I. Structure and functions of biological macromolecules and macromolecular complexes. Biocalalysis.

THE BACTERIAL TOXIN RelE INDUCES SPECIFIC mRNA CLEAVAGE IN THE A SITE OF THE EUKARYOTE RIBOSOME.
Andreev D., Hauryliuk V., Terenin I., Dmitriev S., Ehrenberg M., Shatsky I.
RNA-A PUBLICATION OF THE RNA SOCIETY 14 (2008) 233-239.

RelE/RelB is a well-characterized toxin-anti-toxin pair involved in nutritional stress responses in Bacteria and Archae. RelE lacks any eukaryote homolog, but we demonstrate here that it efficiently and specifically cleaves mRNA in the A site of the eukaryote ribosome. The cleavage mechanism is similar to that in bacteria, showing the feasibility of A-site cleavage of mRNA for regulatory purposes also in eukaryotes. RelE cleavage in the A-site codon of a stalled eukaryote ribosome is precise and easily monitored, making "RelE printing" a useful complement to toeprinting to determine the exact mRNA location on the eukaryote ribosome and to probe the occupancy of its A site.

ONE SmpB MOLECULE ACCOMPANIES tmRNA DURING ITS PASSAGE THROUGH THE RIBOSOMES.
Bugaeva E.Y., Shpanchenko O.V., Felden B., Isaksson L.A., Dontsova O.A.
FEBS LETTERS 582 (2008) 1532-1536.

tmRNA and SmpB are the main participants of trans-translation, a process which rescues the ribosome blocked during translation of non-stop mRNA. While a one-to-one stoichiometry of tmRNA to the ribosome is generally accepted, the number of SmpB molecules in the complex is still under question. We have isolated tmRNA-ribosome complexes blocked at different steps of the tmRNA path through the ribosome and analyzed the stoichiometry of the complexes. Ribosome, tmRNA and SmpB were found in equimolar amount in the tmRNA-ribosome complexes stopped at the position of the 2nd, 4th, 5th or the 11th codons of the coding part of the tmRNA.

NOVEL ISOENZYME OF 2-OXOGLUTARATE DEHYDROGENASE IS IDENTIFIED IN BRAIN, BUT NOT IN HEART.
Bunik V., Kaehne T., Degtyarev D., Shcherbakova T., Reiser G.
FEBS JOURNAL 275 (2008) 4990-5006.

2-Oxoglutarate dehydrogenase (OGDH) is the first and rate-limiting component of the multienzyme OGDH complex (OGDHC) whose malfunction is associated with neurodegeneration. The essential role of this complex in the degradation of glucose and glutamate, which have specific significance in brain, raises questions about the existence of brain-specific OGDHC isoenzyme(s). We purified OGDHC from extracts of brain or heart mitochondria using the same procedure of poly(ethylene glycol) fractionation, followed by size-exclusion chromatography. Chromatographic behavior and the insufficiency of mitochondrial disruption to solubilize OGDHC revealed functionally significant binding of the complex to membrane. Components of OGDHC from brain and heart were identified using nano-high performance liquid chromatography electrospray tandem mass spectrometry after trypsinolysis of the electrophoretically separated proteins. In contrast to the heart complex, where only the known OGDH was determined, the band corresponding to the brain OGDH component was found to also include the novel 2-oxoglutarate dehydrogenase-like (OGDHL) protein. The ratio of identified peptides characteristic of OGDH and OGDHL was preserved during purification and indicated comparable quantities of the two proteins in brain. Brain OGDHC also differed from the heart complex in the abundance of the components, lower apparent molecular mass and decreased stability upon size-exclusion chromatography. The functional competence of the novel brain isoenzyme and different regulation of OGDH and OGDHL by 2-oxoglutarate are inferred from the biphasic dependence of the overall reaction rate versus 2-oxoglutarate concentration. OGDHL may thus participate in brain-specific control of 2-oxoglutarate distribution between energy production and synthesis of the neurotransmitter glutamate.

STRUCTURE-FUNCTION RELATIONSHIPS IN THE 2-OXO ACID DEHYDROGENASE FAMILY: SUBSTRATE- SPECIFIC SIGNATURES AND FUNCTIONAL PREDICTIONS FOR THE 2-OXOGLUTARATE DEHYDROGENASE-LIKE PROTEINS.
Bunik V.I., Degtyarev D.
PROTEINS: STRUCTURE, FUNCTION, AND BIOINFORMATICS 71 (2008) 874-890.

Structural relationship within the family of the thiamine diphosphate-dependent 2-oxo acid dehydrogenases was analyzed by combining different methods of sequence alignment with crystallographic and enzymological studies of the family members. For the first time, the sequence similarity of the homodimeric 2-oxoglutarate dehydrogenase to heterotetrameric 2-oxo acid dehydrogenases is established. The presented alignment of the catalytic domains of the dehydrogenases of pyruvate, branched- chain 2-oxo acids and 2-oxoglutarate unravels the sequence markers of the substrate specificity and the essential residues of the family members without the 3D structures resolved. Predicted dual substrate specificity of some of the 2-oxo acid dehydrogenases was confirmed experimentally. The results were used to decipher functions of the two hypothetical proteins of animal genomes, OGDHL and DHTKD1, similar to the 2-oxoglutarate dehydrogenase. Conservation of all the essential residues confirmed their catalytic competence. Sequence analysis indicated that OGDHL represents a previously unknown isoform of the 2-oxoglutarate dehydrogenase, whereas DHTKD1 differs from the homologs at the N-terminus and substrate binding pocket. The differences suggest changes in heterologous protein interactions and accommodation of more polar and/or bulkier structural analogs of 2-oxoglutarate, such as 2-oxoadipate, 2-oxo-4-hydroxyglutarate, or products of the carboligase reaction between a 2-oxodicarboxylate and glyoxylate or acetaldehyde. The signatures of the Ca²⁺-binding sites were found in the Ca²⁺-activated 2-oxoglutarate dehydrogenase and OGDHL, but not in DHTKD1. Mitochondrial localization was predicted for OGDHL and DHTKD1, with DHTKD1 probably localized also to nuclei. Medical implications of the obtained results are discussed in view of the possible associations of the 2-oxo acid dehydrogenases and DHTKD1 with neurodegeneration and cancer.

Molecular modeling has revealed intimate details of the mechanism of binding of natural substrate, penicillin G (PG), in the penicillin acylase active center and solved questions raised by analysis of available X-ray structures, mimicking Michaelis complex, which substantially differ in the binding pattern of the PG leaving group. Three MD trajectories were launched, starting from PDB complexes of the inactive mutant enzyme with PG (1FXV) and native penicillin acylase with sluggishly hydrolyzed substrate analog penicillin G sulfoxide (1GM9), or from the complex obtained by PG docking. All trajectories converged to a similar PG binding mode, which represented the near-to-attack conformation, consistent with chemical criteria of how reactive Michaelis complex should look. Simulated dynamic structure of the enzyme-substrate complex differed significantly from 1FXV, resembling rather 1GM9; however, additional contacts with residues bG385, bS386, and bN388 have been found, which were missing in X-ray structures. Combination of molecular docking and molecular dynamics also clarified the nature of extremely effective phenol binding in the hydrophobic pocket of penicillin acylase, which lacked proper explanation from crystallographic experiments. Alternative binding modes of phenol were probed, and corresponding trajectories converged to a single binding pattern characterized by a hydrogen bond between the phenol hydroxyl and the main chain oxygen of bS67, which was not evident from the crystal structure. Observation of the trajectory, in which phenol moved from its steady bound to pre-dissociation state, mapped the consequence of molecular events governing the conformational transitions in a coil region a143-a146 coupled to substrate binding and release of the reaction products. The current investigation provided information on dynamics of the conformational transitions accompanying substrate binding and significance of poorly structured and flexible regions in maintaining catalytic framework.

LOCALIZATION OF POST-PROLINE CLEAVING PEPTIDASES IN Tenebrio molitor LARVAL MIDGUT.
Goptar I.A., Filippova I.Y., Lysogorskaya E.N., Oksenoit E.S., Vinokurov K.S., Zhuzhikov D.P., Bulushova N.V., Zalunin I.A., Dunaevsky Y.E., Belozersky M.A., Oppert B., Elpidina E.N.
BIOCHIMIE 90 (2008) 508-514.

Two soluble post-proline cleaving peptidase activities, PPCP1 and PPCP2, were demonstrated in Tenebrio molitor larval midgut with the substrate benzyloxycarbonyl-L-alanyl-L-proline p-nitroanilide. Both activities were serine peptidases. PPCP1 was active in acidic buffers, with maximum activity at pH 5.3, and was located mainly in the more acidic anterior midgut lumen. The dynamics of PPCP1 activity and the total activity of soluble digestive peptidases in the course of food digestion were similar, suggesting that the enzyme participates in protein digestion. PPCP2 is a nondigestive soluble tissue enzyme evenly distributed along the midgut. An increase in the activity of PPCP2 was observed in buffers of pH 5.6-8.6 and was maximal at pH 7.4. The sensitivity of PPCP2 to inhibitors and the effect of pH are similar to prolyl oligopeptidases with a cysteine residue near the substrate binding site.

NEW EVIDENCE FOR COFACTOR'S AMINO GROUP FUNCTION IN THIAMIN CATALYSIS BY TRANSKETOLASE.
Meshalkina L.E., Kochetov G.A., Brauer J., Hubner G., Tittmann K., Golbik R.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 366 (2008) 692-697.

Transketolase from Saccharomyces cerevisiae exhibits a rarely reported activity with a methylated analogue of the native cofactor, 4'-methylamino-thiamin diphosphate. We demonstrated the kinetic stability of the dihydroxyethyl carbanion/enamine intermediate to be dependent on the functionality of the 4'-aminopyrimidine moiety of thiamin diphosphate [R. Golbik, L.E. Meshalkina, T. Sandalova, K. Tittmann, E. Fiedler, H. Neef, S. Konig, R. Kluger, G.A. Kochetov, G. Schneider, G. Hubner, Effect of coenzyme modification on the structural and catalytic properties of wild-type transketolase and of the variant E418A from Saccharomyces cerevisae, FEBS J. (2005) 272 1326-1342]. This paper extends these investigations of the function of the coenzyme's aminopyrimidine in transketolase catalysis exemplified for the 4'-monomethylamino-thiamin diphosphate analogue. Here, we report near UV circular dichroism data and NMR-based analysis of reaction intermediates that give evidence for a strong destabilisation of the carbanion/enamine of DHE-4'-monomethylamino-thiamin diphosphate on the enzyme. A new negative band in near UV circular dichroism arising during turnover is attributed to the conjugate acid of the carbanion/enamine intermediate, an assignment additionally corroborated by 1H NMR-based intermediate analysis. As opposed to the kinetically stabilized carbanion/enamine intermediate in transketolase when reconstituted with the native cofactor, DHE-4'-monomethylamino-thiamin diphosphate is rapidly released from the active centers during turnover and accumulates in the medium on a preparative scale.

Inorganic pyrophosphatase from Mycobacterium tuberculosis (Mt-PPase) is one of the possible targets for the rational design of anti-tuberculosis agents. In this paper, functional properties of this enzyme are characterized in the presence of the most effective activators - Mg²⁺ and Mn²⁺. Dissociation constants of Mt-PPase complexed with Mg²⁺ or Mn²⁺ are essentially similar to those of Escherichia coli PPase. Stability of a hexameric form of Mt-PPase has been characterized as a function of pH both for the metal-free enzyme and for Mg²⁺- or Mn²⁺-enzyme. Hexameric metal-free Mt-PPase has been shown to dissociate, forming monomers at pH below 4 or trimers at pH from 8 to 10. Mg²⁺ or Mn²⁺- shift the hexamer-trimer equilibrium found for the apo-Mt-PPase at pH 8-10 toward the hexameric form by stabilizing intertrimeric contacts. The pKa values have been determined for groups that control the observed hexamer-monomer (pKa 5.4), hexamer-trimer (pKa 7.5), and trimer-monomer (pKa 9.8) transitions. Our results demonstrate that due to the non-conservative amino acid residues His21 and His86 in the active site of Mt-PPase, substrate specificity of this enzyme, in contrast to other typical PPases, does not depend on the nature of the metal cofactor.

N6-Methyladenosine 1618 of Escherichia coli 23 S rRNA is located in a cluster of modified nucleotides 12 A away from the nascent peptide tunnel of the ribosome. Here, we describe the identification of gene ybiN encoding an enzyme responsible for methylation of A1618. Knockout of the ybiN gene leads to loss of modification at A1618. The modification is restored if ybiN knock-out strain has been co-transformed with a plasmid expressing the ybiN gene. On the basis of these results we suggest that ybiN gene should be renamed to rlmF in accordance with the accepted nomenclature for rRNA methyltransferases. Recombinant YbiN protein is able to methylate partially deproteinized 50 S ribosomal subunit, so-called 3.5 M LiCl core particle in vitro, but neither the completely assembled 50 S subunits nor completely deproteinized 23 S rRNA. Both lack of the ybiN gene and it's over-expression leads to growth retardation and loss of cell fitness comparative to the parental strain. It might be suggested that A1618 modification could be necessary for the exit tunnel interaction with some unknown regulatory peptides.

CYCLOOXYGENASE (COX) AND 5-LIPOXYGENASE (5-LOX) SELECTIVITY OF COX INHIBITORS.
Sud'ina G.F., Pushkareva M.A., Shephard P., Klein T.
PROSTAGLANDINS LEUKOT ESSENT FATTY ACIDS 78 (2008) 99-108.

In vitro evaluations of the selectivity of COX inhibitors are based on a great variety of experimental protocols. As a result, data available on cyclooxygenase (COX)-1/COX-2/5- lipoxygenase (LOX) selectivity of COX inhibitors lack consistency. We, therefore, performed a systematic analysis of the COX-1/COX-2/5-LOX selectivity of 14 compounds with selective COX inhibitory activity (Coxibs). The compounds belonged to different structural classes and were analyzed employing the well- recognized whole-blood assay. 5-LOX activity was also tested on isolated human polymorphonuclear leukocytes. Among COX inhibitors, celecoxib and ML-3000 (licofelone) inhibited 5-LOX in human neutrophils at micromolar ranges. Surprisingly, ML-3000 had no effect on 5-LOX product synthesis in whole-blood assay. In addition, we could show that inhibition of COX pathways did not increase the transformation of arachidonic acid by the 5-LOX pathway.

In E. coli cells ribosomal small subunit biogenesis is regulated by RNA-protein interactions involving protein S7. S7 initiates the subunit assembly interacting with 16S rRNA. During shift-down of rRNA synthesis level, free S7 inhibits self-translation by interacting with 96 nucleotides long specific region of streptomycin (str) mRNA between cistrons S12 and S7 (intercistron). Many bacteria do not have the extended intercistron challenging development of specific approaches for searching putative mRNA regulatory regions, which are able to interact with proteins. The paper describes application of SERF approach (Selection of Random RNA Fragments) to reveal regulatory regions of str mRNA. Set of random DNA fragments has been generated from str operon by random hydrolysis and then transcribed into RNA; the fragments being able to bind protein S7 (serfamers) have been selected by iterative rounds. S7 binds to single serfamer, 109 nucleotide long (RNA109), derived from the intercistron. After multiple copying and selection, the intercistronic mutant (RNA109) has been isolated; it has enhanced affinity to S7. RNA109 binds to the protein better than authentic intercistronic str mRNA; apparent dissociation constants are 26 ± 5 and 60 ± 8 nM, respectively. Location of S7 binding site on the mRNA, as well as putative mode of regulation of coupled translation of S12 and S7 cistrons have been hypothesized.

ENZYMATIC SYNTHESIS OF D-XYLULOSE 5-PHOSPHATE FROM HYDROXYPYRUVATE AND d-GLYCERALDEHYDE-3-PHOSPHATE.
Solovjeva O.N., Kochetov G.A.
JOURNAL OF MOLECULAR CATALYSIS B: ENZYMATIC 54 (2008) 90-92.

An enzymatic method for obtaining d-xylulose 5-phosphate has been developed, based on the irreversible reaction catalyzed by transketolase: hydroxypyruvate + d-glyceraldehyde-3-phosphate > d-xylulose 5-phosphate. The preparations of sodium d- xylulose 5-phosphate, obtained using this approach, were 88% pure and contained no aldehyde admixtures.

Cyclic peptides cyclo(-Gly-Asp-Glu-Lys-), cyclo(-Gly-Gly-Asp-Glu-Lys-) and cyclo(-Gly-Gly-Gly-Asp-Glu-Lys-) were synthesized as models of the ?-turn of nerve growth factor loop 4. The corresponding protected linear precursors were obtained in 52-83% yields by the solid-phase method with the use of the Fmoc/But strategy and a chlorotrityl anchor group. The cyclization was carried out with benzotriazolyloxy-tris(dimethylamino)phosphonium (BOP) hexafluorophosphate, N-[(1H- benzotriazole-1-yl)-(dimethy-lamino)methylene]-N-methylmetanaminium-N-oxide (HBTU) hexafluorophosphate, and diphenylphosphory-lazide (DPPA) at a dilution of 10-3 M. The distribution of reaction products was studied for each cyclopeptide in dependence on the type of the coupling agent. The use of DPPA was shown to completely inhibit the formation of cyclodimers in the synthesis of five- and six-membered cyclopeptides; however, in the case of a four-membered peptide, an additional tenfold dilution of the reaction mixture was necessary to achieve the effect. The identification of several byproducts during the synthesis showed that the elongation of the polypeptide chain using the BOP reagent can be complicated by substantial racemization, and the cleavage of the chlorotrityl anchor group by 0.5% TFA in dichloromethane proceeds with insufficient selectivity and is accompanied by the premature Boc deblocking of the lysine side function.

THE GENOME OF THE MODEL BEETLE AND PEST
Tribolium castaneum. Tribolium Genome Sequencing Consortium, including Elpidina E., Vinokurov K.
NATURE 452 (2008) 949-955.

Tribolium castaneum is a member of the most species-rich eukaryotic order, a powerful model organism for the study of generalized insect development, and an important pest of stored agricultural products. We describe its genome sequence here. This omnivorous beetle has evolved the ability to interact with a diverse chemical environment, as shown by large expansions in odorant and gustatory receptors, as well as P450 and other detoxification enzymes. Development in Tribolium is more representative of other insects than is Drosophila, a fact reflected in gene content and function. For example, Tribolium has retained more ancestral genes involved in cell-cell communication than Drosophila, some being expressed in the growth zone crucial for axial elongation in short-germ development. SystemicRNA interference in T. castaneum functions differently from that in Caenorhabditis elegans, but nevertheless offers similar power for the elucidation of gene function and identification of targets for selective insect control.

The interest in proteases secreted by mycelial fungi is due to several reasons of which one of the most important is their involvement in the initiation and development of the pathogenic process. A comparison of saprophytic and phytopathogenic mycelial fungi revealed one characteristic feature, namely, the appearance of a new trypsin-like activity in phytopathogens that is absent in saprophytes. To clear up the question of whether the degree of pathogenicity of a fungus is related to the activity of secreted trypsin-like protease, several species of Fusarium of various pathogenicity were compared. In two species, F. sporotrichioides (which causes ear fusariosis of rye) and F. heterosporum (the causative agent of root rot in wheat), a clear correlation between the activity and pathogenicity was revealed: the more pathogenetic F. sporotrichioides exhibited a higher extracellular trypsin-like activity than the less pathogenetic species F. heterosporum. Thus, the presence of trypsin-like activity in a saprotroph-pathogen pair may be an indicator of the pathogenicity of a fungus; in some cases, the value of this activity may indicate the degree of its pathogenicity. This suggests that trypsin-like proteases specific to phytopathogens are directly involved in the pathogenetic process, probably, through interaction with the "sentry" protein or the product of the resistance gene.

Two post-proline cleaving peptidases PPCP1 and PPCP2 with molecular masses of 101 and 63 kDa, respectively, hydrolyzing Z-AlaAlaPro-pNA were isolated for the first time from the larval midgut of the yellow mealworm Tenebrio molitor and characterized. PPCP1 was active only in acidic media, with a maximum at pH5.6, whereas PPCP2, both in acidic and alkaline media with a maximum at pH 7.9. Using inhibitory analysis, both PPCP1 and PPCP2 were shown to belong to serine peptidases. The data obtained indicate that a Cys residue is located close to the PPCP2 substrate binding site. Z-Pro-prolinal, a specific inhibitor of prolyl oligopeptidases, completely inhibited PPCP2 and partially PPCP1. The substrate specificities of the isolated enzymes were studied. Z-Ala-Ala-Pro-pNA was the best substrate for PPCP1, and Z-Ala-Pro-pNA, for PPCP2. The combination of the properties allows characterization of PPCP2 as a prolyl oligopeptidase.

EFFECT OF BIVALENT CATIONS ON THE INTERACTION OF TRANSKETOLASE WITH ITS DONOR SUBSTRATE.
Sevostyanova I.A., Yurshev V.A., Solovjeva O.N., Zabrodskaya S.V., Kochetov G.A.
PROTEINS: STRUCTURE, FUNCTION, AND BIOINFORMATICS 71 (2008) 541-545.

The effect of the type of the cation cofactor of transketolase (i.e., Ca²⁺ or Mg²⁺) on its interaction with xylulose 5-phosphate (donor substrate) has been studied. In the presence of magnesium, the active centers of the enzyme were functionally equivalent with respect to xylulose 5-phosphate binding and exhibited identical affinities for the donor substrate. Substitution of Ca²⁺ for Mg²⁺ results in the loss of the equivalence. In particular, this becomes apparent on binding of xylulose 5-phosphates to one of the two active centers of the enzyme, which caused the second center to undergo a several fold decrease in the affinity for the donor substrate.

Maturation of immature bovine oocytes requires cytoplasmic polyadenylation and synthesis of a number of proteins involved in meiotic progression and metaphase-II arrest. Aurora serine-threonine kinases-localized in centrosomes, chromosomes, and midbody-regulate chromosome segregation and cytokinesis in somatic cells. in frog and mouse oocytes, Aurora A regulates polyadenylation-dependent translation of several mRNAs such as MOS and CCNB1, presumably by phosphorylating CPEB, and Aurora B phosphorylates histone H3 during meiosis. We analyzed the expression of three Aurora kinase genes-AURKA, AURKB, and AURKC-in bovine oocytes during meiosis by reverse transcription followed by quantitative real-time PCR and immunodetection. Aurora A was the most abundant form in oocytes, both at mRNA and protein levels. AURKA protein progressively accumulated in the oocyte cytoplasm during antral follicle growth and in vitro maturation. AURKB associated with metaphase chromosomes. AURKB, AURKC, and Thr-phosphorylated AURKA were detected at a contractile ring/midbody during the first polar body extrusion. CPEB, localized in oocyte cytoplasm, was hyperphosphorylated during prophase/metaphase-I transition. Most CPEB degraded in metaphase-II oocytes and remnants remained localized in a contractile ring. Roscovitine, U0126, and metformin inhibited meiotic divisions; they all induced a decrease of CCNB1 and phospho-MAPK3/1 levels and prevented CPEB degradation. However, only metformin depleted AURKA. The Aurora kinase inhibitor VX680 at 100 nmol/L did not inhibit meiosis but led to multinuclear oocytes due to the failure of the polar body extrusion. Thus, in bovine oocyte meiosis, massive destruction of CPEB accompanies metaphase-I/II transition, and Aurora kinases participate in regulating segregation of the chromosomes, maintenance of metaphase-II, and formation of the first polar body.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDs) was purified from human sperms and properties of the enzyme were investigated. After sonication of sperms, the most part of GAPDs is associated with the insoluble cell fraction. Trypsin treatment results in the cleavage of part of the N-terminal domain of the enzyme yielding a soluble fragment that was purified by hydrophobic chromatography on Phenyl-Sepharose. The isolated fragment was shown to be a tetramer with molecular weight of approximately 150 kD (according to Blue Native PAGE) and composed of subunits of 40 kD (according to SDS- PAGE). The specific activity of the isolated fragment reached 374 U/mg. It is supposed that GAPDs exists in sperms as the tetrameric molecule bound to the fibrous sheath of the flagellum through the N-terminus of one or two subunits. Comparative study of the amino acid sequences of mammalian GAPDs revealed conservative cysteine residues (C21, C94, and C150) that are specific for the sperm isoenzyme and absent in the somatic isoenzyme. Residue C21 can be involved in the formation of the disulfide bond between the N-terminal domain of GAPDs and fibrous sheath proteins.

The yeast phosphatidylinositol 4-kinase Pik1p is essential for proliferation, and it controls Golgi homeostasis and transport of newly synthesized proteins from this compartment. At the Golgi, phosphatidylinositol 4-phosphate recruits multiple cytosolic effectors involved in formation of post-Golgi transport vesicles. A second pool of catalytically active Pik1p localizes to the nucleus. The physiological significance and regulation of this dual localization of the lipid kinase remains unknown. Here, we show that Pik1p binds to the redundant 14-3-3 proteins Bmh1p and Bmh2p. We provide evidence that nucleocytoplasmic shuttling of Pik1p involves phosphorylation and that 14-3-3 proteins bind Pik1p in the cytoplasm. Nutrient deprivation results in relocation of Pik1p from the Golgi to the nucleus and increases the amount of Pik1p-14-3-3 complex, a process reversed upon restored nutrient supply. These data suggest a role of Pik1p nucleocytoplasmic shuttling in coordination of biosynthetic transport from the Golgi with nutrient signaling.

A green procedure for the kinetic resolution of chiral amines via enzymatic acylation and deacylation has been demonstrated. The fully enzymatic approach obviates the common, waste-generating deacylation under strongly alkaline conditions. The acylating agent was (R)-phenylglycine propyl ester in combination with Candida antarctica lipase B as acylation catalyst. The enantiomerically enriched amides were subsequently deacylated in the presence of the penicillin G acylase from Alcaligenes faecalis. The degree of enantiomer recognition by CaLB in the acylation of aliphatic amines was unexpectedly modest, but a considerable further enantiomeric enrichment could be accomplished in the course of the subsequent enzymatic hydrolysis step.

THERMODYNAMIC AND KINETIC STABILITY OF PENICILLIN ACYLASE FROM Escherichia coli.
Grinberg V.Ya., Burova T.V., Grinberg N.V., Shcherbakova T.A., Guranda D.T., Chilov G.G., Svedas V.K.
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 1784 (2008) 736-746.

Thermal denaturation of penicillin acylase (PA) from Escherichia coli has been studied by high-sensitivity differential scanning calorimetry as a function of heating rate, pH and urea concentration. It is shown to be irreversible and kinetically controlled. Upon decrease in the heating rate from 2 to 0.1 K min(-1) the denaturation temperature of PA at pH 6.0 decreases by about 60C, while the denaturation enthalpy does not change notably giving an average value of 31.6 ± 2.1 J g(-1). The denaturation temperature of PA reaches a maximum value of 64.50C at pH 6.0 and decreases by about of 150C at pH 3.0 and 9.5. The pH induced changes in the denaturation enthalpy follow changes in the denaturation temperature. Increasing the urea concentration causes a decrease in both denaturation temperature and enthalpy of PA, where denaturation temperature obeys a linear relation. The heat capacity increment of PA is not sensitive to the heating rate, nor to pH, and neither to urea. Its average value is of 0.58 ± 0.02 J g(-1) K-1. The denaturation transition of PA is approximated by the Lumry-Eyring model. The first stage of the process is assumed to be a reversible unfolding of the a-subunit. It activates the second stage involving dissociation of two subunits and subsequent denaturation of the beta-subunit. This stage is irreversible and kinetically controlled. Using this model the temperature, enthalpy and free energy of unfolding of the alpha-subunit, and a rate constant of the irreversible stage are determined as a function of pH and urea concentration. Structural features of the folded and unfolded conformation of the alpha-subunit as well as of the transition state of the PA denaturation in aqueous and urea solutions are discussed.

PHYLOGENY AND SYSTEMATICS OF THE GENUS Lophozia s. str. (Dumort.) Dumort. (Hepaticae) AND RELATED TAXA FROM NUCLEAR ITS1-2 AND CHLOROPLAST trnL-F SEQUENCES.
Vilnet A.A., Konstantinova N.A., Troitsky A.V.
MOLECULAR PHYLOGENETICS AND EVOLUTION  47 (2008) 403-418.

Nuclear ITS1-2 and chloroplast trnL-F were sequenced for 21 taxa, of Lophozia s. str., two species of Protolophozia, five species of Schistochilopsis, three species of Barbilophozia and Obtusifolium obtusum. The topologies of phylogenetic trees for 49 taxa constructed from combined sequences of these regions by maximum parsimony, maximum likelihood and Bayesian methods are similar. The species of Lophozia s. str., excluding Lophozia sudetica, combine into two main clades and these contradict subdivisions of Lophozia s. str. based on morphology. The species status of Lophozia lantratoviae is confirmed, whereas Lophozia austro-sibirica is almost identical to Lophozia ventricosa var. guttulata. The genus Schistochilopsis is paraphyletic and occupies basal position to Lophozia s. str., while O. obtusum is clearly separated from Schistochilopsis. A low level of divergence was found between L. sudetica and Protolophozia debiliformes, which are closer to Barbilophozia than to Lophozia s. str. Molecular divergence between geographically remote populations of L. sudetica, Lophozia silvicoloides and Protolophozia debiliformis are low as opposed to those of Lophozia polaris, Lophozia pellucida or Lophozia excisa. Consideration of the trnL intron P8 region indels alone can adequately assign some clades revealed by tree building. A consensus secondary structure of the trnL intron P8 region could not be inferred for taxa studied mainly due to high sequence length diversity originated from deletions.

SYNTHESIS AND CHARACTERISTICS OF MODIFIED DNA FRAGMENTS CONTAINING THYMIDINE GLYCOL RESIDUES.
Fedotova E.A., Yang F., Kubareva E.A., Romanova E.A., Protsenko A.S., Viryasov M.B., Hianik T., Oretskaya T.S.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY 34 (2008) 215-222.

Chemical synthesis of a series of modified oligodeoxyribonucleotides containing one or two residues of thymidine glycol (5,6- dihydro-5,6-dihydroxythymidine), the main product of oxidative DNA damage, is described. The thermal stability of DNA duplexes containing thymidine glycol residues was studied using UV spectroscopy. Introduction of even one thymidine glycol residue into the duplex structure was shown to result in its significant destabilization. Data on the interaction of DNA methyltransferases and type II restriction endonucleases with DNA ligands containing oxidized thymine were obtained for the first time. Introduction of a thymidine glycol residue in the central degenerate position of the recognition site of restriction endonuclease SsoII was found to result in an increase in the initial hydrolysis rate of the modified duplex in comparison with that of unmodified structure. The affinity of C5-cytosine methyltransferase SsoII for the DNA duplex bearing thymidine glycol was found to be twofold higher than for the unmodified substrate. However, such a modification of the DNA ligand prevents its methylation.

The imidazoline BL11282 stimulates insulin release and alters islet proteomes. Subcellular fractions of MIN6 cells showed that the membrane fraction exhibited binding to BL11282 on a Biacore chip and to BL11282-labelled magnetic beads. Bound material extracted from the beads showed a similar to 50 kDa differential band upon SDS-PAGE and a weaker 100 kDa band. The former was sensitive to competitive removal by preincubation of the fraction with BL11282, then highlighting the similar to 100 kDa band. Masspectrometric analysis revealed the similar to 50 kDa band to be EF1A and the similar to 100 kDa band to be glucose regulated P94, both of interest in insulin synthesis and secretion.

Activated lymphocytes and lymphoid-tis centrifugation through a 30% sucrose cushion and the pellet (P1) was resuspended and sedimented through a 5 - 40% sucrose gradient. The gradient separation resulted in two different virus particle populations: a virus fraction (F) that formed a band in the gradient and one that formed a pellet (P2) at the bottom of the gradient. All three preparations contained infectious particles that retained their integrity when visualized by electron microscopy (EM). Western blotting of the P1 particles revealed that the viral RNA helicase, cylindrical inclusion protein (CI), co-purified with virus particles. This result was confirmed with co-immunoprecipitation experiments. Cl was detected in P2 particle preparations, whereas F particles were devoid of detectable amounts of Cl. ATPase activity was detected in all three preparations with the greatest amount in P2. Results from immunogold-labelling EM experiments suggested that a fraction of the Cl present in the preparations was localized to one end of the virion. Atomic force microscopy (AFM) studies showed that P1 and P2 contained intact particles, some of which had a protruding tip structure at one end, whilst F virions were less stable and mostly appeared as beaded structures under the conditions of AFM. The RNA of the particles in F was translated five to ten times more efficiently than RNA from P2 particles when these preparations were subjected to translation in wheat-germ extracts. The results are discussed in the context of a model for Cl-mediated functions.

FACTOR REQUIREMENTS FOR TRANSLATION INITIATION ON THE SIMIAN PICORNAVIRUS INTERNAL RIBOSOMAL ENTRY SITE.
De Breyne, S., Yu Y., Pestova T.V., Hellen C.U.T.
RNA-A PUBLICATION OF THE RNA SOCIETY 14 (2008) 367-380.

The Simian picornavirus type 9 (SPV9) 5'-untranslated region (5' UTR) has been predicted to contain an internal ribosomal entry site (IRES) with structural elements that resemble domains of hepacivirus/pestivirus (HP) IRESs. In vitro reconstitution of initiation confirmed that this 5' UTR contains an IRES and revealed that it has both functional similarities and differences compared to HP IRESs. Like HP IRESs, the SPV9 IRES bound directly to 40S subunits and eukaryotic initiation factor (eIF) 3, depended on the conserved domain IIId for ribosomal binding and consequently for function, and additionally required eIF2/ initiator tRNA to yield 48S complexes that formed elongation-competent 80S ribosomes in the presence of eIF5, eIF5B, and 60S subunits. Toeprinting analysis revealed that eIF1A stabilized 48S complexes, whereas eIF1 induced conformational changes in the 40S subunit, likely corresponding to partial opening of the entry latch of the mRNA-binding channel, that were exacerbated by eIF3 and suppressed by eIF1A. The SPV9 IRES differed from HP IRESs in that its function was enhanced by eIF4A/eIF4F when the [RES was adjacent to the wild-type coding sequence, but was less affected by these factors or by a dominant negative eIF4A mutant when potentially less structured coding sequences were present. Exceptionally, this IRES promoted binding of initiator tRNA to the initiation codon in the P site of 40S subunits independently of eIF2. Although these 40S/IRES/tRNA complexes could not form active 80S ribosomes, this constitutes a second difference between the SPV9 and HP IRESs. eIF1 destabilized the eIF2-independent ribosomal binding of initiator tRNA.

The membrane-spanning protein TGBp3 is one of the three movement proteins (MPs) of Poa semilatent virus. TGBp3 is thought to direct other viral MPs and genomic RNA to peripheral bodies located in close proximity to plasmodesmata. We used the ectopic expression of green fluorescent protein-fused TGBp3 in epidermal cells of Nicotiana benthamia  

The position of mRNA on 40S ribosomal subunits in eukaryotic initiation complexes was determined by UV crosslinking using mRNAs containing uniquely positioned 4-thiouridines. Crosslinking of mRNA positions (+)11 to ribosomal protein (rp) rpS2(S5p) and rpS3(S3p), and (+)9-(+)11 and (+)8-(+)9 to h18 and h34 of 18S rRNA, respectively, indicated that mRNA enters the mRNA-binding channel through the same layers of rRNA and proteins as in prokaryotes. Upstream of the P-site, the proximity of positions (-)3/(-)4 to rpS5(S7p) and h23b,-6/(-)7 to rpS14(S11p), and -8-(-)11 to the 3'-terminus of 18S rRNA (mRNA/ rRNA elements forming the bacterial Shine-Dalgarno duplex) also resembles elements of the bacterial mRNA path. In addition to these striking parallels, differences between mRNA paths included the proximity in eukaryotic initiation complexes of positions (+)7/(+)8 to the central region of h28, (+)4/(+)5 to rpS15(S19p), and F-6 and-7/(-)10 to eukaryote- specific rpS26 and rpS28, respectively. Moreover, we previously determined that eukaryotic initiation factor2 alpha (eIF2 alpha) contacts position-3, and now report that eIF3 interacts with positions (-)8-(-)17, forming an extension of the mRNA- binding channel that likely contributes to unique aspects of eukaryotic initiation.

Unlike bacteria, a specialized eukaryotic initiation factor (eIF)-2, in the form of the ternary complex eIF2-GTP-Met- tRNA(i)(Met), is used to deliver the initiator tRNA to the ribosome in all eukaryotic cells. Here we show that the hepatitis C virus (HCV) internal ribosome entry site (IRES) can direct translation without eIF2 and its GTPase-activating protein eIF5. In addition to the general eIF2- and eIF5-dependent pathway of 80S complex assembly, the HCV IRES makes use of a bacterial- like pathway requiring as initiation factors only eIF5B (an analog of bacterial IF2) and eIF3. The switch from the conventional eukaryotic mode of translation initiation to the eIF2-independent mechanism occurs when eIF2 is inactivated by phosphorylation under stress conditions.

The DHH superfamily human protein h-prune, a binding partner of the metastasis suppressor nm23-H1, is frequently overexpressed in metastatic cancers. From an evolutionary perspective, h-prune is very close to eukaryotic exopolyphosphatases. Here, we show for the first time that h-prune efficiently hydrolyzes short-chain polyphosphates (kcat of 3-40 s-1), including inorganic tripoly- and tetrapolyphosphates and nucleoside 5'-tetraphosphates. Long-chain inorganic polyphosphates (? 25 phosphate residues) are converted more slowly, whereas pyrophosphate and nucleoside triphosphates are not hydrolyzed. The reaction requires a divalent metal cofactor, such as Mg²⁺, Co²⁺, or Mn²⁺, which activates both the enzyme and substrate. Notably, the exopolyphosphatase activity of h-prune is suppressed by nm23-H1, long-chain polyphosphates and pyrophosphate, which may be potential physiological regulators. Nucleoside triphosphates, diadenosine hexaphosphate, cAMP, and dipyridamole (inhibitor of phosphodiesterase) do not affect this activity. Mutation of seven single residues corresponding to those found in the active site of yeast exopolyphosphatase led to a severe decrease in h-prune activity, whereas one variant enzyme exhibited enhanced activity. Our results collectively Suggest that prune is the missing exopolyphosphatase in animals and support the hypothesis that the metastatic effects of h-prune are modulated by inorganic polyphosphates, which are increasingly recognized as critical regulators in cells.

Preparations with different contents of thermolysin were obtained by the immobilization of the enzyme on granulated polyvinyl alcohol cryogel. Their activity and stability in an aqueous medium and in mixtures of polar organic solvents of different composition were investigated. The catalytic properties of the preparations in reactions of peptide bond formation were studied, and the optimal amount of the biocatalyst, the concentrations of initial reagents, and the ratios of organic solvents and water necessary for effective enzymatic peptide synthesis catalyzed by immobilized thermolysin were determined. A series of peptides of the general formula Z-Ala-Ala-Xaa-pNA, where Xaa = Leu, Ile, Phe, Val, or Ala, were synthesized, and the immobilized enzyme was shown to retain substrate specificity in an organic medium.

The PPi-driven sodium pump (membrane pyrophosphatase) of Methanosarcina mazei (Mm-PPase) absolutely requires Na⁺ and Mg²⁺ for activity and additionally employs K⁺ as a modulating cation. Here we explore relationships among Na⁺, K⁺, Mg²⁺, and PPi binding sites by analyzing the dependency of the Mm-PPase PPi-hydrolyzing function on these ligands and protection offered by the ligands against Mm-PPase inactivation by trypsin and the SH-reagent mersalyl. Steady-state kinetic analysis of PPi hydrolysis indicated that catalysis involves random order binding of two Mg²⁺ ions and two Na⁺ ions, and the binding was almost independent of substrate (Mg²⁺PPi complex) attachment. Each pair of metal ions, however, binds in a positively cooperative (or ordered) manner. The apparent cooperativity is lost only when Na⁺ binds to preformed enzyme- Mg²⁺-substrate complex. The binding of K⁺ increases, by a factor of 2.5, the catalytic constant, the Michaelis constant, and the Mg²⁺ binding affinity, and these effects may result from K⁺ binding to either one of the Na⁺ sites or to a separate site. The effects of ligands on Mm-PPase inactivation by mersalyl and trypsin are highly correlated and are strongly indicative of ligand-induced enzyme conformational changes. Importantly, Na⁺ binding induces a conformational change only when completing formation of the catalytically competent enzyme-substrate complex or a similar complex with a diphosphonate substrate analog. These data indicate considerable flexibility in Mm-PPase structure and provide evidence for its cyclic change in the course of catalytic turnover.

BIOSYNTHESIS OF LEUKOTRIENE B4 IN HUMAN POLYMORPHONUCLEAR LEUKOCYTES: REGULATION BY CHOLESTEROL AND OTHER LIPIDS.
Zagryagskaya A.N., Aleksandrov D.A., Pushkareva M.A., Galkina S.I., Grishina Z.V., Sud'ina G.F.
 JOURNAL OF IMMUNOTOXICOLOGY 5 (2008) 347-352.

Leukotriene B4 (LTB4) is one of the most potent chemotactic compounds produced in macrophages and neutrophils. LTB4 is a product of arachidonic acid oxygenation by 5-lipoxygenase pathway. We present here the data on regulation of LT synthesis in human polymorphonuclear leukocytes by cholesterol, cholesterol sulfate and cholesterol phosphate. The addition of Pseudomonas aeruginosa lipopolysaccharides (LPS) with lipid vesicles containing phosphatidylcholine or phosphatidylcholine/cholesterol (70:30) showed that omitting cholesterol abolished the effect of LPS on LT synthesis. We show here the capacity of cholesterol sulfate, the most abundant sulfated sterol in human blood, to suppress LT production in human neutrophils and to neutralize the effect of P. aeruginosa LPS on LT synthesis. We suggest that sulfated lipids serve as specific endogenous regulators of LT synthesis in neutrophils, and anti-inflammatory therapy may be based on modification of cholesterol level and its conversion to anionic derivatives.

This paper presents a kinetic model of phosphofructokinase-1 from Escherichia coli. A complete catalytic cycle has been reconstructed based on available information on the oligomeric structure of the enzyme and kinetic mechanism of its monomer. Applying the generalization of the Monod-Wyman-Changeux approach proposed by Popova and Sel'kov35-37 to the reconstructed catalytic cycle rate equation has been derived. Dependence of the reaction rate on pH, magnesium, and effectors has been taken into account. Kinetic parameters have been estimated via fitting the rate equation against experimentally measured dependencies of initial rate on substrates, products, effectors, and pH available from the literature. The model of phosphofructokinase-1 predicts (1) cooperativity of binding both fructose-6-phosphate and ATPMg2-, (2) significant inhibition of the enzyme resulting from an increase in total concentration of ATP under the condition of fixed concentration of Mg²⁺ ions, and (3) dual effect of ADP consisting of allosteric activation and product inhibition of the enzyme. Moreover, the model developed can be used in the kinetic modeling of biochemical pathways containing phosphofructokinase-1.

A family of kinetic models has been developed that takes into account available experimental information on the regulation of ace operon expression in Escherichia coli. This has allowed us to study and analyze possible versions of regulation of the ace operon and to test their possibilities. Based on literature analysis, we found that there is an ambiguity of properties of IclR (main repressor of ace operon). The main aspect of this ambiguity are two different forms of IclR purified from E. coli K strain and different coeffector sets for IclR purified from E. coli K and B strains. It has been shown that the full-length form of IclR is physiologically relevant and that IclR truncation is a result of purification of the protein from  K strains. We also found that the IclR protein purified from  B strain carries two coeffector binding sites. Using model-developed levels of steady state aceBAK expression against physiologicalranges of coeffectors, concentration has been predicted.

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

MITOCHONDRIA-TARGETED PLASTOQUINONE DERIVATIVES AS TOOLS TO INTERRUPT EXECUTION OF THE AGING PROGRAM. 1. CATIONIC PLASTOQUINONE DERIVATIVES: SYNTHESIS AND in vitro STUDIES.
Antonenko Y.N., Avetisyan A.V., Bakeeva L.E., Chernyak B.V., Chertkov V.A., Domnina L.V., Ivanova O.Y., Izyumov D.S., Khailova L.S., Klishin S.S., Korshunova G.A., Lyamzaev K.G., Muntyan M.S., Nepryakhina O.K., Pashkovskaya A.A., Pletjushkina O.Y., Pustovidko A.V., Roginsky V.A., Rokitskaya T.I., Ruuge E.K., Saprunova V.B., Severina I.I., Simonyan R.A., Skulachev I.V., Skulachev M.V., Sumbatyan N.V., Sviryaeva I.V., Tashlitsky V.N., Vassiliev J.M., Vyssokikh M.Y., Yaguzhinsky L.S., Zamyatnin A.A. Jr, Skulachev V.P.
BIOCHEMISTRY-MOSCOW 73 (2008) 1273-1287.

Synthesis of cationic plastoquinone derivatives (SkQs) containing positively charged phosphonium or rhodamine moieties connected to plastoquinone by decane or pentane linkers is described. It is shown that SkQs (i) easily penetrate through planar, mitochondrial, and outer cell membranes, (ii) at low (nanomolar) concentrations, posses strong antioxidant activity in aqueous solution, BLM, lipid micelles, liposomes, isolated mitochondria, and cells, (iii) at higher (micromolar) concentrations, show pronounced prooxidant activity, the "window" between anti- and prooxidant concentrations being very much larger than for MitoQ, a cationic ubiquinone derivative showing very much lower antioxidant activity and higher prooxidant activity, (iv) are reduced by the respiratory chain to SkQH2, the rate of oxidation of SkQH2 being lower than the rate of SkQ reduction, and (v) prevent oxidation of mitochondrial cardiolipin by OH*. In HeLa cells and human fibroblasts, SkQs operate as powerful inhibitors of the ROS-induced apoptosis and necrosis. For the two most active SkQs, namely SkQ1 and SkQR1, C1/2 values for inhibition of the H2O2-induced apoptosis in fibroblasts appear to be as low as 1x10-11 and 8x10-13 M, respectively. SkQR1, a fluorescent representative of the SkQ family, specifically stains a single type of organelles in the living cell, i.e. energized mitochondria. Such specificity is explained by the fact that it is the mitochondrial matrix that is the only negatively-charged compartment inside the cell. Assuming that the Deltapsi values on the outer cell and inner mitochondrial membranes are about 60 and 180 mV, respectively, and taking into account distribution coefficient of SkQ1 between lipid and water (about 13,000 : 1), the SkQ1 concentration in the inner leaflet of the inner mitochondrial membrane should be 1.3x108 times higher than in the extracellular space. This explains the very high efficiency of such compounds in experiments on cell cultures. It is concluded that SkQs are rechargeable, mitochondria-targeted antioxidants of very high efficiency and specificity. Therefore, they might be used to effectively prevent ROS-induced oxidation of lipids and proteins in the inner mitochondrial membrane in vivo.

MITOCHONDRIA-TARGETED PLASTOQUINONE DERIVATIVES AS TOOLS TO INTERRUPT EXECUTION OF THE AGING PROGRAM. 2. TREATMENT OF SOME ROS- AND AGE-RELATED DISEASES (HEART ARRHYTHMIA, HEART INFARCTIONS, KIDNEY ISCHEMIA, AND STROKE).
Bakeeva L.E., Barskov I.V., Egorov M.V., Isaev N.K., Kapelko V.I., Kazachenko A.V., Kirpatovsky V.I., Kozlovsky S.V., Lakomkin V.L., Levina S.B., Pisarenko O.I., Plotnikov E.Y., Saprunova V.B., Serebryakova L.I., Skulachev M.V., Stelmashook E.V., Studneva I.M., Tskitishvili O.V., Vasilyeva A.K., Victorov I.V., Zorov D.B., Skulachev V.P.

Effects of 10-(6'-plastoquinonyl) decyltriphenylphosphonium (SkQ1) and 10-(6'-plastoquinonyl) decylrhodamine 19 (SkQR1) on rat models of H2O2- and ischemia-induced heart arrhythmia, heart infarction, kidney ischemia, and stroke have been studied ex vivo and in vivo. In all the models listed, SkQ1 and/or SkQR1 showed pronounced protective effect. Supplementation of food with extremely low SkQ1 amount (down to 0.02 nmol SkQ1/kg per day for 3 weeks) was found to abolish the steady heart arrhythmia caused by perfusion of isolated rat heart with H2O2 or by ischemia/reperfusion. Higher SkQ1 (125-250 nmol/kg per day for 2-3 weeks) was found to decrease the heart infarction region induced by an in vivo ischemia/reperfusion and lowered the blood levels of lactate dehydrogenase and creatine kinase increasing as a result of ischemia/reperfusion. In single-kidney rats, ischemia/reperfusion of the kidney was shown to kill the majority of the animals in 2-4 days, whereas one injection of SkQ1 or SkQR1 (1 micromol/kg a day before ischemia) saved lives of almost all treated rats. Effect of SkQR1 was accompanied by decrease in ROS (reactive oxygen species) level in kidney cells as well as by partial or complete normalization of blood creatinine and of some other kidney-controlled parameters. On the other hand, this amount of SkQ1 (a SkQ derivative of lower membrane-penetrating ability than SkQR1) saved the life but failed to normalize ROS and creatinine levels. Such an effect indicates that death under conditions of partial kidney dysfunction is mediated by an organ of vital importance other than kidney, the organ in question being an SkQ1 target. In a model of compression brain ischemia/reperfusion, a single intraperitoneal injection of SkQR1 to a rat (1 micromol/kg a day before operation) effectively decreased the damaged brain area. SkQ1 was ineffective, most probably due to lower permeability of the blood-brain barrier to this compound.

It was proposed that increased level of mitochondrial reactive oxygen species (ROS), mediating execution of the aging program of an organism, could also be critical for neoplastic transformation and tumorigenesis. This proposal was addressed using new mitochondria-targeted antioxidant SkQ1 (10-(6?-plastoquinonyl) decyltriphenylphosphonium) that scavenges ROS in mitochondria at nanomolar concentrations. We found that diet supplementation with SkQ1 (5 nmol/kg per day) suppressed spontaneous development of tumors (predominantly lymphomas) in p53-/- mice. The same dose of SkQ1 inhibited the growth of human colon carcinoma HCT116/p53-/- xenografts in athymic mice. Growth of tumor xenografts of human HPV-16- associated cervical carcinoma SiHa was affected by SkQ1 only slightly, but survival of tumor-bearing animals was increased. It was also shown that SkQ1 inhibited the tumor cell proliferation, which was demonstrated for HCT116 p53-/- and SiHa cells in culture. Moreover, SkQ1 induced differentiation of various tumor cells in vitro. Coordinated SkQ1-initiated changes in cell shape, cytoskeleton organization, and E-cadherin-positive intercellular contacts were observed in epithelial tumor cells. In Ras- and SV40-transformed fibroblasts, SkQ1 was found to initiate reversal of morphological transformation of a malignant type, restoring actin stress fibers and focal adhesion contacts. SkQ1 suppressed angiogenesis in Matrigel implants, indicating that mitochondrial ROS could be important for tumor angiogenesis. This effect, however, was less pronounced in HCT116/p53-/- tumor xenografts. We have also shown that SkQ1 and related positively charged antioxidants are substrates of the P- glycoprotein multidrug resistance pump. The lower anti-tumor effect and decreased intracellular accumulation of SkQ1, found in the case of HCT116 xenografts bearing mutant forms of p53, could be related to a higher level of P-glycoprotein. The effects of traditional antioxidant N-acetyl-L-cysteine (NAC) on tumor growth and tumor cell phenotype were similar to the effects of SkQ1 but more than 1,000,000 times higher doses of NAC than those of SkQ1 were required. Extremely high efficiency of SkQ1, related to its accumulation in the mitochondrial membrane, indicates that mitochondrial ROS production is critical for tumorigenesis at least in some animal models.

Mitochondria-targeted cationic plastoquinone derivative SkQ1 (10-(6?-plastoquinonyl) decyltriphenylphosphonium) has been investigated as a potential tool for treating a number of ROS-related ocular diseases. In OXYS rats suffering from a ROS- induced progeria, very small amounts of SkQ1 (50 nmol/kg per day) added to food were found to prevent development of age_induced cataract and retinopathies of the eye, lipid peroxidation and protein carbonylation in skeletal muscles, as well as a decrease in bone mineralization. Instillation of drops of 250 nM SkQ1 reversed cataract and retinopathies in 3-12-month-old (but not in 24-month-old) OXYS rats. In rabbits, experimental uveitis and glaucoma were induced by immunization with arrestin and injections of hydroxypropyl methyl cellulose to the eye anterior sector, respectively. Uveitis was found to be prevented or reversed by instillation of 250 nM SkQ1 drops (four drops per day). Development of glaucoma was retarded by drops of 5 ?M SkQ1 (one drop daily). SkQ1 was tested in veterinarian practice. A totally of 271 animals (dogs, cats, and horses) suffering from retinopathies, uveitis, conjunctivitis, and cornea diseases were treated with drops of 250 nM SkQ1. In 242 cases, positive therapeutic effect was obvious. Among animals suffering from retinopathies, 89 were blind. In 67 cases, vision returned after SkQ1 treatment. In ex vivo studies of cultivated posterior retina sector, it was found that 20 nM SkQ1 strongly decreased macrophagal transformation of the retinal pigmented epithelial cells, an effect which might explain some of the above SkQ1 activities. It is concluded that low concentrations of SkQ1 are promising in treating retinopathies, cataract, uveitis, glaucoma, and some other ocular diseases.

MITOCHONDRIA-TARGETED PLASTOQUINONE DERIVATIVES AS TOOLS TO INTERRUPT EXECUTION OF THE AGING PROGRAM. 5. SkQ1 PROLONGS LIFESPAN AND PREVENTS DEVELOPMENT OF TRAITS OF SENESCENCE.
Anisimov V.N., Bakeeva L.E., Egormin P.A., Filenko O.F., Isakova E.F., Manskikh V.N., Mikhelson V.M., Panteleeva A.A., Pasyukova E.G., Pilipenko D.I., Piskunova T.S., Popovich I.G., Roshchina N.V., Rybina O.Yu., Saprunova V.B., Samoylova T.A., Semenchenko A.V., Skulachev M.V., Spivak I.M., Tsybul'ko E.A., Tyndyk M.L., Vyssokikh M.Yu., Yurova M.N., Zabezhinsky M.A., Skulachev V.P.
BIOCHEMISTRY-MOSCOW 73 (2008) 1329-1342.

Very low (nano- and subnanomolar) concentrations of 10-(6?-plastoquinonyl) decyltriphenylphosphonium (SkQ1) were found to prolong lifespan of a fungus (Podospora anserina), a crustacean (Ceriodaphnia affinis), an insect (Drosophila melanogaster), and a mammal (mouse). In the latter case, median lifespan is doubled if animals live in a non-sterile vivarium. The lifespan increase is accompanied by rectangularization of the survival curves (an increase in survival is much larger at early than at late ages) and disappearance of typical traits of senescence or retardation of their development. Data summarized here and in the preceding papers of this series suggest that mitochondria-targeted antioxidant SkQ1 is competent in slowing down execution of an aging program responsible for development of age-related senescence.

Proton diffusion along the surface of a planar bilayer lipid membrane was measured by means of acid/base injection with a micropipette and recording of the kinetics of fluorescence changes of fluorescein-labelled lipid on the surface. The dimensionality of the process was assayed by fitting the kinetic curves with two-dimensional (2D) or three-dimensional (3D) diffusion equations. In agreement with Serowy et al. (Biophys J 84:1031-1037, 2003), lateral proton diffusion proceeded via bulk phase by means of buffer molecules as proton carriers (D = 600 microm2/s) under the conditions of 1 mM buffer in the solution. Introduction of proton binding sites on the membrane surface led to the appearance of a considerable contribution of two-dimensional proton diffusion on the membrane surface with D = 1,100 mum2/s. The system described can be used to study the dependence of the proton diffusion rate on the phospholipid and protein composition of the membrane.

The antioxidant activity of mitochondria-targeted small molecules, SkQ1 and MitoQ (conjugates of a lipophilic decyltriphenylphosphonium cation with an antioxidant moiety of a plastoquinone and ubiquinone, respectively), was studied in aqueous solution and in a lipid environment, i.e., micelles, liposomes and planar bilayer lipid membranes. Reactive oxygen species (ROS) were generated by azo initiators or ferrous ions with or without tert-butyl-hydroperoxide (t-BOOH). Chemiluminescence, fluorescence, oxygen consumption and inactivation of gramicidin peptide channels were measured to detect antioxidant activity. In all of the systems studied, SkQ1 was shown to effectively scavenge ROS. The scavenging was inherent to the reduced form of the quinone (SkQ1H2). In the majority of the above model systems, SkQ1 exhibited higher antioxidant activity than MitoQ. It is concluded that SkQ1H2 operates as a ROS scavenger in both aqueous and lipid environments, being effective at preventing ROS-induced damage to membrane lipids as well as membrane-embedded peptides.

Absorption and circular dichroism (CD) spectra of cytochrome bd from Escherichia coli have been compared for the wild type enzyme and an inactive mutant in which a highly conserved E445 in subunit I has been replaced by alanine [Zhang, J., Hellwig, P., Osborne, J. P., Huang, H. W., Moenne-Loccoz, P., Konstantinov, A.A., and Gennis, R. B. (2001) Biochemistry 40, 8548-8556]. The absorption bands of ferrous heme b₅₉₅ are absent from the spectrum of the dithionite-reduced E445A form of cytochrome bd. The difference between the spectra of the dithionite-reduced WT and E445A enzymes indicates that in the mutant, heme b₅₉₅ is present but is not reducible by dithionite. Cytochrome bd reveals intense CD signals dominated by heme d, with almost no contribution from heme b₅₉₅ or heme b558. The CD spectrum of the reduced wild type enzyme in the Soret band indicates strong excitonic interactions between ferrous heme d and ferrous heme b₅₉₅, and these interactions are not observed in dithionite-reduced E445A mutant, in which heme b₅₉₅ remains in the ferric state. Modeling the excitonic interactions in both absorption and CD spectra has been carried out, yielding an estimate of the Fe-to-Fe distance between heme d and heme b₅₉₅ of about 10 A. The physical proximity supports the hypothesis that heme d and heme b₅₉₅ can form a di- heme oxygen reducing site, a unique structure for respiratory oxidases.

Cytochrome bd is a terminal quinol:O2 oxidoreductase of the respiratory chain of Escherichia coli. The enzyme generates protonmotive force without proton pumping and contains three hemes, b₅₅₈, b₅₉₅, and d. A highly conserved glutamic acid residue of transmembrane helix III in subunit I, E107, was suggested to be part of a transmembrane pathway delivering protons from the cytoplasm to the oxygen-reducing site. When E107 is replaced with leucine, the hemes are retained but the ubiquinol- 1-oxidase activity is lost. We compared wild-type and E107L mutant enzymes during single turnover using absorption and electrometric techniques with a microsecond time resolution. Both wild-type and E107L mutant cytochromes ` in the fully reduced state bind O2 rapidly, but the formation of the oxoferryl species in the mutant is dramatically retarded as compared to the wild type. Intraprotein electron redistribution induced by the photolysis of CO bound to ferrous heme d in the one-electron- reduced wild-type enzyme is coupled to the membrane potential generation, whereas the mutant cytochrome bd shows no such potential generation. The E107L mutation also causes decrease of midpoint redox potentials of hemes b₅₉₅ and d by 25-30 mV and heme b558 by approximately 70 mV. There are two protonatable groups redox-linked to hemes b₅₉₅ and d in the active site, one of which has been recently identified as E445, whereas the second group remains unknown. Here we propose that E107 is either the second group or a key residue of a proposed proton delivery pathway leading from the cytoplasm toward this second group.

Many approaches to discovering the interaction energy of molecular transition dipoles use the well-known coefficient ?(?, ? 1 ? 2) = (cos ? ? 3 cos ? 1 cos ? 2)2, where ?, ? 1, and ? 2 are inter-dipole angles. Unfortunately, this formula often yields rather approximate results, in particular, when it is applied to closely positioned molecules. This problem is of great importance when dealing with energy migration in photosynthetic organisms, because the major part of excitation transfers in their chlorophyllous antenna proceed between closely positioned molecules. In this paper, the authors introduce corrected values of the orientation factor for several types of mutual orientation of molecules exchanging with electronic excitations for realistic ratios of dipole lengths and spacing. The corrected magnitudes of interaction energies of neighboring bacteriochlorophyll molecules in LH2 and LH1 light-absorbing complexes are calculated for the class of photosynthetic purple bacteria. Some advantageous factors are revealed in their mutual positions and orientations in vivo.

Discrepancy is revealed between the values of excitation transfer times measured experimentally, and those calculated, for the atomic structures of B800 --> B850 bacteriochlorophylls within the LH2 light-harvesting pigment-protein complex of the purple bacterium Rhodopseudomonas acidophila. The value 2.9-3.2 ps for the B800 --> B850 excitation transfer, calculated on the basis of atomic structure of LH2, is about 4-times longer than that measured for this bacterium (0.7 ps). This discrepancy appears common in at least two purple bacteria. Possible sources responsible for this discrepancy are discussed. It may either signify some drawback/s/ in our notions about the precise in vivo structure of LH2 complexes, for example, possible changes of LH2 structure during crystallization, or it may reflect our ignorance of some mechanisms involved in excitation migration.

Comparative studies on the interaction of the membrane-bound and detergent-solubilized forms of the enzyme in the fully reduced state with carbon monoxide at room temperature have been carried out. CO brings about a bathochromic shift of the heme d band with a maximum at 644 nm and a minimum at 624 nm, and a peak at 540 nm. In the Soret band, CO binding to cytochrome bd results in absorption decrease and minima at 430 and 445 nm. Absorption perturbations in the Soret band and at 540 nm occur in parallel with the changes at 630 nm and reach saturation at 3-5 microM CO. The peak at 540 nm is probably either ?-band of the heme d-CO complex or part of its split ?-band. In both forms of cytochrome bd, CO reacts predominantly with heme d. Addition of high CO concentrations to the solubilized cytochrome bd results in additional spectral changes in the gamma-band attributable to the reaction of the ligand with 10-15% of low-spin heme b558. High-spin heme b₅₉₅ does not bind CO even at high concentrations of the ligand. The apparent dissociation constant values for the heme d-CO complex of the membrane-bound and detergent-solubilized forms of the fully reduced enzyme are about 70 and 80 nM, respectively.

The catalytic properties of sodium-translocating NADH:quinone oxidoreductases (Na⁺-NQRs) from the marine bacterium Vibrio harveyi, the enterobacterium Klebsiella pneumoniae, and the soil microorganism Azotobacter vinelandii have been comparatively analyzed. It is shown that these enzymes drastically differ in their affinity to sodium ions. The enzymes also possess different sensitivity to inhibitors. Na⁺-NQR from A. vinelandii is not sensitive to low 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO) concentrations, while Na⁺-NQR from K. pneumoniae is fully resistant to either Ag⁺ or N-ethylmaleimide. All the Na⁺-NQR-type enzymes are sensitive to diphenyliodonium, which is shown to modify the noncovalently bound FAD of the enzyme.

An electrometrical technique was used to investigate flash-induced electron transfer reactions between Mn-depleted spinach photosystem II core particles incorporated into liposomes and redox mediators. Besides the fast increase in the transmembrane electric potential difference associated with electron transfer between the redox active tyrosine YZ and the primary quinone acceptor QA, an additional electrogenic phase was observed in the presence of N,N,N'N'-tetramethyl-p-phenylenediamine and 2,6-dichlorophenol-indophenol. The latter phase is attributed to vectorial electron transfer from the redox dye(s) to the protein- embedded YZ. The data obtained suggest an electrically isolated location of the YZ from the external water phase.

This review collects data on the influence of intracellular and extracellular acidosis on neuronal viability and the effect of acidosis on neuronal damage progressing under brain ischemia/hypoxia. Particular attention is devoted to the involvement of ionotropic glutamic receptors and acid-sensitive ion channel 1a in these processes.

Using a fluorescent probe for superoxide, hydroethidine, we have demonstrated that glucose deprivation (GD) activates production of reactive oxygen species (ROS) in cultured cerebellar granule neurons. ROS production was insensitive to the blockade of ionotropic glutamate channels by MK-801 (10 microM) and NBQX (10 microM). Inhibitors of mitochondrial electron transport, i.e. rotenone (complex I), antimycin A (complex III), or sodium azide (complex IV), an inhibitor of mitochondrial ATP synthase-oligomycin, an uncoupler of oxidative phosphorylation-CCCP, a chelator of intracellular Ca²⁺- BAPTA, an inhibitor of electrogenic mitochondrial Ca²⁺ transport-ruthenium red, as well as pyruvate significantly decreased neuronal ROS production induced by GD. GD was accompanied by a progressive decrease in the mitochondrial membrane potential and an increase in free cytosolic calcium ions, [Ca²⁺](i). Pyruvate, BAPTA, and ruthenium red lowered the GD- induced calcium overload, while pyruvate and ruthenium red also prevented mitochondrial membrane potential changes induced by GD. We conclude that GD-induced ROS production in neurons is related to potential-dependent mitochondrial Ca²⁺ overload. GD-induced mitochondrial Ca²⁺ overload in neurons in combination with depletion of energy substrates may result in the decrease of the membrane potential in these organelles.

It is known that an addition of FeSO4 in the presence of ascorbic acid to cells or mitochondria can injure energy coupling and some other functions in mitochondria. The present study demonstrates that decrease in ascorbate concentration from 4 to 0.2 mM in the presence of the same low concentrations of FeSO4 accelerates the nonspecific pore opening, while cyclosporin A prevents and under some conditions reverses the pore opening. Hydrophobic cations SkQ1 and MitoQ (structural analogs of plastoquinone and coenzyme Q10, respectively) delay pore opening, SkQ1 being more efficient. It is known that an increase in matrix ADP concentration delays pore opening, while an addition of carboxyatractylate to mitochondria accelerates the beginning of pore opening. Preliminary addition of SkQ1 into a mitochondrial suspension increased the effect of ADP and decreased the effect of carboxyatractylate. These results suggest that under the conditions used SkQ1 protects mitochondria from oxidative damage as an antioxidant when added at extremely low concentrations.

It was shown that separate fragments of the inner mitochondrial compartment (mitoplasts) can exist under a single non- fragmented outer membrane. Here we asked whether fragmentation of the inner mitochondria could prevent rupturing of the outer membrane and release of pro-apoptotic molecules from the mitochondrial intermembrane space into the cytoplasm during mitochondrial swelling. First, we showed that in Saccharomyces cerevisiae yeast addition of amiodarone causes formation of electrically separate compartments within mitochondrial filaments. Moreover, amiodarone treatment of Deltaysp2 mutant produced a higher proportion of cells with electrically discontinuous mitochondria than in the wild type, which correlated with the survival of cells. We confirmed the existence of separated mitoplasts under a single outer membrane using electron microscopy. Mitochondria with fragmented matrixes were also detected in cells of the stationary phase. Our data suggest that such fragmentation acts as a cellular protective mechanism against stress.

Sulfatides - sulfated derivatives of galactocerebroside - are endogenous ligands for P- and L-selectins and are able to induce intracellular signaling in neutrophils through a L-selectin dependent pathway. Sulfatides are implicated in a variety of physiological functions and have been found to suppress the synthesis of 5-lipoxygenase (5-LO) metabolites and impede 5-LO translocation to the nuclear envelope in adherent human polymorphonuclear leukocytes (PMNs) [Sud'ina, G. F., Brock, T. G., Pushkareva, M. A., Galkina, S. I., Turutin, D. V., Peters-Golden, M., et al. (2001). Sulphatides trigger polymorphonuclear granulocyte spreading on collagen-coated surfaces and inhibit subsequent activation of 5-lipoxygenase. The Biochemical Journal, 359, 621-629]. In this study we investigated the mechanism of the leukotriene (LT) synthesis inhibition by sulfatides. Sulfatides neither attenuated the ionophore-induced rise in Ca²⁺(i) nor promoted PKA activation. We demonstrated that sulfatides directly inhibited 5-LO enzyme activity in a cell-free assay. BODIPY-labeled sulfatides were able to rapidly penetrate into the cells. Sulfatides induced rearrangement and redistribution of cytoskeletal components in adherent PMNs. The lipid incorporation as well as sulfatide-induced inhibition of LT synthesis were abolished by cytochalasin D, an inhibitor of actin polymerization and endocytosis. Importantly, sulfatides caused a prominent intracellular cholesterol redistribution, increasing its abundance at the uropod region. On the basis of these data, we suggest that increased cholesterol accumulation in cell compartments represents a novel mechanism by which sulfatides abrogate 5-LO translocation and activation.

Photosynthetic reaction centers of Blastochloris viridis require two quanta of light to catalyse a two-step reduction of their secondary ubiquinone QB to ubiquinol. We employed capacitive potentiometry to follow the voltage changes that were caused by the accompanying transmembrane proton displacements. At pH 7.5 and 200C, the QB-related voltage generation after the first flash was contributed by a fast, temperature-independent component with a time constant of approximately 30 micros and a slower component of approximately 200 micros with activation energy (Ea) of 50 kJ/mol. The kinetics after the second flash featured temperature-independent components of 5 micros and 200 micros followed by a component of 600 micros with Ea approximately 60 kJ/mol.

Energy catastrophe, when mitochondria hydrolyze glycolytic ATP instead of producing respiratory ATP, has been modeled. In highly glycolyzing HeLa cells, 30-50% of the population survived after inhibition of respiration and uncoupling of oxidative phosphorylation for 2-4 days. The survival was accompanied by selective elimination of mitochondria. This type of mitoptosis includes (i) fission of mitochondrial filaments, (ii) clustering of the resulting roundish mitochondria in the perinuclear area, (iii) occlusion of mitochondrial clusters by a membrane (formation of a "mitoptotic body"), (iv) decomposition of mitochondria inside this body to small membrane vesicles, (v) protrusion of the body from the cell, and (vi) disruption of the body boundary membrane. Autophagy was not involved in this mitoptotic program. Increased production of reactive oxygen species (ROS) was necessary for execution of the program, since antioxidants prevent mitoptosis and kill the cells treated with the mitochondrial poisons as if a ROS-linked mitoptosis serves for protection of the cells under conditions of severe mitochondrial stress. It is suggested that exocytosis of mitoptotic bodies may be involved in maturation of reticulocytes and lens fiber cells.

Spectroelectrochemistry was used to determine the midpoint redox potentials of heme cofactors of the caa3-type cytochrome oxidase from the alkaliphilic bacterium Bacillus pseudofirmus FTU. The apparent midpoint potentials (Emapp) for the most prominent transitions of hemes a and a3 (+193 and +334 mV, respectively) were found to be similar to the values reported for other enzymes with high homology to the caa3-type oxidase. In contrast, the midpoint potential of the covalently bound cytochrome c (+89 mV) was 150-170 mV lower than in cytochromes c, either low molecular weight or covalently bound to the caa3 complex in all known aerobic neutralophilic and thermo-neutralophilic bacteria. Such an unusually low redox potential of the covalently bound cytochrome c of the caa3-type oxidase of alkaliphilic bacteria, together with high redox potentials of hemes a and a3, ensures more than twice higher difference in redox potentials inside the respiratory complex compared to the homologous mitochondrial enzyme. The energy released during this redox transition might be stored in the transmembrane H⁺ gradient even under low Deltap in the alkaline environment of the bacteria at the expense of a significant increase in DeltaG of the coupled redox reaction.

Mitochondria-targeted antioxidant 10-(6-plastoquinonyl)decyltriphenyl-phosphonium (SkQ1) as well as insulin and the inhibitor of glycogen-synthase kinase, Li⁺ are shown to (i) protect renal tubular cells from an apoptotic death and (ii) diminish mitochondrial fission (the thread-grain transition) induced by ischemia/reoxygenation. However, SkQ1 and LiCl protected the mitochondrial reticulum of skin fibroblasts from ultraviolet-induced fission but were ineffective in preventing a further cell death. This means that mitochondrial fission is not essential for apoptotic cascade progression.

The midpoint redox potentials (Em) of all cofactors in photosystem I from Synechococcus elongatus as well as of the iron- sulfur (Fe4S4) clusters in two soluble ferredoxins from Azotobacter vinelandii and Clostridium acidiurici were calculated within the framework of a semi-continuum dielectric approach. The widely used treatment of proteins as uniform media with single dielectric permittivity is oversimplified, particularly, because permanent charges are considered both as a source for intraprotein electric field and as a part of dielectric polarizability. Our approach overcomes this inconsistency by using two dielectric constants: optical ?(o)=2.5 for permanent charges pre-existing in crystal structure, and static ?(s) for newly formed charges. We also take into account a substantial dielectric heterogeneity of photosystem I revealed by photoelectric measurements and a liquid junction potential correction for Em values of relevant redox cofactors measured in aprotic solvents. We show that calculations based on a single permittivity have the discrepancy with experimental data larger than 0.7 V, whereas Em values calculated within our approach fall in the range of experimental estimates. The electrostatic analysis combined with quantum chemistry calculations shows that (i) the energy decrease upon chlorophyll dimerization is essential for the downhill mode of primary charge separation between the special pair P700 and the primary acceptor A0; (ii) the primary donor is apparently P700 but not a pair of accessory chlorophylls; (iii) the electron transfer from the A branch quinone QA to the iron-sulfur cluster FX is most probably downhill, whereas that from the B branch quinone QB to FX is essentially downhill.

Mitochondria-targeted antioxidants consisting of a quinone part conjugated with a lipophilic cation via a hydrocarbon linker were previously shown to prevent oxidative damage to mitochondria in vitro and in vivo. In the present work, we studied the permeation of a series of compounds of this type across a planar bilayer phospholipid membrane. For this purpose, relaxation of the electrical current after a voltage jump was measured. With respect to the characteristic time of the relaxation process reflecting the permeation rate, hydrophobic cations can be ranked in the following series: 10(plastoquinonyl) decylrhodamine 19 (SkQR1) > 10-(6'-plastoquinonyl) decyltriphenylphosphonium (SkQ1) > 10-(6'-methylplastoquinonyl) decyltriphenylphosphonium (SkQ3) > 10-(6'-ubiquinonyl) decyltriphenylphosphonium (MitoQ). Thus, the permeation rate increased with (1) an increase in the size of the hydrophobic cation and (2) an increase in hydrophobicity of the quinone moiety. SkQ1 containing plastoquinone was shown to be more permeable through the membrane compared to MitoQ containing ubiquinone, which might be the reason for more pronounced beneficial action of SkQ1 in vitro and in vivo. The above approach can be recommended for the search for new antioxidants or other compounds targeted to mitochondria.

This review is focused on the mechanism of photovoltage generation involving the photosystem II turnover. This large integral membrane enzyme catalyzes the light-driven oxidation of water and reduction of plastoquinone. The data discussed in this work show that there are four main electrogenic steps in native complexes: (i) light-induced charge separation between special pair chlorophylls P680 and primary quinone acceptor QA; (ii) P680⁺ reduction by the redox-active tyrosine YZ of polypeptide D1; (iii) oxidation of Mn cluster by YZox followed by proton release, and (iv) protonation of double reduced secondary quinone acceptor QB. The electrogenicity related to (i) proton-coupled electron transfer between QA- and preoxidized non-heme iron Fe³⁺ in native and (ii) electron transfer between protein-water boundary and YZox in the presence of redox-dye(s) in Mn- depleted samples, respectively, were also considered. Evaluation of the dielectric properties using the electrometric data and the polarity profiles of reaction center from purple bacteria Blastochloris viridis and photosystem II are presented. The knowledge of the profile of dielectric permittivity along the photosynthetic reaction center is important for understanding of the mechanism of electron transfer between redox cofactors.

The existence of cell death program in unicellular organisms has been reported for a number of species. Nevertheless, the question why the ability to commit suicide has been maintained throughout evolution is far from being solved. While it is believed that altruistic death of individual yeast cells could be beneficial for the population, it is generally not known (i) what is wrong with the individuals destined for elimination, (ii) what is the critical value of the parameter that makes a cell unfit and (iii) how the cell monitors this parameter. Studies performed on yeast Saccharomyces cerevisiae allow us to hypothesize on ways of possible solutions of these problems. Here we argue that (a) the main parameter for life-or-death decision measured by the cell is the degree of damage to the genetic material, (b) its critical value is dictated by quorum sensing machinery, and (c) it is measured by monitoring delays in cell division.

ELECTRON AND PROTON TRANSFER IN THE ba3 OXIDASE FROM Thermus thermophilus.
Smirnova I.A., Zaslavsky D., Fee J.A., Gennis R.B., Brzezinski P.
JOURNAL OF BIOENERGETICS AND BIOMEMBRANES 40 (2008) 281-287.

The ba3-type cytochrome c oxidase from Thermus thermophilus is phylogenetically very distant from the aa3-type cytochrome c oxidases. Nevertheless, both types of oxidases have the same number of redox-active metal sites and the reduction of O2 to water is catalysed at a haem a3-CuB catalytic site. The three-dimensional structure of the ba3 oxidase reveals three possible proton-conducting pathways showing very low homology compared to those of the mitochondrial, Rhodobacter sphaeroides and Paracoccus denitrificans aa3 oxidases. In this study we investigated the oxidative part of the catalytic cycle of the ba3- cytochrome c oxidase using the flow-flash method. After flash-induced dissociation of CO from the fully reduced enzyme in the presence of oxygen we observed rapid oxidation of cytochrome b (k congruent with 6.8 x 104 s-1) and formation of the peroxy (PR) intermediate. In the next step a proton was taken up from solution with a rate constant of approximately 1.7 x 104 s-1, associated with formation of the ferryl (F) intermediate, simultaneous with transient reduction of haem b. Finally, the enzyme was oxidized with a rate constant of approximately 1,100 s-1, accompanied by additional proton uptake. The total proton uptake stoichiometry in the oxidative part of the catalytic cycle was approximately 1.5 protons per enzyme molecule. The results support the earlier proposal that the PR and F intermediate spectra are similar (Siletsky et al. Biochim Biophys Acta 1767:138, 2007) and show that even though the architecture of the proton-conducting pathways is different in the ba3 oxidases, the proton-uptake reactions occur over the same time scales as in the aa3-type oxidases.

In the presence of the uncoupler, external zinc ions inhibit rapidly turnover of cytochrome c oxidase reconstituted in phospholipid vesicles or bound to the membrane of intact mitochondria. The effect is promoted by electron leaks into the oxidase during preincubation with Zn²⁺. Inhibition of liposome-bound bovine cytochrome oxidase by external Zn²⁺ titrates with a Ki of 1±0.3 microM. Presumably, the Zn²⁺-binding group at the positively charged side is not reactive in the oxidized enzyme, but becomes accessible to the cation in some partially reduced state(s) of the oxidase; reduction of CuB is tentatively proposed to be responsible for the effect.

Transient absorption difference spectroscopy with approximately 20 femtosecond (fs) resolution was applied to study the time and spectral evolution of low-temperature (90 K) absorbance changes in isolated reaction centers (RCs) of Chloroflexus (C.) aurantiacus. In RCs, the composition of the B-branch chromophores is different with respect to that of purple bacterial RCs by occupying the BB binding site of accessory bacteriochlorophyll by bacteriopheophytin molecule ( PheoВ). It was found that the nuclear wave packet motion induced on the potential energy surface of the excited state of the primary electron donor P* by approximately 20 fs excitation leads to a coherent formation of the states P⁺ PheoВ^- and P⁺BA^- (BA is a bacteriochlorophyll monomer in the A-branch of cofactors). The processes were studied by measuring coherent oscillations in kinetics of the absorbance changes at 900 nm and 940 nm (P* stimulated emission), at 750 nm and 785 nm ( PheoВ absorption bands), and at 1,020-1028 nm (BA- absorption band). In RCs, the immediate bleaching of the P band at 880 nm and the appearance of the stimulated wave packet emission at 900 nm were accompanied (with a small delay of 10-20 fs) by electron transfer from P* to the B-branch with bleaching of the PheoВ absorption band at 785 nm due to PheoВ- formation. These data are consistent with recent measurements for the mutant HM182L Rb. sphaeroides RCs (Yakovlev et al., Biochim Biophys Acta 1757:369-379, 2006). Only at a delay of 120 fs was the electron transfer from P* to the A-branch observed with a development of the BA- absorption band at 1028 nm. This development was in phase with the appearance of the P* stimulated emission at 940 nm. The data on the A-branch electron transfer in C. aurantiacus RCs are consistent with those observed in native RCs of Rb. sphaeroides. The mechanism of charge separation in RCs with the modified B-branch pigment composition is discussed in terms of coupling between the nuclear wave packet motion and electron transfer from P* to PheoВ and BA primary acceptors in the B-branch and A-branch, respectively.

Using method of electron microscopic histochemistry based upon the oxidative polymerization of 3,3'-diaminobenzidine (DAB) to reveal cytochrome c oxidase activity we identified that long hypoxic incubation of isolated small pieces of cardiac tissue during 72 h caused changes in mitochondrial ultrastructure followed by a breach of functional activities of mitochondria, and, in particular, complex IV of the respiratory chain. But for all that, small electron-dense mitochondria appearing inside electron-light mitochondria ("mitochondria inside mitochondria") stained positively for cytochrome c oxidase activity along the full length of cristaes. The results obtained are discussed in connection with conception of changes in the mitochondrial reticulum ultrastructure during mitoptosis.

PEAK INTENSITY ANALYSIS AS A METHOD FOR ESTIMATION OF FLUORESCENT PROBE BINDING TO ARTIFICIAL AND NATURAL NANOPARTICLES: TETRAMETHYLRHODAMINE UPTAKE BY ISOLATED MITOCHONDRIA.
Perevoshchikova I.V., Zorov D.B., Antonenko Y.N.
BIOCHIMICA ET BIOPHYSICA ACTA (BBA) - BIOMEMBRANES 1778 (2008) 2182-2190.

A modified version of fluorescence correlation spectroscopy (FCS) closely related to the photon counting histogram (PCH) method, which is used in the case of a mixture of molecules with similar diffusion coefficients, was applied here for analyzing the binding of the potential-sensitive dye tetramethylrhodamine ethyl ester, TMRE, to isolated mitochondria both in energized and deenergized states. Fluorescence time traces of suspensions of TMRE-doped mitochondria representing sequences of peaks of different intensity appeared to be similar to those of fluorescent beads and TMRE-doped latex particles. The experimental data were obtained under stirring conditions which increased the number of events by about three orders of magnitude thus substantially enhancing the resolution of the method. The statistics of the brightness of identical fluorescent particles reflecting their random walk through the confocal volume was described by a simple analytical equation which enabled us to perform the peak intensity analysis (PIA) of TMRE-doped mitochondria. The validity of PIA was tested with fluorescent beads of different sizes and TMRE-doped latex particles. Mitochondrial energization in the presence of TMRE led to the increase in the number and the intensity of the peaks in fluorescence time traces, the PIA of which allowed us to determine mitochondrial membrane potential and additionally a number of mitochondrial particles per ml of the suspension. The value of the membrane potential on a single mitochondrion was estimated to be about 180 mV in agreement with the data related to mitochondrial suspensions. Importantly, the PIA method required less than 1 microgram of mitochondrial protein per measurement.

Binding of the cationic tetra(tributylammoniomethyl)-substituted hydroxoaluminum phthalocyanine (AlPcN4) to bilayer lipid membranes was studied by fluorescence correlation spectroscopy (FCS) and intramembrane field compensation (IFC) methods. With neutral phosphatidylcholine membranes, AlPcN4 appeared to bind more effectively than the negatively charged tetrasulfonated aluminum phthalocyanine (AlPcS4), which was attributed to the enhancement of the coordination interaction of aluminum with the phosphate moiety of phosphatidylcholine by the electric field created by positively charged groups of AlPcN4. The inhibitory effect of fluoride ions on the membrane binding of both AlPcN4 and AlPcS4 supported the essential role of aluminum-phosphate coordination in the interaction of these phthalocyanines with phospholipids. The presence of negative or positive charges on the surface of lipid membranes modulated the binding of AlPcN4 and AlPcS4 in accord with the character (attraction or repulsion) of the electrostatic interaction, thus showing the significant contribution of the latter to the phthalocyanine adsorption on lipid bilayers. The data on the photodynamic activity of AlPcN4 and AlPcS4 as measured by sensitized photoinactivation of gramicidin channels in bilayer lipid membranes correlated well with the binding data obtained by FCS and IFC techniques. The reduced photodynamic activity of AlPcN4 with neutral membranes violating this correlation was attributed to the concentration quenching of singlet excited states as proved by the data on the AlPcN4 fluorescence quenching.

Modular/chimeric recombinant drugs or drug transporters usually contain a special translocation domain from bacterial toxins, e.g. diphtheria toxin, as a module enabling escape of the chimeric molecules from acidifying endosomes. This approach is limited by the shortage in the knowledge about the precise molecular mechanisms of translocation of diphtheria toxin and the toxin-based chimera across the endosomal membrane and its release into the cytosol. We present experimental data with the modular recombinant drug transporters (MRTs), containing the translocation domain of diphtheria toxin, developed by us earlier, demonstrating that the MRTs interact with lipid membranes (liposomes, free-standing and supported bilayer lipid membranes) and produce defects in them at endosomal pH's which are sufficient for escape of macromolecules: - MRTs induced leakage of calcein-loaded liposomes pH 3-6.5. Large fluctuating conductance states of 2-5 nS are formed in membranes at pH 5.5. Atomic force microscopy revealed two different types of defects in the supported lipid bilayers at pH 5.5: 1) giant pores (50-200 nm diameters) and 2) small depressions/holes (30-50 nm) produced by the MRTs which are inserted into the membranes thus forming the structures described above. The last was shown with the use of cantilever tips modified with anti-MRT antibodies.

Vesicular transport of signaling molecules, specifically neurotrophins, in neurons is essential for their differentiation, survival, and plasticity. Neurotrophins such as neuron growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are internalized by receptor-mediated endocytosis at synaptic terminals and loaded into endosomes for microtubule-based transport along axons to the cell body where they exert their signaling function in the nucleus. The molecular mechanisms underlying this intracellular transport are not only relevant from a basic knowledge viewpoint, but have also important implications for neurodegenerative diseases. Defects in trafficking are increasingly implicated in the pathology of Huntington's disease (HD) and other neurodegenerative disorders. The small GTPases Rab5 and Rab7 play important roles in the endocytic trafficking of neurotrophins. We have recently identified Huntingtin (Htt) and Huntingtin associated protein of 40 kDa (HAP40) as a novel Rab5 effector complex that regulates endosome motility. In HD, we detected higher HAP40 protein levels compared with normal cells. Such increase causes an augmented recruitment of Htt onto Rab5-positive early endosomes that drastically reduces their motility by "switching" these organelles from microtubules to F-actin. These findings suggest a mechanism by which impaired Rab5-mediated trafficking of neurotrophic factors may be a key event of the pathogenetic process leading to neurodegeneration in HD. To dissect the mechanisms by which Htt, HAP40, and Rab5 function in early endosome interactions with the cytoskeleton, we developed assays to investigate endosome-cytoskeleton interactions that can be applied to normal and pathological conditions. We provide here detailed protocols for, first, an assay that measures binding of early endosomes to microtubules and F-actin. Second, we describe an improved protocol for a cell-free assay that recapitulates the motility of early endosomes along microtubules in vitro. These assays provide mechanistic insights into the dysfunction of endosome motility occurring in HD as well as other neurodegenerative disorders

Background - The F- and V-type ATPases are rotary molecular machines that couple translocation of protons or sodium ions across the membrane to the synthesis or hydrolysis of ATP. Both the F-type (found in most bacteria and eukaryotic mitochondria and chloroplasts) and V-type (found in archaea, some bacteria, and eukaryotic vacuoles) ATPases can translocate either protons or sodium ions. The prevalent proton-dependent ATPases are generally viewed as the primary form of the enzyme whereas the sodium-translocating ATPases of some prokaryotes are usually construed as an exotic adaptation to survival in extreme environments. Results - We combine structural and phylogenetic analyses to clarify the evolutionary relation between the proton- and sodium-translocating ATPases. A comparison of the structures of the membrane-embedded oligomeric proteolipid rings of sodium-dependent F- and V-ATPases reveals nearly identical sets of amino acids involved in sodium binding. We show that the sodium-dependent ATPases are scattered among proton-dependent ATPases in both the F- and the V-branches of the phylogenetic tree. Conclusion - Barring convergent emergence of the same set of ligands in several lineages, these findings indicate that the use of sodium gradient for ATP synthesis is the ancestral modality of membrane bioenergetics. Thus, a primitive, sodium- impermeable but proton-permeable cell membrane that harboured a set of sodium-transporting enzymes appears to have been the evolutionary predecessor of the more structurally demanding proton-tight membranes. The use of proton as the coupling ion appears to be a later innovation that emerged on several independent occasions.

All living cells routinely expel Na⁺ ions, maintaining lower concentration of Na⁺ in the cytoplasm than in the surrounding milieu. In the vast majority of bacteria, as well as in mitochondria and chloroplasts, export of Na⁺ occurs at the expense of the proton-motive force. Some bacteria, however, possess primary generators of the transmembrane electrochemical gradient of Na⁺ (sodium-motive force). These primary Na⁺ pumps have been traditionally seen as adaptations to high external pH or to high temperature. Subsequent studies revealed, however, the mechanisms for primary sodium pumping in a variety of non- extremophiles, such as marine bacteria and certain bacterial pathogens. Further, many alkaliphiles and hyperthermophiles were shown to rely on H⁺, not Na⁺, as the coupling ion. We review here the recent progress in understanding the role of sodium- motive force, including (i) the conclusion on evolutionary primacy of the sodium-motive force as energy intermediate, (ii) the mechanisms, evolutionary advantages and limitations of switching from Na⁺ to H⁺ as the coupling ion, and (iii) the possible reasons why certain pathogenic bacteria still rely on the sodium-motive force.

 In Part I of the article, a review of recent data on electron-transfer reactions in photosystem II (PSII) and bacterial reaction center (RC) has been presented. In Part II, transient absorption difference spectroscopy with 20-fs resolution was applied to study the primary charge separation in PSII RC (DI/DII/Cyt b559 complex) excited at 700 nm at 278 K. It was shown that the initial electron-transfer reaction occurs within 0.9 ps with the formation of the charge-separated state P680⁺ChlD1?, which relaxed within 14 ps as indicated by reversible bleaching of 670-nm band that was tentatively assigned to the ChlD1 absorption. The subsequent electron transfer from ChlD1? within 14 ps was accompanied by a development of the radical anion band of PheoD1 at 445 nm, attributable to the formation of the secondary radical pair P680⁺PheoD1?. The key point of this model is that the most blue Q y transition of ChlD1 in RC is allowing an effective stabilization of separated charges. Although an alternative mechanism of charge separation with ChlD1* as a primary electron donor and PheoD1 as a primary acceptor can not be ruled out, it is less consistent with the kinetics and spectra of absorbance changes induced in the PSII RC preparation by femtosecond excitation at 700 nm.

A chemolithoautotrophic sulfur-oxidizing bacterium (SOB) strain ALCO 1 capable of growing at both near-neutral and extremely alkaline pH was isolated from hypersaline soda lakes in S-W Siberia (Altai, Russia). Strain ALCO 1 represents a novel separate branch within the halothiobacilli in the Gammaproteobacteria, which, so far, contained only neutro-halophilic SOB. On the basis of its unique phenotypic properties and distant phylogeny, strain ALCO 1 is proposed as a new genus and species Thioalkalibacter halophilus gen. nov. sp. nov. ALCO 1 was able to grow within a broad range of salinity (0.5-3.5 M of total sodium) with an optimum at around 1 M Na⁺, and pH (7.2-10.2, pHopt at around 8.5). Na⁺ was required for sulfur- dependent respiration in ALCO 1. The neutral (NaCl)-grown chemostat culture had a much lower maximum growth rate (?max), respiratory activity and total cytochrome c content than its alkaline-grown counterpart. The specific concentration of osmolytes (ectoine and glycine-betaine) produced at neutral pH and 3 M NaCl was roughly two times higher than at pH 10 in soda. Altogether, strain ALCO 1 represents an interesting chemolithoautotrophic model organism for comparative investigations of bacterial adaptations to high salinity and pH.

Cytochrome bd catalyzes the two-electron oxidation of either ubiquinol or menaquinol and the four-electron reduction of O2 to H2O. In the current work, the rates of reduction of the fully oxidized and oxoferryl forms of the enzyme by the 2-electron donor ubiquinol-1 and single electron donor N,N,N?,N?-tetramethyl-p-phenylendiamine (TMPD) have been examined by stopped-flow techniques. Reduction of the all-ferric form of the enzyme is 1000-fold slower than required for a step in the catalytic cycle, whereas the observed rates of reduction of the oxoferryl and singly-reduced forms of the cytochrome are consistent with the catalytic turnover. The data support models of the catalytic cycle which do not include the fully oxidized form of the enzyme as an intermediate.

Cytochrome c is one of the key proteins involved in the programmed cell death, and lysine 72 is known to be required for its apoptogenic activity. We have engineered a number of horse and murine cytochrome c single-point mutants with various substitutions at position 72 and compared quantitatively their proapoptotic activity in living cells. Apoptosis was activated by transferring exogenous cytochrome c into the cytoplasm of cells via a nontraumatic electroporation procedure. All mutant proteins studied exhibited significantly reduced proapoptotic activities in comparison with those for the wild type cytochromes. Relative activity of the horse (h(K72X)) and murine (m(K72W)) mutant proteins diminished in the order: h(K72R) > h(K72G) > h(K72A) > h(K72E) > h(K72L) >> h(K72W) > m(K72W). As estimated, the horse and As estimated, the horse and murine K72W mutants were at least 200- and 500-fold less active than corresponding wild type proteins. Thus, the K72W-substituted cytochrome c can serve as an adequate candidate for knock-in studies of cytochrome c-mediated apoptosis. The proapoptotic activity of wild-type cytochrome c from different species in murine monocytic WEHI-3 cells reduced in the order: murine cytochrome c > human cytochrome c ? horse cytochrome c, thus indicating that apoptotic effect of cytochrome c depends on the species compatibility.

Dynamic phase microscopy was used to measure the refractivity of a single mitochondrion. Our previous studies showed that application of an electric potential to artificial and natural mitochondrial membranes sharply increases their refractivity. Under the conditions of proton pump activity, the refractivity of a single mitochondrion is 2 to 4 times higher than an average refractivity of deenergized mitochondria. This study demonstrates that the membrane potential of energized mitochondria varies depending on environmental conditions and is controlled by the mitochondrial osmoregulation system. The refractivity of energized mitochondria, i.e., the difference between the refraction indexes of a single mitochondrion and the medium, is 0.02 0.01, i.e., several times lower than that of the energized mitochondria whose membranes bear an electric charge. Earlier it was shown that refractivity of model multilayer systems formed from purified natural lecithin depends linearly on the electric field strength. These data point to a relationship between the refractivity of a single mitochondrion and the membrane potential generated during operation of the proton pump. Under normal conditions (250 mOsm), the mitochondrion behaves as a dynamic system oscillating on a minute scale between two functional states with different values of the refractivity index and different membrane potentials. The transition time is 10-30 s; the lifetime of both states is several minutes. The histograms reflecting the distribution of refractivities of single energized mitochondria within a population (n = 20-30) revealed the presence of two independent peaks (fractions II and III) with average refractivity values of 0.05 0.01 and 0.09 0.01, respectively; these fractions correspond to two long-lived states of mitochondria. However, under hypotonic conditions (120 mOsm), only one ("static") state was identified, in which oscillations were absent and the refractivity of the overall mitochondrial population does not exceed 0.05 0.01 (fraction II). Studies on mitoplast showed that values of refractivity are related to the inner mitochondrial membrane. It is inferred from these data that there exist two discrete states of mitochondria. Analysis of low-amplitude fluctuations of the refractivity of single mitochondria revealed the presence of frequency components at 1-3 Hz, presumably generated in response to non-uniform functioning of mitochondrial proton pumps. It is suggested that frequency components at 1.8-2.6 Hz are more characteristic of the ATPase pump, while the 1-1.3 Hz frequencies predominate during the functioning of respiratory proton pumps.

Effects of the coenzyme Q analog (MitoQ10) carrying a positively charged decyltetraphenylphosphonium group on functional activity of phosphorylating liver mitochondria were studied. Using inhibitory analysis it was found that at micromolar concentrations this quinone is reduced by NADH-dependent DT-diaphorase. Under conditions of malate oxidation, MitoQ10 stimulates electron transfer from NADH to oxygen by shunting the block of rotenone-induced electron transport in Complex I. Steady-state mitochondrial respiration induced by rotenone and MitoQ10 (1 M), as well as K3 shunt are both blocked by the DT-diaphorase inhibitor dicumarol, the Complex III inhibitor myxothiazole, and the cytochrome oxidase inhibitor cyanide. The electron transport chain induced in liver mitochondria by MitoQ10 in the presence of rotenone appears as follows: NADH DT-diaphorase MitoQ10 Complex III Complex IV O2. Under conditions of malate (but not succinate) oxidation, MitoQ10 and high concentrations of vitamin K3 induce in mitochondria cyanide-resistant respiration and opening of the nonspecific pore eventually resulting in inhibition of oxidative phosphorylation. It is concluded that MitoQ10 should be regarded as an analog of hydrophilic quinones (vitamin K3, duroquinone, etc.) widely known as substrates for mitochondrial DT-diaphorase not interacting with CoQ10 rather than as a natural CoQ10 analog.

TRAIL (Apo2L), a cytokine from the family of tumor necrosis factors (TNF), causes apoptosis in various types of tumor cells but is not toxic for normal cells. Recombinant TRAIL obtained using an original method stimulates the release of cytochrome c from mitochondria into the cytoplasm and apoptosis in HeLa carcinoma cells. Expression of oncoprotein Bcl-2 in these cells blocks both processes. The microtubule inhibitors taxol, nocodazole, and colcemid, as well as an inhibitor of actin microfilaments cytochalasin D, enhance the action of TRAIL and allow it to overcome protection caused by overexpression of Bcl-2. This effect is not associated with enhancement of early steps of TRAIL-dependent apoptosis leading to activation of caspase-8 and Bid protein. The inactivation of Bcl-2 also does not define the effect of cytoskeleton inhibitors. It is supposed that destruction of cytoskeleton alters the mechanism of the TRAIL- (or TNF)-dependent cytochrome c release from mitochondria by making it resistant to Bcl-2. The combined use of cytoskeleton inhibitors, which are antitumor drugs, with the recombinant TRAIL preparations may be efficient in therapy of tumors resistant to traditional chemotherapy.

Борьба со старением - задача масштабная и не под силу какой-то одной научной группе. По сложности она сопоставима с проектом С.П. Королева по запуску первого спутника или "всемирным проектом" по расшифровке генома человека. Над решением этой задачи в мире бьются сотни лабораторий, включая Национальный институт старения США. К настоящему моменту существует более 300 научных теорий старения. С 2005 г. в России в стенах Московского государственного университета им. М.В. Ломоносова проводится междисциплинарный инновационный проект, который может не только составить достойную конкуренцию зарубежным геронтологам, но и имеет реальные шансы на создание первого действенного геропротектора - лекарства против старости. Научный руководитель проекта - академик РАН В.П. Скулачев. Инвестором выступила Русско- азиатская инвестиционная компания (РАИнКо). Для осуществления этого проекта инвестор и руководство МГУ в лице ректора В.А. Садовничего и деканов биомедицинских факультетов приняли решение пойти по пути, достаточно распространенному на Западе, но все еще необычному для России - создать "приуниверситетскую" биотехнологическую компанию. Так была создана "Митотехнология", уже три года ведущая проект "Практическое использование ионов Скулачева"

M1 protein binding to the lipid bilayer membrane (BLM) was recorded by the inner field compensation technique as a change of the boundary potential. After the protein was added to the bulk solution, the M1 adsorption produced a slow increase in boundary potential to a stationary value that was reached within the time period dependent on the quantity of the added protein. The stationary value of the potential grew with the decrease of pH or KCl concentration in the medium and was higher in the presence of negatively charged lipids in the BLM. It was shown that the potential growth with the decrease of pH is due to an increase of M1 molecule charge and not due to the increase of the M1 surface concentration or to the change of lipid charge. As the potential did not change after the removal of the protein from the bulk solution, we consider the protein adsorption on the BLM irreversible. The obtained results suggest that the protein adsorption is influenced by both electrostatic and hydrophobic interactions of M1 molecules with each other and with lipid membrane. We offer a mechanism of dissociation of the viral shell formed by M1 matrix protein. The protein shell is destabilized due to electrostatic repulsion of protein molecules caused by the increase of their positive charge.

Protein aggregation is intimately linked to a number of neurodegenerative diseases. Expansion of the huntingtin polyglutamine-rich domain causes protein aggregation and neuronal degeneration. Recently we found that, similar to neurons, yeast expressing the expanded domain show markers of programmed cell death. Here we showed that deletion of yeast metacaspase gene YCA1 partly rescues the toxic effect of the domain overexpression. We also performed genetic screen for other genes deletions alleviating the toxic effect and found ASE1. Ase1 is a substrate of the Cdh1 form of anaphase promoting complex, APC/Cdh1. We tested Cdh1 overexpression and the deletion of CLB2 (mitotic cyclin, substrate of APC/Cdh1) and found that both mutations had a rescuing effect on the expanded polyglutamine toxicity. Our data suggest that the toxic effect of aggregated proteins is partly indirect. We speculate that cellular attempt to degrade the aggregates overloads the proteasome, and this leads to pathological accumulation of APC substrates.

Reactive oxygen species (ROS) generation in mitochondria as a side product of electron and proton transport through the inner membrane is important for normal cell operation as well as development of pathology. Matrix and cytosol alkalization stabilizes semiquinone radical, a potential superoxide producer, and we hypothesized that proton deficiency under the excess of electron donors enhances reactive oxygen species generation. We tested this hypothesis by measuring pH dependence of reactive oxygen species released by mitochondria. The experiments were performed in the media with pH varying from 6 to 8 in the presence of complex II substrate succinate or under more physiological conditions with complex I substrates glutamate and malate. Matrix pH was manipulated by inorganic phosphate, nigericine, and low concentrations of uncoupler or valinomycin. We found that high pH strongly increased the rate of free radical generation in all of the conditions studied, even when Delta pH = 0 in the presence of nigericin. In the absence of inorganic phosphate, when the matrix was the most alkaline, pH shift in the medium above 7 induced permeability transition accompanied by the decrease of ROS production. ROS production increase induced by the alkalization of medium was observed with intact respiring mitochondria as well as in the presence of complex I inhibitor rotenone, which enhanced reactive oxygen species release. The phenomena revealed in this report are important for understanding mechanisms governing mitochondrial production of reactive oxygen species, in particular that related with uncoupling proteins.

Optimal values of the quantum yield of charge separation in the reaction centers (RCs) of photosynthetic purple bacteria Rhodospirillum rubrum and carotenoidless mutant Rhodobacter sphaeroides R-26 were found to be 0.915 ± 0.045 and 0.978 ± 0.006 respectively. The calculations of these parameters were based on the present-day model of primary events in bacterial RCs, a complete set of kinetic and energy parameters now available for these species as well as temperature dependences of the nanosecond luminescence component(s) emitted by the RCs. The range of the quantum yield value for Rhodobacter sphaeroides R-26 partially overlaps with that obtained earlier in a classical work of Wright and Clayton (BBA. 1974. V. 333. P. 246-260), i.e., 1.02 ± 0.04.

OPTICAL PROPERTIES OF THE MONOMERIC RED FLUORESCENCT PROTEIN mRFP1.
Vrzheshch E.P., Dmitrienko D.V., Rudanov G.S., Zagidullin V.E., Paschenko V.Z., Razzhivin A.P. Saletsky A.M., Vrzheshch P.V.
VESTNIK OF MOSCOW UNIVERSITY, SERIA 16. BIOLOGY (2008) № 3, 21-24.

For the expression in , the PCR-amplified fragment encoding mRFP1 from vector pMT-mRFP1 (Fungal Genetic Stock Center) was placed to the pQE-60 vector. Chemically competent  ER1821 were transformed and grown overnight at 37°C. The protein was purified by Ni-NTA chromatography and dialyzed in 67 mM Na2HPO4, 67 mM KH2PO4, and pH 7.5. There are two peaks (at 503 nm and at 584 nm) in the mRFP1 absorption spectrum. The green component (503 nm) may be corresponded to a green fraction of the protein (a fraction that never matures beyond the green intermediate or a fraction which trapped as a dead-end product such as the nonproductive trans conformation for the F65-Q66 peptide bond). The mRFP1's extinction coefficient equals to 42 mM-1 cm-1 at 584 nm; the emission maximum is at 607 nm; the excitation maximum is at 584-586 nm; the Stocks' shift equals to 23 nm; the fluorescence lifetime is about 1.8 ns; the quantum yield equals to 0.27; pKa is 4.0. Analysis of the absorption spectrum of mRFP1 by means of the high-order derivative spectroscopy shows that the electron transition system of fully matured protein form (its absorption maximum is at 584 nm) and the same one of the green fraction (its absorption maximum is at 503 nm) are practically identical.

EET between the two circular bacteriochlorophyll compartments B800 and B850 in native (containing the carotenoid rhodopin) and carotenoidless LH2 isolated from the photosynthetic purple sulfur bacterium Allochromatium minutissimum was investigated by femtosecond time-resolved transient absorption spectroscopy. Both samples were excited with 120-fs laser pulses at 800 nm, and spectral evolution was followed in the 720-955 nm range at different delay times. No dependence of transient absorption in the B800 band on the presence of the carotenoid rhodopin was found. Together with the likewise virtually unchanged absorption spectra in the bacteriochlorophyll Q(y) region, these observations suggest that absence of rhodopin does not significantly alter the structure of the pigment-protein complex including interactions between bacteriochlorophylls. Apparently, rhodopin does also not accelerate B800 to B850 EET in LH2, contrary to what has been suggested previously. Moreover, "carotenoid-catalyzed internal conversion" can also be excluded for the bacteriochlorophylls in LH2 of A. minutissimum. Together with previous results obtained with two-photon fluorescence excitation spectroscopy, it can also be concluded that there is neither EET from rhodopin to B800 nor (back-)EET from B800 to rhodopin.

Key cellular functions and developmental processes rely on cascades of GTPases. GTPases of the Rab family provide a molecular ID code to the generation, maintenance and transport of intracellular compartments. Here, we addressed the molecular design principles of endocytosis by focusing on the conversion of early endosomes into late endosomes, which entails replacement of Rab5 by Rab7. We modelled this process as a cascade of functional modules of interacting Rab GTPases. We demonstrate that intermodule interactions share similarities with the toggle switch described for the cell cycle. However, Rab5-to-Rab7 conversion is rather based on a newly characterized 'cut-out switch' analogous to an electrical safety- breaker. Both designs require cooperativity of autoactivation loops when coupled to a large pool of cytoplasmic proteins. Live cell imaging and endosome tracking provide experimental support to the cut-out switch in cargo progression and conversion of endosome identity along the degradative pathway. We propose that, by reconciling module performance with progression of activity, the cut-out switch design could underlie the integration of modules in regulatory cascades from a broad range of biological processes.

A comparative study of thermal denaturation and inactivation of aspartate aminotransferase from pig heart mitochondria (mAAT) has been carried out (10 mM Na phosphate buffer, pH 7.5). Analysis of the data on differential scanning calorimetry shows that thermal denaturation of mAAT follows the kinetics of irreversible reaction of the first order. The kinetics of thermal inactivation of mAAT follows the exponential law. It has been shown that the inactivation rate constant (kin) is higher than the denaturation rate constant (k(den)). The k(in)/k(den) ratio decreases from 28.8 ± 0.1 to 1.30 ± 0.09 as the temperature increases from 57.5 to 770C. The kinetic model explaining the discrepancy between the inactivation and denaturation rates has been proposed. The size of the protein aggregates formed at heating of mAAT at a constant rate (10C min-1) has been characterized by dynamic light scattering.

The investigated green sulfur bacterium, strain M, was isolated from a sulfidic spring on the Black Sea Coast of the Caucasus. The cells of strain M are straight or curved rods 0.6-0.9 x 1.8-4.2 gm in size. According to the cell wall structure, the bacteria are gram-negative. Chlorosomes are located along the cell periphery. Strain M is an obligate anaerobe capable of photoautotrophic growth on sulfide, thiosulfate, and H-2. Acetatate is utilized as an additional carbon source. It utilizes ammonium, urea, casein hydrolysate, and N-2 as nitrogen sources and sulfide, thiosulfate, and elemental sulfur as sulfur sources. Bacteriochlorophyll c and the carotenoid chlorobactene are the main pigments. The optimal growth temperature is 25- 280C; the optimal pH is 6.8. The strain does not require NaCl. Vitamin B 12 stimulates growth. The content of the G+C base pairs in the DNA of strain M is 58.3 mol %. In the phylogenetic tree constructed on the basis of analysis of nucleotide sequences of 16S rRNA genes, strain M forms a separate branch, which occupies an intermediate position between the phylogenetic cluster containing representatives of the genus Chlorobaculum (94.9-96.8%) and the cluster containing species of the genus Chlorobium (94.1-96.5%). According to the results of analysis of the amino acid sequence corresponding to the fmo gene, strain M represents a branch which, unlike that in the "ribosomal" tree, falls into the cluster of the genus Chlorobaculum (95.8-97.2%). Phylogenetic analysis of the amino acid sequence corresponding to the nifH gene placed species of the genera Chlorobaculum and Chlorobium into a single cluster, whereas strain M formed a separate branch. The results obtained allow us to describe strain M as a new species of the genus ChlorobacChlorobaculum - Chlorobaculum macestae sp. nov.

Typical signs of cardiomyoblasts were determined: high mitochondrial membrane potential and high number of mitochondria in these cells compared to fibroblasts. These signs can be used for identification of these cells. Energy-dependent accumulation of highly specific mitochondrial fluorescent probes applied for visual detection of energized mitochondria allows clear-cut separation of the mixed population: cardiomyocyte population is characterized by higher transmembrane potential than concomitant cells. Using flow cytometry and cell sorting we obtained a population enriched with cardiomyocytes and cardiomyoblasts. Taking into account the fact that the dye has no toxic effect on cells and is rapidly eliminated, the risk of cell damage and death during isolation is considerably reduced compared to traditional methods. These results open possibilities for the development of a new specific method for isolation of cardiomyocyte culture from fetal and embryonic sources for their further use in clinical practice.

MitoQ - a positively charged analogue of CoQ - has been studied as an electron transport cofactor in liver mitochondria. NADH-dependent DT-diaphorase is able to reduce MitoQ at a high rate. MitoQH(2) in the presence of malate can restore an electron flow from NADH to oxygen blocked by rotenone. Respiration restored by MitoQ is blocked by dicumarol, mixothiazol, and antimycin A. Therefore, in the presence of MitoQ the following electron transfer chain is operating in mitochondria: NADH -> DT-diaphorase -> MitoQ -> complex III -> complex IV -> oxygen. It is shown also that MitoQH(2) in the presence of malate (but not succinate) reduces oxygen in the o-center of mitochondrial bc(l)-complex giving superoxide anion. This reactive oxygen species induces opening of non-specific pore, which leads to the block of oxidative phosphorylation. The data obtained allow considering MitoQ as an analogue of hydrophilic quinones, such as duroquinone and K3, which are well-known substrates of DT-diaphorase, but not as an analogue of a natural ubiquinone.

Ionophoric activity of the N-terminus-truncated gramicidin A (gA) analogue mini-gramicidin and its covalent dimer was studied in planar phospholipid bilayer membranes (BLM) by measuring macroscopic currents and by single channel recording. Mini-gramicidin-induced macroscopic current across BLM, similarly to the gA-induced one, appeared to undergo sensitized photoinactivation, i.e. it was suppressed by illumination with visible light in the presence of a photosensitizer generating singlet oxygen. Sensitivity to the photo inactivation decreased in a series: mini-gramicidin dimer, mini-gramicidin monomer, gA. Based on single-channel measurements and the analysis of the flash-induced kinetics of the photoinactivation at different levels of the macroscopic current, it was concluded that mini-gramicidin and its covalent dimer are able to form a variety of conducting states, the preference of which is determined by the membrane thickness. Experiments performed with natural membranes (mitochondria and erythrocytes) showed that the ionophoric activity of the mini-gramicidin dimer and monomer essentially depends on the kind of membranes.

Each of two hydrophobic subunits of Na⁺-translocating NADH:quinone oxidoreductase (NQR), NqrD and NqrE, contain a pair of strictly conserved cysteine residues within their transmembrane alpha-helices. Site-directed mutagenesis showed that substitutions of these residues in NQR of Vibrio harveyi blocked the Na⁺-dependent and 2-n-heptyl-4-hydroxyquinoline N- oxide-sensitive quinone reductase activity of the enzyme. However, these mutations did not affect the interaction of NQR with NADH and menadione. It was demonstrated that these conserved cysteine residues are necessary for the correct folding and/or the stability of the NQR complex. Mass and EPR spectroscopy showed that NQR from V. harveyi bears only a 2Fe-2S cluster as a metal-containing prosthetic group.

A review of genomic and proleomics methods for studying of cyanobacteria is given. These bacteria are model organisms for studying of photosynthesis and its regulation, cell division and differentiation, nitrogen fixation, metabolism of carbon, nitrogen, and hydrogen, as well as a stress response to different environmental factors and evolution. Many genetic tools and available data on the complete genomic sequences for unicellular and filamentous cyanobacteria strains provide the opportunity (1) for global monitoring of changes in gene expression at the transcriptional level as a response to variation in environmental conditions; (2) to conduct proteomic studies of various biological processes at the protein level; (3) to identify, study, modify, and compare cyanobacteria genes; (4) to analyze evolutionary interrelations, including relations between chloroplasts and ancient cyanobacteria. The methods of molecular genetics elaborated enable to use different biosynthetic potentialities of these photoautotrophic organisms in biotechnology.

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

TRIFLURALIN-INDUCED DISORGANIZATION OF MICROTUBULAR CYTOSKELETON ALTERS THE DEVELOPMENT OF ROOTS IN Hordeum vulgare.
Sheval E.V., Kazhura Y.I., Poleshuk N.A., Lazareva E.M., Smirnova E.A., Maximova N.P., Polyakov V.Y.
ACTA BIOLOGICA HUNGARICA 59 (2008) 465-478.

The extensive use of herbicides in agriculture becomes an important factor in environmental pollution, especially in case of slowly degradable compounds. Some agents act on plants during a long period of time, even if a very low concentration of the herbicide remains in the soil. Here, we investigated the toxicological effect of a low concentration of dinitroaniline herbicide, trifluralin, on growing seedlings of Hordeum vulgare L. Trifluralin in concentration of 1 microg/ml inhibited root growth. The mitotic activity of meristematic cells was suppressed due to the retardation of metaphase progression-alteration that can be caused by cytoskeleton disorder. Using antibodies to ?-tubulin, we investigated the distribution of microtubules in root meristem cells. During all stages of mitosis, the highly regular system of microtubular cytoskeleton observed in control cells was slightly disorganized. An examination of root structure using light and electron microscopy demonstrated that the cell walls did not form normally during cell division that led to the appearance of large multinucleated cells. Also, the premature (pathological) cell differentiation was induced by trifluralin. A part of differentiating cells showed intracellular structural changes that are consistent with programmed cell death. It seems that the development of alterations in trifluralin-treated roots was due to the microtubular cytoskeleton disorganization.

Water molecules immobilized on a protein or DNA surface are known to play an important role in intramolecular and intermolecular interactions. Comparative analysis of related three-dimensional (3D) structures allows to predict the locations of such water molecules on the protein surface. We have developed and implemented the algorithm WLAKE detecting "conserved" water molecules, i.e. those located in almost the same positions in a set of superimposed structures of related proteins or macromolecular complexes. The problem is reduced to finding maximal cliques in a certain graph. Despite exponential algorithm complexity, the program works appropriately fast for dozens of superimposed structures. WLAKE was used to predict functionally significant water molecules in enzyme active sites (transketolases) as well as in intermolecular (ETS-DNA complexes) and intramolecular (thiol-disulfide interchange protein) interactions. The program is available online at http://monkey.belozersky.msu.ru/~evgeniy/wLake/wLake.html.

Contemporary knowledge about centrosome proteins and their ensembles, which can be divided into several functional groups- microtubule-nucleating proteins, microtubule-anchoring proteins, centriole-duplication proteins, cell cycle control proteins, primary cilia growth regulation proteins, and proteins of regulation of cytokinesis-is reviewed. Structural-temporal classification of centrosomal proteins and the scheme of interconnection between the different centrosomal protein complexes are presented.

S acylation of cysteines located in the transmembrane and/or cytoplasmic region of influenza virus hemagglutinins (HA) contributes to the membrane fusion and assembly of virions. Our results from using mass spectrometry (MS) show that influenza B virus HA possessing two cytoplasmic cysteines contains palmitate, whereas HA-esterase-fusion glycoprotein of influenza C virus having one transmembrane cysteine is stearoylated. HAs of influenza A virus having one transmembrane and two cytoplasmic cysteines contain both palmitate and stearate. MS analysis of recombinant viruses with deletions of individual cysteines, as well as tandem-MS sequencing, revealed the surprising result that stearate is exclusively attached to the cysteine positioned in the transmembrane region of HA.

Influenza A virus matrix M1 protein is membrane associated and plays a crucial role in virus assembly and budding. The N- terminal two thirds of M1 protein was resolved by X-ray crystallography. The overall 3D structure as well as arrangement of the molecule in relation to the viral membrane remains obscure. Now a proteolytic digestion of virions with bromelain was used as an instrument for the in situ assessment of the M1 protein structure. The lipid bilayer around the subviral particles lacking glycoprotein spikes was partially disrupted as was shown by transmission electron microscopy. A phenomenon of M1 protein fragmentation inside the subviral particles was revealed by SDS-PAGE analysis followed by in-gel trypsin hydrolysis and MALDI-TOF mass spectrometry analysis of the additional bands. Putative bromelain-digestion sites appeared to be located at the surface of the M1 protein globule and could be used as landmarks for 3D molecular modeling.

A 25-kD movement protein (25K protein) encoded by the first gene of the potexvirus Potato virus X triple gene block of transport genes is essential for the viral movement in infected plants. The 25K protein belongs to superfamily 1 of NTPase/helicases and exhibits in vitro RNA helicase, Mg²⁺-dependent NTPase, and RNA-binding activities. In the present work, the ability of 25K protein for homologous interactions was studied using the yeast two-hybrid system, protein chemical cross-linking in the presence of glutaraldehyde, far-Western blotting, and ultracentrifugation in sucrose density gradients. The 25K protein was shown to form homodimers and homooligomers. Sites of homologous protein-protein interactions were found in both the N- and C-terminal portions of the protein.

The interactions of non-ionic surfactant Triton X-100 and the coat protein of tobacco mosaic virus, which is an established model for both ordered and non-ordered protein aggregation, were studied using turbidimetry, differential scanning calorimetry, isothermal titration calorimetry, and dynamic light scattering. It was found that at the critical aggregation concentration (equal to critical micelle concentration) of 138 x 10-6 M, Triton X-100 induces partial denaturation of tobacco mosaic virus coat protein molecules followed by protein amorphous aggregation. Protein aggregation has profound ionic strength dependence and proceeds due to hydrophobic sticking of surfactant-protein complexes (start aggregates) with initial radii of 46 nm. It has been suggested that the anionic surfactant sodium dodecyl sulfate forms mixed micelles with Triton X- 100 and therefore reverses protein amorphous aggregation with release of protein molecules from the amorphous aggregates. A stoichiometric ratio of 5 was found for Triton X-100-sodium dodecyl sulfate interactions.

The goals of the study were: (1) to explore the communication between human mesenchymal stem cells (MSC) and rat cardiac myocytes resulting in differentiation of the stem cells and, (2) to evaluate the role of mitochondria in it. Light and fluorescence microscopy as well as scanning electron microscopy revealed that after co-cultivation, cells formed intercellular contacts and transient exchange with cytosolic elements could be observed. The transport of cytosolic entity had no specific direction. Noticeably, mitochondria also could be transferred to the recipient cells in a unidirectional fashion (towards cardiomyocytes only). Transmission electron microscopy revealed significant variability in both the diameter of intercellular contacting tubes and their shape. Inside of these nanotubes mitochondria-resembling structures were identified. Moreover, after co-cultivation with cardiomyocytes, expression of human-specific myosin was revealed in MSC. Thus, we speculate that: (1) transport of intracellular elements to MSC possibly can determine the direction of their differentiation and, (2) mitochondria may be involved in the mechanism of the stem cell differentiation. It looks plausible that mitochondrial transfer to recipient cardiomyocytes may be involved in the mechanism of failed myocardium repair after stem cells transplantation.

Using an original high-salt extraction protocol, we observed a novel chromosome substructure, referred to as the peripheral chromosome scaffold. This chromosome domain contained the perichromosomal layer proteins pKi-67, B23/nucleophosmin and fibrillarin, but no DNA fragments (i.e., the loop domain bases were not associated with the peripheral scaffold). Modern models of chromosome organization do not predict the existence of a peripheral chromosome scaffold domain, and thus our observations have conceptual implications for understanding chromosome architecture.


A method for isolation of constitutive heterochromatin (chromocenters) from nuclei of mouse liver cells is described. This method is based on the higher resistance of chromocenters to low ionic strength treatment as compared with that of nucleoli and euchromatin. The method allows separation of chromocenters that are essentially free of nucleoli and other nuclear contaminants. In contrast to nuclei and nucleoli, isolated chromocenters are characterized by a simpler protein composition and contain a smaller number of proteins (especially of high molecular weight proteins). They possess telomeric DNA and telomerase activity that suggests a tight association of chromocenters with the telomerase complex in mouse hepatocyte nuclei.

The terahertz time-domain and Raman spectra of sulfur-containing cystein-based peptides in the region of the low-frequency infrared vibrations have been measured at room temperature. The low-frequency bands that can be assigned to the S-S bridges are observed. The vibrational modes found in molecular crystalline materials should be described as phonon modes with strong coupling to the intra molecular vibrations.

Microtubules spatial organization is essential for different cellular processes to proceed normally. It is supposed traditionally, that the fibroblasts have radial microtubule array consisting of long microtubules running from the centrosome. However, the detailed analysis of the microtubule array in the internal cytoplasm has never been performed. In the current study we used laser photobleaching for the analysis of the spatial organization of microtubules in the internal cytoplasm of cultured 3T3 fibroblasts. Cells were injected with Cy-3-labeled tubulin, and then in the bleached zone growth of microtubules in the centrosome region and in the peripheral parts of cytoplasm was analyzed. In most cases microtubules growth in the bleached zone occurred rectilinearly, on the distance up to 5 microm they seldom bend more than 10-15 degrees. We considered a growing fragment of the microtubule as a vector with the beginning in the point of occurrence and with the end in a point where growth terminated (or the end point after 30 s if microtubule's persistent growth proceeded longer). We defined the direction of microtubules growth in different parts of the cell using these vectors and measured the angle of their deviation from the vector of comparison. In the area of the centrosome we directed the vector of comparison inside of the bleached zone from the centrosome to the beginning of the growing microtubule segment; in fibroblast lamella and in fibroblast trailing part we used, the vector of comparison was directed along the long axis of the cell from its geometrical center to periphery. The microtubules growing immediately from the centrosome grew along the cell radius. However at a distance of 10 microm from the centrosome radially growing microtubules gave 40% from the overall number, and at a distance of 20 microm--only 25%. The rest of microtubules grew in different directions, with the preferred angle between their growth direction and cell radius around 90 degrees. Fibroblast lamella and trailing part 80% of all microtubules grew along the cell long axis or at the angle no more than 20 degrees, and 10-15% of microtubules grew along cell axis but towards the centrosome. Thus, in 3T3 fibroblasts the radial system of microtubules is perturbed starting from the distance of several microns from the centrosome. In the internal cytoplasm the microtubule system is completely disordered, and in the stretched parts of the polarized cell (lamella, trailing edge) the microtubule system again becomes well organized--microtubules are preferentially oriented along the long cell axis. From the results obtained we conclude that orderliness of microtubules at the periphery of the fibroblast is not a consequence of their growth from the centrosome, but their orientation is preset by local factors.

In interphase cells, microtubules (MT) are long and form extended radial array. The length of individual MTs in living cells exhibits substantial stochastic fluctuations while the average length distribution in a cell remains nearly constant. We present a quantitative model that describes relation of the MT length and dynamics in the steady state in the cell using the minimal set of parameters (cell radius, tubulin concentration, critical concentration for plus end elongation, and the number of nucleation sites). The MT array is approximated as a radial system, where MT minus ends are associated with the nucleation sites on the centrosome, while plus ends grow and shorten. Dynamic instability of MT plus ends is approximated as a random walk process with boundary conditions and the behavior of MT array is quantified using diffusion and drift coefficients (Vorobjev et al., 1997, 1999). We show that establishment of the extended steady-state array could be accomplished solely by the limitation of the MT growth by the cell margin. We determined for the cell radius, tubulin concentration, critical concentration for plus end elongation, and number of nucleation sites the reference point in the parameter space where plus ends of individual MT on average neither elongate nor shorten. In this case average length of MT is equal to the half of cell radius. When any parameter is shifted from its reference value MTs become longer or shorter and consequently acquire positive or negative drift of their ends. In the vicinity of reference point, change in any parameter has major effect on the MT length and rather small effect on the drift. When mean length of the MTs is close to the cell radius the drift of the free plus ends becomes substantial, resulting in processive growth of individual MTs in the internal cytoplasm accompanied by apparent stabilization of the plus ends at the cell margin. Under these conditions small changes in parameters have significant impact on the magnitude of drift. Experimental analysis of the MT plus ends dynamics in different cultured cells shows that in most cases plus ends display positive drift, which, in the framework of the presented model, is in agreement with the simultaneous presence of long MTs.

The effects of DNA methylation inhibitor 5-azacytidine (5-aza-C) and histone acetylation inhibitor trichostatine A (TSA) on the structure of pericentric heterochromatin in cultured mouse cells (L929) has been studied. After 48 h of 5-aza-C treatment, about 85% of the cells demonstrate transformation of chromocenters from ovoid to elongated structures. Hypotonic treatment of these cells reveals tandemly arranged DAPI-positive globules, well distinguishable by light microscopy. The same globular units can be revealed in hypotonic-treated control cells. 48 h of TSA treatment causes dramatic decrease in HP 1alpha content in the cells. Chromocenters in 25% of treated cells became highly decondenced and can not be reliably detected by light and electron microscopy. 85% of cells demonstrate globular chromocenters with low HP 1alpha content. Hypotonic treatment causes transformation of compact chromocenters into ring-like structures, which can be either single or clustered. Rings are formed by uniform fiber, in which no globular subunits are detected. The data obtained are discussed concerning several mechanisms of heterochromatin structure maintenance and the roles of epigenetic marks in them.

Endothelial cell barrier dysfunction is often associated with dramatic cytoskeletal reorganization, activation of actomyosin contraction and finally gap formation. At present time the role of microtubules in endothelial cell barrier regulation is not fully understood, however a number of observations allow to assume that microtubules reaction is the extremely important part in development of endothelial dysfunction. These observations have been forced us to examine the role of microtubule system reorganization in endothelial cell barrier regulation. In quiescent endothelial cells microtubule density is the highest in the centrosome region and insignificant near the cell margin. The analysis of microtubules distribution after specific antibodies staining using the method of measurement of their fluorescence intensity has shown that in control endothelial cells the reduction of fluorescence intensity from the cell center to its periphery is described by the equation of an exponential regression. The hormone agent, thrombin (25 nM), causes rapid increase of endothelial cell barrier permeability accompanied by fast decrease in quantity of peripheral microtubules and reorganization of microtubule system in internal cytoplasm of endothelial cells (the decrease of fluorescence intensity is described by the equation of linear regress already through 10 min after the beginning of the treatment). Both effects are reversible -- through 60 min after the beginning of the treatment the microtubule network does not differ from normal one, so the microtubule system is capable to adapt for influence of a natural regulator thrombin. The microtubules reaction develops more quickly, than reorganization of the actin filaments system, which responsible for the subsequent changes in the cell shape during barrier dysfunction. Apparently, the microtubules are the first part in a circuit of the reactions leading to the pulmonary endothelial cell barrier compromise.

In the present review the description of history of the centrosome investigation is given and the current state of knowledge of this cellular structure in morphological, biochemical, and functional aspects is presented. Besides of the classical functions of the centrosome as a MT nucleating, MT ancoriging, and MT organizing center, the idea about the centrosome as a cellular regulating center and as a structural part of the mechanism operating dynamic morphology of a cell is discussed.

It is well known that at the beginning of mitosis the nucleolus disassembles but then reassembles at the end of mitosis. However, the mechanisms of these processes are still unclear. In the present work, we show for the first time that selective inhibition of cyclin B-dependent kinase 1 (CDK1) by roscovitine induces premature assembly of the nucleolus in mammalian cells in metaphase. Treatment of metaphase cells with roscovitine induces formation of structures in their cytoplasm that contain major proteins of the mature nucleolus participating in rRNA processing, such as B23/nucleophosmin, C23/nucleolin, fibrillarin, Nop52, as well as partially processed (immature) 46-45S pre-rRNA. This effect is reproducible in cells of various types; this indicates that general mechanisms regulate early stages of the nucleolus reassembly with CDK1 participation in mammalian cells. Based on our and literature data, we suggest that inactivation of the CDK1-cyclin B complex at the end of mitosis results in dephosphorylation of B23/nucleophosmin and C23/nucleolin; this facilitates their interaction with pre-rRNA and leads to formation of insoluble supramolecular complexes-nucleolus-derived foci.

The worldwide campaign to eradicate poliomyelitis is nearing its 20th anniversary, and is 8 years over its original target date of the year 2000. During the past decade, the programme has encountered a number of unanticipated obstacles,1-3 which have led to renewed discussions about whether eradication of poliomyelitis is feasible and, if so, what is the best way to achieve it.4-6 To address these issues, we engaged more than 100 scientists from 18 countries-virologists, epidemiologists, public- health workers, policy makers, and representatives from the pharmaceutical industry-to review current progress and propose directions for the future.

A yeast plasmid was constructed to contain a hybrid GAL-CYC promoter, the NPTII neomycin phosphotransferase gene, and the FRT sequence between them. The CYC part of the GAL-CYC promoter harbored four upstream activating sequences (UASs) and two close TATA boxes. NPTII was efficiently expressed upon induction with galactose, conferring G418 resistance on yeast cells. Nucleosome positioning was studied in repressed and induced NPTII in transformed cells. A stable positioning of three nucleosomes was detected under repressive conditions (growth on glucose). Two nucleosomes were on the CYC part of the promoter, one including both of the TATA boxes. The third nucleosome overlapped the FRT sequence and the start of the NPTII coding region. Each of the three nucleosomes displayed multiple positions, suggesting their sliding along DNA. After induction of NPTII expression with galactose, a sliding of two nucleosomes was detected, exposing the TATA box and a long promoter segment. The 5'-distal nucleosome moved closer to the UASs, bringing them closer to the TATA box, which was assumed to facilitate the assembly of the preinitiation complex. The two nucleosomes slid independently of each other. The second nucleosome moved towards the FRT sequence and repositioned at its nucleosome positioning signal. Galactose-induced expression did not affect the nucleosome positioning in the coding region of NPTII. Unidirectional sliding and repositioning were detected without induction after deacetylase inhibition with trichostatin A. Basal NPTII expression was observed without activation of the GAL-CYC promoter and after a spatial uncoupling of the coding sequence and promoter via gene inversion and was probably driven by the FRT TATA-like element, which is in the region permanently exposed in vivo.

Conformational changes in chromatin structure are nowadays the object of intensive research due to its importance for proper regulation of intranuclear processes. The fine structure of chromatin within the DNA replication sites was studied in in situ fixed cells and cells permebilized by low ionic strength solutions in the presence of divalent cations. The latter method provides visualization of higher level chromatin structures such as globular chromomeres and chromonema fibres. Nascent DNA was detected immunochemically using anti-BrdU antibodies on the surface of ultrathin sections prepared from Epon- embedded material. It was shown that newly replicated DNA preferentially localized within the zones filled with globular and fibrillar elements with characteristic diameter of 30 nm, and not in chromonema fibres, while after replication had been completed DNA became embedded into as thick as 60-80 nm chromonema elements. The results obtained are discussed in the context of conception of hierarchical folding of chromatin fibers.

The lecture covers three main topics: (i) Viruses: properties, place in the living world, and possible origin; (ii) Molecular basis of viral variability and evolution; and (iii) Evolution of viral pathogenicity and emerging viral infections.

Рассмотрены различные аспекты молекулярной эволюции вакцинных штаммов полиовируса: мутационная и рекомбинационная изменчивость, закономерности фиксации возникающих генетических изменений. Освещены вопросы, касающиеся политики вакцинации населения против полиомиелита.

We found that a 2-h incubation of potato virus X (PVX) virions in 10 mM Tris-HCl buffer pH 7.5 at ?20°C results in a strong but reversible drop in virion stability. Under these conditions, the PVX virions are completely disrupted by low (starting from 50 mM) concentrations of LiCl and CaCl2 but not of NaCl. Incubation of PVX samples with 0.05?2 M LiCl at +4 C did not result in virion disassembly and the virions were not disrupted upon incubation at ?20°C in 10 mM Tris-HCl buffer pH 7.5 without LiCl. We suggest that a 2-h incubation of the PVX virions at ?20°C in 10 mM Tris-HCl pH 7.5 results in a structural transition in the virus particles. A revised model of the three-dimensional organization of coat protein subunits in the PVX virions is proposed. This two-domain model explains better the high plasticity of the PVX CP structure.

Intracellular disk-like lamellar structure (DLS), which we had found earlier in the motility apparatus of Halobacterium salinarum, was studied by means of electron microscopy. The details of the insertion of the flagella proximal ends in the DLS were also investigated. Analysis of ultra-thin sections after the fixation with the help of potassium permanganate suggests that the DLS includes a membrane structure. Electron micrographs of cell "ghosts" obtained in solutions with low concentration of NaCl made possible to study both the Structure of DLS and the way of fitting flagella in it. The organization of the motility apparatus in two representatives of separate taxonomic domains, bacteria and archaeae, is discussed.

The intracellular structure found earlier in Halobacterium salinarum cells and named "polar organelle" was Studied in detail by electron microscopy of single and serial ultrathin sections. This structure was found near the cell pole under membrane on each side of disk-like lamellar structure (DLS) in the motility apparatus of Hb. salinarum, Morphologic features of this structure are identical to those described in literature as bacterial "polar organelle". The structure, like the bacterial "polar organelle", is stained cytochemically by reagents detecting ATPase activity. The role of this structure in Bacteria and Archaea is discussed.

The density of glycoprotein (GP) distribution on the virion surface substantially influences the virus infectivity and pathogenicity. A method to quantitatively determine the area occupied by surface GP spikes was proposed for influenza virus (Flu) strain A/PR/8/34 on the basis of data of tritium bombardment and dynamic light scattering. The latter was used to measure the diameter of intact virions and subviral particles (Flu virions lacking GP spikes after bromelain digestion). Intact virions and subviral particles were bombarded with a hot tritium atom flux, and the specific radioactivity of the matrix M1 protein was analyzed. The tritium label was incorporated into the amino acid residues of a thin exposed protein layer and partly penetrated through the lipid bilayer of the viral envelope, labeling M1, located under the lipid bilayer. The tritium label distribution among different amino acid residues was the same in M1 isolated from subviral particles and M1 isolated from intact virions, demonstrating that the M1 spatial structure remained unchanged during proteolysis of GP spikes. The difference in specific radioactivity between the M1 proteins isolated from intact virions and subviral particles was used to calculate the GP-free portion of the viral surface. Approximating the Flu virion as a sphere, the GP-covered area was estimated at 1.4 x 104 nm2, about 40% of the total virion surface. This was consistent with the cryoelectron tomography data published for Flu strain A/X-31. The approach can be applied for other enveloped high pathogenic viruses, such as HIV and the Ebola virus.

Insulin maintains homeostasis of glucose by promoting its uptake into cells from the blood. Hyperglycemia triggers secretion of insulin from pancreatic beta-cells. This process is mediated by secretory granule exocytosis. However, how beta-cells keep granule stores relatively constant is still unknown. ICA512 is an intrinsic granule membrane protein, whose cytosolic domain binds beta 2-syntrophin, an F-actin-associated protein, and is cleaved upon granule exocytosis. The resulting cleaved cytosolic fragment, ICA512-CCF, reaches the nucleus and up-regulates the transcription of granule genes, including insulin and ICA512. Here, we show that ICA512-CCF also dimerizes with intact ICA512 on granules, thereby displacing it from beta 2-syntrophin. This leads to increased granule mobility and insulin release. Based on these findings, we propose a model whereby the generation of ICA512-CCF first amplifies insulin secretion. The ensuing reduction of granule stores would then increase the probability of newly generated ICA512-CCF to reach the nucleus and enhance granule biogenesis, thus allowing beta-cells to constantly adjust production of granules to their storage size and consumption. Pharmacological modulation of these feedback loops may alleviate deficient insulin release in diabetes.

Actin filaments that serve as "rails" for the myosin-based transport of membrane organelles [1-4] continuously turn over by concurrent growth and shortening at the opposite ends [5]. Although it is known that dynamics of actin filaments is essential for many of the actin cytoskeleton functions, the role of such dynamics in myosin-mediated organelle transport was never studied before. Here, we addressed the role of turnover of actin filaments in the myosin-based transport of membrane organelles by treating cells with the drugs that suppress actin-filament dynamics and found that such a suppression significantly inhibited organelle transport along the actin filaments without inhibiting their intracellular distribution or the activity of the myosin motors. We conclude that dynamics of actin filaments is essential for myosin-based transport of membrane organelles and suggest a previously unknown role of actin-filament dynamics in providing the "rails" for continuous organelle movement resulting in the increased distances traveled by membrane organelles along the actin filaments.

Membrane solubilization with a mixture of cold non-ionic detergents has been applied to isolate detergent-resistant membranes from intact virus A lipid bilayer. Association of the viral envelope glycoproteins and M1 into a raft lipid-protein complex was verified via detergent insolubility experiments, and the M1:HA stoichiometry of the proposed supramolecular complex was estimated via amino acid analysis. Electron microscopy and dynamic light scattering data revealed that these lipid-protein rafts form unilamellar vesicles with HA spikes on their surfaces similar to influenza virus virions. Together, our data suggest that the cold co-extraction technique visualizes the raft-like nature of the viral envelope and demonstrates the interaction of matrix M1 protein with the envelope.

Interphase microtubules are organized into a radial array with centrosome in the center. This organization is a subject of cellular regulation that can be driven by protein phosphorylation. Only few protein kinases that regulate microtubule array in interphase cells have been described. Ste20-like protein kinase LOSK (SLK) was identified as a microtubule and centrosome- associated protein. In this study we have shown that the inhibition of LOSK activity by dominant-negative mutant K63R-Delta T or by LOSK depletion with RNAi leads to unfocused microtubule arrangement. Microtubule disorganization is prominent in Vero, CV-1, and CHO-K1 cells but less distinct in HeLa cells. The effect is a result neither of microtubule stabilization nor of centrosome disruption. In cells with suppressed LOSK activity centrosomes are unable to anchor or to cap microtubules, though they keep nucleating microtubules. These centrosomes are depleted of dynactin. Vero cells overexpressing K63R-Delta T have normal dynactin "comets" at microtubule ends and unaltered morphology of Golgi complex but are unable to polarize it at the wound edge. We conclude that protein kinase LOSK is required for radial microtubule organization and for the proper localization of Golgi complex in various cell types.

Phosphatidylinositol 4-phosphate (PI4P) is a key regulator of membrane transport required for the formation of transport carriers from the trans-Golgi network (TGN). The molecular mechanisms of PI4P signaling in this process are still poorly understood. In a search for PI4P effector molecules, we performed a screen for synthetic lethals in a background of reduced PI4P and found the gene GGA2. Our analysis uncovered a PI4P-dependent recruitment of the clathrin adaptor Gga2p to the TGN during Golgi-to-endosome trafficking. Gga2p recruitment to liposomes is stimulated both by PI4P and the small GTPase Arf1p in its active conformation, implicating these two molecules in the recruitment of Gga2p to the TGN, which ultimately controls the formation of clathrin-coated vesicles. PI4P binding occurs through a phosphoinositide-binding signature within the N-terminal VHS domain of Gga2p resembling a motif found in other clathrin interacting proteins. These data provide an explanation for the TGN-specific membrane recruitment of Gga2p.

Endothelium lining the internal surface of blood vessels carries out the barrier function and regulates vascular membrane permeability and thus provides an exchange of nutrients and metabolites between blood circulating in vessels and tissue liquids. Disturbances of the barrier function of endothelium are related with cytoskeletal reorganization, activation of actomyosin contraction, and gap formation. Microtubules are the first effector part in the reaction chains leading to the barrier disorder. Increased vascular permeability caused by endothelial dysfunction is observed in many human diseases and, in particular, emerges as a by-effect of mitosis-blocking drugs used for treatment of oncological diseases. In this study, we attempted to find a concentration of a mitostatic agent that would affect microtubules without significant interference with the endothelial barrier function. The population of microtubules in endothelial cells is heterogeneous; in the cytoplasm, alongside with dynamic microtubules, there present post-translationally modified microtubules, less dynamic and less susceptible to external influences. The area occupied by such stable microtubules in endothelial cells is rather significant (about one third of the cell area), and we have assumed that this may be a base of the resistance of the endothelial microtubule system to factors inducing barrier dysfunction. This assumption was examined using nocodazole as an inducer of the barrier dysfunction in endothelial cells. The effect of nocodazole on endothelial cell cytoskeleton is dose-dependent. At micromolar concentrations nocodazole causes an incurable damage of the barrier function and irreversibly upsets vital functions of cells. Treatment with nocodazole in nanomolar concentrations also causes an increase of endothelial monolayer permeability. However, in the concentration range 100200 nM this effect is reversible. Treatment with 100 nM nocodazole leads to a partial disruption of microtubules near the cell margin without significant influence on the quantity of acetylated microtubules and actin filaments. Increase of the concentration up to 200 nM leads to a notable destruction of dynamic (but not acetylated) microtubules and also to an increase of the actin filament quantity in the central area of the cell. We suggest that the destruction of peripheral microtubules triggers the reaction cascade leading to the endothelial barrier dysfunction, but the presence of a significant amount of stable microtubules resistant to nanomolar concentrations of nocodazole helps to keep cell viability and also to restore functional activity.

Actin is one of the most abundant proteins in nature. It is found in all eukaryotes and plays a fundamental role in many diverse and dynamic cellular processes. Also, actin is one of the most ubiquitous proteins because actin-like proteins have recently been identified in bacteria. Actin filament (F-actin) is a highly dynamic structure that can exist in different conformational states, and transitions between these states may be important in cytoskeletal dynamics and cell motility. These transitions can be modulated by various factors causing the stabilization or destabilization of actin filaments. In this review, we look at actin stabilization and destabilization as expressed by changes in the thermal stability of actin; specifically, we summarize and analyze the existing data on the thermal unfolding of actin as measured by differential scanning calorimetry. We also analyze in vitro data on the heat-induced aggregation of actin, the process that normally accompanies actin thermal denaturation. In this respect, we focus on the effects of small heat shock proteins, which can prevent the aggregation of thermally denatured actin with no effect on actin thermal unfolding. As a result, we have proposed a mechanism describing the thermal denaturation and aggregation of F-actin. This mechanism explains some of the special features of the thermal unfolding of actin filaments, including the effects of their stabilization and destabilization; it can also explain how small heat shock proteins protect the actin cytoskeleton from damage caused by the accumulation of large insoluble aggregates under heat shock conditions.

The proteolysis or flu virions of the strain A/Puerto Rico/8/34 (subtype H 1N1) by enzymes of various classes was studied to develop an approach to the study of the structural organization and interaction of the major protein components of the virion: hemagglutinin (HA), transmembrane homotrimeric glycoprotein, and matrix protein M I forming a layer under the lipid membrane. Among the tested proteolytic enzymes and enzymic preparations (thermolysin, trypsin, chymotrypsin, subtilisin Carlsberg, pronase, papain, and bromelain), the cysteine proteases bromelain and papain and the enzymic preparation pronase efficiently removed HA ectodomains, while chymotrypsin, trypsin, and subtilisin Carlsberg deleted only a part of them. An analysis by MALDI TOF mass spectrometry allowed us to locate the sites of HA hydrolysis by various enzymic preparations. Bromelain, papain, trypsin, and pronase split the polypeptide chain after the K177 residue located before the transmembrane domain (HA(2) 185-211). Subtilisin Carlsberg hydrolyzed the peptide bond at other neighboring points: after L178 (a major site) or V176. The hydrolytic activity of bromelain measured by a highly specific chromogenic substrate of cysteine proteases Glp-Phe-Ala-pNA was almost three times higher in the presence of 5 mM beta-mercaptoethanol than in the presence of 50 mM. However, the complete removal of ectodomains of HA by the high- and low-activity enzyme required identical time intervals. In the absence of the reducing reagent, the removal of HA by bromelain proceeded a little more slowly and was accompanied by significant fragmentation of protein M1. The action of trans-epoxysuccinyl-L-leucylamido)(4- guanidino)butane (E-64), a specific inhibitor of cysteine proteases, and HgCl2 on the hydrolysis of proteins HA and M1 by bromelain was investigated.

We applied different methods, such as turbidity measurements, dynamic light scattering, differential scanning calorimetry and co-sedimentation assay, to analyze the interaction of small heat shock protein Hsp27 with isolated myosin head ( myosin subfragment 1, S1) under heat-stress conditions. Upon heating at 430C, Hsp27 effectively suppresses S1 aggregation, and this effect is enhanced by mutations mimicking Hsp27 phosphorylation. However, Hsp27 was unable to prevent thermal unfolding of myosin heads and to maintain their ATPase activity under heat-shock conditions.

Specific interactions of the classical swine fever virus internal ribosomal entry site (IRES) with 40S ribosomal subunits and eukaryotic translation initiation factor (eIF) 3 enable 43S preinitiation complexes containing eIF3 and eIF2-GTP-Met- tRNA(i)(Met) to bind directly to the initiation codon, yielding 48S initiation complexes. We report that eIF5B or eIF5B/eIF3 also promote Met-tRNA(i)(Met) binding to IRES-40S complexes, forming 48S complexes that can assemble elongation- competent ribosomes. Although 48S complexes assembled both by eIF2/eIF3- and eIF5B/eIF3-mediated Met-tRNA(i)(Met) recruitment were destabilized by eIF1, dissociation of 48S complexes formed with eIF2 could be out-competed by efficient subunit joining. Deletion of IRES domain II, which is responsible for conformational changes induced in 40S subunits by IRES binding, eliminated the sensitivity of 48S complexes assembled by eIF2/eIF3- and eIF5B/eIF3-mediated mechanisms to eIF1-induced destabilization. However, 48S complexes formed by the eIF5B/eIF3-mediated mechanism on the truncated IRES could not undergo efficient subunit joining, as reported previously for analogous complexes assembled with eIF2, indicating that domain II is essential for general conformational changes in 48S complexes, irrespective of how they were assembled, that are required for eIF5-induced hydrolysis of eIF2-bound GTP and/or subunit joining.

Cytoplasmic dynein is known to be involved in the establishment of radial microtubule (MT) arrays. During mitosis, dynein activity is required for tethering of the MTs at the spindle poles. In interphase cells, dynein inhibitors induce loss of radial MT organization; however, the exact role of dynein in the maintenance of MT arrays is unclear. Here, we examined the effect of dynein inhibitors on MT distribution and the centrosome protein composition in cultured fibroblasts. We found that while these inhibitors induced rapid (t(1/2) similar to 20 min) loss of radial MT organization, the levels of key centrosomal proteins or the rates of MT nucleation did not change significantly in dynein-inhibited cells, suggesting that the loss of dynein activity does not affect the structural integrity of the centrosome or its capacity to nucleate MTs. Live observations of the centrosomal activity showed that dynein inhibition enhanced the detachment of MTs from the centrosome. We conclude that the primary role of dynein in the maintenance of a radial MT array in interphase cells consists of retention of MTs at the centrosome and hypothesize that dynein has a role in the MT retention, separate from the delivery to the centrosome of MT-anchoring proteins.

Potato virus A (PVA) particles were purified byna leaves to study the TGBp3 intracellular trafficking pathway. Treatment with inhibitors was used to reveal that the targeting of TGBp3 to plasmodesmata does not require a functional cytoskeleton or secretory system. In addition, the suppression of endoplasmic reticulum-derived vesicle formation by a dominant negative mutant of small GTPase Sar1 had no detectable effect on TGBp3 trafficking to peripheral bodies. Collectively, these results suggested the involvement of an unconventional pathway in the intracellular transport of TGBp3. The determinants of targeting to plasmodesmata were localized to the C-terminal region of TGBp3, including the conserved hydrophilic and terminal membrane-spanning domains.

Cytological and molecular genetics methods were used to study sperm from patients with sperm infected with herpes simplex virus (HSV) as indicated by virological and immunocytochemical tests. The following methods were used: (1) sperm analysis to evaluate the morphology and functional properties of sperm; (2) fluorescence in situ hybridization (FISH) with DNA probes specific for chromosomes 1, X, and Y to evaluate nondisjunction frequencies of these chromosomes in sperm; and (3) quantitative analysis of immature germ cells in the ejaculate to identify spermatogenic abnormalities. The total sperm count and the count of sperm with normal motility proved similar to the norm. FISH analysis demonstrated no difference in the nondisjunction frequency of chromosomes 1, X, and Y between infertile patients with HSV-infected sperm and fertile donors. Comparative quantitative analysis of immature germ cells from the ejaculate has demonstrated a significant and considerable (threefold) increase in the number of spermatocytes I in pachytene and diplotene, at the prepachytene stages of prophase I (preleptotene, leptotene, and zygotene) in HSV patients compared to normal donors. At the same time, HSV patients demonstrated a significant decrease in the number of spermatocytes I, in pachytene and diplotene, decrease in the proportion of spermatocytes II and spermatids, and a twofold increase in the number of unidentifiable immature germ cells. The data obtained indicate a partial spermatogenic arrest at the early stages of meiotic prophase I in HSV patients, which prompts further research into the cellular mechanisms of abnormal spermatogenesis after viral infection in humans.

The difficulty in localizing specific cellular proteins by immuno-electron microscopy techniques limits applications of electron microscopy to cell biology. We found that in vivo immunogold labeling improves epitope accessibility, ultrastructural preservation and three-dimensional visualization, and allows correlated light and electron microscopy. We detected large-scale chromatin folding motifs within intact interphase nuclei of CHO cells and visualized the ultrastructure of DNA replication 'factories' labeled with GFP-proliferating cell nuclear antigen (PCNA).

Previously, we have shown that encapsidated Potato virus X (PVX) RNA was non-translatable in vitro, but could be converted into a translatable form by binding of the PVX movement protein TGBp1 to one end of the virion or by coat protein (CP) phosphorylation. Here, a mutagenic analysis of PVX CP and TGBp1 was used to identify the regions involved in TGBp1-CP binding and translational activation of PVX RNA by TGBp1. It was found that the C-terminal (C-ter) 10/18 amino acids region was not essential for virus-like particle (VP) assembly from CP and RNA. However, the VPs assembled from the CP lacking C-ter 10/18 amino acids were incapable of TGBp1 binding and being translationally activated. It was suggested that the 10- amino-acid C-ter regions of protein subunits located at one end of a polar helical PVX particle contain a domain accessible to TGBp1 binding and PVX remodelling. The non-translatable particles assembled from the C-ter mutant CP could be converted into a translatable form by CP phosphorylation. The TGBp1-CP binding activity was preserved unless a conservative motif IV was removed from TGBp1. By contrast, TGBp1-dependent activation of PVX RNA translation was abolished by deletions of various NTPase/helicase conservative motifs and their combinations. The motif IV might be essential for TGBp1-CP binding, but insufficient for PVX RNA translation activation. The evidence to discriminate between these two events, i.e. TGBp1 binding to the CP-helix and TGBp1-dependent RNA translation activation, is discussed.

Potato virus A is studied by atomic force microscopy. Topographic images of virus particles deposited onto mica are obtained. Geometric characteristics of potato virus A are determined by computer-aided analysis of the images obtained.

IV. Genomics, proteomics. Protein and gene ingeneering. transgenosis, gene terapy. molecular medicine.

IMPORT OF HYBRID FORMS OF CYP11A1 INTO YEAST MITOCHONDRIA.
Minenko A.N., Novikova L.A., Luzikov V.N., Kovaleva I.E.
BIOCHIMICA ET BIOPHYSICA ACTA 1780 (2008) 1121-1130.

Agrobacterium tumefaciens is the main plant biotechnology gene transfer tool with host range which can be extended to non- plant eukaryotic organisms under laboratory conditions. Known medical cases of Agrobacterium species isolation from bloodstream infections necessitate the assessment of biosafety-related risks of A. tumefaciens encounters with mammalian organisms. Here, we studied the survival of A. tumefaciens in bloodstream of mice injected with bacterial cultures. Bacterial titers of 108 CFU were detected in the blood of the injected animals up to two weeks after intravenous injection. Agrobacteria carrying Cauliflower mosaic virus (CaMV) 35S promoter-based constructs and isolated from the injected mice retained their capacity to promote green fluorescent protein (GFP) synthesis in Nicotiana benthamiana leaves. To examine whether or not the injected agrobacteria are able to express in mouse organs, we used an intron-containing GFP (GFPi) reporter driven either by a cytomegalovirus (CMV) promoter or by a CaMV 35S promoter. Western and northern blot analyses as well as RT-PCR analysis of liver, spleen and lung of mice injected with A. tumefaciens detected neither GFP protein nor its transcripts. Thus, bacteraemia induced in mice by A. tumefaciens does not lead to detectable levels of genetic transformation of mouse organs.

Complete and incomplete transitions of epitheliocytes into cells of mesenchymal type, so-called epithelial-mesenchymal transitions (EMT), take place in many types of normal morphogenesis and in epithelial carcinogenesis. Connective tissue cells (fibroblasts) also undergo considerable morphological changes during normal morphogenesis and carcinogenesis, but their dynamics are less known. It is suggested that EMT and fibroblast dynamics may have some common step that is some united precursor cell type. The program for normal EMT can be activated in the course of multistep progression of epithelial carcinogenesis; this activation can be supported by cell selection as it provides a basis for dissemination of neoplastic cells from original tumor.

The appearance of antibodies to cancer-associated antigens in biological fluids (particularly, in blood sera) of cancer patients is now a well-established fact, and their detection by immunochemical methods is a promising approach to diagnostics of malignant neoplasms. In this review, we consider some immunobiological aspects of the most extensively studied cancer- associated B-cell antigens, various applications of autoantibodies as cancer biomarkers, and prospects for the use of antigen arrays for improving diagnostic sensitivity.

An insufficient number of insulin-producing beta-cells is a major cause of defective control of blood glucose in both type 1 and type 2 diabetes. The aim of this study was to clarify whether the insulinotropic imidazolines can affect the survival of highly proliferating insulin-secreting cells, here exemplified by the MIN6 cell line. Our data demonstrate that RX871024, but not efaroxan, triggered MIN6 cell death and potentiated death induced by a combination of the pro-inflammatory cytokines interleukin-1 beta, interferon- gamma and tumor necrosis factor-alpha. These effects did not involve changes in nitric oxide production but correlated with stimulation of c-jun N-terminal kinase (JNK) activity and activation of caspases-1, -3, -8 and -9. Our results suggest that the imidazoline RX871024 causes death of highly proliferating insulin-secreting cells, putatively via augmentation of JNK activity, a finding that may impact on the possibility of using compounds of similar activity in the treatment of diabetes.

Senescence and wilting of the leaves of pea (Pisum sativum L.) of normal (AfAf) and aphyllous (afaf) genotypes were accompanied by DNA degradation. In young (12th-9th) subapical leaves of AfAf plants, total DNA was high-polymeric; in the 6th leaf, DNA degradation was appreciable; and in the 4th and 3rd leaves, hydrolysis of DNA was pronounced. Similar degradation of DNA was also observed in senescing leaves of aphyllous plants, but there it started later than in the plants of normal type. The extent of DNA degradation was closely related to the elevation of total nuclease activity in pea leaves associated with the age. The leaves of plants of different genotypes distinctly differed in the activity of acid and alkaline nucleases. Senescence of the leaves was accompanied by the induction of Ca²⁺ and Mg²⁺-dependent nucleases with mol wts of 42, 37, 34, 26, and 21 kD. In different stages of leaf senescence, different sets of nucleases were detected.

Dimeric bisbenzimidazole DB(5) fluorescing in the blue spectral area (lambda(em) 476 nm) within the DNA complex was synthesized. DB(5) is bound by a double-strand DNA and can differentially stain human and plant (flax) chromosomes. According to preliminary data, it provides a considerably more intensive contrast of chromosome staining than DAPI and Hoechst 33258 dyes. It was also found that DB(5) is an in vitro inhibitor of HIV-1 integrase in the reaction of 3'-processing.

Proteins involved in the visual signaling cascade show light-dependent expression levels in photoreceptor cells. Recently, these proteins have been described to be expressed in neuroectodermal tumors and to function as cancer-retina antigens. Here, we show that light can down-regulate gene expression of rhodopsin, transducin, and cyclic guanosine 3',5'-monophosphate phosphodiesterase 6 (PDE6) and up-regulate guanylyl cyclase 1, recoverin, and arrestin in human melanoma cells in vitro, comparable to physiologic changes earlier observed in photoreceptor cells. Similar modulation can be detected at the protein level in melanoma cells except for no changes in PDE6 protein levels. Two regulatory pathways have been identified: Sp1/Sp3/Sp4 proteins for rhodopsin and PDE6, and mitogen-activated protein kinases for recoverin and arrestin. The visual cascade and retinoic acid as its derivate do not play any role in this process. Putative explanations for light-dependent modulation of cancer-retina antigen expression in melanoma cells are discussed.

Nuclear ITS1-2 and chloroplast trnL-F were sequenced for 21 taxa, of Lophozia s. str., two species of Protolophozia, five species ofsue inducer cells express lymphotoxins (LTs), which are essential for the organogenesis and maintenance of lymphoreticular microenvironments. Here we describe that T-cell-restricted overexpression of LT induces fulminant thymic involution. This phenotype was prevented by ablation of the LT receptors tumor necrosis factor receptor (TNFR) 1 or LT beta receptor (LT beta R), representing two non redundant pathways. Multiple lines of transgenic Lt alpha beta and Lt alpha mice show such a phenotype, which was not observed on overexpression of LT beta alone. Reciprocal bone marrow transfers between LT-overexpressing; and receptor-ablated mice show that involution was not due to a T cell-autonomous defect but was triggered by TNFR1 and LT beta R signaling to radioresistant stromal cells. Thymic involution was partialty prevented by the removal of one allele of LT beta R but not of TNFR1, establishing a hierarchy in these signaling events. infection with the lymphocytic choriomeningitis virus triggered a similar thymic pathology in wit, but not in Tnfr1(-/-) mice. These mice displayed elevated TNF alpha in both thymus and plasma, as well as increased Us on both CD8(+) and CD4(-)CD8(-) thymocytes. These findings suggest that enhanced T cell-derived LT expression helps to control the physiological size of the thymic stroma and accelerates its involution via TNFR1/LT beta R signaling in pathological conditions and possibly also in normal aging.

We have isolated and purified endogenous cytosolic tankyrase from human embryonic kidney cells of line 293. Our data confirm a model of De Rycker and Price who consider that tankyrase is a master scaffolding protein capable of regulating assembly of large protein complexes. We have also studied kinetic characteristics of tankyrase in the complex, pH dependence of the enzyme activity, and its physicochemical properties.

PURPOSE: Tankyrases 1 and 2 are telomere-associated poly(ADP-ribose) polymerases (PARP) that can positively regulate telomere elongation and interact with multiple cellular proteins. Recent reports implicated tankyrases as tumor antigens and potential targets of anticancer treatment. We examined expression of tankyrases in colon tumors and immune response to these enzymes in patients with different types of cancer. METHODS: mRNA and protein expression was evaluated by quantitative real-time RT-PCR and Western blotting, respectively. Humoral immune response to recombinant tankyrases was investigated by modified enzyme-linked immunoassays. Cellular immune response was analysed by ELISPOT and 51Cr release assays. RESULTS: We found that both mRNA and protein levels of tankyrase 2 (TNKL) are upregulated in colon tumors. In contrast, protein level of tankyrase 1 (TNKS) is downregulated, while mRNA level shows variable changes. More than a quarter of colon cancer patients develop humoral immune response to at least one of the two tankyrases. In this study we mapped common and unique B-cell epitopes located in different domains of the two proteins. Additionally, we present evidence for T- cell responses both to epitopes that are unique for TNKL and to those shared between TNKL and TNKS. CONCLUSION: Our study favors a biomarker usage of antibody response to tankyrases. Spontaneous CD8⁺ T-cell responses to these enzymes are rare and further investigation is needed to evaluate tankyrases as potential targets for cancer immunotherapy.

The nuclear oncoprotein prothymosin ? (ProT ?) was tested for the ability to regulate the p53 activity with the use of a reporter gene controlled by a p53-responsive promoter. Overexpression of the ProT ? gene stimulated the p53 activity, while downregulation of the endogenous ProT ? level via RNA interference suppressed transcription of the reporter gene. An increase in ProT ? activated p53-dependent transcription and increased the intracellular p53 content in human HeLa, but not HCT116, cells. N-terminal deletions had almost no effect on the ability of ProT ? to activate p53-dependent transcription, while deletions from the central region and C-terminal mutations distorting the active transport of ProT ? into the cell nucleus prevented its transactivating effect. Mutations affecting Keap1 binding did not impair the ProT ? ability to activate the p53- responsive reporter gene. Based on the results, stimulation of p53-dependent transcription was ascribed to the central acidic region of ProT ? . The conclusion was supported by the fact that parathymosin, another protein containing an extended acidic region, was also capable of activating p53.

Recently, several caspase-like proteases have been described in plants. In particular, we have identified a tobacco caspase-like protease (CLP) that has a caspase cleavage specificity, that becomes activated in the course of plant programmed cell death (PCD), and that is essential for implementation of the cell death programme. The enzyme cleaves a peptide bond next to the Asp (D) residue within the TATD motif in the substrate VirD2 protein. Here we found that activation of tobacco CLP does occur in the course of healthy leaf tissue disruption as well, possibly through zymogen activation or enzyme de-sequestration. CLPs with identical cleavage specificity were demonstrated to be ubiquitous in mono- and dicotyledonous plants. Purified CLPs of tobacco and rice were shown to possess similar biochemical properties. Furthermore, inhibitor analysis demonstrated that CLP is sensitive to a number of peptide aldehyde inhibitors of animal caspases, with a notable exception of DEVD-CHO. Since these inhibitors have previously been employed in suppression of PCD mediated by different stress inducers, this result suggests that their inhibitory effect could be due, at least in part, to inactivation of the CLP under study.

Heracleum is a large and taxonomically complex genus of the Umbelliferae-Tordylieae. The phylogenetic relationships of West Asian Heracleum species and related taxa were explored using data from sequences of the internal transcribed spacer (ITS1 and ITS2) region of nuclear ribosomal DNA. The data set consists of 56 species, of which 47 were analyzed for the first time; it represents all subdivisions of the genus Heracleum, as well as some representatives of Pastinaca complex. Heracleum was shown to be a polyphyletic genus, as its species fall into two different clades, one of which comprises also Symphyoloma and Mandenovia. Section Pubescentia was confirmed, in contrast to the sections Villosa and Heracleum being polyphyletic. A separate position of the section Wendia was supported. H. marashicum was shown to be a member of a clade comprising Pastinaca and related genera. The sequences of chloroplast psbA-trnH intergenic spacer, the region recently proposed for DNA barcoding in plants, were also analyzed for 33 species, representing all principal clades within Heracleum and its relatives. They have been proven to be very similar and not suitable for DNA barcoding in this group. However, some sequence variation was revealed. This variation could be explained by the combination of such evolutionary events as inversion and duplication. It was shown that these events are rather common in Tordylieae and can occur independently in different lineages. The evolution patterns of psbA-trnH spacer are hypothesized.

The studies on floral homeotic mutants of the model plant species Arabidopsis thaliana (L.) Heynh. and Antirrhinum majus L. have clarified many important aspects of the genetic control of flower development. However, the details of this process can vary in species representing different lineages of flowering plants. The studies on floral homeotic mutants of nonmodel plant species may significantly improve the understanding of the mechanisms of morphological evolution of flowers. We report here the results of the genetic and morphological analysis of two floral homeotic mutants of common buckwheat (Fagopyrum esculentum Moench.). The mutant, tepal-like bract (tlb), is characterized by the transformation of bracts into petaloid organs, whereas fagopyrum apetala (fap), has a carpelloid perianth. Both mutant phenotypes are caused by a single recessive nuclear mutation. The double mutant fap tlb combines the features of tlb and fap. Our results show that single gene mutations are sufficient to convert the buckwheat bract into a tepal and to confer carpel identity on first whorl organs. These results are consistent with the premise that variations on the ABC model can be used to explain a wide range of floral architectures.

The complete nucleotide sequence of the duckweed (Lemna minor) chloroplast genome (cpDNA) was determined. The cpDNA is a circular molecule of 165,955 bp containing a pair of 31,223-bp inverted repeat regions (IRs), which are separated by small and large single-copy regions of 89,906 and 13,603 bp, respectively. The entire gene pool and relative positions of 112 genes (78 protein-encoding genes, 30 tRNA genes, and 4 rRNA genes) are almost identical to those of Amborella trichopoda cpDNA; the minor difference is the absence of infA and ycf15 genes in the duckweed cpDNA. The inverted repeat is expanded to include ycf1 and rps15 genes; this pattern is unique and does not occur in any other sequenced cpDNA of land plants. As in basal angiosperms and eudicots, but not in other monocots, the borders between IRs and a large single-copy region are located upstream of rps19 and downstream of trnH, so that trnH is not included in IRs. The model of rearrangements of the chloroplast genome during the evolution of monocots is proposed as the result of the comparison of cpDNA structures in duckweed and other monocots. The phylogenetic analyses of 61 protein-coding genes from 38 plastid genome sequences provided strong support for the monophyly of monocots and position of Lemna as the next diverging lineage of monocots after Acorales. Our analyses also provided support for Amborella as a sister to all other angiosperms, but in the bayesian phylogeny inference based on the first two codon positions Amborella united with Nymphaeales.

Background - Chloroplast genome sequences are extremely informative about species-interrelationships owing to its non- meiotic and often uniparental inheritance over generations. The subject of our study, Fagopyrum esculentum, is a member of the family Polygonaceae belonging to the order Caryophyllales. An uncertainty remains regarding the affinity of Caryophyllales and the asterids that could be due to undersampling of the taxa. With that background, having access to the complete chloroplast genome sequence for Fagopyrum becomes quite pertinent. Results - We report the complete chloroplast genome sequence of a wild ancestor of cultivated buckwheat, Fagopyrum esculentum ssp. ancestrale. The sequence was rapidly determined using a previously described approach that utilized a PCR- based method and employed universal primers, designed on the scaffold of multiple sequence alignment of chloroplast genomes. The gene content and order in buckwheat chloroplast genome is similar to Spinacia oleracea. However, some unique structural differences exist: the presence of an intron in the rpl2 gene, a frameshift mutation in the rpl23 gene and extension of the inverted repeat region to include the ycf1 gene. Phylogenetic analysis of 61 protein-coding gene sequences from 44 complete plastid genomes provided strong support for the sister relationships of Caryophyllales (including Polygonaceae) to asterids. Further, our analysis also provided support for Amborella as sister to all other angiosperms, but interestingly, in the bayesian phylogeny inference based on first two codon positions Amborella united with Nymphaeales. Conclusion - Comparative genomics analyses revealed that the Fagopyrum chloroplast genome harbors the characteristic gene content and organization as has been described for several other chloroplast genomes. However, it has some unique structural features distinct from previously reported complete chloroplast genome sequences. Phylogenetic analysis of the dataset, including this new sequence from non-core Caryophyllales supports the sister relationship between Caryophyllales and asterids.

Some new nrITS sequences of Lotus are produced and added to the data set analysed ill DEGTJAREVA et al. (2006). Lotus burttii and L. filicaulis are revealed as members of the /Lotus corniculatus clade. Lotus conimbricensis is found to be sister to the entire/Lotus corniculatus clade; the /Lotus pedunculatus clade is more distantly related. The New Caledonian Lotus anfractuosus is closest among species sampled to two Australian endemics, though its molecular divergence is considerable. The NE African Lotus torulosus is close to some other red-flowered species from the same region; it does not group with any other Lotus species with dimorphic leaflets. In general, dimorphic vs. monomorphic leaflets is a quite homoplastic character in Lotus. Molecular divergence is weak within the /Pedrosia clade, where the morphological divergence is especially high. In contrast, molecular divergence is considerable but morphological differentiation is weak in the /Lotus corniculatus clade.

The nuclear factor NF-kappa B is an inducible transcription factor activating expression of genes the products of which play a key role in cardiovascular pathologies, carcinogenesis, inflammatory and viral diseases. The review discribes the processes inducing NF-kappa B activation and determines the components of a signal cascade that could constitute targets for NF-kappa B inhibition. The low-molecular-weight inhibitors of the NF-kappa B factor described systematically, and their properties and molecular action mechanism are considered.

To study the interrelation between the spectral and structural properties of fluorescent proteins, structures of mutants of monomeric red fluorescent protein mRFP1 with all possible point mutations of Glu66 (except replacement by Pro) were simulated by molecular dynamics. A global search for correlations between geometrical structure parameters and some spectral characteristics (absorption maximum wavelength, integral extinction coefficient at the absorption maximum, excitation maximum wavelength, emission maximum wavelength, and quantum yield) was performed for the chromophore and its 6 angstrom environment in mRFP1, Q66A, Q66L, Q66S, Q66C, Q66H, and Q66N. The correlation coefficients (0.81-0.87) were maximal for torsion angles in phenolic and imidazolidine rings as well as for torsion angles in the regions of connection between these rings and chromophore attachment to beta-barrel. The data can be used to predict the spectral properties of fluorescent proteins based on their structures and to reveal promising positions for directed mutagenesis.

 Endonuclease WEN2 with an apparent molecular mass 21.5 kD was isolated from subcellular vesicular fraction obtained from aging apoptotic coleoptiles of 8-day-old etiolated wheat seedlings and partially characterized. Similar to wheat endonuclease WEN1 of the same origin described earlier, the WEN2 enzyme is a neutral Ca²⁺, Mg²⁺, Mn²⁺-dependent endonuclease. Both enzymatic activities were found also in nuclei from the same cells. Mn²⁺ activates WEN2 more efficiently than Mg²⁺ or Ca²⁺. High ionic strength, Zn²⁺, and EDTA inhibit the enzyme completely. In the presence of Mg²⁺, elevated WEN2 activity was observed at pH between 5.5 and 7.7 and at 370C, and without Mg²⁺ added it was observed in narrower pH range (from pH 6.8 to pH 7.7). The enzyme is active even at high temperature (650C). WEN2 splits preferentially unmethylated, but WEN1 - methylated lambda phage DNA. Double-stranded DNA is a preferential substrate to be hydrolyzed with WEN2. S-Adenosyl- L-methionine (SAM) significantly activates endonuclease WEN2 (the optimal SAM concentration is 0.3 mM). Contrary to strong stimulating action on WEN1, the competitive inhibitors of the DNA methylation reaction (SAM analogs S-adenosyl-L- homocysteine and S-isobutyladenosine) at concentration 0.3 mM increase WEN2 activity slightly. It is suggested that WEN2 may take part in apoptotic DNA degradation. Thus, in plants there are endonucleases that recognize methylation status of substrate DNAs and are modulated by the methyl group donor, SAM, in different fashions. Therefore, all this may indicate the presence of a restriction-modification (R-M) system in higher plants.

The CC3252 gene product, DmNAT1, represents the first Nutrient Amino acid Transporter cloned from Drosophila. It absorbs a broader set of neutral amino acids versus earlier characterized insect NATs and mammalian NATs-B-0 system transporters from the Sodium Neurotransmitter symporter Family (SNF, a.k.a. solute carrier family 6, SLC6). In addition to Bo-specific L- Substrates, DmNAT1 equally or more effectively transports D-amino acids with sub-millimolar affinities and 1:1 sodium:amino acid transport stoichiometry. DmNAT1 is strongly transcribed in the absorptive and secretory regions of the larval alimentary canal and larval brain, revealing its roles in the primary absorption and redistribution of large neutral L- amino acids as well as corresponding D-isomers. The absorption Of D-amino acids via DmNAT1 may benefit the acquisition of fermented and symbiotic products, and may support the unique capacity of fruit fly larvae to utilize a diet with substitution of essential amino acids by D-isomers. It also suggests a remarkable adaptive plasticity of NAT-SLC6 mechanisms via alterations of a few identifiable sites in the substrate-binding pocket. The strong transcription in the brain suggests roles for DmNAT1 in neuronal nutrition and clearance Of L-neutral amino acids from the fly brain. In addition, neuronal DmNAT1 may absorb synaptic D-serine and modulate NMDA receptor-coupled signal transduction. The characterization of the first invertebrate B-0-like transporter extends the biological roles of the SLC6 family, revealing adaptations for the absorption of D- isomers of the essential amino acids. These findings suggest that some members of the NAT-SLC6 subfamily are evolving specific properties which contribute to nutrient symbiotic relationships and neuronal functions.

Pectin methylesterase (PME) is a cell-wall enzyme that acts as a growth and morphogenesis factor in higher plants and is involved in gene silencing, plant virus reproduction, and transgenesis. A study was made of the role of PME as a stress protein in host plant-virus interactions. PME enzymatic activity was induced, not only by an additional PME gene copy, but also by an empty vector. PME suppressed tobacco mosaic virus (TMV) reproduction, including short-and long-distance virus movement in plants. Surprisingly, elevated PME activity was observed in intact stably transformed transgenic plants. For example, PME activity was increased in transgenic Nicotiana tabacum and N. benthamiana plants expressing the genes for the TMV movement protein and GFP and in tomato plants with cosuppression of the polygalacturonase gene. Activation of light- inducible psbO induced transcription of the PME gene. It was suggested that PME is involved in maintaining the stability of the plant transcriptome and restores its status quo upon viral infection, transformation with a foreign gene, or excess transcription of the cell genome. TIP GROWTH OF Neurospora crassa

On thin agar or non-penetrating surface such as cellophane, a mature mycelium of Neurospora crassa grows by tip elongation at rates (V) up to 40 mu m/min (240C) with periodic branching. During growth the mycelium forms a two-dimensional tree with a constant ratio of total hyphal length to the number of growing tips. This ratio, the "hyphal growth unit" (HGU), is about 350 mu m for N. crassa. In this work mycelium was preconditioned to steady-state growth in 0.35% glucose (2 mM) by inoculation onto a cellophane sheet a top agar containing Vogel's medium plus glucose. After 15-18 h of growth at 240C, a small square of cellophane with mycelium attached was cut, washed, and transferred onto similar agar, having 2 mM sorbitol (not metabolized by Neurospora) to replace glucose. Four hours later, hyphae were filled with vacuoles, while the measured mean V and HGU remained close to the control values (36 mu m/min and 367 mu m in sorbitol, 37 mu m/min and 344 mu m in glucose, respectively). Glucose deprivation had similarly little effect on V of isolated apical segments, at least for periods up to 3 hours after severing, but did significantly reduce the frequency of branching. Isolated fragments of mycelium (mean length, 843 ± 83 mu m; n = 13) maintained on glucose-containing medium elongated at V = 11 ± 4 mu m/min, with HGU = 396 ± 74 mu m; segments deprived of glucose (mean length, 807 ± 86 mu m; n = 16) elongated at V = 10 ± 2 mu m/min, with HGU = 545 ± 55 mu m. Using the phase-contrast microscopy we observed an enormous proliferation of vacuoles after glucose deprivation. Experiments using Calcofluor White (10 mu g/ml), a widely used cell wall stain did not reveal substantial changes in the hyphal cell wall or septa under the glucose deprivation for 4-5 h. Independence of V of the Neurospora growth from extracellular sugar presumably relies upon the organism's ability to utilize stored polysaccharides (e.g., glycogen) over a short term.

The molecular organization, copy number, and chromosome location of the human TNF/LT transgenes were studied in the genomes of two transgenic mouse strains. One strain proved to carry two transgene copies arranged head-to-tail and detected on chromosome 8 by karyotyping. The other strain had one transgene copy observed on chromosome 5. The strains provide a model for studying the physiological functions of the tumor necrosis factor and lymphotoxin.

The modeling of the mechanical process of hearing requires an accurate geometrical model of the inner ear (cochlea). The purpose of this study was the creation of a 3-D model of the fluid chambers of Guinea pig cochlea, which could serve as a basis for further mechanical modeling. Micro computer tomography used in this study is a noninvasive method to visualize bony structures. The visualization of the membranous labyrinth was achieved by additional staining of the specimen with OSO4. The resulting stack of images has been transformed into a cylindrical coordinate system. To suppress noise on tomography images, a nonlinear smoothing method, anisotropic diffusion, Were applied. A new approach has been proposed to estimate algorithm parameters automatically. Then, a segmentation using active contours (snakes) was performed. In this study, a new energy linking the contours on adjacent slices has been added to the standard approach. This compensates the inconsistencies between adjacent contours. The images segmented in this way were used as a basis for a 3-D reconstruction of the hearing organ.

Structural and functional features of the HIV-1 integrase are examined and the current state in the search for effective inhibitors of this enzyme is reviewed. Main classes of integrase inhibitors with known mechanisms of action and in vitro and in vivo inhibiting activity are presented.

TNF is an exciting cytokine which has helped to establish many paradigms in immunology. Although TNF itself has found only very limited use in the clinic, anti-cytokine therapy, which targets this single molecule, has enjoyed astounding success in treatment of a growing number of human diseases. However, since TNF mediates unique physiologic functions, in particular those related to host defense, TNF blockade may result in unwanted consequences. Much of our understanding about TNF intrinsic functions in the body, as well as about consequences of its overexpression and ablation, is based on studying phenotypes of various genetically engineered mice. Here we review mouse studies aimed at understanding TNF physiologic functions using transgenic and knockout models, and we discuss additional mouse models that may be helpful in the future.

V. Matematical models in biology.
 
THE COMPARATIVE ANALYSIS OF STATISTICS, BASED ON THE LIKELIHOOD RATIO CRITERION, IN THE AUTOMATED ANNOTATION PROBLEM.
Leontovich A.M., Tokmachev K.Y., van Houwelingen H.C.
BMC BIOINFORMATICS 9 (2008) 31.

BACKGROUND: This paper discusses the problem of automated annotation. It is a continuation of the previous work on the A4-algorithm (Adaptive algorithm of automated annotation) developed by Leontovich and others. RESULTS: A number of new statistics for the automated annotation of biological sequences is introduced. All these statistics are based on the likelihood ratio criterion. CONCLUSION: Some of the statistics yield a prediction quality that is significantly higher (up to 1.5 times higher) in comparison with the results obtained with the A4-procedure.

Life-threatening RNA viruses emerge regularly, and often in an unpredictable manner. Yet, the very few drugs available against known RNA viruses have sometimes required decades of research for development. Can we generate preparedness for outbreaks of the, as yet, unknown viruses? The VIZIER (VIral enZymes InvolvEd in Replication) (http://www.vizier- europe.org/) project has been set-up to develop the scientific foundations for countering this challenge to society. VIZIER studies the most conserved viral enzymes (that of the replication machinery, or replicases) that constitute attractive targets for drug-design. The aim of VIZIER is to determine as many replicase crystal structures as possible from a carefully selected list of viruses in order to comprehensively cover the diversity of the RNA virus universe, and generate critical knowledge that could be efficiently utilized to jump-start research on any emerging RNA virus. VIZIER is a multidisciplinary project involving (i) bioinformatics to define functional domains, (ii) viral genomics to increase the number of characterized viral genomes and prepare defined targets, (iii) proteomics to express, purify, and characterize targets, (iv) structural biology to solve their crystal structures, and (v) pre-lead discovery to propose active scaffolds of antiviral molecules.