are important for AdoMet binding and catalysis. D96A (motif III) showed reduced AdoMet binding but increased activity under conditions of saturation with S-adenosyl-L-methionine (AdoMet), indicating that the contact of Asp96 to AdoMet is not required for catalysis. R130A (following motif IV), R241A and R246A (in the TRD), R292A, and R297A (both located in front of motif X) showed reduced DNA binding. R130A displayed a strong reduction in catalytic activity and a complete change in flanking sequence preferences, indicating that Arg130 has an important role in the DNA interaction of Dnmt3a. R292A also displayed reduced activity and changes in the flanking sequence preferences, indicating a potential role in DNA contacts farther away from the CG target site. N167A (motif VI) and R202A (motif VIII) have normal AdoMet and DNA binding but reduced catalytic activity. While Asn167 might contribute to the positioning of residues from motif VI, according to structural data Arg202 has a role in catalysis of cytosine-C5 methyltransferases. The R295A variant was catalytically inactive most likely because of destabilization of the hinge sub-domain of the protein.

Regulation of Microtubule Dynamics in 3T3 Fibroblasts by Rho Family GTPases

Grigoriev I., Borisy G., Vorobjev I.

Cell Motility and the Cytoskeleton 63 (2006) 29-40.

To get insight into the action of Rho GTPases on the microtubule system we investigated the effects of Cdc42, Rac1, and RhoA on the dynamics of microtubules in Swiss 3T3 fibroblasts. In control cells microtubule ends were dynamic:plus ends frequently switched between growth, shortening and pauses; the growth phase predominated over shortening. Free minus ends of microtubules depolymerized rapidly and never grew. Free microtubules were short-lived, and the microtubule network was organized into a radial array. In serum-starved cells microtubule ends became more stable:although Plus ends still transited between growth and shortening, polymerization and depolymerization excursions became shorter and balanced each other. Microtubule minus ends were also stabilized. Consequently lifespan of free microtubules increased and microtubule, array changed its radial pattern into a random one. Activation of Cdc42 and Rac1 in serum-starved cells promoted dynamic behavior of microtubule plus and minus ends, while inhibition of these GTPases in serum-grown cells suppressed microtubule dynamics and mimicked all effects of serum starvation. Activation of RhoA in serum-grown cells had effects similar to Cdc42/Rac1 inactivation:it suppressed the dynamics of plus and minus ends, reduced the length of growth and shrinking episodes, and disrupted the radial organization of microtubules. However, in contrast to Cdc42 and Rac1 inactivation, active RhoA had no effect on the balance between microtubule growth and shortening. We conclude that Cdc42 and Rac1 have similar stimulating effects on microtubule dynamics while RhoA acts in an opposite way.

Relationship Between the Oligomeric Status of HIV-1 Integrase on DNA and Enzymatic Activity

Guiot E., Carayon K., Delelis O., Simon F., Tauc P., Zubin E., Gottikh M., Mouscadet J.F., Brochon J.C., Deprez E.

Journal of Biological Chemistry 281 (2006) 22707-22719.

The 3'-processing of the extremities of viral DNA is the first of two reactions catalyzed by HIV-1 integrase (IN). High order IN multimers (tetramers) are required for complete integration, but it remains unclear which oligomer is responsible for the 3'-processing reaction. Moreover, IN tends to aggregate, and it is unknown whether the polymerization or aggregation of this enzyme on DNA is detrimental or beneficial for activity. We have developed a fluorescence assay based on anisotropy for monitoring release of the terminal dinucleotide product in real-time. Because the initial anisotropy value obtained after DNA binding and before catalysis depends on the fractional saturation of DNA sites and the size of IN center dot DNA complexes, this approach can be used to study the relationship between activity and binding/multimerization parameters in the same assay. By increasing the IN:DNA ratio, we found that the anisotropy increased but the 3'-processing activity displayed a characteristic bell-shaped behavior. The anisotropy values obtained in the first phase were predictive of subsequent activity and accounted for the number of complexes. Interestingly, activity peaked and then decreased in the second phase, whereas anisotropy continued to increase. Time-resolved fluorescence anisotropy studies showed that the most competent form for catalysis corresponds to a dimer bound to one viral DNA end, whereas higher order complexes such as aggregates predominate during the second phase when activity drops off. We conclude that a single IN dimer at each extremity of viral DNA molecules is required for 3'-processing, with a dimer of dimers responsible for the subsequent full integration.

Thermal Energy Dissipation in Reaction Centres and in the Antenna of Photosystem II Protects Desiccated Poikilohydric Mosses Against Photo-Oxidation

Heber U., Bilger W., Shuvalov V.A.

Journal of Experimental Botany 57 (2006) 2993-3006.

Seasonal differences have been observed in the ability of desiccated mosses to dissipate absorbed light energy harmlessly into heat. During the dry summer season desiccation-tolerant mosses were more protected against photo-oxidative damage in the dry state than during the more humid winter season. Investigation of the differences revealed that phototolerance could be acquired or lost even under laboratory conditions. When a desiccated poikilohydric moss such as Rhytidiadelphus squarrosus is in the photosensitive state, the primary quinone, Q_A, in the reaction centre of photosystem II is readily reduced even by low intensity illumination as indicated by reversibly increased chlorophyll fluorescence. No such reduction is observed even under strong illumination in desiccated mosses after phototolerance has been acquired. In this state, reductive charge stabilization is replaced by energy dissipation. As a consequence, chlorophyll fluorescence is quenched. Different mechanisms are responsible for quenching. One is based on the presence of zeaxanthin provided drying occurs in the light. This mechanism is known to be controlled by a protonation reaction which is based on proton-coupled electron transport while the moss is still hydrated. Another mechanism which also requires light for activation, but no protonation, is activated during desiccation. While water is slowly lost, fluorescence is quenched. In this situation, an absorption band formed at 800 nm in the light is stabilized. It loses reversibility on darkening. Comparable kinetics of fluorescence quenching and 800 nm signals as well as the linear relationship between non-photochemical fluorescence quenching (NPQ) and loss of stable charge separation in photosystem II reaction centres suggested that desiccation-induced quenching is a property of photosystem II reaction centres. During desiccation, quenchers accumulate which are stable in the absence of water but revert to non-quenching molecular species on hydration. Together with zeaxanthin-dependent energy dissipation, desiccation-induced thermal energy dissipation protects desiccated poikilohydric mosses against photo-oxidation, ensuring survival during drought periods.

Conservation and Dissipation of Light Energy As Complementary Processes:Homoiohydric and Poikilohydric Autotrophs

Heber U., Lange O.L., Shuvalov V.A.

Journal of Experimental Botany 57 (2006) 1211-1223.

The relationship between photosynthetic energy conservation and thermal dissipation of light energy is considered, with emphasis on organisms which tolerate full desiccation without suffering photo-oxidative damage in strong light. As soon as water becomes available to dry poikilohydric organisms, they resume photosynthetic water oxidation. Only excess light is then thermally dissipated in mosses and chlorolichens by a mechanism depending on the protonation of a thylakoid protein and availability of zeaxanthin. Upon desiccation, another mechanism is activated which requires neither protonation nor zeaxanthin although the zeaxanthin-dependent mechanism of energy dissipation remains active, provided desiccation occurs in the light. Increased thermal energy dissipation under desiccation finds expression in the loss of variable, and in the quenching of, basal chlorophyll fluorescence. Spectroscopical analysis revealed the activity of photosystem II reaction centres in the absence of water. Oxidized beta-carotene (Car⁺) and reduced chlorophyll (Chl^-), perhaps ChlD1 next to P680 within the D1 subunit, accumulates reversibly under very strong illumination. Although recombination between Car⁺ and Chl^- is too slow to contribute significantly to thermal energy dissipation, a much faster reaction such as the recombination between P680⁺ and the neighbouring Chl^- is suggested to form the molecular basis of desiccation-induced energy dissipation in photosystem II reaction centres. Thermal dissipation of absorbed light energy within a picosecond time domain deactivates excited singlet chlorophyll, thereby preventing triplet accumulation and the consequent photo-oxidative damage by singlet oxygen.

The Depolymerizing Kinesin MCAK Uses Lattice Diffusion to Rapidly Target Microtubule Ends

Helenius J., Brouhard G., Kalaidzidis Y., Diez S., Howard J.

Nature 441 (2006) 115-119.

The microtubule cytoskeleton is a dynamic structure in which the lengths of the microtubules are tightly regulated. One regulatory mechanism is the depolymerization of microtubules by motor proteins in the kinesin-13 family(1). These proteins are crucial for the control of microtubule length in cell division(2-4), neuronal development(5) and interphase microtubule dynamics(6,7). The mechanism by which kinesin-13 proteins depolymerize microtubules is poorly understood. A central question is how these proteins target to microtubule ends at rates exceeding those of standard enzyme-substrate kinetics(8). To address this question we developed a single-molecule microscopy assay for MCAK, the founding member of the kinesin-13 family(9). Here we show that MCAK moves along the microtubule lattice in a one-dimensional (1D) random walk. MCAK-microtubule interactions were transient:the average MCAK molecule diffused for 0.83 s with a diffusion coefficient of 0.38 mu m(2) s(-1). Although the catalytic depolymerization by MCAK requires the hydrolysis of ATP, we found that the diffusion did not. The transient transition from three-dimensional diffusion to 1D diffusion corresponds to a "reduction in dimensionality"(10) that has been proposed as the search strategy by which DNA enzymes find specific binding sites(11). We show that MCAK uses this strategy to target to both microtubule ends more rapidly than direct binding from solution.

DNA-Duplexes Containing Abasic Sites:Correlation Between Thermostability and Acoustic Wave Properties

Hianik T., Wang X., Andreev S., Dolinnaya N., Oretskaya T., Thompson M.

Analyst 131 (2006) 1161-1166.

Aldehydic apurinic or apyrimidinic sites that lack a nucleobase moiety are one of the most common forms of toxic lesions in DNA. In the present study, a close structural analog of such a site, the 2-(hydroxymethyl) tetrahydrofuranyl residue, was synthesized in order to act as a model for damaged nucleic acid probes. Prepared oligodeoxyribonucleotides containing one, two or three abasic sites were hybridized to complementary sequences immobilized on a gold surface using the neutravidin - biotin interaction for study by thickness shear mode acoustic wave detector. Measurement of the complex electrical impedance of an AT-cut quartz device with immobilized biotinylated nucleotide allowed the detection of changes of series resonance frequency, Delta f(s), and motional resistance, R-m, associated with duplex formation. The changes as detected by the acoustic wave method correlated well with the thermostability of DNA duplexes in solution. With respect to the latter, UV-monitored melting curves indicate that both the number of sites and their localization in the double-stranded structure influence the amount by which a 19 b.p. duplex is destabilized. The presence of 3 abasic sites completely destabilized the DNA duplex.

On Two East Asian Species of Brachythecium (Brachytheciaceae, Musci)

Ignatov M.S., Milyutina I.A., Huttunen S.

Journal of the Hattori Botanical Laboratory (2006) 191-199.

Brachythecium auriculatum A. Jaeger was synonymized with Palamocladium leskeoides by Hoffman (1998). The present study shows their independence. Brachythecium complanatum Broth. is reported from Japan for the first time; this species was confused before with the superficially similar B. garovaglioides (=B. wichurae). Descriptions and illustrations of both species are provided. Japanese species of Brachytheciaceae were carefully revised by Takaki (1955a,b; 1956), and later the taxonomic concepts of Takaki were followed by Noguchi (1991) with relatively minor changes. In the course of study of Brachythecium species of Asia we found new data which are important for understanding two of the species occurring in Japan.

Stress Granules:RNP-Containing Cytoplasmic Bodies Arising in Stress:Structure and Mechanism of Organization

Ivanov P.A. and Nadezhdina E.S.

Molecular Biology 40 (2006) 844-850.

The review considers recent data on stress granules, which are dense RNP-containing cytoplasmic bodies that arise under stress conditions, e.g., in beat shock, UV irradiation, energy depletion, and oxidative stress. There is evidence that stress granules accumulate incomplete initiation complexes containing mRNA associated with proteins, small ribosomal subunits, and some translation initiation factors, and that stress granules are formed when cells are depleted of the ternary complex (eIF2-tRNA(Met)-GTP), in particular, upon eIF2A phosphorylation or a decrease in GTP. Large ribosomal subunits and the ternary complex are absent from stress granules. The structural basis of stress granules is known. It is probable, however, that RNA-binding protein TIA-1, which normally occurs in the nucleus, forms prion-like aggregates that serve as scaffolds for other components of stress granules. The cytoskeleton facilitates the accumulation of stress granule components in local cytoplasmic sites. Studies of the formation and composition of stress granules are important for abetter understanding of the regulation of translation initiation in vivo and the mechanisms of the cell response to stress factors.

Potato Virus X RNA-Mediated Assembly of Single-Tailed Ternary 'Coat Protein-RNA-Movement Protein' Complexes

Karpova O.V., Zayakina O.V., Arkhipenko M.V., Sheval E.V., Kiselyova O.I., Poljakov V.Y., Yaminsky I.V., Rodionova N.P., Atabekov J.G.

Journal of General Virology 87 (2006) 2731-2740.

Different models have been proposed for the nature of the potexvirus transport form that moves from cell to cell over the infected plant:(i) genomic RNA moves as native virions; or (ii) in vitro-assembled non-virion ribonucleoprotein (RNP) complexes consisting of viral RNA, coat protein (CP) and movement protein (MP), termed TGBp1, serve as the transport form in vivo. As the structure of these RNPs has not been elucidated, the products assembled in vitro from potato virus X (PVX) RNA, CP and TGBp1 were characterized. The complexes appeared as single-tailed particles (STPs) with a helical, head-like structure composed of CP subunits located at the 5'-proximal region of PVX RNA; the TGBp1 was bound to the terminal CP molecules of the head. Remarkably, no particular non-virion RNP complexes were observed. These data suggest that the CP-RNA interactions resulting in head formation prevailed over TGBp1-RNA binding upon STP assembly from RNA, CP and TGBp1. STPs could be assembled from the 5' end of PVX RNA and CP in the absence of TGBp1. The translational ability of STPs was characterized in a cell-free translation system. STPs lacking TGBp1 were entirely non-translatable; however, they were rendered translatable by binding of TGBp1 to the end of the head. It is suggested that the RNA-mediated assembly of STPs proceeds via two steps. Firstly, non-translatable CP-RNA STPs are produced, due to encapsidation of the 5'-terminal region. Secondly, the TGBp1 molecules bind to the end of a polar head, resulting in conversion of the STPs into a translatable form.

Participation of ATP/ADP Antiporter in Oleate- and Oleate Hydroperoxide-Induced Uncoupling Suppressed by GDP and Carboxyatractylate

Khailova L.S., Prikhodko E.A., Dedukhova V.I., Mokhova E.N., Popov V.N., Skulachev V.P.

Biochimica et Biophysica Acta-Bioenergetics 1757 (2006) 1324-1329.

In experiments on isolated kidney and liver mitochondria, it is shown that oleate hydroperoxide induces a much smaller increase in the controlled respiration rate and Delta psi decrease than the same concentrations of oleate. Palmitate appears to be less efficient than oleate but more efficient than oleate hydroperoxide. In all cases, GDP and CAtr cause some recoupling, CAtr being more effective. Addition of 0.2 mM GDP before CAtr does not prevent further AV increase by subsequent CAtr addition. On the other hand, GDP added after CAtr is without any effect. GDP partially prevents the Delta psi lowering by ADP at the State 4 - State 3 transition if small amounts of CAtr are present. The data are consistent with the suggestion of F. Goglia and V.P. Sk-ulachev (FASEB J 17, 1585-1591, 2003) that fatty acid anions are translocated by mitochondrial anion carriers much better than their hydroperoxides. As to GDP recoupling, it cannot be regarded as a specific probe for uncoupling by UCPs since it can be mediated by the ATP/ADP antiporter.

Structural Changes in the Ribosome During the Elongation Cycle

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

Molecular Biology 40 (2006) 675-687.

Ample data on structural changes that arise in the ribosome during translation have been accumulated. The most interesting information on such changes has been obtained by cryoelectron microscopy of ribosome complexes with various ligands and by rRNA site-directed mutagenesis combined with a structural analysis of the ribosome by a chemical modification technique (chemical probing). The review considers the best-known structural changes that arise in the translating ribosome upon its interactions with tRNA and the elongation factors. The changes are discussed in the context of interactions between the functional centers of the ribosome. A universal system of rRNA helices and proteins is described in detail. The system integrates the functional centers of the ribosome and allows transduction of allosteric conformational signals. Biochemical data are considered in terms of the structures and interactions of ribosomal elements, and a hypothesis is advanced that the position of the GTPase-associated center in the ribosome regulates the binding of the elongation factors.

Structure of the Intracellular Part of the Motility Apparatus of Halobacteria

Kireev I.I., Novikova T.M., Sheval E.V., Metlina A.L.

Microbiology 75 (2006) 306-311.

The electron microscopic study of the structure of the motility apparatus of the archaea Halobacterium salinarium 4W12 and Natronobacterium magadii confirmed our earlier observation that the motility apparatus of halobacteria contains an intracellular disk-shaped lamellar structure (DLS). Polar cap structures (PCSs) isolated from the halobacterium were preliminarily identified as the DLSs. The PCSs in complexes with flagella C, were also isolated from the haloalkaliphilic bacterium N. magadii. The specific structure of the archaeal motility apparatus is discussed.

New Viral Vector for Efficient Production of Target Proteins in Plants

Komarova T.V., Skulachev M.V., Zvereva A.S., Schwartz A.M., Dorokhov Y.L., Atabekov J.G.

Biochemistry-Moscow 71 (2006) 846 -850.

A new potato virus X (PVX)-based viral vector for superproduction of target proteins in plants has been constructed. The triple gene block and coat protein gene of PVX were substituted by green fluorescent protein. This reduced viral vector was delivered into plant cells by agroinjection (injection of Agrobacterium tumefaciens cells, carrying viral vector cDNA within T-DNA, into plant leaves), and this approach allowed to dramatically reduce the size of the vector genome. The novel vector can be used for production of different proteins including pharmaceuticals in plants.

Surface Plasmon Resonance Study of G Protein/Receptor Coupling in a Lipid Bilayer-Free System

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

Analytical Chemistry 78 (2006) 1228-1234.

Surface plasmon resonance (SPR) spectroscopy is a technique to study protein-protein interactions in real time; however, application of SPR spectroscopy for investigations of membrane receptors is difficult with respect to functional and uniform immobilization of receptors on a biosensor surface. In the current study, we developed a simple, direct, biosensor-based approach to monitor the molecular interactions between G protein transducin (G) and rhodopsin (Rho), a prototypical G protein-coupled receptor (GPCR). Detergent-solubilized dark-adapted Rho was captured onto a biosensor surface via lectin interaction, enabling site-directed immobilization of the receptor that made its cytoplasmic surface accessible to a coupling G protein. The system resembled the natural system with respect to receptor density, binding of G(t) following flash or constant light application, fast GTP-dependent dissociation of Gt from Rho, regeneration of Rho, and dependence of Gt binding on light intensity and on concentration of Gt. The apparent K-D of the G(t)/Rho interaction was 13.6 nM. Our results validate the use of SPR spectroscopy as a tool to study G protein activation in GPCR systems and could be extended for application to other interaction partners of GPCRs.

Thermal Unfolding of Smooth Muscle and Nonmuscle Tropomyosin Alpha-Homodimers With Alternatively Spliced Exons

Kremneva E., Nikolaeva O., Maytum R., Arutyunyan A.M., Kleimenov S.Y., Geeves M.A., Levitsky D.I.

FEBS Journal 273 (2006) 588-600.

We used differential scanning calorimetry (DSC) and circular dichroism (CD) to investigate thermal unfolding of recombinant fibroblast isoforms of alpha-tropomyosin (Tm) in comparison with that of smooth muscle Tm. These two nonmuscle Tm isoforms 5a and 5b differ internally only by exons 6b/6a, and they both differ from smooth muscle Tm by the N-terminal exon 1b which replaces the muscle-specific exons 1a and 2a. We show that the presence of exon 1b dramatically decreases the measurable calorimetric enthalpy of the thermal unfolding of Tm observed with DSC, although it has no influence on the alpha-helix content of Tm or on the end-to-end interaction between Tm dimers. The results suggest that a significant part of the molecule of fibroblast Tm (but not smooth muscle Tm) unfolds noncooperatively, with the enthalpy no longer visible in the cooperative thermal transitions measured. On the other hand, both DSC and CD studies show that replacement of muscle exons 1a and 2a by nonmuscle exon 1b not only increases the thermal stability of the N-terminal part of Tm, but also significantly stabilizes Tm by shifting the major thermal transition of Tm to higher temperature. Replacement of exon 6b by exon 6a leads to additional increase in the alpha-Tm thermal stability. Thus, our data show for the first time a significant difference in the thermal unfolding between muscle and nonmuscle alpha-Tm isoforms, and indicate that replacement of alternatively spliced exons alters the stability of the entire Tm molecule.

Determination of Concentration and Aggregate Size in Influenza Virus Preparations From the True UV-Absorption Spectra

Ksenofontov A.L., Kozlovsky V.S., Kordyukova L.V., Radyukhin V.A., Timofeeva A.V., Dobrov E.N.

Molecular Biology 40 (2006) 172-179.

It is well-known that influenza virus (IV) preparations are characterized by very large contribution of light-scattering to their UV absorption spectra. With the help of so called extrapolation method we managed to measure true absorption spectra of IV preparations and to determine absorption coefficients (E-1 cm, 280(0.1%)) for the intact IV virions and for IV subviral particles. These coefficients turned out to equal 1.26 +/- 0.17 and 0.96 +/- 0.11 for the virions and subviral particles respectively. The knowledge of exact IV concentration is necessary for quantitative physico-chemical studies of IV virions and their components. It is also shown that UV absorption spectra measurements allow to register IV virion aggregation. Aggregation properties of IV subviral particles were also studied.

Proteins Surrounding Hairpin IIIe of the Hepatitis C Virus Internal Ribosome Entry Site on the Human 40S Ribosomal Subunit

Laletina E., Graifer D., Malygin A., Ivanov A., Shatsky I., Karpova G.

Nucleic Acids Research 34 (2006) 2027-2036.

Binding of the internal ribosome entry site (IRES) of the hepatitis C virus (HCV) RNA to the eIF-free 40S ribosomal subunit is the first step of initiation of translation of the viral RNA. Hairpins IIId and IIIe comprising 253-302 nt of the IRES are known to be essential for binding to the 40S subunit. Here we have examined the molecular environment of the HCV IRES in its binary complex with the human 40S ribosomal subunit. For this purpose, two RNA derivatives were used that bore a photoactivatable perfluorophenyl azide cross-linker. In one derivative the cross-linker was at the nucleotide A296 in hairpin IIIe, and in the other at G87 in domain II. Site-specific introduction of the cross-linker was performed using alkylating derivatives of oligodeoxyribonucleotides complementary to the target RNA sequences. No cross-links with the rRNA were detected with either RNA derivative. The RNA with the photoactivatable group at A296 cross-linked to proteins identified as S5 and S16 (major) and p40 and S3a (minor), while no cross-links with proteins were detected with RNA modified at G87. The results obtained indicate that hairpin IIIe is located on the solvent side of the 40S subunit head on a site opposite the beak.

Molecular Environment of the Subdomain IIIe Loop of the RNA IRES Element of Hepatitis C Virus on the Human 40S Ribosomal Subunit

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

Russian Journal of Bioorganic Chemistry 32 (2006) 280-287.

The molecular environment of the internal ribosome entry site (IRES element) of hepatitis C viral (HCV) RNA in the binary complex with the human 40S ribosomal subunit was studied. To this end, RNA derivatives bearing mild UV-reactive perfluorophenylazide groups at nucleotide G87 in IRES domain II and at nucleotide A296 in the subdomain IIIe loop were used, which were prepared by the RNA complementarily-addressed modification with alkylating oligonucleotide derivatives. None of the RNA derivatives were shown to be crosslinked to the 18S IRNA of the 40S subunit. It was found that the photoreactive group of IRES nucleotide A296 crosslinked to the 40S subunit S2/S3a, S5, and p40 (SOA) proteins. No protein crosslinking was observed for the RNA derivative containing the same photoreactive group at nucleotide G87. It was concluded that the subdomain IIIe loop of the HCV RNA IRES element in the complex with the 40S subunit is located on the subunit between the head and the body aside the "beak" near the exit from the mRNA-binding channel.

Methods for Improving the Quality of Prediction in the Process of Automatic Annotating A(4)

Leontovich A.M. and Tokmachev K.Y.

Biofizika 51 (2006) 593-601.

Modifications of the previously described adaptive algorithm of automatic annotating A(4) have been considered, which make it possible to improve the quality of prediction. First, the direct use of the so-called basis statistics eta refines the quality of prediction compared with the previously proposed statistics gamma. Second, the quality is improved if not all similar sequences found but only part of them are used. This decreases the noising of the data, which in turn improves the quality of prognosis.

A Minimal Region in the NTPase/Helicase Domain of the TGBp1 Plant Virus Movement Protein Is Responsible for ATPase Activity and Cooperative RNA Binding

Leshchiner A.D., Solovyev A.G., Morozov S.Y., Kalinina N.O.

Journal of General Virology 87 (2006) 3087-3095.

The TGBp1 protein, encoded in the genomes of a number of plant virus genera as the first gene of the 'triple gene block', possesses an NTPase/helicase domain characterized by seven conserved sequence motifs. It has been shown that the TGBp1 NTPase/helicase domain exhibits NTPase, RNA helicase and RNA-binding activities. In this paper, we have analysed a series of deletion and point mutants in the TGBp1 proteins encoded by Potato virus X (PVX, genus Potexvirus) and Poa semilatent virus (PSLV, genus Hordeivirus) to map functional regions responsible for their biochemical activities in vitro. It was found that, in both PVX and PSLV, the N-terminal part of the TGBp1 NTPase/helicase domain comprising conserved motifs 1, la. and 11 was sufficient for ATP hydrolysis, RNA binding and homologous protein-protein interactions. Point mutations in a single conserved basic amino acid residue upstream of motif I had little effect on the activities of C-terminally truncated mutants of both TGBp1 proteins. However, when introduced into the full-length NTPase/helicase domains, these mutations caused a substantial decrease in the ATPase activity of the protein, suggesting that the conserved basic amino acid residue upstream of motif I was required to maintain a reaction-competent conformation of the TGBp1 ATPase active site.

Identification of Escherichia coli M(2) G Methyltransferases:I. The YcbY Gene Encodes a Methyltransferase Specific for G2445 of the 23 S RRNA

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

Journal of Molecular Biology 364 (2006) 20-25.

N-2-methylguanosine 2445 of the 23 S rRNA is located in a cluster of modified nucleotides concentrated at the peptidyl transferase center of the ribosome. Here we describe the identification of a gene, ycbY, as encoding an enzyme responsible for methylation of G2445. Knock-out of the ycbY gene leads to loss of modification at G2445 as revealed by reverse transcription. The modification is restored in the ycbY knock-out strain if co-transformed with a plasmid expressing the ycbY gene product. Recombinant YcbY protein is able to methylate 23 S rRNA purified from the ycbY knock-out strain in vitro, assembled 50 S subunits are not a substrate for the methylase. Knock-out of the ycbY gene leads to growth retardation. Growth competition with the parental wild-type strain leads to a gradual decrease in the knock-out strain cells proportion in the media. It is likely that the G2445 modification is necessary for prevention of nonfunctional secondary or tertiary structure formation at the peptidyl transferase center. Based on these results we suggest that YcbY be renamed to RlmL in accordance with the accepted nomenclature for rRNA methyltransferases.

What Is Hidden in the Pannexin Treasure Trove:the Sneak Peek and the Guesswork

Litvin O., Tiunova A. , Connell-Alberts Y., Panchin Y. , Baranova A.

Journal of Cellular and Molecular Medicine 10 (2006) 613-634.

Connexins had been considered to be the only class of the vertebrate proteins capable of gap junction formation; however, new candidates for this function with no homology to connexins, termed pannexins were discovered. So far three pannexins were described in rodent and human genomes:Panx1, Panx2 and Panx3. Expressions of pannexins can be detected in numerous brain structures, and now found both in neuronal and glial cells. Hypothetical roles of pannexins in the nervous system include participating in sensory processing, hippocampal. plasticity, synchronization between hippocampus and cortex, and propagation of the calcium waves supported by glial cells, which help maintain and modulate neuronal metabolism. Pannexin also may participate in pathological reactions of the neural cells, including their damage after ischemia and subsequent cell death. Recent study revealed non-gap junction function of Panx1 hemichannels in erythrocytes, where they serve as the conduits for the ATP release in response to the osmotic stress. High-throughput studies produced some evidences of the pannexin involvement in the process of tumorigenesis. According to brain cancer gene expression database REMBRANDT, PANX2 expression levels can predict post diagnosis survival for patients with glial tumors. Further investigations are needed to verify or reject hypotheses listed.

The Fidelity of Translation Initiation:Reciprocal Activities of EIF1, IF3 and YciH

Lomakin I.B., Shirokikh N.E., Yusupov M.M., Hellen C.U.T., Pestova T.V.

Embo Journal 25 (2006) 196-210.

Eukaryotic initiation factor eIF1 and the functional C-terminal domain of prokaryotic initiation factor IF3 maintain the fidelity of initiation codon selection in eukaryotes and prokaryotes, respectively, and bind to the same regions of small ribosomal subunits, between the platform and initiator tRNA. Here we report that these nonhomologous factors can bind to the same regions of heterologous subunits and perform their functions in heterologous systems in a reciprocal manner, discriminating against the formation of initiation complexes containing codon - anticodon mismatches. We also show that like IF3, eIF1 can influence initiator tRNA selection, which occurs at the stage of ribosomal subunit joining after eIF5-induced hydrolysis of eIF2-bound GTP. The mechanisms of initiation codon and initiator tRNA selection in prokaryotes and eukaryotes are therefore unexpectedly conserved and likely involve related conformational changes induced in the small ribosomal subunit by factor binding. YciH, a prokaryotic eIF1 homologue, could perform some of IF3's functions, which justifies the possibility that YciH and eIF1 might have a common evolutionary origin as initiation factors, and that IF3 functionally replaced YciH in prokaryotes.

Voltage Changes Involving Photosystem II Quinone-Iron Complex Turnover

Mamedov M.D., Tyunyatkina A.A., Siletsky S.A., Semenov A.Y.

European Biophysics Journal with Biophysics Letters 35 (2006) 647-654.

An electrometrical technique was used to investigate proton-coupled electron transfer between the primary plastoquinone acceptor Q_A^- and the oxidized non-heme iron Fe³⁺ on the acceptor side of photosystem II core particles incorporated into phospholipid vesicles. The sign of the transmembrane electric potential difference Delta psi (negative charging of the proteoliposome interior) indicates that the iron-quinone complex faces the interior surface of the proteoliposome membrane. Preoxidation of the non-heme iron was achieved by addition of potassium ferricyanide entrapped into proteoliposomes. Besides the fast unresolvable kinetic phase (tau similar to 0.1 mu s) of Delta psi generation related to electron transfer between the redox-active tyrosine Y-Z and Q_A, an additional phase in the submillisecond time domain (tau similar to 0.1 ms at 23 degrees C, pH 7.0) and relative amplitude similar to 20% of the amplitude of the fast phase was observed under exposure to the first flash. This phase was absent under the second laser flash, as well as upon the first flash in the presence of DCMU, an inhibitor of electron transfer between Q_A and the secondary quinone Q_B. The rate of the additional electrogenic phase is decreased by about one-half in the presence of D₂O and is reduced with the temperature decrease. On the basis of the above observations we suggest that the submillisecond electrogenic reaction induced by the first flash is due to the vectorial transfer of a proton from external aqueous phase to an amino acid residue(s) in the vicinity of the non-heme iron. The possible role of the non-heme iron in cyclic electron transfer in photosystem II complex is discussed.

Mechanism of Thermal Aggregation of Rabbit Muscle Glyceraldehyde-3-Phosphate Dehydrogenase

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

Biochemistry 45 (2006) 13375-13384.

Thermal denaturation and aggregation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have been studied using differential scanning calorimetry (DSC), dynamic light scattering (DLS), and analytical ultracentrifugation. The maximum of the protein thermal transition (T-m) increased with increasing the protein concentration, suggesting that the denaturation process involves the stage of reversible dissociation of the enzyme tetramer into the oligomeric forms of lesser size. The dissociation of the enzyme tetramer was shown by sedimentation velocity at 45 degrees C. The DLS data support the mechanism of protein aggregation that involves a stage of the formation of the start aggregates followed by their sticking together. The hydrodynamic radius of the start aggregates remained constant in the temperature interval from 37 to 55 degrees C and was independent of the protein concentration (R-h,R-0 approximate to 21 nm; 10 mM sodium phosphate, pH 7.5). A strict correlation between thermal aggregation of GAPDH registered by the increase in the light scattering intensity and protein denaturation characterized by DSC has been proved.

Molecular Characterization of the First Aromatic Nutrient Transporter From the Sodium Neurotransmitter Symporter Family

Meleshkevitch E.A., ssis-Nascimento P., Popova L.B., Miller M.M., Kohn A.B., Phung E.N., Mandal A., Harvey W.R., Boudko D.Y.

Journal of Experimental Biology 209 (2006) 3183-3198.

Nutrient amino acid transporters (NATs, subfamily of sodium neurotransmitter symporter family SNF, a.k.a. SLC6) represent a set of phylogenetically and functionally related transport proteins, which perform intracellular absorption of neutral, predominantly essential amino acids. Functions of NATs appear to be critical for the development and survival in organisms. However, mechanisms of specific and synergetic action of various NAT members in the amino acid transport network are virtually unexplored. A new transporter, agNAT8, was cloned from the malaria vector mosquito Anopheles gambiae (SS). Upon heterologous expression in Xenopus oocytes it performs high-capacity, sodium-coupled (2:1) uptake of nutrients with a strong preference for aromatic catechol-branched substrates, especially phenylalanine and its derivatives tyrosine and L-DOPA, but not catecholamines. It represents a previously unknown SNF phenotype, and also appears to be the first sodium-dependent B-0 type transporter with a narrow selectivity for essential precursors of catecholamine synthesis pathways. It is strongly and specifically transcribed in absorptive and secretory parts of the larval alimentary canal and specific populations of central and peripheral neurons of visual-, chemo- and mechano-sensory afferents. We have identified a new SNF transporter with previously unknown phenotype and showed its important role in the accumulation and redistribution of aromatic substrates. Our results strongly suggest that agNAT8 is an important, if not the major, provider of an essential catechol group in the synthesis of catecholamines for neurochemical signaling as well as ecdysozoan melanization and sclerotization pathways, which may include cuticle hardening/coloring, wound curing, oogenesis, immune responses and melanization of pathogens.

Structure-Based Cross-Linking of NF-Kappa B P50 Homodimer and Decoy Bearing a Novel 2 '-Disulfide Trapping Site

Metelev V., Romanenkov A., Kubareva E., Zubin E., Polouchine N., Zatsepin T., Molochkov N., Oretskaya T.

Iubmb Life 58 (2006) 654-658.

Double-stranded oligodeoxyribonucleotides with engineered disulfide units were successfully used for covalent trapping of cysteine containing proteins. In particular, an efficient cross-linking of NF-kappa B p50 homodimer to a sequence-specific decoy was demonstrated. The results suggest that the synthetic oligonucleotides bearing a novel 2'-disulfide trapping site can be used as new tools to study and manipulate biological systems.

Mathematical Modeling of Mitochondrial Adenine Nucleotide Translocase

Metelkin E., Goryanin I., Demin O.

Biophysical Journal 90 (2006) 423 -432.

We have developed a mathematical model of adenine nucleotide translocase ( ANT) function on the basis of the structural and kinetic properties of the transporter. The model takes into account the effect of membrane potential, pH, and magnesium concentration on ATP and ADP exchange velocity. The parameters of the model have been estimated from experimental data. A satisfactory model should take into account the influence of the electric potential difference on both ternary complex formation and translocation processes. To describe the dependence of translocation constants on electric potential we have supposed that ANT molecules carry charged groups. These groups are shifted during the

behavior of ANT under physiological conditions.

Dissociative Mechanism of F-Actin Thermal Denaturation

Mikhailova V.V., Kurganov B.I., Pivovarova A.V., Levitsky D.I.

Biochemistry-Moscow 71 (2006) 1261-1269.

We have applied differential scanning calorimetry to investigate thermal unfolding of F-actin. It has been shown that the thermal stability of F-actin strongly depends on ADP concentration. The transition temperature, T-m, increases with increasing ADP concentration up to 1 mM. The T-m value also depends on the concentration of F-actin:it increases by almost 3 degrees C as the F-actin concentration is increased from 0.5 to 2.0 mg/ml. Similar dependence of the T-m value on protein concentration was demonstrated for F-actin stabilized by phalloidin, whereas it was much less pronounced in the presence of AlF4-. However, T-m was independent of protein concentration in the case of monomeric G-actin. The results suggest that at least two reversible stages precede irreversible thermal denaturation of F-actin; one of them is dissociation of ADP from actin subunits, and another is dissociation of subunits from the ends of actin filaments. The model explains why unfolding of F-actin depends on both ADP and protein concentration.

Mammalian CLASPs Are Required for Mitotic Spindle Organization and Kinetochore Alignment

Mimori-Kiyosue Y., Grigoriev I., Sasaki H., Matsui C. , Akhmanova A., Tsukita S., Vorobjev I.

Genes to Cells 11 (2006) 845-857.

CLASP1 and CLASP2 are homologous mammalian proteins, which associate with the ends of growing microtubules, as well as the cell cortex and the kinetochores of mitotic chromosomes. Previous studies have shown that in interphase cells CLASPs can attach microtubule plus ends to the cortex and stabilize them by repeatedly rescuing them from depolymerization. Here we show that CLASP1 and 2 play similar and redundant roles in organizing the mitotic apparatus in HeLa cells. Simultaneous depletion of both CLASPs causes mitotic spindle defects and a significant metaphase delay, which often results in abnormal exit from mitosis. Metaphase delay is associated with decreased kinetochore tension, increased kinetochore oscillations and more rapid microtubule growth. We show that the association of CLASP2 with the kinetochores relies on its C-terminal domain, but is independent of microtubules or association with CLIP-170. We propose that CLASPs exhibit at the kinetochores an activity similar to that at the cortex, providing apparent stabilization of microtubules by locally reducing the amplitude of growth/shortening episodes at the microtubule ends. This local stabilization of microtubules is essential for the formation of normal metaphase spindle, completion of anaphase and cytokinesis.

Use of the Addressing Sequence of Yeast D-Lactate Dehydrogenase for Insertion of CYP11A1p into the Inner Membrane of Yeast Mitochondria

Minenko A.N., Luzikov V.N., Kovaleva I.E.

Biochemistry-Moscow 71 (2006) 32-38.

Mammalian cytochrome P450scc (CYPIIAlp) is a pseudointegral protein of the inner membrane of mitochondria with the active center exposed in the matrix. Upon import of the CYPIIAlp precursor into yeast mitochondria, only a minor part was incorporated into the inner mitochondrial membrane and acquired catalytic activity (Kovaleva, I. E., Novikova, L. A., Nazarov, P. A., Grivennikov, S. I., and Luzikov, V. N. (2003) Far. J. Biochem., 270, 222-229). The present work is an attempt to increase the efficiency of this process by substitution of the inherent N-terminal presequence of CYPIIAlp by the addressing signal of D-lactate dehydrogenase (D-LD) of the yeast Saccharomyces cerevisiae. D-LD is known to be inserted into the inner membrane of mitochondria through its transmembrane domain located close to the N-terminus of the polypeptide chain in such a way that the protein globule is exposed in the intermembrane space. The hybrid protein D-LD(1-72)-mCYPIIAlp synthesized in yeast cells was imported into yeast mitochondria, underwent processing, and was inserted into the inner membrane on the side of the intermembrane space. In the presence of adrenodoxin and adrenodoxin reductase, the hybrid protein exhibited cholesterol side-chain cleavage activity Thus, CYPIIAlp insertion into the inner membrane of mitochondria mediated by the D-LD topogenic signal resulted in the catalytically active mCYPIIAlp domain in the hybrid protein.

Kinetic Model of Mitochondrial Krebs Cycle:Unraveling the Mechanism of Salicylate Hepatotoxic Effects

Mogilevskaya E., Demin O., Goryanin I.

Journal of Biological Physics 32 (2006) 245-271.

This paper studies the effect of salicylate on the energy metabolism of mitochondria using in silico simulations. A kinetic model of the mitochondrial Krebs cycle is constructed using information on the individual enzymes. Model parameters for the rate equations are estimated using in vitro experimental data from the literature. Enzyme concentrations are determined from data on respiration in mitochondrial suspensions containing glutamate and malate. It is shown that inhibition in succinate dehydrogenase and alpha-ketoglutarate dehydrogenase by salicylate contributes substantially to the cumulative inhibition of the Krebs cycle by salicylates. Uncoupling of oxidative phosphorylation has little effect and coenzyme A consumption in salicylates transformation processes has an insignificant effect on the rate of substrate oxidation in the Krebs cycle. It is found that the salicylate-inhibited Krebs cycle flux can be increased by flux redirection through addition of external glutamate and malate, and depletion in external alpha-ketoglutarate and glycine concentrations.

Starving Tetrahymena Pyriformis Responds to Sound Frequency Irradiation of Cells and Mitochondria

Mokhova E.N., Brailovskaya I.V., Larionov V.N., Prikhodko E.A.

Biochimica et Biophysica Acta-Bioenergetics (2006) 383-383.

Probing Biological Interfaces by Tracing Proton Passage Across Them

Mulkidjanian A.Y. and Cherepanov D.A.

Photochemical & Photobiological Sciences 5 (2006) 577-587.

The properties of water at the surface, especially at an electrically charged one, differ essentially from those in the bulk phase. Here we survey the traits of surface water as inferred from proton pulse experiments with membrane enzymes. In such experiments, protons that are ejected (or captured) by light-triggered enzymes are traced on their way between the membrane surface and the bulk aqueous phase. In several laboratories it has been shown that proton exchange between the membrane surface and the bulk aqueous phase takes as much as about 1 ms, but could be accelerated by added mobile pH-buffers. Since the accelerating capacity of the latter decreased with increase in their electric charge, it was suggested that the membrane surface is separated from the bulk aqueous phase by a barrier of electrostatic nature. In terms of ordinary electrostatics, the barrier could be ascribed to dielectric saturation of water at a charged surface. In terms of nonlocal electrostatics, the barrier could result from the dielectric overscreening in the surface water layers. It is discussed how the interfacial potential barrier can affect the reactions at interface, especially those coupled with biological energy conversion and membrane transport.

Proton in the Well and Through the Desolvation Barrier

Mulkidjanian A.Y.

Biochimica et Biophysica Acta-Bioenergetics 1757 (2006) 415-427.

The concept of the membrane proton well was suggested by Peter Mitchell to account for the energetic equivalence of the chemical (Delta pH) and electrical (Delta psi) components of the proton-motive force. The proton well was defined as a proton-conducting crevice passing down into the membrane dielectric and able to accumulate protons in response to the generation either of Delta psi or of Delta pH. In this review, the concept of proton well is contrasted to the desolvation penalty of > 500 meV for transferring protons into the membrane core. The magnitude of the desolvation penalty argues against deep proton wells in the energy-transducing enzymes. The shallow Delta pH- and Delta psi-sensitive proton traps, mechanistically linked to the functional groups in the membrane interior, seem more realistic. In such constructs, the draw of a trapped proton into the membrane core can happen at the expense of some exergonic reaction, e.g., release of another proton from the membrane into the aqueous phase. It is argued that the proton transfer in the ATP synthase and the cytochrome bc complex could proceed in this way.

Protons @ Interfaces:Implications for Biological Energy Conversion

Mulkidjanian A.Y., Heberle J., Cherepanov D.A.

Biochimica et Biophysica Acta-Bioenergetics 1757 (2006) 913-930.

The review focuses on the anisotropy of proton transfer at the surface of biological membranes. We consider (i) the data from "pulsed" experiments, where light-triggered enzymes capture or eject protons at the membrane surface, (ii) the electrostatic properties of water at charged interfaces, and (iii) the specific structural attributes of proton-translocating enzymes. The pulsed experiments revealed that proton exchange between the membrane surface and the bulk aqueous phase takes as much as about I ms, but could be accelerated by added mobile pH-buffers. Since the accelerating capacity of the latter decreased with the increase in their electric charge, it was concluded that the membrane surface is separated from the bulk aqueous phase by a barrier of electrostatic nature. The barrier could arise owing to the water polarization at the negatively charged membrane surface. The barrier height depends linearly on the charge of penetrating ions; for protons, it has been estimated as about 0.12 eV. While the proton exchange between the surface and the bulk aqueous phase is retarded by the interfacial barrier, the proton diffusion along the membrane, between neighboring enzymes, takes only microseconds. The proton spreading over the membrane is facilitated by the hydrogen-bonded networks at the surface. The membrane-buried layers of these networks can eventually serve as a storage/buffer for protons (proton sponges). As the proton equilibration between the surface and the bulk aqueous phase is slower than the lateral proton diffusion between the "sources" and "sinks", the proton activity at the membrane surface, as sensed by the energy transducing enzymes at steady state, might deviate from that measured in the adjoining water phase. This trait should increase the driving force for ATP synthesis, especially in the case of alkaliphilic bacteria.

The Cyanobacterial Genome Core and the Origin of Photosynthesis

Mulkidjanian A.Y., Koonin E.V., Makarova K.S., Mekhedov S.L., Sorokin A., Wolf Y.I. , Dufresne A., Partensky F., Burd H., Kaznadzey D., Haselkorn R., Galperin M.Y.

Proceedings of the National Academy of Sciences of the United States of America 103 (2006) 13126-13131.

Comparative analysis of 15 complete cyanobacterial genome sequences, including "near minimal" genomes of five strains of Prochlorococcus spp., revealed 1,054 protein families [core cyanobacterial clusters of orthologous groups of proteins (core CyOGs)] encoded in at least 14 of them. The majority of the core CyOGs are involved in central cellular functions that are shared with other bacteria; 50 core CyOGs are specific for cyanobacteria, whereas 84 are exclusively shared by cyanobacteria and plants and/or other plastid-carrying eukaryotes, such as diatoms or apicomplexans. The latter group includes 35 families of uncharacterized proteins, which could also be involved in photosynthesis. Only a few components of cyanobacterial photosynthetic machinery are represented in the genomes of the anoxygenic phototrophic bacteria Chlorobium tepidum, Rhodopseudomonas palustris, Chloroflexus aurantiacus, or Heliobacillus mobilis. These observations, coupled with recent geological data on the properties of the ancient phototrophs, suggest that photosynthesis originated in the cyanobacterial lineage under the selective pressures of UV light and depletion of electron donors. We propose that the first phototrophs were anaerobic ancestors of cyanobacteria ("procyanobacteria") that conducted anoxygenic photosynthesis using a photosystem I-like reaction center, somewhat similar to the heterocysts of modern filamentous cyanobacteria. From procyanobacteria, photosynthesis spread to other phyla by way of lateral gene transfer.

Phenomenon of Activation of Cytochrome P450 by Nonionic Detergents

Myasoedova K.N., Arutyunyan A.M., Magretova N.N.

Bioscience Reports 26 (2006) 69-78.

Mechanism of substitution of nonionic detergent Emulgen 913 for phospholipid as an activator of N-demethylase activity of cytochrome P450 form 2B4 (LM2) has been studied. It is shown that such an activation takes place at the detergent concentrations below values critical for micelle formation. Under these conditions, Emulgen does not affect the hexameric state of the cytochrome. The stimulating effect proved to be similar in reconstituted monooxygenase systems containing (a) cytochrome P450 2B4 and NADPH-cytochrome P-450 reductase and (b) cytochrome 2B4 and organic hydroperoxides. These results indicate that the activation is due to an effect of the detergent upon P450 2B4 per se rather than upon P450/flavoprotein complex formation. The above conclusion is supported by the sedimentation data and measurement of the CD spectra of cytochrome P450 2B4 at 380-450 nm.

Unfolded, Oxidized, and Thermoinactivated Forms of Glyceraldehyde-3-Phosphate Dehydrogenase Interact With the Chaperonin GroEL in Different Ways

Naletova I.N., Muronetz V.I., Schmalhausen E.V.

Biochimica et Biophysica Acta-Proteins and Proteomics 1764 (2006) 831-838.

The interaction of GroEL with different denatured forms of glyceraldehyde-3-phosphate dehydrogenase* (GAPDH) has been investigated. GroEL does not prevent thermal denaturation of GAPDH, but effectively interacts with the thermodenatured enzyme, thus preventing the aggregation of denatured molecules. Binding of the thermodenatured GAPDH shifts the T-m value of the GroEL thermodenaturation curve by 3 degrees towards higher temperatures and increases the Delta H-cal value 1.44-fold. indicating a significant increase in the thermal stability of the resulting complex. GAPDH thermodenatured in the presence of GroEL cannot be reactivated by the addition of GroES, Mg²⁺, and ATP. In contrast, GAPDH denatured in guanidine hydrochloride (GAPDH(den)) is reactivated in the presence of GroEL, GroES, Mg²⁺, and ATP, yielding 11-15% of its original activity, while the spontaneous reactivation yields only 2-3%. The oxidation of GAPDH with hydrogen peroxide in the presence of 4 M guanidine hydrochloride results in the formation of the enzyme (GAPDH(ox)) that cannot acquire its native conformation and binds to GroEL irreversibly. Binding of GAPDH(ox) to one of the GroEL rings completely inhibits the GroEL-assisted reactivation of GAPDH,,,,,, but does not affect the GroEL-assisted reactivation of lactate dehydrogenase (LDH). The data suggest that LDH can be successfully reactivated due to the binding of the denatured molecules to the apical domain of the opposite GroEL ring with their subsequent release into the Solution Without encapsulation (trans-mechanism). In contrast, GAPDH requires the hydrophilic cavity for the reactivation (cis-mechanism).

Phylogenetic Position of Multicilia Marina and the Evolution of Amoebozoa

Nikolaev S.I., Berney C., Petrov N.B., Mylnikov A.P., Fahrni J.F., Pawlowski J.

International Journal of Systematic and Evolutionary Microbiology 56 (2006) 1449-1458.

Recent molecular phylogenetic studies have led to the erection of the phylum Amoebozoa, uniting naked and testate lobose amoebae, the mycetozoan slime moulds and amitochondriate amoeboid protists (Archamoebae). Molecular data together with ultrastructural evidence have suggested a close relationship between Mycetozoa and Archamoebae, classified together in the Conosea, which was named after the cone of microtubules that, when present, is characteristic of their kinetids. However, the relationships of conoseans to other amoebozoans remain unclear. Here, we obtained the complete small-subunit (SSU) rRNA gene sequence (2746 bp) of the enigmatic, multiflagellated protist Multicilia marina, which has formerly been classified either in a distinct phylum, Multiflagellata, or among lobose amoebae. Our study clearly shows that Multicilia marina belongs to the Amoebozoa. Phylogenetic analyses including 60 amoebozoan SSU rRNA gene sequences revealed that Multicilia marina branches at the base of the Conosea, together with another flagellated amoebozoan, Phalansterium solitarium, as well as with Gephyramoeba sp., Filamoeba nolandi and two unidentified amoebae. This is the first report showing strong support for a clade containing all flagellated amoebozoans and we discuss the position of the root of the phylum Amoebozoa in the light of this result.

The Kinetic Model of the Shikimate Pathway As a Tool to Optimize Enzyme Assays for High-Throughput Screening

Noble M., Sinha Y., Kolupaev A., Demin O., Earnshaw D., Tobin F., West J., Martin J.D., Qiu C.Y., Liu W.S., Dewolf W.E. , Tew D., Goryanin I.I.

Biotechnology and Bioengineering 95 (2006) 560-573.

Four-enzyme section of the shikimate pathway (Aro B, D, E, and K) of Streptococcus pneumoniae has been studied. Kinetic properties of the individual enzymes and three- and four-enzyme linked reactions have been characterized in vitro. On the basis of the data measured in spectrophotometric and LC-MS experiments, kinetic mechanisms of the enzymes have been suggested and all kinetic parameters have been identified. Kinetic models for these three- and four-enzyme sections of the shikimate pathway have been constructed and validated. The model of the four-enzyme section of shikimate pathway has been employed to design an inhibition-sensitive reconstituted pathway for a high-throughput screening effort on the shikimate pathway. It was demonstrated that using the model it was possible to optimize this reconstituted pathway in such a way to provide equal sensitivity of the enzymes to inhibition. (c) 2006 Wiley Periodicals, Inc.

Dynamics of the Emission Spectrum of a Single LH2 Complex:Interplay of Slow and Fast Nuclear Motions

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

Biophysical Journal 90 (2006) 2890-2902.

We have studied the relationship between the realizations of static disorder and the emission spectra observed for a single LH2 complex. We show that the experimentally observed spectral fluctuations reflect realizations of the disorder in the B850 ring associated with different degrees of exciton delocalization and different effective coupling of the excitons to phonon modes. The main spectral features cannot be explained using models with correlated disorder associated with elliptical deformations of the ring. A quantitative explanation of the measured single-molecule spectra is obtained using the modified Redfield theory and a model of the B850 ring with uncorrelated disorder of the site energies. The positions and spectral shapes of the main exciton components in this model are determined by the disorder-induced shift of exciton eigenvalues in combination with phonon-induced effects (i.e., reorganization shift and broadening, that increase in proportion to the inverse delocalization length of the exciton state). Being dependent on the realization of the disorder, these factors produce different forms of the emission pro. le. In addition, the different degree of delocalization and effective couplings to phonons determines a different type of excitation dynamics for each of these realizations. We demonstrate that experimentally observed quasistable conformational states are characterized by excitation energy transfer regimes varying from a coherent wavelike motion of a delocalized exciton (with a 100-fs pass over half of the ring) to a hopping-type motion of the wavepacket (with a 350-fs jump between separated groups of 3-4 molecules) and self-trapped excitations that do not move from their localization site.

Theoretical Model of Interactions Between Ligand-Binding Sites in a Dimeric Protein and Its Application for the Analysis of Thiamine Diphosphate Binding to Yeast Transketolase

Ospanov R., Kochetov G., Kurganov B.

Biophysical Chemistry 124 (2006) 106-114.

The binding of thiamin diphosphate (ThDP) to yeast dimeric apotransketolase (apoTK) is accompanied by the appearance of a band in the absorption spectrum with maximum at 320 nm. The saturation function has been analyzed using a scheme that involves binding of ThDP to each subunit followed by the conformational transition of this subunit. It is assumed that the binding of ThDP to one subunit may affect the conformational transition of the other subunit. Rigorous mathematical expressions describing the dependence of the optical absorption on the total concentration of ThDP are first developed. Equilibrium constants and corresponding rate constants for the binding of ThDP to apoTK have been estimated. The negative cooperativity in the ThDP binding has been characterized by the function reflecting the dependence of the conformational change on the saturation of apoTK by ThDP.

At-4/1, an Interactor of the Tomato Spotted Wilt Virus Movement Protein, Belongs to a New Family of Plant Proteins Capable of Directed Intra- and Intercellular Trafficking

Paape M., Solovyev A.G., Erokhina T.N., Minina E.A., Schepetilnikov M.V., Lesemann D.E., Schiemann J., Morozov S.Y., Kellmann J.W.

Molecular Plant-Microbe Interactions 19 (2006) 874-883.

The Tomato spotted wilt virus (TSWV) encoded NSm movement protein facilitates cell-to-cell spread of the viral genome through structurally modified plasmodesmata. NSm has been utilized as bait in yeast two-hybrid interaction trap screenings. As a result, a protein of unknown function, called At-4/1, was isolated from an Arabidopsis thaliana GAL4 activation domain-tagged cDNA library. Using polyclonal antibodies against bacterially expressed At-4/1, Western blot analysis of protein extracts isolated from different plant species as well as genome database screenings showed that homologues of At-4/1 seemed to be encoded by many vascular plants. For subcellular localization studies, At-4/1 was fused to green fluorescent protein, and corresponding expression vectors were used in particle bombardment and agroinfiltration assays. Confocal laser scannings revealed that At-4/1 assembled in punctate spots at the cell periphery. The protein accumulated intracellularly in a polarized fashion, appearing in only one-half of a bombarded epidermal cell, and, moreover, moved from cell to cell, forming twin-structured bodies seemingly located at both orifices of the plasmodesmatal pore. In coexpression studies, At-4/1 collocalized with a plant virus movement protein TGBp3 known to reside in endoplasmic reticulum-derived membrane structures located in close vicinity to plasmodesmata. Thus, At-4/1 belongs to a new family of plant proteins capable of directed intra- and intercellular trafficking.

Low Cetyltrimethylammonium Bromide Concentrations Induce Reversible Amorphous Aggregation of Tobacco Mosaic Virus and Its Coat Protein at Room Temperature

Panyukov Y.V., Nemykh M.A., Rafikova E.R., Kurganov B.I., Yaguzhinsky L.S., Arutyunyan A.M., Drachev V.A., Dobrov E.N.

International Journal of Biochemistry & Cell Biology 38 (2006) 533-543.

Ordered and amorphous protein aggregation causes numerous diseases. Tobacco mosaic virus coat protein for many decades serves as the classical model of ordered protein aggregation ("polymerization"). It was also found to be highly prone to heat-induced amorphous aggregation and the rate of this aggregation could be easily manipulated by changes in solution ionic strength and temperature. Here, we report that rapid amorphous aggregation of this protein call be induced at 25 degrees C in phosphate buffer by low micromolar (start at about 15 mu M) concentrations of cationic surfactant cetyltrimethylammonium bromide. At equilibrium four surfactant molecules bound to the protein subunit. As judged by circular dichroism and fluorescence spectroscopy data, the coat protein molecules retained their native structure upon the cetyltrimethylammonium bromide induced aggregation. No aggregation was observed at the higher surfactant concentrations (above 300 mu M). Micromolar concentrations of anionic surfactant sodium dodecylsulfate rapidly reversed the cetyltrimethylammonium bromide induced aggregation of the coat protein due to formation of mixed surfactant-surfactant micelles. Cetyltrimethylammonium bromide (100-300 mu M) also induced the reversible intact tobacco mosaic virus virion aggregation. The possible liability to the cetyltrimethylamnionium bromide induced amorphous aggregation of other ordered aggregate-producing proteins has been discussed.

Grafting of Polylysine With Polyethylenoxide Prevents Demixing of O-Pyromellitylgramicidin in Lipid Membranes

Pashkovskaya A.A., Lukashev E.P., Antonov P.E., Finogenova O.A., Ermakov Y.A., Melik-Nubarov N.S., Antonenko Y.N.

Biochimica et Biophysica Acta-Biomembranes 1758 (2006) 1685-1695.

Both natural and synthetic polycations can induce demixing of negatively charged components in artificial and possibly in natural membranes. This process can result in formation of clusters (binding of several components to a polycation chain) and/or domains (aggregation of clusters and formation of a separate phase enriched in some particular component). In order to distinguish between these two phenomena, a model lipid membrane system containing ion channels, formed by a negatively charged peptide, beta-pyromellitylgramicidin, and polycations of different structures was used. Microelectrophoresis of liposomes, changes in boundary potential of planar bilayers, the shape of compression curves and potentials of lipid and lipid/peptide monolayers were used to monitor the electrostatic factors in polymer adsorption to the membrane and peptide-polymer interactions. The synthesized PEO-grafted polylysine, PLL-PEO20000, did not induce peptide demixing monitored by stabilization of the gramicidin channels, in contrast to parent polylysine (PLL). Both polymers were shown to bind effectively to negatively charged liposomes and lipid monolayers, suggesting that the ineffectiveness of PLL-PEO20000 was not due to reduction of its binding. It was hypothesized that PLL-PEO20000 could not induce domain formation due to steric hindrance of long PEO chains preventing lateral fusion of clusters. Another copolymer, PLL-PEO4000, having four PEO chains of 4000 Da, exhibited intermediate effect between PLL and PLL-PEO20000, which shows the importance of the copolymer architecture for the effect on the lateral distribution of OPg channels. The model system can be relevant to regulation of lateral organization of ion channels and other components in natural membrane systems.

A New Method for Spectrophotometric Assay of Activity of Cross-Linked Penicillin Acylase Aggregates

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

Biochemistry-Moscow 71 (2006) 315 -319.

A new method for monitoring reactions catalyzed by an immobilized enzyme, cross-linked penicillin acylase aggregates (PA CLEA), is suggested. Appropriate chromogenic substrates for spectrophotometric assay of catalytic activity of immobilized enzyme were chosen and their kinetic parameters determined. Active sites in PA CLEA preparations were titrated by the suggested method; it is shown that almost all active sites are retained during immobilization. This method is characterized as highly expressive, simple, and precise, and may be used for control of PA immobilization efficiency as well as for study of operational, thermal, and pH stability immobilized enzyme preparations.

Enigmatic Genus Haussknechtia (Umbelliferae):Systematic Relationships Based on Molecular and Carpological Data

Pimenov M.G., Valiejo-Roman C.M., Terentieva E.I., Samigullin T.H., Mozaffarian V.

Nordic Journal of Botany 24 (2006) 555-565.

After its description in 1872 on the basis of incomplete C. Haussknecht's collections without mature fruits the genus Haussknechtia remains a true botanical enigma in Iranian flora. As late as 1980s Iranian botanists repeated H. elymaitica gatherings in Zagros Mts and found some new localities. The new sheets have mature fruits and green leaves. This allowed to check the systematic position of Haussknechtia based both on morphological (carpological) and molecular (ITS sequences) data in comparison with all putatively related taxa. Both kinds of data showed a good correlation. Earlier hypotheses on Haussknechtia affinity with Oenanthe, Seseli, Doreina, Aethusa and Opopanax have been rejected. The Iranian endemic Denlavendia and Irano-Turanian endemic Zeravschania were found to be the closest relatives of Haussknechtia.

Kinetic Analysis of Interaction of Eukaryotic Release Factor 3 With Guanine Nucleotides

Pisareva V.P., Pisarev A.V., Hellen C.U.T., Rodnina M.V., Pestova T.V.

Journal of Biological Chemistry 281 (2006) 40224-40235.

Eukaryotic translation termination is mediated by two release factors:eRF1 recognizes stop codons and triggers peptidyl-tRNA hydrolysis, whereas eRF3 accelerates this process in a GTP-dependent manner. Here we report kinetic analysis of guanine nucleotide binding to eRF3 performed by fluorescence stopped-flow technique using GTP/GDP derivatives carrying the fluorescent methylanthraniloyl (mant-) group, as well as thermodynamic analysis of eRF3 binding to unlabeled guanine nucleotides. Whereas the kinetics of eRF3 binding to mant-GDP is consistent with a one-step binding model, the double-exponential transients of eRF3 binding to mant-GTP indicate a two-step binding mechanism, in which the initial eRF3.mant-GTP complex undergoes subsequent conformational change. The affinity of eRF3 for GTP (K-d, similar to 70 mu M) is about 70-fold lower than for GDP (K-d, similar to 1 mu M) and both nucleotides dissociate rapidly from eRF3 (k(-1)(mant-GDP)similar to 2.4 s(-1); k(-2)(mant-GTP) similar to 3.3 s(-1)). Whereas not influencing eRF3 binding to GDP, association of eRF3 with eRF1 at physiological Mg²⁺ concentrations specifically changes the kinetics of eRF3/mant-GTP interaction and stabilizes eRF3.GTP binding by two orders of magnitude (K-d similar to 0.7 mu M) due to lowering of the dissociation rate constant similar to 24-fold (k(-1)(mant-GTP)similar to 0.14 s(-1)). Thus, eRF1 acts as a GTP dissociation inhibitor (TDI) for eRF3, promoting efficient ribosomal recruitment of its GTP-bound form. 80 S ribosomes did not influence guanine nucleotide binding/exchange on the eRF1.eRF3 complex. Guanine nucleotide binding and exchange on eRF3, which therefore depends on stimulation by eRF1, is entirely different from that on prokaryotic RF3 and unusual among GTPases.

Effect of Oxidative Stress on Dynamics of Mitochondrial Reticulum

Pletjushkina O.Y., Lyamzaev K.G., Popova E.N., Nepryakhina O.K., Ivanova O.Y., Domnina L.V., Chernyak B.V., Skulachev V.P.

Biochimica et Biophysica Acta-Bioenergetics 1757 (2006) 518-524.

Fission of the mitochondrial reticulum (the thread-grain transition) and following gathering of mitochondria in the perinuclear area are induced by oxidative stress. It is shown that inhibitors of the respiratory chain (piericidin and myxothiazol) cause fission of mitochondria in HeLa cells and fibroblasts, whereas a mitochondria-targeted antioxidant (MitoQ) inhibits this effect. Hydrogen peroxide also induced the fission, which was stimulated by the inhibitors of respiration and suppressed by MitoQ. In untreated cells, the mitochondrial reticulum consisted of numerous electrically-independent fragments. Prolonged treatment with MitoQ resulted in drastic increase in size and decrease in number of these fragments. Local photodamage of mitochondria caused immediate depolarization of a large fraction of the mitochondrial network in MitoQ-treated cells. Our data indicate that the thread-grain transition of mitochondria depends on production of reactive oxygen species (ROS) in initial segments of the respiratory chain and is a necessary step in the process of elimination of mitochondria (mitoptosis).

Hydrogen Peroxide Produced Inside Mitochondria Takes Part in Cell-to-Cell Transmission of Apoptotic Signal

Pletjushkina O.Y., Fetisova E.K., Lyamzaev K.G., Ivanova O.Y., Domnina L.V., Vyssokikh M.Y., Pustovidko A.V., Alexeevski A.V., Alexeeski D.A., Vasiliev J.M., Murphy M.P., Chernyak B.V., Skulachev V.P.

Biochemistry-Moscow 71 (2006) 60-67.

In monolayer of HeLa cells treated with tumor necrosis factor (TNF), apoptotic cells formed clusters indicating possible transmission of apoptotic signal via the culture media. To investigate this phenomenon, a simple method of enabling two cell cultures to interact has been employed. Two coverslips were placed side by side in a Petri dish, one coverslip covered with apoptogen -treated cells (the inducer) and another with non-treated cells (the recipient). TNF, staurosporine, or H₂O₂ treatment of the inducer cells is shown to initiate apoptosis on the recipient coverslip. This effect is increased by a catalase inhibitor aminotriazole and is arrested by addition of catalase or by pre-treatment of either the inducer or the recipient cells with nanomolar concentrations of mitochondria-targeted cationic antioxidant MitoQ (10-(6'ubiquinolyl)decyltriplienylpliosphonium), which specifically arrests H₂O₂-induced apoptosis. The action of MitoQ is abolished by an uncoupler preventing accumulation of MitoQ in mitochondria. It is concluded that reactive oxygen species (ROS) produced by mitochondria in the apoptotic cells initiate the release of H₂O₂ from these cells. The H₂O₂ released is employed as a long-distance cell suicide messenger. In processing of such a signal by the recipient cells, mitochondrial ROS production is also involved. It is suggested that the described phenomenon may be involved in expansion of the apoptotic region around a damaged part of the tissue during heart attack or stroke as well as in "organoptosis", i.e. disappearance of organs during ontogenesis.

Structural-Functional Model of the Mitotic Chromosome

Polyakov V.Y., Zatsepina O.V., Kireev I.I., Prusov A.N., Fais D.I., Sheval E.V., Koblyakova Y.V., Golyshev S.A., Chentsov Y.S.

Biochemistry-Moscow 71 (2006) 1-9.

In the present review the structural role of noncoding DNA, mechanisms of differential staining of mitotic chromosomes, and structural organization of different levels of DNA compactization are discussed. A structural-functional model of the mitotic chromosome is proposed based on the principle of discreteness of structural levels of DNA compactization.

Tip Growth of Neurospora crassa

Potapova T.V.

Biologicheskie Membrany 23 (2006) 436-452.

The filamentous fungus Neurospora crassa is a popular experimental model organism. The defining characteristic of filamentous fungi is the development of hyphae, tip-growing tubular branching structure. Tip growth of N. crassa has been studied intensively for over some tens years. It is clear now that the tip-growing hyphae can use the biosynthetic resources of many tens and hundreds of microns of hyphal trunk to drive forward the apex. Cell-wall biosynthesis and cell extension are located at the apical region and the membrane and enzymes required to expand the apex are packaged in microvesicles that are translocated through the hypha to apex via actin microfilaments and microtubules. Secretory vesicles then fuse with the tip to deliver membrane and materials required for the continuous cell-wall synthesis. This is thought to be coordinated by the Schpitzencorper, a fungal specific organelle, which is localized in the hyphal apex and serves as a vesicle-supply center. A lot of molecular and genetic aspects of the tip growth have become clear now, especially those concerning the activity of chitin synthesis, molecular motors and signal transduction systems. Electrophysiological studies of N. crassa indicate membrane polarization, pH- and Ca²⁺-gradients and electrical currents at the growing apex. Some theories based on the mathematical modeling and computer simulation approaches attempt to account for various aspects of tip growth. The genome sequence of N. crassa was recently reported. Thus, the next few years should clarify the problem of the hyphal growth and integrate the whole basis of physiological, cytological and molecular biology details into a generally accepted picture.

Isolation of Influenza Virus A Hemagglutinin C-Terminal Domain by Hemagglutinin Proteolysis in Octylglucoside Micelles

Radyukhin V.A., Serebryakova M.V., Ksenofontov A.L., Lukashina E.V., Baratova L.A.

Protein and Peptide Letters 13 (2006) 907-913.

A method of isolation of hydrophobic membrane-bound C-terminal domain of influenza virus A hemagglutinin (HA) is suggested. The method is based on the insertion of HA into octylglucoside micelles followed by pepsin or thermolysin hydrolysis. Subsequent treatment of proteolytic digests with chloroform-hexafluoroisopropanol mixture resulted in the extraction of a few hydrophobic peptides into organic phase. Mass-spectrometry (MALDI-TOF) analysis revealed that the peptides with ion masses corresponding to the anchoring C-terminal domain with or without modifications predominated in the organic solution. The data obtained confirmed our speculation on the possibility of the suggested isolation scheme following from the strong interactions of anchoring domains in compact trimeric structure of HA spikes combined with micelle protection effect. Several appropriate peptides presence in the organic phase apparently arises from the presence of a few accessible proteolytic sites in HA transmembrane region.

Zinc Ions Stimulate the Cooperative RNA Binding of Hordeiviral Gamma b Protein

Rakitina D.V., Yelina N.E., Kalinina N.O.

FEBS Letters 580 (2006) 5077-5083.

A small regulatory gamma b protein of the Poa semilatent hordeivirus (PSLV) contains two zinc finger-like motifs separated by a basic motif in the N-terminal part and a C-terminal coiled-coil motif. Interactions of the recombinant PSLV gamma b protein and its mutants with various RNAs (ssRNA, dsRNA, ssRNA oligonucleotides) and ssDNA were studied in gel-shift assays. The results demonstrated that zinc ions are essential for effective nucleic-acid-binding activity of the gamma b protein, suggesting the important role of zinc finger motifs in these interactions. Deletion of the C-proximal coiled-coil region did not affect highly cooperative RNA-protein binding, indicating that the N-terminal part of the protein contributes to the protein-protein interactions needed for the protein-RNA cooperativity. .

Mitochondria As a Critical Element of Heat Shock Response in Yeasts With Different Types of Energy Metabolism

Rikhvanov E.G., Lukina E.A., Varakina N.N., Rusaleva T.M., Gamburg K.Z., Knorre D.A., Borovskii G.B., Voinikov V.K.

Russian Journal of Plant Physiology 53 (2006) 615-621.

Yeasts with different types of energy metabolism were examined with fluorescent imicroscopy in the presence of tetramethylrhodamine methyl ester in order to investigate the role of mitochondria in the development of a defense response to heat treatment. Heat shock was found to increase the electrochemical polarization of the inner mitochondrial membrane in an obligate aerobe Debaryomyces vanrijiae. In D. vanrijiae yeasts grown on a glucose medium, the addition of azide, 2,4-dinitrophenol, and antimycin A during "mild" heat shock treatment suppressed the development of induced thermotolerance and inhibited synthesis of a heat shock protein Hsp60. In a facultative anaerobe Saccharomyces cerevisiae grown on a glucose medium (fermentative energy metabolism), antimycin A had no effect on the development of induced thermotolerance and accumulation of a heat shock protein Hsp104. However, after growing S. cerevisiae on ethanol-containing medium under conditions of effective oxidative metabolism, antimycin A blocked the fulfillment of the defense program and prevented the enhanced synthesis of Hsp104. The results provide evidence that mitochondria play a principal role in the development of stress response and support the notion that activated transcription of stress genes depends on changes in electrochemical polarization of the inner mitochondrial membrane.

DNA-Methyltransferase SsoII As a Bifunctional Protein:Features of the Interaction With the Promoter Region of SsoII Restriction-Modification Genes

Romanenkov A.S., Kisil O.V., Zatsepin T.S., Yamskova O.V., Karyagina A.S., Metelev V.G., Oretskaya T.S., Kubareva E.A.

Biochemistry-Moscow 71 (2006) 1341-1349.

DNA duplexes bearing an aldehyde group at the 2'-position of the sugar moiety were used for affinity modification of (cytosine-5)-DNA methyltransferase SsoII. It is shown that lysine residues of M.SsoII N-terminal region are located in proximity to DNA sugar-phosphate backbone of a regulatory sequence of promoter region of SsoII restriction-modification enzyme coding genes. The ability of the two M.SsoII subunits to interact with DNA regulatory sequence has been demonstrated by affinity modification using DNA duplexes with two 2'-aldehyde groups. Changes in nucleotide sequence of one half of the regulatory region prevented cross-linking of the second M.SsoII subunit. The results on sequential affinity modification of M.SsoII by two types of modified DNA ligands (i.e. by 2'-aldehyde-containing and phosphoryldisulfide-containing) have demonstrated the possibility of covalent attachment of the protein to two different DNA recognition sites:regulatory sequence and methylation site.

Accumulation of Lipophilic Dications by Mitochondria and Cells

Ross M.F., Da Ros T., Blaikie F.H., Prime T.A., Porteous C.M., Severina I.I., Skulachev V.P., Kjaergaard H.G., Smith R.A.J., Murphy M.P.

Biochemical Journal 400 (2006) 199-208.

Lipophilic monocations can pass through phospholipid bilayers and accumulate in negatively-charged compartments such as the mitochondrial matrix, driven by the membrane potential. This property is used to visualize mitochondria, to deliver therapeutic molecules to mitochondria and to measure the membrane potential. In theory, lipophilic dications have a number of advantages over monocations for these tasks, as the double charge should lead to a far greater and more selective uptake by mitochondria, increasing their therapeutic potential. However, the double charge might also limit the movement of lipophilic dications through phospholipid bilayers and little is known about their interaction with mitochondria. To see whether lipophilic dications could be taken up by mitochondria and cells, we made a series of bistriphenylphosphonium cations comprising two triphenylphosphonium moieties linked by a 2-,4-, 5-,6- or 10-carbon methylene bridge. The 5-. 6- and 10-carbon dications were taken up by energized mitochondria, whereas the 2- and 4-carbon dications were not. The accumulation of the dication was greater than that of the monocation methyltriphenylphosphonium. However, the uptake of dications was only described by the Nernst equation at low levels of accumulation, and beyond a threshold membrane potential of 90-100 mV there was negligible increase in dication uptake. Interestingly, the 5- and 6-carbon dications were not accumulated by cells, due to lack of permeation through the plasma membrane. These findings indicate that conjugating compounds to dications offers only a minor increase over monocations in delivery to mitochondria. Instead, this suggests that it may be possible to form dications within mitochondria that then remain within the cell.

Spectral Trends in the Fluorescence of Single Bacterial Light-Harvesting Complexes:Experiments and Modified Redfield Simulations

Rutkauskas D., Novoderezhkin V., Gall A., Olsen J., Cogdell R.J., Hunter C.N., van Grondelle R.

Biophysical Journal 90 (2006) 2475-2485.

In this work we present and discuss the single-molecule fluorescence spectra of a variety of species of light-harvesting complexes:LH2 of Rhodopseudomonas acidophila, Rhodobacter sphaeroides, and Rhodospirillum molischianum and LH1 of Rhodobacter sphaeroides. The emission spectrum of these complexes varies as a function of time as was described in earlier work. For each type of complex, we observe a pronounced and well-reproducible characteristic relationship between the fluorescence spectral parameters of the peak wavelength, width, and asymmetry. This dependence for the LH2 complexes can be quantitatively explained on the basis of a disordered exciton model by varying the static disorder and phonon coupling parameters. In addition, a correlation of the pigment site energies has to be assumed to interpret the behavior of the LH1 complex.

Two Soluble Pyrophosphatases in Vibrio Cholerae:Transient Redundancy or Enduring Cooperation?

Salminen A., Ilias M. , Belogurov G.A., Baykov A.A., Lahti R., Young T.

Biochemistry-Moscow 71 (2006) 978 -9U1.

Soluble pyrophosphatases (PPases), which are essential for cell life, comprise two evolutionarily unrelated families (I and II). Prokaryotic genomes generally contain a single PPase gene encoding either family I or family II enzyme. Surprisingly, four Vibrionales species, including the human pathogen Vibrio cholerae, contain PPase genes of both families. Here we show that both genes are transcriptionally active in V. cholerae, and encode functional PPases when expressed in Escherichia coli. In contrast, only the family I PPase protein is detected in V. cholerae under our experimental conditions. Phylogenetic analyses indicate that family II enzymes are not native to gamma-proteobacteria, but are of benefit to the marine species of this bacterial class. In this context, we favor the hypothesis that in the course of evolution, family II PPase was laterally transferred to the Vibrionales ancestor and partially degenerated due to functional redundancy, but nevertheless remained fixed as an adjunct to the family I enzyme.

Inhibitors of the Alpha-Ketoglutarate Dehydrogenase Complex Alter [1-C-13] Glucose and [U-C-13] Glutamate Metabolism in Cerebellar Granule Neurons

Santos S.S., Gibson G.E., Cooper A.J.L., Denton T.T., Thompson C.M., Bunik V.I. , Alves P.M., Sonnewald U.

Journal of Neuroscience Research 83 (2006) 450-458.

Diminished activity of the alpha-ketoglutarate dehydrogenase complex (KGDHC), an important component of the tricarboxylic acid (TCA) cycle, occurs in several neurological diseases. The effect of specific KGDHC inhibitors [phosphonoethyl ester of succinyl phosphonate (PESP) and the carboxy ethyl ester of succinyl phosphonate (CESP)] on [1-C-13]glucose and [U-C-13]glutamate metabolism in intact cerebellar granule neurons was investigated. Both inhibitors decreased formation of [4-C-13]glutamate from [1-C-13]glucose, a reduction in label in glutamate derived from [1-C-13]glucose/[U-C-13]glutamate through a second turn of the TCA cycle and a decline in the amounts of gamma-aminobutyric acid (GABA), aspartate, and alanine. PESP decreased formation of [U-C-13]aspartate and total glutathione, whereas CESP decreased concentrations of valine and leucine. The findings are consistent with decreased KGDHC activity; increased alpha-ketoglutarate formation; increased transamination of alpha-ketoglutarate with valine, leucine, and GABA; and new equilibrium position of the aspartate aminotransferase reaction. Overall, the findings also suggest that some carbon derived from alpha-ketoglutarate may bypass the block in the TCA cycle at KGDHC by means of the GABA shunt and/or conversion of valine to succinate. The results suggest the potential of succinyl phosphonate esters for modeling the biochemical and pathophysiological consequences of reduced KGDHC activity in brain diseases. (c) 2006 Wiley-Liss, Inc.

A New Model of Weak Acid Permeation Through Membranes Revisited:Does Overton Still Rule?

Saparov S.M., Antonenko Y.N., Pohl P.

Biophysical Journal 90 (2006) L86 -L88.

According to a recent publication by Thomae, A. V., H. Wunderli-Allenspach, and S. D. Kramer (2005. Biophys. J. 89:1802 - 1811), membrane bilayers are well-permeable to the charged species of aromatic carboxylic acids. At physiological pH, the anions were claimed to be the major diffusing species. In contrast, calculation of the Born energy barrier predicts a 10(5)-fold higher permeability for the uncharged (protonated) form. To test the new model, we now have measured both the current carried by the salicylate anion through solvent-free planar membranes and the amount of protons transported by the neutral species. The corresponding membrane permeabilities of the charged and protonated forms were 4 x 10(-7) cm/s and 1.2 cm/s. These data are in perfect agreement with literature data gathered in the last three decades (compare, e. g., Gutknecht, J., and D. C. Tosteson. 1973. Science. 182:1258 - 1261). They indicate that the report by Thomae at al. represents an experimental artifact. The well-documented role of neutral species in the permeation process of weak acids and bases across artificial and natural membranes is not in question. Overton still rules.

Long-Lived Coherent Oscillations of the Femtosecond Transients in Cyanobacterial Photosystem I

Sarkisov O.M., Gostev F.E., Shelaev I.V., Novoderezhkin V.I., Gopta O.A., Mamedov M.D., Semenov A.Y., Nadtochenko V.A.

Physical Chemistry Chemical Physics 8 (2006) 5671-5678.

The pulsed excitation of electronic levels coupled to specific nuclear modes by a 26 fs laser pulse at 706 nm creates a wavepacket in the nuclear space of photopystem I (PS I) of Synechocystis sp. strain PCC 6803 both in the ground state and in the one-exciton manifold. Fourier transform of transient decay curves shows several low frequency peaks. The most prominent Power Spectral Density (PSD) peaks are at omega = 49 cm(-1) and omega = 88 cm(-1). The peculiarity of the coherent wavepacket in the PS I of S. sp. strain PCC 6803 is the unique, long-lived 49 cm(-1) and 88 cm(-1) oscillations with decay times up to 10 ps. It was suggested that such a long-lived coherence is determined by a contribution of the ground state wavepacket. The dependence of these two PSD peaks on the probe wavelength resembles the profile of the transient absorption spectra of PS I. The pump-probe signal in the Soret region reflects the dynamics of the ground state wavepacket created by pulsed excitation of the Q(y)-band. It was shown that the multimode Brownian oscillator model allows a quantitative fit of the oscillatory patterns of the pump-probe signal to be obtained.

Stability of Plant MRNAs Depends on the Length of the 3 '-Untranslated Region

Schwartz A.M., Komarova T.V., Skulachev M.V., Zvereva A.S., Dorokhov Y.L., Atabekov J.G.

Biochemistry-Moscow 71 (2006) 1377-1384.

Eukaryotic mRNAs that prematurely terminate translation are recognized and degraded by nonsense mediated decay (NMD). This degradation pathway is well studied in animal and yeast cells. The data available imply that NMD also takes place in plants. However, the molecular mechanism of recognition and degradation of plant RNAs containing premature terminator codon (PTC) is not known. Here we report that in plant cells this mechanism involves the recognition of the sizes of the 3'-untranslated regions (3'UTR). Plant 3'UTRs longer than 300 nucleotides induce mRNA instability. Contrary to mammalian and yeast cells, this destabilization does not depend on the presence of any specific sequences downstream of the terminator codon. Unlike nuclear-produced mRNAs, plant virus vector long 3'UTR-containing RNAs, which are synthesized directly in the cytoplasm, are stable and translated efficiently. This shows that RNAs produced in the cytoplasm by viral RNA-dependent RNA polymerase are able to avoid the proposed mechanism.

Ca²⁺/Recoverin Dependent Regulation of Phosphorylation of the Rhodopsin Mutant R135L Associated With Retinitis Pigmentosa

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

Biochemical and Biophysical Research Communications 349 (2006) 345-352.

No single molecular mechanism accounts for the effect of mutations in rhodopsin associated with retinitis pigmentosa. Here we report on the specific effect of a Ca²⁺/recoverin upon phosphorylation of the autosomal dominant retinitis pigmentosa R135L rhodopsin mutant. This mutant shows specific features like impaired G-protein signaling but enhanced phosphorylation in the shut-off process. We now report that R135L hyperphosphorylation by rhodopsin kinase is less efficiently inhibited by Ca²⁺/recoverin than wild-type rhodopsin. This suggests an involvement of Ca²⁺/recoverin into the molecular pathogenic effect of the mutation in retinitis pigmentosa which is the cause of rod photoreceptor cell degeneration. This new proposed role of Ca²⁺/recoverin may be one of the specific features of the proposed new Type III class or rhodopsin mutations associated with retinitis pigmentosa.

Mass Spectrometric Sequencing and Acylation Character Analysis of the C-Terminal Anchoring Segment From Influenza A Hemagglutinin

Serebryakova M.V., Kordyukova L.V., Baratova L.A., Markushin S.G.

European Journal of Mass Spectrometry 12 (2006) 51-62.

Influenza A virus hemagglutinin (HA) is a major envelope glycoprotein mediating viral and cell membrane fusion. HA is anchored in the viral envelope by a light HA, chain containing one transmembrane domain and a cytoplasmic tail. Three cysteine residues in the C-terminal region, one in the transmembrane domain and two in the cytoplasmic tail, are highly conserved and potentially palmitoylated in all HA subtypes. The HA, C-terminal anchoring segments were extracted to organic phase from the bromelain-digested viruses (subviral particles) of three strains:A/X-31 (H3 subtype), A/Puerto Rico/8/34 (HI subtype) and A/ FPV/Weybridge/34 (H7 subtype). Their primary structures were assessed by matrix-assisted laser desorption /ionization time-of-flight time-of-flight mass spectrometry (MALDI-ToF-ToF MS). Trypsin-type protease-cleaved peptides prevailed over bromelain-cleaved ones in the peptide mixtures. All of them included transmembrane domains. Several distinctive features of the C-terminal HA, peptides acylation character were discovered by MALDI-ToF MS:1) the peptides isolated from the viruses, which were digested by bromelain in the absence of P-mercaptoethanol, were predominantly triply acylated; 2) the peptides were acylated not only by palmitic, but also by stearic acid residues; 3) the palmitate/stearate ratio was different for the three strains studied; 4) the A/FPV/NVeybridge/34 strain has a priority to stearate binding. This fatty acid residue was discovered at the first of three conservative cysteine residues located in the transmembrane domain. It was found that presence of thiol reagent during preparation of subviral particles led to the appearence of the C-terminal HA, peptides acylated to different degrees. Triply, doubly, mono-and even unacylated peptides were detected. It was demonstrated that the thioester bond in the isolated acylpeptides was extremely sensitive to thiol reagents.

Identification of Escherichia coli M(2) G Methyltransferases:II. The YgjO Gene Encodes a Methyltransferase Specific for G1835 of the 23 S rRNA

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

Journal of Molecular Biology 364 (2006) 26-31.

Escherichia coli ribosomal RNA contains five guanosine residues methylated at N2. The ygjO gene was previously predicted to methylate 16 S rRNA residue G966 due to its. high sequence homology with the protein RsmC, responsible for G1207 methylation. We have identified the target of YgjO as being m2G1835 of the 23 S rRNA and not m2G966 of the 16 S rRNA as expected. Knock-out of the ygjO gene leads to loss of modification at G1835, as revealed by reverse transcription. Moreover, the modification could be restored by in vivo complementation of the ygjO knock-out strain with a plasmid expressing ygjO. Recombinant YgjO protein is able to methylate in vitro protein-free 23 S rRNA, but not assembled 50 S subunits purified from the ygjO knock-out strain. The nucleotide m2G1835 is located in a functionally extremely important region of the ribosome, being located within intersubunit bridges of group B2. Growth competition assays reveal that the lack of the G1835 methylation causes growth retardation, especially at temperatures higher than optimal and in poor media. Based on these results we suggest that YgjO be renamed to RlmG in accordance with the accepted nomenclature for rRNA methyltransferases.

Two Methods for Determination of Transketolase Activity

Sevostyanova I.A., Solovjeva O.N., Kochetov G.A.

Biochemistry-Moscow 71 (2006) 560 -562.

Two new optical methods for transketolase activity assay using only one substrate, xylulose 5-phosphate or glycol aldehyde, have been developed. For transketolase activity assay in the first method, it is necessary to add auxiliary enzyme, glyceraldehyde phosphate dehydrogenase. It is not needed in the second method. The range of transketolase concentration in the activity assay is 0.036-0.144 U/ml for the first method and 1.8-6.8 U/ml for the second one.

Effect of Some Carcinogenic and Non-Carcinogenic Polycyclic Aromatic Hydrocarbons on Gap Junction Intercellular Communication in Hepatoma Cell Cultures

Sharovskaya J., Kobliakova I., Solomatina N., Kobliakov V.

European Journal of Cell Biology 85 (2006) 387-397.

One of the systems that regulate tissue homeostasis is gap junction intercellular communication (GJIC). It is accepted that the down-regulation of GJIC is linked to the tumor-promoting properties of carcinogens. In this study, the effect of some carcinogenic and non-carcinogenic polycyclic aromatic hydrocarbons (PAH) on GJIC was investigated. It was found that in hepatoma cell culture (Hep G2) carcinogenic PAH inhibited GJIC after 24h exposure by 75-100% depending on the PAH structure. The inhibition effect on GJIC is reversible because removing the PAH by changing of culture medium restores the GJIC. The non-carcinogenic PAH do not significantly influence GJIC. alpha-Naphthoflavone, an inhibitor of PAH metabolism, has no effect on inhibition of GJIC by carcinogenic PAH. 2,3,7,8-Tetrachloro-p-dibenzodioxin, an aryl hydrocarbon (Ah) receptor ligand, inhibits GJIC by about 50% only after 48 h exposure. To clarify the role of formation of PAH metabolites and interaction with Ah receptor on inhibition of GJIC, we determined the effect of benzo/a/pyrene on hepatoma G27 cells in which neither mRNA of CYP1A1 nor Ah receptor was determined. As in Hep G2 cells, benzo/a/pyrene, unlike non-carcinogenic benzo/e/pyrene, inhibits GJIC. We conclude that in the studied hepatoma cells carcinogenic PAH inhibit GJIC directly (that is, not via their metabolites) and this effect is not associated with Ah receptor interaction.

Coexpression of All Constituents of the Cholesterol Hydroxylase/Lyase System in Escherichia coli Cells

Shashkova T.V., Luzikov V.N., Novikova L.A.

Biochemistry-Moscow 71 (2006) 810 -814.

Using the pTrc99A/P450scc vector, a plasmid was constructed in which cDNAs for cytochrome P450scc, adrenodoxin reductase, and adrenodoxin are situated in a single expression cassette. This plasmid was shown to direct the synthesis of all the above proteins in Escherichia coli. Their localization in the E. coli cells and stoichiometry were determined. Cell homogenates exhibited cholesterol hydroxylase/lyase activity, due to catalytically active forms of all three proteins. Thus, the full set of constituents of the mammalian cholesterol hydroxylase/lyase system was shown to be synthesized in bacterial cells for the first time.

Visualization of the Chromosome Scaffold and Intermediates of Loop Domain Compaction in Extracted Mitotic Cells

Sheval E.V. and Polyakov V.Y.

Cell Biology International 30 (2006) 1028-1040.

A novel extraction protocol for cells cultured on coverslips is described. Observations of the extraction process in a perfusion chamber reveal that cells of all mitotic stages are not detached from coverslips during extraction, and all stages can be recognized using phase contrast images. We studied the extracted cell morphology and distribution of a major scaffold component-topoisomerase II alpha, in extracted metaphase and anaphase cells. An extraction using 2 M NaCl leads to destruction of chromosomes at the light microscope level. Immunogold studies demonstrate that the only residual structure observed is an axial chromosome scaffold that contains topoisomerase II alpha. In contrast, mitotic chromosomes are swelled only partially after an extraction using dextran sulphate and heparin, and it appears that this treatment does not lead to total destruction of loop domains. In this case, the chromosome scaffold and numerous structures resembling small rosettes are revealed inside extracted cells. The rosettes observed condense after addition of Mg²⁺-ions and do not contain topoisomerase II alpha suggesting that these structures correspond to intermediates of loop domain compaction. We propose a model of chromosome structure in which the loop domains are condensed into highly regular structures with rosette organization. (c) 2006 International Federation for Cell Biology.

Expression of Cytochrome P450scc in Escherichia coli Cells:Intracellular Location and Interaction With Bacterial Redox Proteins

Shkumatov V.M., Radyuk V.G., Falertov Y.V., Vinogradova A.A., Luzikov V.N., Novikova L.A.

Biochemistry-Moscow 71 (2006) 884 -892.

Escherichia coli cells producing the mature form of adrenal cytochrome P450scc were used as a model for study of cytochrome P450scc topogenesis. By disruption of transformed E. coli cells and centrifugation of the homogenate under conventional conditions, we obtained membrane and soluble (high-speed supernatant) fractions both containing the recombinant protein. Gel-permeation high performance liquid chromatography showed that in the high-speed supernatant the native cytochrome P450scc exists exclusively as a component of membrane fragments exceeding 400 kD. These data supported by kinetic assays suggest that the > 400-kD particles containing P450scc are lipoprotein associates. In total, we failed to detect a genuine soluble cytochrome P450scc in the E. coli cells, which suggests that membrane insertion is an obligatory stage of holoenzyme formation. In the high-speed supernatant supplemented with NADPH, cytochrome P450scc underwent one-electron reduction and could convert 22R-hydroxycholesterol into pregnenolone. Thus, we have for the first time observed functional coupling of cytochrome P450scc with the bacterial electron transfer system.

Population Genetic Structure of the Char Species of the Northern Kuril Islands and the Rank of the Dolly Varden Char in the System of the Genus Salvelinus (Salmonidae:Teleostei)

Shubina E.A., Ponomaryova E.V., Gritzenko O.F.

Zhurnal Obshchei Biologii 67 (2006) 280-297.

Analysis of the taxonomic position of most species and forms of the char (genus Salvelinus, Salmonidae:Teleostei) was made based on RAPD-PCR. The material was represented by samples from 29 populations from the Kuril Islands, coast of the Sea of Okhotsk, Kamchatka, Chukotka, Taymyr, Transbaikalia, the Kola Peninsula, Svalbard, Finland, and North America. It was shown that the genus Salvelinus splits into three well-justified clusters:(1) all the forms assigned to the Salvelinus alpinus - S. malma complex; (2) two samples of the White-Spotted Char from the Southern Kuril Islands and from Kamchatka; (3) two North American species, S. fontinalis and S. namaycush (samples of the North American species S. confluentis were absent from the collection). Analysis of the absolute values of genetic disctances of the S. alpinus - S. malma forms relative to S. leucomaenis, S. fontinalis, and S. namaycush revealed distances approaching the species rank between the following isolates:Frolikh Char, Mountain Char, Black Lake Char, Goggle-Eyed Char, and Neyva Char. Samples of Dolly Varden currently considered as "S. malma", do not constitute a separate cluster, falling within the group of the Arctic char S. alpinus. This conclusion is supported by the analysis of the results of three series of experiments by R. Phillips on ITS l ribosome genes (Pleute et al., 1992; Phillips et al., 1995; Phillips et al., 1999). This indicates the infraspecific rank of malma within S. alpinus. Isolated populations of "Salvethymus svetovidovi" from the lake Elgygytgyn (Chukotka Peninsula) and of the char from the lake Chyomoye (Onekotan Island), recently described as S. gritzenkoi (Vasil'eva, Stygar, 2000), fell withing the S. alpinus - S. malma complex, the Onekotan char grouped together with another isolate from the same island. Comparison of genetic distances between the samples showed that the differences between the two isolated of Onekotan and migratory forms of the Kuril Islands are approximately equal, yet the homogeneity of the Chyomoye sample is higher than that of the other samples. The revealed 330-nucleotide diagnostic sequence of the Onekotan take isolate showed identity of part of the fragment with a section of expressed DNA from the library of EST clones derived from the gills of Salmo salar, this possibly indicates the adaptive character of the evolution.

Immunohistochemical Detection of Tankyrase 2 in Human Breast Tumors and Normal Renal Tissue

Sidorova N., Zavalishina L., Kurchashova S., Korsakova N., Nazhimov V., Frank G. , Kuimov A.

Cell and Tissue Research 323 (2006) 137-145.

Tankyrase, which functions at telomeres and other cellular compartments, is thought to be a positive regulator of telomerase; its isoenzyme tankyrase 2 has been cloned as a putative cancer antigen. This pilot immunohistochemical study was designed to examine whether tumors overexpress tankyrase 2. An antibody was generated by using synthetic peptide specific for tankyrase 2 and was tested by Western blot and immunocytochemically; no cross-reaction with isoenzyme 1 was revealed. Among tissue sections, two tumors of 18 specimens were positive for tankyrase 2. Others were negative or contained barely detectable protein. The surrounding normal tissues were negative. Tankyrase 2 was also revealed in epithelial cells of a limited number of normal renal tubules, whereas other renal tissues were negative. These data suggest that tankyrase 2 is not expressed ubiquitously in human tissues. To determine whether the up-regulation of tankyrase 2 is associated with tissue regeneration and cell proliferation, we compared the activity and concentration of the enzyme in a model human embryonic kidney cell line 293 arrested by serum deprivation and restimulated with serum. The serum-starved quiescent cell culture exhibited detectable protein as did the proliferating cells; enzyme activity dramatically increased in the latter. We conclude that pathologic overexpression of tankyrase 2 in some tumors may be a result of the cancer-related adaptation of the malignant cells dependent on tankyrase activity. Under normal conditions, the protein might be up-regulated during cell differentiation and also posttranslationally in proliferating cells.

Single-Electron Photoreduction of the P-M Intermediate of Cytochrome c Oxidase

Siletsky S.A., Han D. , Brand S., Morgan J.E., Fabian M., Geren L., Millett F., Durham B., Konstantinov A.A., Gennis R.B.

Biochimica et Biophysica Acta-Bioenergetics 1757 (2006) 1122-1132.

The P-M -> F transition of the catalytic cycle of cytochrome c oxidase from bovine heart was investigated using single-electron photoreduction and monitoring the subsequent events using spectroscopic and electometric techniques. The P-M state of the oxidase was generated by exposing the oxidized enzyme to CO Plus O-2. Photoreduction results in rapid electron transfer from heme a to oxoferryl heme a(3) with a time constant of about 0.3 ms, as indicated by transients at 605 nm and 580 nm. This rate is similar to 5-fold more rapid than the rate of electron transfer from heme a to heme a(3) in the F -> O transition, but is significantly slower than formation of the F state from the PR intermediate in the reaction of the fully reduced enzyme with O-2 to form state F (70-90 mu s). The similar to 0.3 ms P-M -> F transition is coincident with a rapid photonic phase of transmembrane voltage generation, but a significant part of the voltage associated with the P-M -> F transition is generated much later, with a time constant of 1.3 ms. In addition, the P-M -> F transition of the R. sphaeroides oxidase was also measured and also was shown to have two phases of electrogenic proton transfer, with r values of 0.18 and 0.85 ms.

Uncoupling Effect of Lauryl Sulfate on Mitochondria Can Be Mediated by Release of Bound Endogenous Fatty Acids

Simonyan R.A., Pustovidko A.V., Vyssokikh M.Y., Skulachev V.P.

Biochemistry-Moscow 71 (2006) 1365-1369.

The mechanism of uncoupling by lauryl sulfate (LS) has been studied. The very fact that uncoupling by low concentration of LS (a strong acid) resembles very much that by fatty acids (weak acids) was used as an argument against the fatty acid cycling scheme of uncoupling where protonated fatty acids operate as a protonophore. We have found that rat liver and heart muscle mitochondria can be uncoupled by low (70 mu M) LS concentration in a fashion completely arrested by the ATP/ADP antiporter inhibitor carboxyatractylate (CAtr). On the other hand, uncoupling by two-fold higher LS concentration is not sensitive to CAtr. Addition of oleate desensitizes mitochondria to low LS so that addition of bovine serum albumin becomes necessary to recouple mitochondria. The data are accounted for assuming that low LS releases endogenous fatty acids from some mitochondrial depots, and these fatty acids are responsible for uncoupling. As to high LS, it causes a nonspecific (CAtr-insensitive) damage to the mitochondrial membrane.

Activity of the Motor Cortex During Scratching

Sirota M.G., Pavlova G.A., Beloozerova I.N.

Journal of Neurophysiology 95 (2006) 753-765.

In awake cats sitting with the head restrained, scratching was evoked using stimulation of the ear. Cats scratched the shoulder area, consistently failing to reach the ear. Kinematics of the hind limb movements and the activity of ankle muscles, however, were similar to those reported earlier in unrestrained cats. The activity of single neurons in the hind limb representation of the motor cortex, including pyramidal tract neurons (PTNs), was examined. During the protraction stage of the scratch response, the activity in 35% of the neurons increased and in 50% decreased compared with rest. During the rhythmic stage, the motor cortex population activity was approximately two times higher compared with rest, because the activity of 53% of neurons increased and that of 33% decreased in this stage. The activity of 61% of neurons was modulated in the scratching rhythm. The average depth of frequency modulation was 12.1 +/- 5.3%, similar to that reported earlier for locomotion. The phases of activity of different neurons were approximately evenly distributed over the scratch cycle. There was no simple correlation between resting receptive field properties and the activity of neurons during the scratch response. We conclude that the motor cortex participates in both the protraction and the rhythmic stages of the scratch response.

Aging As a Mitochondria-Mediated Atavistic Program - Can Aging Be Switched Off?

Skulachev V.P. and Longo V.D.

Reversal of Aging:Resetting the Pineal Clock 1057 (2006) 145-164.

Programmed death phenomena have been demonstrated on subcellular (mitoptosis), cellular (apoptosis), and supracellular (collective apoptosis) levels. There are numerous examples of suicide mechanisms at the organismal level (phenoptosis). In yeast, it was recently shown that the death of aging cells is programmed. Many of the steps of programmed cell death are shown to be common for yeast and animals, including mammals. In particular, generation of the mitochondrial reactive oxygen species (ROS) is involved in the suicide programs. Aging of higher animals is accompanied by an increase in damage induced by mitochondrial ROS. Perhaps prevention of such damage by scavenging of mitochondrial ROS might slow down or even switch off the aging programs.

Bioenergetic Aspects of Apoptosis, Necrosis and Mitoptosis

Skulachev V.P.

Apoptosis 11 (2006) 473-485.

In this review I summarize interrelations between bioenergelc processes and such programmed death phenomena as cell suicide (apoptosis and necrosis) and mitochondrial suicide (miloptosis). The following conclusions are made. (I) ATP and rather often mitochondrial hyperpolarization (i.e. an increase in membrane potential, Delta Psi) are required for certain steps of apoptosis and necrosis. (II) Apoptosis, even if it is accompanied by Delta Psi and [ATP] Increases at its early stage, finally results in a Delta Psi collapse and ATP decrease. (III) Moderate (about threw-fold) lowering of [ATP] for short and long periods of time induces apoptosis and necrosis, respectively. In some types of apoptosis and necrosis, the cell death is mediated by a Delta Psi-dependent overproduction of ROS by the initial (Complex I) and the middle (Complex III) spans of the respiratory chain. ROS initiate mitoptosis which is postulated to rid the intracellular population of mitochondria from those that are ROS overproducing. Massive mitoptosis can result in cell death due to release to cytosol of the cell death proteins normally hidden in the mitochondrial Intermembrane space.

Zinc and Copper Content in Developing and Aging Coleoptiles of Wheat Seedlings

Smirnova T.A., Kolomiitseva G.Y., Prusov A.N., Vanyushin B.F.

Russian Journal of Plant Physiology 53 (2006) 535-540.

The content of zinc and copper in coleoptiles of etiolated wheat (Triticum aestivum L.) seedlings was measured after seed germination. The content of these metals changed differently during coleoptile development and aging:the content of zinc increased substantially from the 8th to 14th day of seedling development, whereas the content of copper slightly increased on day 8 and later slightly decreased. These changes coincided with the period of increased proteolytic activity and the signs of coleoptile cell apoptotic death. Zinc accumulation and copper amount reduction in the aging coleoptile were most pronounced in the oldest apical segment of the coleoptile, which was most enriched in apoptotic cells. The modulations in the zinc and copper amounts observed might be related to the induction and continuation of terminal stages of apoptosis.

Kinetic Study of the HIV-1 DNA 3 '-End Processing - Single-Turnover Property of Integrase

Smolov M., Gottikh M. , Tashlitskii V., Korolev S., Demidyuk I., Brochon J.C., Mouscadet J.F., Deprez E.

FEBS Journal 273 (2006) 1137-1151.

The 3'-processing of viral DNA extremities is the first step in the integration process catalysed by human immunodeficiency virus (HIV)-1 integrase (IN). This reaction is relatively inefficient and processed DNAs are usually detected in vitro under conditions of excess enzyme. Despite such experimental conditions, steady-state Michaelis-Menten formalism is often applied to calculate characteristic equilibrium/kinetic constants of IN. We found that the amount of processed product was not significantly affected under conditions of excess DNA substrate, indicating that IN has a limited turnover for DNA cleavage. Therefore, IN works principally in a single-turnover mode and is intrinsically very slow (single-turnover rate constant = 0.004 min^-1), suggesting that IN activity is mainly limited at the chemistry step or at a stage that precedes chemistry. Moreover, fluorescence experiments showed that IN-DNA product complexes were very stable over the time-course of the reaction. Binding isotherms of IN to DNA substrate and product also indicate tight binding of IN to the reaction product. Therefore, the slow cleavage rate and limited product release prevent or greatly reduce subsequent turnover. Nevertheless, the time-course of product formation approximates to a straight line for 90 min (apparent initial velocity), but we show that this linear phase is due to the slow single-turnover rate constant and does not indicate steady-state multiple turnover. Finally, our data ruled out the possibility that there were large amounts of inactive proteins or dead-end complexes in the assay. Most of complexes initially formed were active although dramatically slow.

Lipid Dependence of the Channel Properties of a Colicin E1-Lipid Toroidal Pore

Sobko A.A., Kotova E.A., Antonenko Y.N., Zakharov S.D., Cramer W.A.

Journal of Biological Chemistry 281 (2006) 14408-14416.

Colicin E1 belongs to a group of bacteriocins whose cytotoxicity toward Escherichia coli is exerted through formation of ion channels that depolarize the cytoplasmic membrane. The lipid dependence of colicin single-channel conductance demonstrated intimate involvement of lipid in the structure of this channel. The colicin formed "small" conductance 60-picosiemens (pS) channels, with properties similar to those previously characterized, in 1,2-dieicosenoyl-sn-glycero3- phosphocholine (C20) or thinner membranes, whereas it formed a novel "large" conductance 600-pS state in thicker 1,2-dierucoyl-sn-glycero-3-phosphocholine (C22) bilayers. Both channel states were anion-selective and voltage-gated and displayed a requirement for acidic pH. Lipids having negative spontaneous curvature inhibited the formation of both channels but increased the ratio of open 600 pS to 60 pS conductance states. Different diameters of small and large channels, 12 and 16 angstrom, were determined from the dependence of single-channel conductance on the size of nonelectrolyte solute probes. Colicin-induced lipid "flip-flop" and the decrease in anion selectivity of the channel in the presence of negatively charged lipids implied a significant contribution of lipid to the structure of the channel, most readily described as toroidal organization of lipid and protein to form the channel pore.

Lipid-Mediated Inactivation of Colicin E1 Channels by Calcium Ions

Sobko A.A., Kotova E.A., Zakharov S.D., Cramer W.A., Antonenko Y.N.

Biochemistry-Moscow 71 (2006) 99-103.

Based on the model of a toroidal protein-lipid pore, the effect of calcium ions on colicin El channel was predicted. In electrophysiological experiments Ca²⁺ Suppressed the activity of colicin El channels in membranes formed of diphytanoylphosphatidylglycerol, whereas no desorption of the protein Occurred from the membrane surface. The effect of Ca²⁺ was not observed on membranes formed of dipliytanoyipliospilatidyiclioline. Single-channel measurements revealed that Ca²⁺-induced reduction of the colicin-induced Current across the negatively charged membrane was due to a decrease. in the number of open colicin channels and not changes in their properties. In line with the toroidal model, the effect of Ca²⁺ on the colicin E I channel-forming activity is explained by alteration of the membrane lipid curvature caused by electrostatic interaction of Ca²⁺ with negatively charged lipid head groups.

Ysp2 Mediates Death of Yeast Induced by Amiodarone or Intracellular Acidification

Sokolov S., Knorre D. , Smirnova E., Markova O., Pozniakovsky A., Skulachev V., Severin F.

Biochimica et Biophysica Acta-Bioenergetics 1757 (2006) 1366-1370.

Recently we have found that the drug amiodarone induces apoptosis in yeast, which is mediated by reactive oxygen species (ROS). Here we have used this finding as a tool to screen for genes involved in the death program. We have described a novel mitochondrial protein, Ysp2, acting in the amiodarone-induced death cascade. After amiodarone addition both the control and amiodarone-resistant ysp2-deleted cells formed ROS, but the mutant (unlike the control) did not undergo the mitochondrial thread-to-grain transition. To test whether the action of Ysp2 is amiodarone-specific we tried to induce PCD by other agents. We have found that acetic acid-induced PCD also depends on Ysp2. We also demonstrate that, like acetic acid, propionic acid or nigericin triggered intracellular acidification causing ROS-dependent death. We suggest that intracellular acidification results in the protonation of superoxide anion (O-2(-.)) to form HO2, one of the most aggressive ROS, which in turn induces Ysp2-mediated PCD.

Expression of an Expanded Polyglutamine Domain in Yeast Causes Death With Apoptotic Markers

Sokolov S., Pozniakovsky A., Bocharova N., Knorre D., Severin F.

Biochimica et Biophysica Acta-Bioenergetics 1757 (2006) 660-666.

Huntington's disease is caused by specific mutations in huntingtin protein. Expansion of a polyglutamine (polyQ) repeat of huntingtin leads to protein aggregation in neurons followed by cell death with apoptotic markers. The connection between the aggregation and the degeneration of neurons is poorly understood. Here, we show that the physiological consequences of expanded polyQ domain expression in yeast are similar to. those in neurons. In particular, expression of expanded polyQ in yeast causes apoptotic changes in mitochondria, caspase activation, nuclear DNA fragmentation and death. Similar to neurons, at the late stages of expression the expanded polyQ accumulates in the nuclei and seems to affect the cell cycle of yeast. Interestingly, nuclear localization of the aggregates is dependent on functional caspase Yca1. We speculate that the aggregates in the nuclei disturb the cell cycle and thus contribute to the development of the cell death process in both systems. Our data show that expression of the polyQ construct in yeast can be used to model patho-physiological effects of polyQ expansion in neurons.

The Pathway Editor:A Tool for Managing Complex Biological Networks

Sorokin A., Paliy K., Selkov A., Demin O.V., Dronov S., Ghazal P., Goryanin I.

Ibm Journal of Research and Development 50 (2006) 561-573.

Biological networks are systems of biochemical processes inside a cell that involve cellular constituents such as DNA, RNA, proteins, and various small molecules. Pathway maps are often used to represent the structure of such networks with associated biological information. Several pathway editors exist, and they vary according to specific domains of knowledge. This paper presents a review of existing pathway editors, along with an introduction to the Edinburgh Pathway Editor (EPE). EPE was designed for the annotation, visualization, and presentation of a wide variety of biological networks that include metabolic, genetic, and signal transduction pathways. EPE is based on a metadata-driven architecture. The editor supports the presentation and annotation of maps, in addition to the storage and retrieval of reaction kinetics information in relational databases that are either local or remote. EPE also has facilities for linking graphical objects to external databases and Web resources, and is capable of reproducing most existing graphical notations and visual representations of pathway maps. In summary, EPE provides a highly flexible tool,for combining visualization, editing, and database manipulation of information relating to biological networks. EPE is open-source software, distributed under the Eclipse open-source application platform license.

A Comparative Study of Functional Properties of Calf Chymosin and Its Recombinant Forms

Starovoitova V.V., Velichko T.I., Baratova L.A., Filippova I.Y., Lavrenova G.I.

Biochemistry-Moscow 71 (2006) 320 -324.

The action of calf chymosin obtained from transgenic sheep milk and the recombinant protein expressed in yeast Kluyveromyces lactis (Maxiren) on fluorogenic peptide substrates, namely Abz-A-A-F-F-A-A-Ded, Abz-A-A-F-F-A-A-pNA, Abz-A-F-F-A-A-Ded, Abz-A-A-F-F-A-Ded, Abz-A-A-F-F-Ded, Abz-A-A-F-F-pNA, and heptapeptide L-S-F-M-A-I-P-NH2, a fragment of K-casein (the native chymosin substrate), was investigated. It has been established that transgenic chymosin and recombinant chymosin (Maxiren) differ from the native enzyme in their action on low molecular weight substrates, whereas there was no difference in enzymatic action on protein substrates. Pepstatin, a specific inhibitor of aspartic proteinases, inhibits the recombinant chymosin forms less efficiently than the native enzyme. Perhaps this is associated with local conformational changes in the substrate binding site of recombinant chymosin occurring during the formation of the protein globule.

Some Factors Controlling the Biosynthesis of Chlorosome Antenna Bacteriochlorophylls in Green Filamentous Anoxygenic Phototrophic Bacteria of the Family Oscillochlofidaceae

Taisova A.S., Keppen O.I., Novikov A.A., Naumova M.G., Fetisova Z.G.

Microbiology 75 (2006) 129-135.

We determined the concentrations of bacteriochlorophylls (BChl) in the light-harvesting antennae of Oscillochloris trichoides (of the family Oscillochloridaceae belonging to green filamentous mesophilic bacteria) cultivated either with gabaculine, an inhibitor of the C-5 pathway of BChl biosynthesis in a number of bacteria, or at various illumination intensities,. We determined the BChl c:BChl a molar ratios in intact cells, in chlorosome-membrane complexes, and in isolated chlorosomes. We revealed that BChl c synthesis in Osc. trichoides was more gabaculine-sensitive than BChl a synthesis. Accordingly, an increase in gabaculine concentrations in the medium resulted in a decrease in the BChl c:BChl a ratio in the tested samples. We suggest that BChl synthesis in Ose. trichoides proceeds via the C-5 pathway, similar to representatives of other fan-Lilies of green bacteria (Chlorobium limicola and Chloroflexus aurantiacus). We demonstrated that the BChl c:BChl a ratio in the chlorosomes varied from 55:1 to 110:1, depending on light intensity. This ratio is, therefore, closer to that of Chlorobiaceae, and it significantly exceeds the BChl c:BChl a ratio in Chloroflexaceae.

The Higher Toxicity of Cereulide Relative to Valinomycin Is Due to Its Higher Affinity for Potassium at Physiological Plasma Concentration

Teplova V.V., Mikkola R., Tonshin A.A., Saris N.E.L., Salkinoja-Salonen M.S.

Toxicology and Applied Pharmacology 210 (2006) 39-46.

Valinomycin and cereulide are bacterial toxins with closely similar chemical structure and properties but different toxic effects. Emetic poisoning is induced by cereulide but not by valinomycin. Both are specific potassium ionophores. Such compounds may affect mitochondrial functions. Both compounds cause a potassium-dependent drop in the transmembrane inner membrane potential due to the uptake of K⁺ as positively charged ionophore complex. Valinomycin is more potent than cereulide at high [K⁺] (> 80 mM), whereas cereulide in contrast to valinomycin is active already at < 1 mM. With cereulide, there is a substantial lag, while valinomycin acts without lag. Both ionophores induce mitochondrial swelling in the presence of K⁺, in the case of cerculide with a lag. These toxins strongly inhibited respiration at the level of complex IV when used at higher concentrations than that used for detection of ionophoretic transport of K⁺. At high [KCI] (120 mM), valinomycin was more potent than cerculide both as ionophore and inhibitor, but at low [KCI] (2.5 mM), cereulide was much more potent. Thus. valinomycin needed 20-30 mM KCI for Substantial effects, cerculide only 1-3 mM K⁺, which is close to its level in blood serum. This explains the higher toxicity of cerculide at low concentrations with the positively charged potassium complex being accumulated in the cell by transport through the plasma membrane driven by the membrane potential. Furthermore, with similar concentrations, the final concentration of cereulide in the cells may become higher than that of valinomycin.

Modified DNA Fragments Specifically and Irreversibly Bind Transcription Factor NF-Kappa B in Lysates of Human Tumor Cells

Timchenko M.A., Rybalkina E.Y., Lomakin A.Y., Evlakov K.I., Serdyuk I.N., Ivanovskaya M.G.

Biochemistry-Moscow 71 (2006) 454 -460.

Covalent binding of a synthetic DNA fragment with eukaryotic transcription factor NF-kappa B has been studied in lysates of human colon carcinoma HCT-116 cells. For binding we used P-32-labeled 17/19 bp nucleotide DNA duplex containing an NF-kappa B recognition site (kappa B-site) in which one of internucleoticle phosphate groups was replaced by a chemically active trisubstituted pyrophosphate group. Using gel electrophoresis under denaturing conditions (Laemmli electrophoresis) followed by immunoblotting revealed selective irreversible binding of 12 P-labeled DNA duplex with NF-kappa B in lysates of tumor cells in the presence of other cell components. Experiment on delivery of this DNA duplex containing rhodamine at 3'-end of the modified chain in an intact cell revealed that rhodamine-labeled DNA penetrated through the plasma membrane of tumor cells without any additional delivery systems. Using fluorescent microscopy, we found that the rhodamine-labeled DNA is initially localized in the cytoplasm. Confocal laser scanning microscopy revealed that subsequent treatment of the cells with TNF-alpha promoted partial translocation of the DNA reagent into the nucleus.

The Interrelation of Specific Changes in Mitochondrial Membranes Permeability and Internuclearsomal DNA Fragmentation in Heart Tissue Incubated Under Anoxia Conditions

Tonshin A.A., Lobysheva N.V., Yaguzhinsky L.S.

Biologicheskie Membrany 23 (2006) 394-401.

Specific changes in mitochondrial membrane permeability were found in heart tissue incubated under anoxia conditions. Some of these changes were shown to be a necessary step to induce internuclearsomal DNA fragmentation, an apoptosis hallmark which was observed in such conditions. The outer membrane of mitochondria isolated from tissue incubated in anoxia was characterized by increased permeability for cytochrome c. However, it was found that cytochrome c was not released from intermembrane space of mitochondria in vivo and consequently did not participate in apoptosis induction. In was shown that apoptosis depends on the permeability transition pore (PTP) opening. The addition of PTP inhibitor, cyclosporine A, prevents the drop of membrane potential of inner membrane and DNA fragmentation. However, this inhibitor does not prevent the increase of outer membrane permeability for cytochrome c. The scheme of biochemical reactions of apoptosis induced by anoxia in heart tissue is discussed.

Block Copolymers of Pluronics and Poly-(2-Dimethylamino Plus Ethyl Methacrylate) for Delivery of Oligonucleotides into Tumor Cells

Valeeva Y.K., Dorodnykh T.Y., Alexandrova N.A., Zubin E.M., Kachatova A.V., Volkov E.M., Gottikh M.B., Melik-Nubarov N.S.

Journal of Drug Delivery Science and Technology 16 (2006) 245-251.

Poly-(2-dimethylaminoethlmethacrylate) (pDMAEMA) and its block copolymers with Pluronics L61, P85 and F127 were synthesized via controlled atom-transfer radical polymerization (ATRP) using 2-bromopropionyl groups as initiators. Being a weak polycation, pDMAEMA contains only 10-20% of charged repeat units atpH 7.0. Therefore, complete binding of a short 11-mer oligonucleotide was achieved only at 5-10-fold excess of pDMAEMA repeat units as against oligonucleotide phosphate groups. Ability of the copolymers to favor penetration of the oligonucleotide into immortalized NIH/3T3 mouse fibroblasts transfected with the EWS-Fli1 cDNA under the control of the LTR retroviral promoter, which mimic spontaneous transformations occurring in patients with Ewing sarcomas, was investigated. It was found that the copolymers facilitated the penetration of the oligonucleotide into the cell nuclei with the efficacy increased in the series P85-pDMAEMA<homo-pDMAEMA<F127-pDMAEMA<L61-pDMAEMA. The results obtained indicate that the hydrophobicity of amphiphilic copolymers favors their ability to provide oligonucleotides delivery into cells.

An Attempt to Clarify Taxonomic Relationships in "Verwandtschaftskreis Der Gattung Ligusticum" (Umbelliferae-Apioideae) by Molecular Analysis

Valiejo-Roman C.M., Shneyer V.S., Samigullin T.H., Terentieva E.I., Pimenov M.G.

Plant Systematics and Evolution 257 (2006) 25-43.

Relationships among the taxa of Umbelliferae, presumably close to Ligusticum and Selinum were investigated by two independent molecular taxonomic methods. 134 ITS 1-2 sequences were analyzed (29 new and 2 reinvestigated species) and immunochemical comparison of storage seed proteins for 38 species of Apioideae of Ligusticum affinity was performed, eight reference systems (antisera) were used. Both approaches yield similar results, showing the extremely polyphyletic nature of this group and some large genera (Ligusticum s.l., Selinum s.l., Pachypleurum) in the Umbelliferae. The independent status of the genera Magadania, Sphaenolobium, Arafoe, Lomatocarpa, Dimorphosciadium and some other segregates of Ligusticum, Cnidium and Selinum have been confirmed, but Cnidium proved to be unnatural even as currently circumscribed. In the group of East Asian taxa the genera Oreocome, Ligusticopsis, Cortia and Cortiella appeared to be closely related. Haplosphaera was shown to be a genus of Hansenia-Notopterygium group.

Energy Transfer in Photosynthesis:Experimental Insights and Quantitative Models

van Grondelle R. and Novoderezhkin V.I.

Physical Chemistry Chemical Physics 8 (2006) 793-807.

We overview experimental and theoretical studies of energy transfer in the photosynthetic light-harvesting complexes LH1, LH2, and LHCII performed during the past decade since the discovery of high-resolution structure of these complexes. Experimental findings obtained with various spectroscopic techniques makes possible a modelling of the excitation dynamics at a quantitative level. The modified Redfield theory allows a precise assignment of the energy transfer pathways together with a direct visualization of the whole excitation dynamics where various regimes from a coherent motion of delocalized exciton to a hopping of localized excitations are superimposed. In a single complex it is possible to observe the switching between these regimes driven by slow conformational motion (as we demonstrate for LH2). Excitation dynamics under quenched conditions in higher-plant complexes is discussed.

DNA Methylation in Plants

Vanyushin B.F.

Dna Methylation:Basic Mechanisms 301 (2006) 67-122.

DNA in plants is highly methylated, containing 5-methylcytosine (m(5)C) and N-6-methyladenine (m(6)A); m(5)C is located mainly in symmetrical CG and CNG sequences but it may occur also in other non-symmetrical contexts. m(6)A but not m(5)C was found in plant mitochondrial DNA. DNA methylation in plants is species-, tissue-, organelle- and age-specific. It is controlled by phytohormones and changes on seed germination, flowering and under the influence of various pathogens (viral, bacterial, fungal). DNA methylation controls plant growth and development, with particular involvement in regulation of gene expression and DNA replication. DNA replication is accompanied by the appearance of under-methylated, newly formed DNA strands including Okazaki fragments; asymmetry of strand DNA methylation disappears until the end of the cell cycle. A model for regulation of DNA replication by methylation is suggested. Cytosine DNA methylation in plants is more rich and diverse compared with animals. It is carried out by the families of specific enzymes that belong to at least three classes of DNA methyltransferases. Open reading frames (ORF) for adenine DNA methyltransferases are found in plant and animal genomes, and a first eukaryotic (plant) adenine DNA methyltransferase (wadmtase) is described; the enzyme seems to be involved in regulation of the mitochondria replication. Like in animals, DNA methylation in plants is closely associated with histone modifications and it affects binding of specific proteins to DNA and formation of respective transcription complexes in chromatin. The same gene (DRM2) in Arabidopsis thaliana is methylated both at cytosine and adenine residues; thus, at least two different, and probably interdependent, systems of DNA modification are present in plants. Plants seem to have a restriction-modification (R-M) system. RNA-directed DNA methylation has been observed in plants; it involves de novo methylation of almost all cytosine residues in a region of siRNA-DNA sequence identity; therefore, it is mainly associated with CNG and non-symmetrical methylations (rare in animals) in coding and promoter regions of silenced genes. Cytoplasmic viral RNA can affect methylation of homologous nuclear sequences and it may be one of the feedback mechanisms between the cytoplasm and the nucleus to control gene expression.

DNA Methylation and Epigenetics

Vanyushin B.F.

Russian Journal of Genetics 42 (2006) 985-997.

In eukaryotic cells, nuclear DNA is subject to enzymatic methylation with the formation of 5-methylcytosine residues, mostly within the CG and CNG sequences. In plants and animals this DNA methylation is species-, tissue-, and organelle-specific. It changes (decreases) with age and is regulated by hormones. On the other hand, genome methylation can control hormonal signal. Replicative and post-replicative DNA methylation types are distinguished. They are mediated by multiple DNA methyltransferases with different site-specificity. Replication is accompanied by the appearance of hemimethylated DNA sites. Pronounced asymmetry of the DNA strand methylation disappears to the end of the cell cycle. A model of methylation-regulated DNA replication is proposed. DNA methylation controls all genetic processes in the cell (replication, transcription, DNA repair, recombination, and gene transposition). It is the mechanism of cell differentiation, gene discrimination and silencing. In animals, suppression of DNA methylation stops development (embryogenesis), switches on apoptosis, and is usually lethal. Disruption of DNA methylation pattern results in the malignant cell transformation and serves as one of the early diagnostic features of carcinogenesis. In malignant cell the pattern of DNA methylation, as well as the set of DNA methyltransferase activities, differs from that in normal cell. In plants inhibition of DNA methylation is accompanied by the induction of seed storage and florescence genes. In eukaryotes one and the same gene can be simultaneously methylated both at cytosine and adenine residues. It can be thus suggested, that the plant cell contains at least two different, and probably, interdependent systems of DNA methylation. The first eukaryotic adenine DNA methyltransferase was isolated from plants. This enzyme methylates DNA with the formation of N-6-methyladenine residues in the sequence TGATCA (TGATCA -> TGm(6)ATCA). Plants possess AdoMet-dependent endonucleases sensitive to DNA methylation. It seems likely that plants, similarly to microorganisms and some lower eukaryotes, have restriction-moditication (R-M) system. Discovery of the essential role of DNA methylation in regulation of genetic processes served as a principle basis and materialization of epigenetics and epigenomics.

Cellular Search Migrations in Normal Development and Carcinogenesis

Vasiliev J.M. and Gelfand I.M.

Biochemistry-Moscow 71 (2006) 821 -826.

This review describes the large group of morphogenetic processes designated as search migrations. Search migrations typically include two stages:i) search, when a group of cells or of the cytoplasmic processes migrate over the cell-free spaces, and ii) choice, the stage when migrating cells reach specific loci where they stop and undergo specific differentiations induced by local factors such as cell-cell contacts and humoral agents. Migrating cells that do not meet their targets usually undergo apoptosis. Numerous examples of search migrations range from gastrulation to formation of axon-muscle connections. Critical stages of carcinogenesis such as acquisition of cell ability for invasion may be regarded as the genetic aberration of normal search migration:cancer cells perform an endless search but cannot make final choice.

Diversity of Digestive Proteinases in Tenebrio molitor (Coleoptera:Tenebrionidae) Larvae

Vinokurov K.S., Elpidina E.N., Oppert B., Prabhakar S., Zhuzhikov D.P., Dunaevsky Y.E., Belozersky M.A.

Comparative Biochemistry and Physiology B-Biochemistry & Molecular Biology 145 (2006) 126-137.

The spectrum of Tenebrio molitor larval digestive protemases was studied in the context of the spatial organization of protein digestion in the midgut. The pH of midgut contents increased from 5.2-5.6 to 7.8-8.2 from the anterior to the posterior. This pH gradient was reflected in the pH optima of the total proteolytic activity, 5.2 in the anterior and 9.0 in the posterior midgut. When measured at the pH and reducing conditions characteristic of each midgut section, 64% of the total proteolytic activity was in the anterior and 36% in the posterior midgut. In the anterior midgut, two-thirds of the total activity was due to cysteine proteinases, whereas the rest was from serine protemases. In contrast, most (76%) of the proteolytic activity in the posterior midgut was from serine proteinases. Cysteine protemases from the anterior were represented by a group of anionic fractions with similar electrophoretic mobility. Trypsin-like activity was predominant in the posterior midgut and was due to one cationic and three anionic protemases. Chymotrypsin-like protemases also were prominent in the posterior midgut and consisted of one cationic and four anionic protemases, four with an extended binding site. Latent protemase activity was detected in each midgut section. These data support a complex system of protein digestion, and the correlation of protemase activity and pH indicates a physiological mechanism of enzyme regulation in the gut.

Fractionation of Digestive Proteinases From Tenebrio molitor (Coleoptera:Tenebrionidae) Larvae and Role in Protein Digestion

Vinokurov K.S., Elpidina E.N., Oppert B., Prabhakar S., Zhuzhikov D.P., Dunaevsky Y.E., Belozersky M.A.

Comparative Biochemistry and Physiology B-Biochemistry & Molecular Biology 145 (2006) 138-146.

Tenebrio molitor larval digestive proteinases were purified and characterized by gel filtration chromatography combined with activity electrophoresis. Cysteine proteinases, consisting of at least six distinct activities, were found in three chromatographic peaks in anterior and posterior midgut chromatographics. The major activity in the anterior midgut, peak cys II, consisted of cysteine proteinases with Mm of 23 kDa. The predominant peak in the posterior, cys 1, was represented by 38 kDa proteinases. The activities of all cysteine proteinases were maximal in buffers from pH 5.0 to 7.0, with 80% stability at pH values from 4.0 to 7.0. In the conditions of the last third of the midgut, the activity and stability of cysteine proteinases was sharply decreased. Trypsin-like activity included a minor peak of "heavy" trypsins with Mm 59 kDa, located mainly in the anterior midgut. An in vitro study of the initial stages of digestion of the main dietary protein, oat 12S globulin, by anterior midgut proteinases revealed that hydrolysis occurred through the formation of intermediate high-Mm products, similar to those formed during oat seed germination. Cysteine proteinases from the cys III peak and heavy trypsins were capable of only limited proteolysis of the protein, whereas incubation with cys II proteinases resulted in substantial hydrolysis of the globulin.

Tuning of a Neuronal Calcium Sensor

Weiergraber O.H., Senin I.I., Zernii E.Y., Churumova V.A., Kovaleva N.A., Nazipova A.A., Permyakov S.E., Permyakov E.A., Philippov P.P., Granzin J., Koch K.W.

Journal of Biological Chemistry 281 (2006) 37594-37602.

Recoverin is a Ca²⁺-regulated signal transduction modulator expressed in the vertebrate retina that has been implicated in visual adaptation. An intriguing feature of recoverin is a cluster of charged residues at its C terminus, the functional significance of which is largely unclear. To elucidate the impact of this segment on recoverin structure and function, we have investigated a mutant lacking the C-terminal 12 amino acids. Whereas in myristoylated recoverin the truncation causes an overall decrease in Ca²⁺ sensitivity, results for the non-myristoylated mutant indicate that the truncation primarily affects the high affinity EF-hand 3. The three-dimensional structure of the mutant has been determined by x-ray crystallography. In addition to significant changes in average coordinates compared with wild-type recoverin, the structure provides strong indication of increased conformational flexibility, particularly in the C-terminal domain. Based on these observations, we propose a novel role of the C-terminal segment of recoverin as an internal modulator of Ca²⁺ sensitivity.

On the Localized Coupling of Respiration and Phosphorylation in Mitochondria

Yaguzhinsky L.S., Yurkov V.I., Krasinskaya I.P.

Biochimica et Biophysica Acta-Bioenergetics 1757 (2006) 408-414.

This paper is an overview of the theoretical and experimental studies performed in our laboratory to answer the question whether there exist conditions where the hypothetical mechanism of the localized coupling of respiration and phosphorylation postulated by R. Williams in 1961 operates. These studies were undertaken to verify the earlier suggestion that mitochondria may exist in two structural and functional states. Correspondingly, there are two operation modes of oxidative phosphorylation, one of which corresponds to the Williams' mechanism of localized coupling and the other, to the Mitchell's mechanism of delocalized coupling. The paper considers the principle of the energy conservation of oxidative reactions in mitochondrial membranes in the form of the thermodynamic potential of hydrogen ions (Delta mu(sol)) lacking, in part, the solvation shell. We present experimental evidence for the existence of the mechanism of localized coupling and describes the conditions favorable for its implementation. The experiments described in this paper show that the aforementioned models for proton coupling are not necessarily alternative. A conclusion is made that, depending on the particular conditions, either localized or delocalized coupling mechanisms of oxidative phosphorylation may come into operation.

Antigenic Evolution of Vaccine-Derived Polioviruses:Changes in Individual Epitopes and Relative Stability of the Overall Immunological Properties

Yakovenko M.L., Cherkasova E.A., Rezapkin G.V., Ivanova O.E., Ivanov A.P., Eremeeva T.P., Baykova O.Y., Chumakov K.M., Agol V.I.

Journal of Virology 80 (2006) 2641-2653.

The Sabin oral poliovirus vaccine (OPV) readily undergoes changes in antigenic sites upon replication in humans. Here, a set of antigenically altered descendants of the three OPV serotypes (76 isolates) was characterized to determine the driving forces behind these changes and their biological implications. The amino acid residues of OPV derivatives that lie within or close to the known antigenic sites exhibited a marked tendency to be replaced by residues characteristic of homotypic wild polioviruses, and these changes may occur very early in OPV evolution. The specific amino acid alterations nicely correlated with serotype-specific changes in the reactivity of certain individual antigenic sites, as revealed by the recently devised monoclonal antibody-based enzyme-linked immunosorbent assay. In comparison to the original vaccine, small changes, if any, in the neutralizing capacity of human or rabbit sera were observed in highly diverged vaccine polioviruses of three serotypes, in spite of strong alterations of certain epitopes. We propose that the common antigenic alterations in evolving OPV strains largely reflect attempts to eliminate fitness-decreasing mutations acquired either during the original selection of the vaccine or already present in the parental strains. Variability of individual epitopes does not appear to be primarily caused by, or lead to, a significant immune evasion, enhancing only slightly, if at all, the capacity of OPV derivatives to overcome immunity in human populations. This study reveals some important patterns of poliovirus evolution and has obvious implications for the rational design of live viral vaccines.

Vibrational Coherence in Bacterial Reaction Centers With Genetically Modified B-Branch Pigment Composition

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

Biochimica et Biophysica Acta-Bioenergetics 1757 (2006) 369-379.

Femtosecond absorption difference spectroscopy was applied to study the time and spectral evolution of low-temperature (90 K) absorbance changes in isolated reaction centers (RCs) of the HM182L mutant of Rhodobacter (Rb.) sphaeroides. In this mutant, the composition of the B-branch RC cofactors is modified with respect to that of wild-type RCs by replacing the photochemically inactive B-B accessory bacteriochlorophyll (BChl) by a photoreducible bacteriopheophytin molecule (referred to as Phi(B)). We have examined vibrational coherence within the first 400 fs after excitation of the primary electron donor P with 20-fs pulses at 870 nm by studying the kinetics of absorbance changes at 785 nm (Phi(B) absorption band), 940 nm (P*-stimulated emission), and 1020 nm (B-A(-) absorption band). The results of the femtosecond measurements are compared with those recently reported for native Rb. sphaeroides R-26 RCs containing an intact B-B BChl. At delay times longer than similar to 50 fs (maximum at 120 fs), the mutant RCs exhibit a pronounced BChl radical anion (B-A(-)) absorption band at 1020 nm, which is similar to that observed for Rb. sphaeroides R-26 RCs and represents the fort-nation of the intermediate charge-separated state P+BA-. Femtosecond oscillations are revealed in the kinetics of the absorption development at 1020 nm and of decay, of the P*-stimulated emission at 940 nm, with the oscillatory components of both kinetics displaying a generally synchronous behavior. These data are interpreted in terms of coupling of wave packet-like nuclear motions on the potential energy surface of the P* excited state to the primary electron-transfer reaction P* -> P+BA- in the A-branch of the RC cofactors. At very early delay times (up to 80 fs), the mutant RCs exhibit a weak absorption decrease around 785 nm that is not observed for Rb. sphaeroides R-26 RCs and can be assigned to a transient bleaching of the Q(y) ground-state absorption band of the Phi(B) molecule. In the range of 740-795 nm, encompassing the Qy optical transitions of bacteriopheophytins H-A, H-B, and Phi(B), the absorption difference spectra collected for mutant RCs at 30-50 fs resemble the difference spectrum of the P+Phi(-)(B) charge-separated state previously detected for this mutant in the picosecond time domain (E. Katilius, Z. Katiliene, S. Lin, A.K.W. Taguchi, N.W. Woodbury, J. Phys. Chem., B 106 (2002) 1471-1475). The dynamics of bleaching at 785 nm has a non-monotonous character, showing a single peak with a maximum at 40 fs. Based on these observations, the 785-nm bleaching is speculated to reflect reduction of 1% of Phi(B) in the B-branch within about 40 fs, which is earlier by similar to 80 fs than the reduction process in the A-branch, both being possibly linked to nuclear wave packet motion in the P* state.

RX871024 Reduces NO Production but Does Not Protect Against Pancreatic Beta-Cell Death Induced by Proinflammatory Cytokines

Zaitseva I.I., Sharoyko V., Storling J., Efendic S., Guerin C., Mandrup-Poulsen T., Nicotera P., Berggren P.O., Zaitsev S.V.

Biochemical and Biophysical Research Communications 347 (2006) 1121-1128.

The imidazoline compound RX871024 reduces IL-I beta-induced NO production thereby protecting against IL-I beta-induced beta-cell apoptosis. The aim of this study was to evaluate whether imidazolines RX871024 and efaroxan protect beta-cells against death in the presence of a combination of the cytokines IL-1 beta, IFN gamma, and TNF alpha. To address this issue, experiments involving different methods for detection of cell death, different concentrations of the cytokines, and a variety of conditions of preparation and culturing of ob/ob mouse islets and P-cells have been carried out. Thoroughly performed experiments have not been able to demonstrate a protective effect of RX871024 and efaroxan on beta-cell death induced by the combination of cytokines. However, the inhibitory effect of RX871024 on NO production in ob/ob mouse islets and P-cells was still observed in the presence of all three cytokines and correlated with the decrease in p38 MAPK phosphorylation. Conversely, efaroxan did not affect cytokine-induced NO production. Our data indicate that a combination of pro-inflammatory cytokines IL-I beta, IFN gamma, and TNF alpha conditions modelling those that take place in type 1 diabetes, induces pancreatic P-cell death that does not directly correlate with NO production and cannot be counteracted with imidazoline compounds.

Export of Metabolites by the Proteins of the DMT and RhtB Families and Its Possible Role in Intercellular Communication

Zakataeva N.P., Kutukova E.A., Gronskiy S.V., Troshin P.V., Livshits V.A., Aleshin V.V.

Microbiology 75 (2006) 438-448.

The earlier published and new experimental data are summarized on the properties of the genes encoding the membrane proteins of the DMT family (RhtA (YbiF), EamA (YdeD), YijE, YddG, YedA, PecM, eukaryotic nucleotide sugar, triose phosphate/phosphate, and hexose phosphate transporters), the RhtB/LysE family (RhtB, RhtC, LeuE, YahN, EamB (YfiK), ArgO (YggA), CmaU), as well as some other families (YicM, YdhC, YdeAB, YdhE (NorE)). These proteins are involved in the export of amino acids, purines, and other metabolites from the cell. The expression of most of the genes encoding these proteins is not induced by the substrates they transport but is controlled by the global regulation systems, such as the Lrp protein, and activated by the signal compounds involved in the intracellular communication. The level of expression, assessed in experiments on translational fusion of the corresponding bacterial genes with the beta-galactosidase gene, depends on the growth phase of the bacterial culture, composition of the medium, and some stress factors, such as pH, osmolarity or decreased aeration. The efflux of normal cell metabolites is assumed to be the natural function of these proteins. This function may play a role in density-dependent behavior of cell populations (quorum sensing). It may have been enhanced in the course of evolution via specialization of these proteins in the efflux of compounds derived from metabolic intermediates and adjusted to the role of transmitters.

Assessment of the Integral Membrane Protein Topology in Living Cells

Zamyatnin A.A., Solovyev A.G., Bozhkov P.V., Valkonen J.P.T., Morozov S.Y., Savenkov E.I.

Plant Journal 46 (2006) 145-154.

The bimolecular fluorescence complementation ( BiFC) phenomenon has been successfully applied for in vivo protein - protein interaction studies and protein tagging analysis. Here we report a novel BiFC- based technique for investigation of integral membrane protein topology in living plant cells. This technique relies on the formation of a fluorescent complex between a non- fluorescent fragment of the yellow fluorescent protein ( YFP) targeted into a specific cellular compartment and a counterpart fragment attached to the integral membrane protein N- or C- terminus or inserted into the internal loop( s). We employed this technique for topological studies of beet yellows virus- encoded p6 membrane- embedded movement protein, a protein with known topology, and the potato mop- top virus- encoded integral membrane TGBp2 protein with predicted topology. The results confirm that p6 is a type III integral transmembrane protein. Using a novel method, the central hydrophilic region of TGBp2 was localized into the ER lumen, whereas the N- and C- termini localized to the cytosol. We conclude that the BiFC- based reporter system for membrane protein topology analysis is a relatively fast and efficient method that can be used for high- throughput analysis of proteins integrated into the endoplasmic reticulum in living plant cells.

Mitochondrial ROS-Induced ROS Release:An Update and Review

Zorov D.B., Juhaszova M., Sollott S.J.

Biochimica et Biophysica Acta-Bioenergetics 1757 (2006) 509-517.

Unstable mitochondrial membrane potential and redox transitions can occur following insults including ischemia/reperfusion injury and toxin exposure, with negative consequences for mitochondrial integrity and cellular survival. These transitions can involve mechanisms such as the recently described process, "Reactive Oxygen Species (ROS)-induced ROS-release" (RIRR), and be generated by circuits where the mitochondrial permeability transition (MPT) pore and the inner membrane anion channel (IMAC) are involved. The exposure to excessive oxidative stress results in an increase in ROS reaching a threshold level that triggers the opening of one of the requisite mitochondrial channels. In turn, this leads to the simultaneous collapse of the mitochondrial membrane potential and a transient increased ROS generation by the electron transfer chain. Generated ROS can be released into cytosol and trigger RIRR in neighboring mitochondria. This mitochondrion-to-mitochondrion ROS-signaling constitutes a positive feedback mechanism for enhanced ROS production leading to potentially significant mitochondrial and cellular injury. This review and update considers a variety of RIRR mechanisms (involving MPT, INIAC and episodes of mitochondrial transient hyperpolarization). RIRR could be a general cell biology phenomenon relevant to the processes of programmed mitochondrial destruction and cell death, and may contribute to other mechanisms of post-ischemic pathologies, including arrhythmias.