Biochemistry (Moscow) (v.75, #9)

Role of 4-hydroxy-trans-2-nonenal in cell functions by E. E. Dubinina; V. A. Dadali (1069-1087).
The role of lipid peroxidation product 4-hydroxy-trans-2-nonenal (4-HNE) in functional activity of cells under normal and different pathological conditions is discussed. Different pathways of 4-HNE metabolism in tissues are analyzed, with particular focus on the role the glutathione system in this process. 4-HNE is implicated in regulation of cell growth, proliferation, differentiation, and apoptosis. 4-HNE and metabolic products of other antioxidants (carotenoids) resemble each other in chemical nature of the product and influence general pathways of signal transduction. Manifestation of 4-HNE toxicity under oxidative stress conditions is regarded as a link to many diseases whose pathogenesis is connected with modifications of proteins and nucleic acids.
Keywords: 4-hydroxy-trans-2-nonenal; lipid peroxidation; apoptosis; glutathione system; carotenoids; oxidative stress

Lysophosphatidic acid is a lipid mediator with wide range of biological activities. Biosynthetic pathways and mechanism of action by I. N. Berdichevets; T. V. Tyazhelova; Kh. R. Shimshilashvili; E. I. Rogaev (1088-1097).
Lysophosphatidic acid (LPA) is a lipid mediator required for maintaining homeostasis of numerous physiological functions and also involved in development of some pathological processes through interactions with G protein-coupled receptors. Recently many data have appeared about the role of this phospholipid in humans, but pathways of LPA biosynthesis and mechanisms of its action remain unclear. This review presents modern concepts about biosynthesis, reception, and biological activity of LPA in humans. Natural and synthetic LPA analogs are considered in the view of their possible use in pharmacology as agonists and/or antagonists of G protein-coupled receptors of LPA.
Keywords: lysophosphatidic acid; LPA analogs; autotaxin; G protein-coupled receptors; expression of human phospholipase genes

Toll-like receptors and their adapter molecules by A. I. Tukhvatulin; D. Yu. Logunov; D. N. Shcherbinin; M. M. Shmarov; B. S. Naroditsky; A. V. Gudkov; A. L. Gintsburg (1098-1114).
Toll-like receptors (TLR) are among key receptors of the innate mammalian immune system. Receptors of this family are able to recognize specific highly conserved molecular regions (patterns) in pathogen structures, thus initiating reactions of both innate and acquired immune response finally resulting in the elimination of the pathogen. In this case every individual TLR type is able to bind a broad spectrum of molecules of microbial origin characterized by different chemical properties and structures. Recent data demonstrate the existence of a multistep mechanism of the TLR recognition of the pathogen in which, in addition to receptors proper, the involvement of different adapter molecules is necessary. However, functions of separate adapter molecules as well as the principles of formation of a multicomponent system of ligand-specific recognition are still not quite understandable. We describe all identified as well as possible (candidate) adapter TLR molecules by giving their brief characteristics, and we also propose generalized possible variants of the TLR ligand-specific recognition with involvement of adapter molecules.
Keywords: Toll-like receptors; adapter molecules; ligand-receptor recognition

Here we studied the inhibition of the catalytic domain of Dnmt3a methyltransferase (Dnmt3a-CD) by DNA duplexes containing the mechanism-based inhibitor pyrimidine-2(1H)-one (P) instead of the target cytosine. It has been shown that conjugates of Dnmt3a-CD with P-DNA (DNA containing pyrimidine-2(1H)-one) are not stable to heating at 65°C in 0.1% SDS. The yield of covalent intermediate increases in the presence of the regulatory factor Dnmt3L. The importance of the DNA minor groove for covalent intermediate formation during the methylation reaction catalyzed by Dnmt3a-CD has been revealed. P-DNA was shown to inhibit Dnmt3a-CD; the IC50 is 830 nM. The competitive mechanism of inhibition of Dnmt3a-CD by P-DNA has been elucidated. It is suggested that therapeutic effect of zebularine could be achieved by inhibition of not only Dnmt1 but also Dnmt3a.
Keywords: pyrimidine-2(1H)-one; covalent intermediate; competitive inhibition; eukaryotic DNA methyltransferase

Ornithine decarboxylase activity in rat organs and tissues under artificial hypobiosis by G. E. Aksyonova; O. S. Logvinovich; L. A. Fialkovskaya; V. N. Afanasyev; D. A. Ignat’ev; I. K. Kolomiytseva (1126-1131).
The influence of hypothermia-hypoxia-hypercapnia on ornithine decarboxylase (ODC, EC activities in rat organs and tissues and also on the thymocyte distribution throughout the cell cycle stages was studied. The state of artificial hypobiosis in rats on decrease in the body temperature to 14.4–18.0°C during 3.0–3.5 h was accompanied by drops in the ODC activities in the neocortex and liver by 50–60% and in rapidly proliferating tissues (thymus, spleen, and small intestine mucosa) by 80% of the control value. In kidneys the ODC activity raised to 200% of the control level. Twenty-four hours after termination of the cooling and replacing the rats under the standard conditions, the ODC activities in the neocortex, liver, kidneys, spleen, and intestinal mucosa returned to the control values, but remained decreased in the thymus. Forty-eight hours later the ODC activities in the thymus and spleen exceeded the normal level. The distribution of thymocytes throughout the cell cycle stages did not change in rats in the state of hypothermia (hypobiosis); 24 and 48 h after termination of the cooling the fraction of thymocytes in the S stage was decreased and the fraction of the cells in the G0+G1 stage was increased. The normal distribution of thymocytes throughout the cell cycle stages recovered in 72 h. Thus, in the thymus the diminution of the ODC activity preceded the suppression of the cell proliferation rate. The tissue-specific changes in the ODC activity are suggested to reflect adaptive changes in the functional and proliferative activities of organs and tissues during the development of hypobiosis under conditions of hypothermia-hypoxia-hypercapnia.
Keywords: artificial hypobiosis; rats; ornithine decarboxylase; thymocytes; cell cycle

Lipids of nuclear fractions from neurons and glia of rat neocortex under conditions of artificial hypobiosis by I. K. Kolomiytseva; L. N. Markevich; D. A. Ignat’ev; O. V. Bykova (1132-1138).
Lipid contents were studied in tissue and nuclei isolated from neurons and glia of neocortex of rats under conditions of normothermia and in the state of artificial hypobiosis caused by hypothermia-hypoxia-hypercapnia. Compared to the neocortex tissue, both nuclear fractions were fivefold impoverished in phospholipids and cholesterol and strongly enriched with mono- and diglycerides and fatty acids. The nuclear fractions from neurons and glia contained similar amounts of phospholipids, and only the cardiolipin content in the neuronal nuclei was lower than in the glial nuclei. The state of artificial hypobiosis in rats led to an increase in the cholesterol/phospholipids ratio (mol/mol) in the nuclei from the neurons and glia; amounts of cholesterol and sphingomyelin in the nuclei from the glia were increased. The increases in the cholesterol and sphingomyelin contents and in the cholesterol/phospholipids ratio suggest an involvement of lipid-dependent signaling systems of the nuclei in the functional response of mammalian neocortex cells to artificial hypobiosis.
Keywords: lipids; rats; hypobiosis; nuclei; neurons; glia

Influence of ATP-dependent K+-channel opener on K+-cycle and oxygen consumption in rat liver mitochondria by O. V. Akopova; V. I. Nosar; V. A. Bouryi; I. N. Mankovskaya; V. F. Sagach (1139-1147).
The influence of the K ATP + -channel opener diazoxide on the K+ cycle and oxygen consumption has been studied in rat liver mitochondria. It was found that diazoxide activates the K ATP + -channel in the range of nanomolar concentrations (50–300 nM, K 1/2 ∼ 140 nM), which results in activation of K+/H+ exchange in mitochondria. The latter, in turn, accelerates mitochondrial respiration in respiratory state 2. The contribution of K ATP + -channel to the mitochondrial potassium cycle was estimated using the selective K ATP + -channel blocker glibenclamide. The data show that the relative contribution of K ATP + -channel in the potassium cycle of mitochondria is variable and increases only with the decrease in the ATP-independent component of K+ uptake. Possible mechanisms underlying the observed phenomena are discussed. The experimental results more fully elucidate the role of K ATP + -channel in the regulation of mitochondrial functions, especially under pathological conditions accompanied by impairment of the mitochondrial energy state.
Keywords: ATP-dependent K+-channel; mitochondria; potassium; transport; diazoxide; oxygen consumption; potassium cycle

Identification of proteins overexpressed in papillary thyroid tumors by L. V. Sipina; Yu. A. Bukurova; I. G. Nikitina; G. S. Krasnov; S. A. Sergeev; N. A. Lisitsyn; V. L. Karpov; S. F. Beresten (1148-1152).
A modified method of proteome comparative analysis based on preliminary removal of cell structural proteins by extraction using salt buffer and subsequent separation of extracts by two-dimensional gel electrophoresis was developed. Identification of differentially expressed proteins by mass spectrometry has revealed three proteins with noticeably increased level of synthesis in most samples of papillary thyroid tumors compared to normal tissues. An increase in ubiquitin content was found for the first time. Oncomarker search efficiencies by two-dimensional gel electrophoresis and bioinformatic search were compared.
Keywords: differential proteomics; two-dimensional gel electrophoresis; thyroid cancer; protein oncomarkers

Effect of enteropeptidase on survival of cultured hippocampal neurons under conditions of glutamate toxicity by A. M. Makarova; L. R. Gorbacheva; I. V. Savinkova; A. G. Mikhailova; L. D. Rumsh; V. G. Pinelis; S. M. Strukova (1153-1159).
The effects of full-size bovine enteropeptidase (BEK) and of human recombinant light chain enteropeptidase (L-HEP) on survival of cultured hippocampal neurons were studied under conditions of glutamate excitotoxicity. Low concentrations of L-HEP or BEK (0.1–1 and 0.1–0.5 nM, respectively) protected hippocampal neurons against the death caused by 100 μM glutamate. Using the PAR1 (proteinase-activated receptor) antagonist SCH 79797, we revealed a PAR1-dependent mechanism of neuroprotective action of low concentrations of enteropeptidase. The protective effect of full-size enteropeptidase was not observed at the concentrations of 1 and 10 nM; moreover, 10 nM of BEK caused death of 88.9% of the neurons, which significantly exceeded the cell death caused by glutamate (31.9%). Under conditions of glutamate cytotoxicity the survival of neurons was 26.8% higher even in the presence of 10 nM of L-HEP than in the presence of 10 nM BEK. Pretreatment of cells with 10 nM of either form of enteropeptidase abolished the protective effect of 10 nM thrombin under glutamate cytotoxicity. High concentrations of BEK and L-HEP caused the death of neurons mainly through necrosis.
Keywords: enteropeptidase; glutamate toxicity; apoptosis; hippocampal neurons; proteinase activated receptor

Enzymes of SPZ7 phage: Isolation and properties by P. A. Levashov; D. V. Popov; V. M. Popova; E. L. Zhilenkov; O. A. Morozova; N. G. Belogurova; S. A. Sedov; I. A. Dyatlov; N. L. Klyachko; A. V. Levashov (1160-1164).
Bacteriophage enzyme preparations exolysin and endolysin were studied. Exolysin (a phage-associated enzyme) was obtained from tail fraction and endolysin from phage-free cytoplasmic fraction of disintegrated Salmonella enteritidis cells. A new method for purification of these enzymes was developed, and their molecular masses were determined. The main catalytic properties of the studied enzymes (pH optimum and specificity to bacterial substrates) were found to be similar. Both enzymes lyse Escherichia coli cells like chicken egg lysozyme, but more efficiently lyse S. enteritidis cells and cannot lyse Micrococcus luteus, a good substrate for chicken egg lysozyme. Similar properties of exolysin and endolysin suggest that these enzymes are structurally similar or even identical.
Keywords: bacteriophage enzyme; lysis of bacteria; bacteriolytic activity

Study of peptide fractions from hemolymph of Galleria mellonella by O. S. Sribnaya; P. P. Purygin; N. A. Klenova; A. K. Buryak; E. G. Litvinova (1165-1172).
Changes in the peptide composition of hemolymph of Galleria mellonella larvae induced by their immunization have been studied, and some new peptides have been found. The composition of fractions exhibiting antibacterial activity was investigated. Known antibacterial peptides have been found in the hemolymph of control larvae and those immunized with bacteria.
Keywords: Galleria mellonella ; peptides; HPLC; MALDI

Destabilase-lysozyme of medicinal leech. Multifunctionality of recombinant protein by L. L. Zavalova; V. N. Lazarev; S. A. Levitsky; T. G. Yudina; I. P. Baskova (1173-1181).
Preparation and purification of a recombinant protein are described along with characteristics of its specific (for ɛ-(γ-Glu)-Lys and D-dimer substrates) and nonspecific (for L-γ-Glu-pNA) isopeptidase activities; the absence of peptidase function for α-(α-Glu)-Lys substrate is noted. It is shown that the protein exhibits muramidase (cell walls of Micrococcus lysodeikticus) and specific glycosidase activities. The latter was determined towards the fluorogenic substrate 4-methylum-belliferyl-tetra-N-acetyl-β-chitotetraoxide. Antimicrobial activity of recombinant destabilase-lysozyme protein (recDest-Lys) and its 11-membered amphipathic peptide was revealed towards cells of the strict anaerobic Archaean Methanosarcina barkeri, whose cell walls contain no murein. Possible mechanisms of the effect of recDest-Lys on these cells are discussed.
Keywords: endo-ɛ-(γ-Glu)-Lys- and exo-ɛ-(γ-Glu)-Lys-isopeptidolysis; specific glycosidase activity; 4-methylumbelliferyl-N-acetyl-β-chitotetraoxide; muramidase activity; antibacterial activity; Methanosarcina barkeri

Microarray methods and protocols by G. Ya. Wiederschain (1182-1182).