Biochemistry (Moscow) (v.75, #3)

It was previously found that 20 pM SkQ1 (10-(6′-plastoquinonyl) decyltriphenylphosphonium) solution increased lifespan of virgin females and males of the fruit fly Drosophila melanogaster with maximal and highly reproducible effect on early survival of females. In this paper we demonstrate that SkQ1 solution of the same concentration does not increase lifespan of mated females and males, early effect on female survival being absent, whereas early fertility and the total number of progeny are elevated in treated flies. Increase in fertility observed in young mated females instead of increase of survival typical for young virgin females might illustrate the trade-off between the fly’s lifespan and reproduction.
Keywords: SkQ1; antioxidant; lifespan; reproduction

Antimutagenic activity of mitochondria-targeted plastoquinone derivative by V. A. Chistyakov; M. A. Sazykina; A. A. Alexandrova; N. I. Belichenko; E. V. Mashkina; L. V. Gutnikova; P. V. Zolotukhin; T. P. Shkurat (269-273).
The ability of cationic plastoquinone derivative 10-(6′-plastoquinonyl) decyltriphenylphosphonium (SkQ1) to modify processes of spontaneous and induced mutagenesis was studied. It is shown that daily introduction of this compound into male Wistar rats in doses of 25 and 250 nmol/kg during two weeks decreases spontaneous level of chromosome aberrations in anaphase in the eye cornea from 0.39 ± 0.09 to 0.13 ± 0.08 and 0.14 ± 0.05, respectively. The level of 8-hydroxy-2′-deoxyguanosine in blood serum of the investigated animals decreases from 32.12 ± 1.55 to 25.90 ± 2.26 and 25.76 ± 1.50 ng/ml, respectively. These facts indicate that the decrease in spontaneous clastogenesis is caused by decreased level of DNA damage by endogenous reactive oxygen species. A higher dose of SkQ1 also decreases to control level chromosome aberrations caused by oxygen under pressure of 0.5 MPa for 60 min. It is also shown in experiments with bacterial biosensors that SkQ1 is able to efficiently protect cells against genotoxic effect of UV radiation at 300–400 nm.
Keywords: SkQ1; penetrating cations; antioxidants; mutagenesis; 8-OH-dG; chromosome aberrations; DNA damage; hyperbaric oxygenation; UV radiation

Novel mitochondria-targeted antioxidants, “Skulachev-Ion” derivatives, accelerate dermal wound healing in animals by I. A. Demianenko; T. V. Vasilieva; L. V. Domnina; V. B. Dugina; M. V. Egorov; O. Y. Ivanova; O. P. Ilinskaya; O. Y. Pletjushkina; E. N. Popova; I. Y. Sakharov; A. V. Fedorov; B. V. Chernyak (274-280).
It is shown that the novel mitochondria-targeted antioxidant SkQ1, (10-(6′-plastoquinonyl) decyltriphenylphosphonium) stimulates healing of full-thickness dermal wounds in mice and rats. Treatment with nanomolar doses of SkQ1 in various formulations accelerated wound cleaning and suppressed neutrophil infiltration at the early (7 h) steps of inflammatory phase. SkQ1 stimulated formation of granulation tissue and increased the content of myofibroblasts in the beginning of regenerative phase of wound healing. Later this effect caused accumulation of collagen fibers. Local treatment with SkQ1 stimulated re-epithelization of the wound. Lifelong treatment of mice with SkQ1 supplemented with drinking water strongly stimulated skin wounds healing in old (28 months) animals. In an in vitro model of wound in human cell cultures, SkQ1 stimulated movement of epitheliocytes and fibroblasts into the “wound”. Myofibroblast differentiation of subcutaneous fibroblasts was stimulated by SkQ1. It is suggested that SkQ1 stimulates wound healing by suppression of the negative effects of oxidative stress in the wound and also by induction of differentiation. Restoration of regenerative processes in old animals is consistent with the “rejuvenation” effects of SkQ1, which prevents some gerontological diseases.
Keywords: mitochondria-targeted antioxidants; SkQ1; myofibroblasts; inflammation; skin wounds

Oxidative stress in yeast by V. I. Lushchak (281-296).
The mechanisms of production and elimination of reactive oxygen species in the cells of the budding yeast Saccharomyces cerevisiae are analyzed. Coordinative role of special regulatory proteins including Yap1p, Msn2/4p, and Skn7p (Pos9p) in regulation of defense mechanisms in S. cerevisiae is described. A special section is devoted to two other well-studied species from the point of view of oxidative stress — Schizosaccharomyces pombe and Candida albicans. Some examples demonstrating the use of yeast for investigation of apoptosis, aging, and some human diseases are given in the conclusion part.
Keywords: yeast; Saccharomyces cerevisiae ; Schizosaccharomyces pombe ; Candida albicans ; oxidative stress response; regulatory proteins

Induction of permeability of the inner membrane of yeast mitochondria by M. V. Kovaleva; E. I. Sukhanova; T. A. Trendeleva; K. M. Popova; M. V. Zylkova; L. A. Uralskaya; R. A. Zvyagilskaya (297-303).
The current view on apoptosis is given, with a special emphasis placed on apoptosis in yeasts. Induction of a non-specific permeability transition pore (mPTP) in mammalian and yeast mitochondria is described, particularly in mitochon-dria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts, which are aerobes possessing the fully competent respiratory chain with all three points of energy conservation and well-structured mitochondria. They were examined for their ability to induce an elevated permeability transition of the inner mitochondrial membrane, being subjected to virtually all conditions known to induce the mPTP in animal mitochondria. Yeast mitochondria do not form Ca2+-dependent pores, neither the classical Ca2+/Pi-dependent, cyclosporin A-sensitive pore even under deenergization of mitochondria or depletion of the intramitochondrial nucleotide pools, nor a pore induced in mammalian mitochondria upon concerted action of moderate Ca2+ concentrations (in the presence of the Ca2+ ionophore ETH129) and saturated fatty acids. No pore formation was found in yeast mitochondria in the presence of elevated phosphate concentrations at acidic pH values. It is concluded that the permeability transition in yeast mitochondria is not coupled with Ca2+ uptake and is differently regulated compared to the mPTP of animal mitochondria.
Keywords: mitochondria; yeasts; Yarrowia lipolytica ; Dipodascus (Endomyces) magnusii ; Ca2+ ; inorganic phosphate; saturated fatty acids; pH; membrane potential; swelling; pore; apoptosis

Protein kinase C-δ is involved in induction of NOX1 gene expression by aldosterone in rat vascular smooth muscle cells by Haiyan Wei; Xuhua Mi; Ling Ji; Lichuan Yang; Qingjie Xia; Yuquan Wei; Isamu Miyamori; ChunYuan Fan (304-309).
In this study, we focused on the relationship between aldosterone and NOX1 expression in vascular smooth muscle cells (VSMCs). For the first time, with the use of specific inhibitors of protein kinase C (PKC), we report that PKCδ mediates upregulation of NOX1 induced by 10 nM aldosterone in cultured VSMCs. Participation of PKC in the mediation of NOX1 regulation was further confirmed by the effect of diacylglycerol, a PKC agonist, on the NOX1 RNA in A7r5 cells with Northern blot analysis. To establish cause and effect, we next silenced the PKCδ gene partly by RNA interference and found knockdown of PKCδ gene attenuated aldosterone-induced NOX1 expression, generation of superoxide, as well as protein synthesis in VSMCs. Taken together, these data indicated PKCδ might mediate aldosterone-dependent NOX1 upregulation in VSMCs. In addition, we showed that the cascade from aldosterone to PKCδ activation had the participation of the mineralocorticoid receptor.
Keywords: aldosterone; NOX1; PKCδ

Solid-phase assays for study of carbohydrate specificity of galectins by E. M. Rapoport; T. V. Pochechueva; O. V. Kurmyshkina; G. V. Pazynina; V. V. Severov; E. A. Gordeeva; I. M. Belyanchikov; S. Andre; H. -J. Gabius; N. V. Bovin (310-319).
We have recently shown that the carbohydrate-binding pattern of galectins in cells differs from that determined in artificial (non-cellular) test-systems. To understand the observed discrepancy, we compared several test-systems differing in the mode of galectin presentation on solid phase. The most representative system was an assay where the binding of galectin (human galectins-1 and -3 were studied) to asialofetuin immobilized on solid phase was inhibited by polyacrylamide glycoconjugates, Glyc-PAA. This approach permits us to range quantitatively glycans (Glyc) by their affinity to galectin, i.e. to study both high and low affinity ligands. Our attempts to imitate the cell system by solid-phase assay were not successful. In the cell system galectin binds glycoconjugates by one carbohydrate-recognizing domain (CRD), and after that the binding to the remaining non-bound CRD is studied by means of fluorescein-labeled Glyc-PAA. In an “imitation” variant when galectins are loaded on adsorbed asialofetuin or Glyc-PAA followed by revealing of binding by the second Glyc-PAA, the interaction was not observed or glycans were ordered poorly, unlike in the inhibitory assay. When galectins were adsorbed on corresponding antibodies (when all CRDs were free for recognition by carbohydrate), a good concentration dependence was observed and patterns of specificities were similar (though not identical) for the two methods; notably, this system does not reflect the situation in the cell. Besides the above-mentioned, other variants of solid-phase analysis of galectin specificity were tested. The results elucidate the mechanism and consequence of galectin CRD cis-masking on cell surface.
Keywords: galectins; glycoconjugates; masking; solid-phase assay; oligosaccharides; carbohydrate specificity

To understand regulatory mechanisms of cotton fiber development, microarray analysis has been performed for upland cotton (Gossypium hirsutum). Based on this, a cDNA (GhKCR3) encoding a polypeptide belonging to short-chain alcohol dehydrogenase/reductase family was isolated and cloned. It contains an open reading frame of 987 bp encoding a polypeptide of 328 amino acid residues. Following its overexpression in bacterial cells, the purified recombinant protein specifically uses NADPH to reduce a variety of short-chain aldehydes. A fragment between Gly180 and Gly191 was found to be essential for its catalytic activity. Though the GhKCR3 gene shares low sequence similarities to the ortholog of Saccharomyces cerevisiae YBR159w that encodes 3-ketoacyl-CoA reductase (KCR) catalyzing the second step of fatty acid elongation, it was surprisingly able to complement the yeast ybr159wδ mutant. Gas chromatography-mass spectrometry analysis showed that very long-chain fatty acids, especially C26:0, were produced in the ybr159wδ mutant cells expressing GhKCR3. Applying palmitoyl-CoA and malonyl-CoA as substrates, GhKCR3 showed KCR activity in vitro. Quantitative real time-PCR analysis indicated GhKCR3 transcripts accumulated in rapidly elongating fibers, roots, and stems. Our results suggest that GhKCR3 is probably a novel KCR contributing to very long-chain fatty acid biosynthesis in plants.
Keywords: Gossypium hirsutum ; short-chain alcohol dehydrogenase/reductase family; 3-ketoacyl-CoA reductase; fatty acid elongation; very long-chain fatty acids

Binding of MgADP and MgATP to Escherichia coli F1-ATPase (EcF1) has been assessed by their effects on extent of the enzyme inhibition by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl). MgADP at low concentrations (K d 1.3 μM) promotes the inhibition, whereas at higher concentrations (K d 0.7 mM) EcF1 is protected from inhibition. The mutant βY331W-EcF1 requires much higher MgADP, K d of about 10 mM, for protection. Such MgADP binding was not revealed by fluorescence quenching measurements. MgATP partially protects EcF1 from inactivation by NBD-Cl, but the enzyme remains sensitive to NBD-Cl in the presence of MgATP at concentrations as high as 10 mM. The activating anion selenite in the absence of MgATP partially protects EcF1 from inhibition by NBD-Cl. A complete protection of EcF1 from inhibition by NBD-Cl has been observed in the presence of both MgATP and selenite. The results support a bi-site catalytic mechanism for MgATP hydrolysis by F1-ATPases and suggest that stimulation of the enzyme activity by activating anions is due to the anion binding to a catalytic site that remains unoccupied at saturating substrate concentration.
Keywords: anion activation; ATP synthase; catalytic cooperativity; bi-site catalysis; nucleotide binding; tri-site catalysis

Studies on amino acid replacement and inhibitory activity of a β-lactamase inhibitory peptide by Liping Xie; Mingfei Xu; Tao Yang; Chunbao Zhu; Baoquan Zhu; Youjia Hu (336-341).
An SHV β-lactamase gene was amplified from a β-lactam resistant Klebsiella pneumoniae K-71 genomic DNA. After expression and purification, we demonstrated that peptide P1 could inhibit the hydrolysis activity of both TEM-1 and SHV β-lactamase in vitro. Three mutations were introduced into P1 in which the first residue S was replaced by F, the 18th residue V was mutated to Y, and the 15th residue Y was substituted with A, C, G, and R to obtain the mutants of P1-A, P1- C, P1-G, and P1-R, respectively. The mutant peptides were purified and their inhibitory constants against TEM-1 and SHV β-lactamase were determined. All these β-lactamase inhibitory peptides could inhibit the activity of both β-lactamases, while the mutant peptides showed stronger inhibitory activities against TEM-1 β-lactamase than against SHV β-lactamase. Inhibition data suggested that P1-A improved the β-lactamase inhibitory activity by over 3-fold compare to P1. When P1-A was incubated with K. pneumoniae K-71 in Luria-Bertani medium containing ampicillin, it showed a much stronger growth of inhibition ratio over P1. This study gives us a good candidate for development of novel β-lactamase inhibitors.
Keywords: β-lactamase inhibitory peptide; β-lactamase; amino acid replacement; Klebsiella pneumoniae ; inhibitory constant

Peculiarities of cyanide binding to the ba 3-type cytochrome oxidase from the thermophilic bacterium Thermus thermophilus by A. V. Kalinovich; N. V. Azarkina; T. V. Vygodina; T. Soulimane; A. A. Konstantinov (342-352).
Cytochrome c oxidase of the ba 3-type from Thermus thermophilus does not interact with cyanide in the oxidized state and acquires the ability to bind heme iron ligands only upon reduction. Cyanide complexes of the reduced heme a 3 in cytochrome ba 3 and in mitochondrial aa 3-type cytochrome oxidase are similar spectroscopically, but the a 3 2+ -CN complex of cytochrome ba 3 is strikingly tight. Experiments have shown that the K d value of the cytochrome ba 3 complex with cyanide in the presence of reductants of the enzyme binuclear center does not exceed 10−8 M, which is four to five orders of magnitude less than the K d of the cyanide complex of the reduced heme a 3 of mitochondrial cytochrome oxidase. The tightness of the cytochrome ba 3 complex with cyanide is mainly associated with an extremely slow rate of the ligand dissociation (k off ≤ 10−7 sec−1), while the rate of binding (k on ∼ 102 M−1·sec−1) is similar to the rate observed for the mitochondrial cytochrome oxidase. It is proposed that cyanide dissociation from the cytochrome ba 3 binuclear center might be hindered sterically by the presence of the second ligand molecule in the coordination sphere of Cu B 2+ . The rate of cyanide binding with the reduced heme a 3 does not depend on pH in the neutral area, but it approaches linear dependence on H+ activity in the alkaline region. Cyanide binding appears to be controlled by protonation of an enzyme group with pK a = 8.75.
Keywords: cytochrome oxidase ba 3 ; cyanide; hemoproteins; oxygen reducing center; Thermus thermophilus

Glucocorticoid hormones directly or indirectly control virtually all metabolic and physiological processes. Glucocorticoids are also shown to act on a multitude of genes, enzyme systems, and proinflammatory factors, but for these hormones there is no representative index of action on metabolism similar to glucose content in blood for insulin. The absence of such an index prevents the assessment of tissue provision with these hormones under various conditions and seems to be an essential cause of complications associated with the clinical use of glucocorticoid preparations. Considering specific features of tyrosine metabolism and data obtained experimentally and on a clinical model (adrenalectomy in rats and substitution therapy in endocrine disease), blood content of this amino acid seems promising as such an index. Based on comparing results of glucocorticoid treatment in patients with systemic lupus erythematosus with changes in their blood tyrosine contents, the pharmacological effect of glucocorticoid preparations is suggested to be mainly due to compensating a relative shortage of these hormones.
Keywords: glucocorticoids; hormonal provision of tissues; blood tyrosine; tyrosine aminotransferase

We have studied the effect of 2,2,2-trifluoroethanol (TFE), an α-helix inducer, versus methyl cyanide (MeCN), a β-sheet inducer, on acid-denatured human serum albumin (HSA) using far-UV circular dichroism, intrinsic fluorescence, 1-anilino-8-naphthalene sulfonate binding, and acrylamide quenching studies. Interestingly, at pH 2.0, where the recovery and resolution of the protein in reverse phase chromatography is high, its secondary structure remains unchanged even in the presence of very high concentration (76% v/v) of MeCN. Gain of 23 and 34% α-helicity was observed in the presence of 20 and 50% TFE, respectively. At pH 7.3, HSA aggregates in the presence of 40% MeCN, but it remains soluble up to 75% MeCN at pH 2.0. The results seem to be important for HSA isolation and purification.
Keywords: acrylamide quenching; aggregation; circular dichroism; E-state; human serum albumin

Structural stability and functional analysis of L-asparaginase from Pyrococcus furiosus by S. Bansal; D. Gnaneswari; P. Mishra; B. Kundu (375-381).
We report studies on an L-asparaginase from Pyrococcus furiosus, cloned and expressed in Escherichia coli and purified to homogeneity. Protein stability and enzyme kinetic parameters were determined. The enzyme was found to be thermostable, natively dimeric, and glutaminase-free, with optimum activity at pH 9.0. It showed a K m of 12 mM and a substrate inhibition profile above 20 mM L-asparagine. Urea could not induce unfolding and enzyme inactivation; however, with guanidine hydrochloride (GdnCl) a two-state unfolding pattern was observed. Reduced activity and an altered near-UV-CD signal for protein at low GdnCl concentration (1 M) suggested tertiary structural changes at the enzyme active site. A homology three-dimensional model was developed and the structural information was combined with activity and stability data to give functional clues about the asparaginase.
Keywords: Pyrococcus furiosus ; L-asparaginase; hyperthermophile; chemical denaturation; homology model; stability; enzyme kinetics