Biochemistry (Moscow) (v.75, #6)

This review analyzes the available information concerning mechanisms of non-genotoxic action of reactive oxygen species (ROS) during tumor promotion and pathways of their generation under the influence of chemical compounds. Special attention is given to the ability of ROS to induce pseudohypoxia through inhibition of prolyl oxidase, which is an oxygen sensor in the cell. Functions of HIF-1α as a main contributor to the ROS-induced promotion are analyzed. Data suggest that an unregulated high level of HIF-1α in the cell could induce the development of tumors. Hypothetical possibilities of ROS production under the influence of different environmental pollutants, which are promoters of tumorigenesis, include functioning of cytochrome P450 during oxidation of substrates, functioning of the mitochondrial respiratory chain, and action of peroxisome proliferators.
Keywords: carcinogenesis; reactive oxygen species; hypoxia; receptors of xenobiotics; cytochrome P450

Cell surface of leishmaniasis causal agent, a parasitic member of Protozoa of Leishmania genus, is covered by thick glycocalix consisting of various phosphatidylinositol-anchored molecules. This review deals with the structure and biosynthesis of the main phosphoglycans and glycoproteins of Leishmania cell surface, many of which incorporate the rare natural D-arabinopyranose, and the problem concerning the involvement of these molecules in support of Leishmania survival during their intricate life cycle is discussed.
Keywords: D-arabinopyranose; Leishmania ; glycosylphosphatidylinositol; lipophosphoglycan; glycosylinositol phospholipids; proteophosphoglycan

Minor groove dimeric bisbenzimidazoles inhibit in vitro DNA binding to eukaryotic DNA topoisomerase I by O. Yu. Susova; A. A. Ivanov; S. S. Morales Ruiz; E. A. Lesovaya; A. V. Gromyko; S. A. Streltsov; A. L. Zhuze (695-701).
The potential of six dimeric bisbenzimidazoles bound to scDNA to inhibit eukaryotic DNA topoisomerase (topo-I) was studied chemically; the tested compounds differed in linker structure and length. All the compounds inhibited topo-I, DB(7) being the most efficient; its inhibitory activity in vitro was 50-fold higher than that of camptothecin. It is noteworthy that inhibitory properties of nearly all the tested compounds increased many times if they were preincubated with scDNA for three days.
Keywords: dimeric bisbenzimidazoles; topoisomerase I; inhibition

Role of heat shock protein Hsp90 in formation of protective reactions in acute toxic stress by O. V. Glushkova; T. V. Novoselova; M. O. Khrenov; S. B. Parfenyuk; S. M. Lunin; E. E. Fesenko; E. G. Novoselova (702-707).
The involvement of heat shock protein Hsp90 in pro-inflammatory response in male NMRI mice under conditions of acute toxic stress, caused by lipopolysaccharide from Gram negative bacteria, was studied using geldanamycin, a specific blocker of the activity of this protein. It is shown that the introduction of geldanamycin lowers total intoxication of the organism upon acute toxic stress caused by endotoxin. Thus, a decrease in cytokine TNF-α, IFN-γ, IL-1, and IL-10 concentrations in blood serum of the geldanamycin-treated animals with acute toxic stress was found along with normalization of functional activity of nitric oxide producing peritoneal macrophages. Studying expression of receptor protein Tlr-4 as well of proteins of two signal cascades, NF-κB and SAPK/JNK, has shown that mechanisms of the geldanamycin protective effect are realized at the level of inhibition of Tlr-4 receptor expression, which provides for endotoxin-to-cell binding, and due to lowering the endotoxin-stimulated activation of signal cascades NF-κB and SAPK/JNK. The results suggest Hsp90 might be a therapeutic target in diseases accompanied by acute toxic stress.
Keywords: heat shock protein 90; geldanamycin; toxic stress; lipopolysaccharide; NF-κB and SAPK/JNK signal pathways; cytokines; nitric oxide; Tlr-4

Hexadecanoid pathway in plants: Lipoxygenase dioxygenation of (7Z,10Z,13Z)-hexadecatrienoic acid by E. V. Osipova; N. V. Lantsova; I. R. Chechetkin; F. K. Mukhitova; M. Hamberg; A. N. Grechkin (708-716).
7,10,13-Hexadecatrienoic acid (16:3) is abundant in many plant species. However, its metabolism through the lipoxygenase pathway is not sufficiently understood. The goal of present work was to investigate the oxygenation of 16:3 by different plant lipoxygenases and to study the occurrence of oxygenated derivatives of 16:3 in plant seedlings. The recombinant maize 9-lipoxygenase specifically converted 16:3 into (7S)-hydroperoxide. Identification of this novel oxylipin was substantiated by data of GC-MS, LC-MS/MS, 1H-NMR, and 2D-COSY as well as by deuterium labeling from [2H6]16:3. Soybean lipoxygenase 1 produced 91% (11S)-hydroperoxide and 6% racemic 14-hydroperoxide. Recombinant soybean lipoxygenase 2 (specifically oxidizing linoleate into 13-hydroperoxide) lacked any specificity towards 16:3. Lipoxygenase 2 produced 7-, 8-, 10-, 11-, 13-, and 14-hydroperoxides of 16:3, as well as a significant amount of bis-allylic 9-hydroperoxide. Seedlings of several examined plant species possessed free hydroxy derivatives of 16:3 (HHTs), as well as their ethyl esters. Interestingly, HHTs occur not only in “16:3 plants”, but also in typical “18:3 plants” like pea and soybean seedlings.
Keywords: (7Z,10Z,13Z)-hexadecatrienoic acid; lipoxygenases; oxygenation; fatty acid hydroperoxides; hexadecanoids; higher plants

Statistical analysis of protein folding rates has been done for 84 proteins with available experimental data. A surprising result is that the proteins with multi-state kinetics from the size range of 50–100 amino acid residues (a.a.) fold as fast as proteins with two-state kinetics from the same size range. At the same time, the proteins with two-state kinetics from the size range 101–151 a.a. fold faster than those from the size range 50–100 a.a. Moreover, it turns out unexpectedly that usually in the group of structural homologs from the size range 50–100 a.a., proteins with multi-state kinetics fold faster than those with two-state kinetics. The protein folding for six proteins with a ferredoxin-like fold and with a similar size has been modeled using Monte Carlo simulations and dynamic programming. Good correlation between experimental folding rates, some structural parameters, and the number of Monte Carlo steps has been obtained. It is shown that a protein with multi-state kinetics actually folds three times faster than its structural homologs.
Keywords: protein folding; intermediates of folding; two-state transition; folding nucleus; folding rates

Induction of lipid flip-flop by colicin E1 — a hallmark of proteolipidic pore formation in liposome membranes by A. A. Sobko; S. I. Kovalchuk; E. A. Kotova; Y. N. Antonenko (728-733).
The addition of the channel-forming domain of colicin E1 to liposomes elicited the transmembrane diffusion (flip-flop) of lipids concomitant to the release of the fluorescent dye from liposomes. Good correlation was found between kinetic and concentration dependences of the two processes. Both the liposome leakage and the lipid flip-flop were stimulated upon alkalinization of the buffer solution after colicin binding at acidic pH. These results in combination with the analysis of the data on colicin binding to liposomes provide evidence in favor of the validity of the toroidal (proteolipidic) pore model as the mechanism of colicin channel formation.
Keywords: colicin; ion channel; liposome leakage; proteolipidic pore; flip-flop; fluorescence quenching

A method for transmembrane protein thromboplastin (tissue factor) immobilization on polystyrene surface is described. Tissue factor is the main activating factor launching the blood coagulation process. It is a cofactor of factor VIIa, the first protease in the cascade of coagulation reactions. The proposed method preserves kinetic characteristics specific for native tissue factor on the fibroblast surface. The kinetics of binding to factor VIIa and enzymic activity of the formed complex follow Michaelis-Menten kinetics, which is also characteristic of native complex. A small difference is that dissociation constant for tissue factor immobilized on polystyrene surface exceeds 2.7-fold that for native factor. The proposed technique of immobilization provides for protein density on the activating surface corresponding to the tissue factor density on the fibroblast surface. The immobilized tissue factor can be used to activate blood coagulation in methods simulating spatial dynamics of in vitro clot growth. Investigation in this direction will make it possible to register both hypo- and hypercoagulation states of the system. This approach is advantageous over traditional methods of estimation of the coagulation system conditions, which mainly register only hypocoagulation. Investigation of the storage time has shown that activators containing immobilized tissue factor can be stored and used during for at least 100 days in the method studying spatial dynamics of fibrin clot formation.
Keywords: thromboplastin; tissue factor; immobilization; kinetics of factor VIIa-thromboplastin complex; spatial dynamics of blood coagulation

A novel nitrate reductase (NR) was isolated from cell extract of the haloalkaliphilic bacterium Thioalkalivibrio nitratireducens strain ALEN 2 and characterized. This enzyme is a classical nitrate reductase containing molybdopterin cofactor in the active site and at least one iron-sulfur cluster per subunit. Mass spectrometric analysis showed high homology of NR with the catalytic subunit NarG of the membrane nitrate reductase from the moderately halophilic bacterium Halomonas halodenitrificans. In solution, NR exists as a monomer with a molecular weight of 130–140 kDa and as a homotetramer of about 600 kDa. The specific nitrate reductase activity of NR is 12 μmol/min per mg protein, the maximal values being observed within the neutral range of pH. Like other membrane nitrate reductases, NR reduces chlorate and is inhibited by azide and cyanide. It exhibits a higher thermal stability than most mesophilic enzymes.
Keywords: Thioalkalivibrio nitratireducens ; haloalkaliphilic bacteria; nitrate reductase; NarG

The internal domain of hordeivirus movement protein TGB1 forms in vitro filamentous structures by V. V. Makarov; E. A. Obraztsova; A. G. Solovyev; S. Yu. Morozov; M. E. Taliansky; I. V. Yaminsky; N. O. Kalinina (752-758).
The 63 kDa hordeivirus movement protein TGB1 of poa semilatent virus (the PSLV TGB1 protein) forms viral ribonucleoprotein for virus transport within a plant. It was found using the dynamic laser light scattering technique that the internal domain of TGB1 protein forms in vitro high molecular weight complexes. According to results of atomic force microscopy, a part of these complexes is represented by globules of different sizes, while another part consists of extended filamentous structures. Similar properties are also characteristic of the N-terminal half of the protein and are obviously due to its internal domain moiety. The data support the hypothesis that upon viral ribonucleoprotein complex formation, the N-terminal half of the PSLV TGB1 protein plays a structural role and exhibits the ability to form multimeric filamentous structures (the ability for self-assembly).
Keywords: hordeivirus; TGB1 movement protein; internal domain; filamentous structures

Synthesis of sulfated pectins and their anticoagulant activity by F. V. Vityazev; V. V. Golovchenko; O. A. Patova; N. N. Drozd; V. A. Makarov; A. S. Shashkov; Yu. S. Ovodov (759-768).
The following pectins were sulfated: bergenan BC (the pectin of Bergenia crassifolia L), lemnan LM (the pectin of Lemna minor L), and galacturonan as a backbone of pectins. Pyridine monomethyl sulfate, pyridine sulfotrioxide, and chlorosulfonic acid were used as reagents for sulfation. Chlorosulfonic acid proved to be the optimal reagent for sulfation of galacturonan and other pectins. Galacturonan and pectin derivatives with different degrees of sulfation were synthesized and their anticoagulant activities were shown to depend on the quantity of sulfate groups in the pectin macromolecules.
Keywords: lemnan; bergenan; sulfated pectin; sulfated galacturonan; anticoagulant activity; Bergenia crassifolia L; Lemna minor L

Purification of core enzyme of Escherichia coli RNA polymerase by affinity chromatography by Yu. A. Khodak; O. N. Koroleva; V. L. Drutsa (769-776).
A method for isolation of a highly purified preparation of E. coli RNA polymerase core enzyme was developed based on IMPACT technology and dissociation of the RNA polymerase complex with σ70 subunit. Washing of the immobilized RNA polymerase with 5–10 mM solution of glutamate (pH 5.0–5.5) completely removed the σ70 subunit from the holoenzyme and decreased amounts of protein admixtures. The possibility of reconstruction of the RNA polymerase holoenzyme directly on the affinity column was demonstrated. Activities of the resulting RNAP core enzyme preparations were tested by in vitro transcription. Some amino acids and their mixtures were shown to influence the in vitro transcription. The findings indicate that changes in the transcription efficiency in the presence of amino acids should be associated with a specific destruction of the interaction between σ70 subunit and the core enzyme.
Keywords: E. coli RNA polymerase; subunits; affinity chromatography; IMPACT system; transcription; amino acids

Using quantitative realtime PCR, the levels of mitochondrial DNA transcripts in murine tissues (skeletal muscle, liver, and brain) were determined at different time points (1, 5, and 24 h) following X-ray irradiation at the dose of 10 Gy. One hour after irradiation the levels of mitochondrial transcripts ND2, ND4, CYTB, and ATP6 dramatically decreased by 85–95% and remained at the same minimum level for 24 h in all analyzed tissues. This decrease was not associated with depletion of mtDNA as a matrix for transcription, since mtDNA copy number increased after irradiation in all tissues. The decrease in mitochondrial transcription in liver, brain, and skeletal muscle did not generally result from the damage of cell transcription apparatus, because the transcription level of nuclear housekeeping gene BETA-ACTIN remained virtually unchanged after irradiation. The mitochondrial gene transcription decreased after irradiation in the same manner as that of the nuclear gene TFB2M encoding mitochondrial transcription factor, whose regulatory role under normal conditions is well understood.

Two types of ammonium uncoupling in pea chloroplasts by V. K. Opanasenko; L. A. Vasyukhina; I. A. Naydov (784-791).
The effect of ammonium on ATP synthesis, electron transfer, and light-induced uptake of hydrogen ions in pea chloroplasts was studied. It is shown that the dependence of these reactions on ammonium concentration could be due to effects of two different uncoupling processes. The first process is induced by low ammonium concentrations (<0.2 mM); the second one is observed in the NH4Cl concentration interval of 0.5–5.0 mM. The first type of uncoupling is stimulated by palmitic acid or by N,N′-dicyclohexylcarbodiimide, while the second is stimulated by chloroplast thylakoid swelling caused by energy-dependent osmotic gradients. In the presence of the fluorescent dye sulforhodamine B, which does not penetrate through the cell membrane, this swelling causes the dye to enter the lumens. It is supposed that ammonium activates two different routes of cation leakage from the lumen. The first route involves channel proteins, while the second is a mechanosensitive pore that opens in response to osmotic gradients.
Keywords: ammonium uncoupling; ion regulation; DCCD; chloroplasts; electron transfer; ATP synthesis