Biochemistry (Moscow) (v.78, #5)

The processes that lead to violation of genome integrity are known to increase with age. This phenomenon is caused both by increased production of reactive oxygen species and a decline in the efficiency of antioxidant defense system as well as systems maintaining genome stability. Accumulation of different unrepairable genome damage with age may be the cause of many age-related diseases and the development of phenotypic and physiological signs of aging. It is also clear that there is a close connection between the mechanisms of the maintenance of genome stability, on one hand, and the processes of spontaneous tumor formation and lifespan, on the other. In this regard, the system of protein poly(ADP-ribosyl)ation activated in response to a variety of DNA damage seems to be of particular interest. Data accumulated to date suggest it to be a kind of focal point of cellular processes, guiding the path of cell survival or death depending on the degree of DNA damage. This review summarizes and analyzes data on the involvement of poly(ADP-ribosyl)ation in various mechanisms of DNA repair, its interaction with progeria proteins, and the possible role in the development of spontaneous tumors and lifespan determination. Special attention is given to the relationship between various polymorphisms of the human poly(ADP-ribose) polymerase-1 gene and longevity.
Keywords: poly(ADP-ribosyl)ation; poly(ADP-ribose) polymerases; poly(ADP-ribose) glycohydrolase; genome stability; aging; DNA repair; lifespan

Topology of mRNA chain in isolated eukaryotic double-row polyribosomes by Zh. A. Afonina; A. G. Myasnikov; N. F. Khabibullina; A. Yu. Belorusova; J. -F. Menetret; V. D. Vasiliev; B. P. Klaholz; V. A. Shirokov; A. S. Spirin (445-454).
In the process of protein synthesis, the translating ribosomes of eukaryotic cells form polyribosomes that are found to be multiplex functional complexes possessing elements of ordered spatial organization. As revealed by a number of electron microscopy studies, the predominant visible configurations of the eukaryotic polyribosomes are circles (circular polyribosomes) and two-stranded formations (so-called double-row polyribosomes). The “long” (i.e. heavy loaded) polyribosomes are usually represented by double-row structures, which can be interpreted as either topologically circular (“col-lapsed rings”), or topologically linear (zigzags or helices). In the present work we have analyzed the mRNA path within the eukaryotic polyribosomes, isolated from a wheat germ cell-free translation system, by integrating two approaches: the visualization of mRNA ends in polyribosomes by marking them with gold nanoparticles (3′-end) and initiating 40S subunits (5′-end), as well as by the cryoelectron tomography. Examination of the location of the mRNA markers in polyribosomes and mutual orientation of ribosomes in them has shown that the double-row polyribosomes of the same sample can have both circular and linear arrangements of their mRNA.
Keywords: eukaryotic polyribosomes; mRNA; circular translation; cryoelectron tomography

Method for isolation of intact titin (connectin) molecules from mammalian cardiac muscle by I. M. Vikhlyantsev; A. D. Okuneva; U. V. Shumilina; N. N. Salmov; A. G. Bobylev; N. V. Molochkov; Z. A. Podlubnaya (455-462).
Cardiac titin was isolated from rabbit and ground squirrel ventricular muscles by a method that was used earlier to obtain myofibrils with intact minor proteins located in A-bands of sarcomeres (Podlubnaya, Z. A., et al. (1989) J. Mol. Biol., 210, 655–658). Small pieces of cardiac muscle were incubated for 2–3 weeks at 4°C in Ca2+-depleting solution before their homogenization to decrease activity of Ca2+-dependent proteases. Then the muscle was homogenized, and titin was isolated by the method of Soteriou, A., et al. (1993) J. Cell Sci., 14, 119–123. In control experiments, titin was isolated from cardiac muscle without its preincubation in Ca2+-depleting solution. Sometimes control titin preparations contained only T2-fragment, but generally they contained ∼5–20% N2B-isoform of titin along with its T2-fragment. Preparations of titin obtained from rabbit cardiac muscle by our method contained ∼30–50% of N2BA- and N2B-titin isoforms along with its T2-fragment. The content of α-structures in titin isolated by our method was increased. Actomyosin ATPase activity in vitro increased in the presence of titin preparations containing more intact molecules. This result confirms the significant role of titin in the regulation of actin-myosin interaction in muscles. The method used by us to preserve titin might be used for isolation of other proteins that are substrates of Ca2+-dependent proteases.
Keywords: striated muscles of mammals; NT-; N2A-; N2BA- and N2B-isoforms of titin; T2-fragments; actin-activated ATPase activity of myosin; Ca2+-depleting solution

Changes in levels of gene expression in human aortal intima during atherogenesis by T. A. Shchelkunova; I. A. Morozov; P. M. Rubtsov; L. M. Samokhodskaya; I. V. Andrianova; I. A. Sobenin; A. N. Orekhov; A. N. Smirnov (463-470).
Changes in the contents of 36 mRNAs species related to lipid turnover, inflammation, metabolism and the action of sex hormones in samples of aortal intima along the “intact tissue — lesions of type I — lesions of type II — lesions of type Va” sequence were analyzed using quantitative PCR. The expression of several mRNAs coding for components of the vesicular transfer and lipid turnover machinery was found to be resistant to atherogenesis or even decline in the course of atherogenesis. Decrease in expression was also recorded for steroid sulfatase, androgen receptor, and low density lipoprotein receptor mRNAs. However, the contents of the majority of other mRNA species increased gradually during disease progression. The earliest changes found as early as in lesions of type I were characteristic for estrogen sulfotransferase, apolipoprotein E, scavenger receptor SR-BI, collagen COL1A2, as well as chemokine CCL18 mRNAs. The contents of several mRNAs in intact tissue and atherosclerotic injuries had gender differences. Additionally, responses of two mRNAs, for aromatase and sterol regulatory element binding protein 2, to atherosclerotic lesion were also sex-differentiated. The contents of the majority of analyzed mRNAs in peripheral blood monocyte-derived macrophages were higher than in intact aorta. The correlations found in atherosclerotic lesions between mRNA species that predominant in macrophages and those expressed at comparable levels in macrophages and intact aorta or mainly in aorta suggest that the observed rise in the content of the majority of mRNAs during atherogenesis is determined by increase in expression in resident cells. The data suggest that the revealed absence of homeostatic regulation of expression of a number of genes associated with vesicular transfer and lipid turnover can serve as one of the reasons for lysosomal function insufficiency that leads to foam cell formation in atheroma. The observed sex differences in expression of a number of mRNAs suggest that estrogens in women perform their atheroprotective effects starting with predisposition to the disease and finishing with advanced stages of the pathologic process.
Keywords: mRNA; PCR; gene expression; atherogenesis; aorta; gender differences; macrophages

The level of nitric oxide (NO) in roots of 2-day-old etiolated pea (Pisum sativum L.) seedlings was investigated by fluorescence microscopy using the fluorescent probe 4,5-diaminofluorescein diacetate. Segments representing transversal (cross) cuts of the roots having thickness of 100 to 150 μm (a segment of the root located 10 to 15 mm from the apex) were analyzed. A substantial concentration of NO in the roots was registered when the seedlings were grown in water (control). Addition of 4 mM sodium nitroprusside, 20 mM KNO3, 2 mM NaNO2, 2 mM L-arginine into the growth medium increased NO concentration with respect to the control by 1.7- to 2.3-fold. Inhibitors of animal NO-synthase — 1 mM Nω-nitro-L-arginine methyl ester hydrochloride and 1 mM aminoguanidine hydrochloride — reduced the intensity of fluorescence in the root segments in the presence of all the studied compounds. In medium with KNO3, the inhibitor of nitrate reductase −150 μM sodium tungstate -lowered the fluorescence intensity by 60%. Scavengers of nitric oxide — 100 μM 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and 4 μM hemoglobin — lowered NO concentration in all the studied variants. Potassium ferrocyanide (4 mM) as the inactive analog of sodium nitroprusside inhibited generation of NO. These results are discussed regarding possible pathways of NO synthesis in plants.
Keywords: Pisum sativum L.; etiolated seedlings of pea; fluorescence probe; nitric oxide (NO); inhibitors of animal NO-synthase (NOS) and plant nitrate reductase; NO scavengers

Protein profiles of the basidiomycete Trametes hirsuta grown on standard medium without laccase as an inducer and on medium supplemented with CuSO4 were analyzed using a differential proteomics approach. Protocols developed for isolation and purification of extracellular and intracellular proteins of the mycelium allowed us to show extensive extraction of protein components. Simultaneously, components hampering two-dimensional electrophoresis (pigments, low molecular mass metabolites) were removed from the samples, and high-resolution protein maps were obtained. Analysis of the basidiomycete secretomes revealed qualitative changes in the protein profile: the addition of CuSO4 as an inducer resulted in increase in the produced laccase isoforms and/or isozymes from 7 to 11, whereas its pI range change exceeded 2 units. The number of separated intracellular protein components was 552 and 502 for the control medium and medium with the inducer, respectively. Comparative analysis of the protein maps revealed five regions with the most pronounced differences in the protein profiles. The proteins of interest were identified using MALDI-TOF/TOF mass spectrometry with subsequent peptide fingerprinting. Some intracellular proteins (β-subunits of ATP synthase, molecular chaperones, chaperone activator) upregulated during the growth on the inducer-containing medium were identified. These proteins are supposed to be involved in the regulation of laccase biosynthesis during folding and secretion of the enzyme.
Keywords: laccase biosynthesis; proteomic analyses; intracellular proteins; two-dimensional electrophoresis; Trametes hirsuta

Identification and differential expression of two dehydrin cDNAs during maturation of Jatropha curcas seeds by S. A. Omar; N. I. Elsheery; H. M. Kalaji; M. K. H. Ebrahim; S. Pietkiewicz; C. -H. Lee; S. I. Allakhverdiev; Zeng-Fu Xu (485-495).
Plant dehydrin proteins (DHNs) are known to be important for environmental stress tolerance and are involved in various developmental processes. Two full-length cDNAs JcDHN-1 and JcDHN-2 encoding two dehydrins from Jatropha curcas seeds were identified and characterized. JcDHN-1 is 764 bp long and contains an open reading frame of 528 bp. The deduced JcDHN-1 protein has 175 a.a. residues that form a 19.3-kDa polypeptide with a predicted isoelectric point (pI) of 6.41. JcDHN-2 is 855 bp long and contains an open reading frame of 441 bp. The deduced JcDHN-2 protein has 156 a.a. residues that form a 17.1-kDa polypeptide with a predicted pI of 7.09. JcDHN-1 is classified as type Y3SK2 and JcDHN-2 is classified as type Y2SK2 according to the YSK shorthand for structural classification of dehydrins. Homology analysis indicates that both JcDHN-1 and JcDHN-2 share identity with DHNs of other plants. Analysis of the conserved domain revealed that JcDHN-2 has glycoside hydrolase GH20 super-family activity. Quantitative real time PCR analysis for JcDHN-1 and JcDHN-2 expression during seed development showed increasing gene expression of both their transcript levels along with the natural dehydration process during seed development. A sharp increase in JcDHN-2 transcript level occurred in response to water content dropping from 42% in mature seeds to 12% in dry seeds. These results indicate that both JcDHNs have the potential to play a role in cell protection during dehydration occurring naturally during jatropha orthodox seed development.
Keywords: dehydrin; Jatropha curcas ; real time PCR; seed development

OmpC-like porin from outer membrane of Yersinia enterocolitica: Molecular structure and functional activity by O. P. Vostrikova; M. P. Isaeva; G. N. Likhatskaya; O. D. Novikova; N. Yu. Kim; V. A. Khomenko; T. F. Solov’eva (496-504).
OmpC-like porin was isolated from the outer membrane (OM) of Yersinia enterocolitica cultured at 37°C (the “warm” variant) and its physicochemical and functional properties were studied. The amino acid sequence of OmpC porin was established, and the primary structure and transmembrane topology of this protein were analyzed in comparison with the OmpF porin isolated from Y. enterocolitica cultured at 6°C (the “cold” variant). Both porins of Y. enterocolitica had a high homology degree (65%) between themselves and with OmpC and OmpF porins from OM of Escherichia coli (58 and 76% homology, respectively). The secondary structure of OmpC and OmpF porins from OM of Y. enterocolitica consists of 16 β-strands connected by short “periplasmic” and longer “extracellular” loops with disordered structure, according to the topological model developed for porins of E. coli. The molecular structures of OmpC and OmpF porins of Y. enterocolitica have significant differences in the structure of the “extracellular” loops and in the position of one of three tryptophan residues. Using the bilayer lipid membrane (BLM) technique, pores formed by OmpC porin of Y. enterocolitica were shown to differ in electrophysiological characteristics from channels of OmpF protein of this microorganism. The isolated OmpC porin reconstructed into BLM displayed functional plasticity similarly to OmpF protein and nonspecific porins of other enterobacteria. The conductivity level of the channels formed by this protein in the BLM was regulated by value of the applied potential.
Keywords: Yersinia enterocolitica ; pore-forming proteins; structure; functional activity

Modulation of action of wheat seedling endonucleases WEN1 and WEN2 by histones by L. I. Fedoreyeva; T. A. Smirnova; G. Ya. Kolomijtseva; B. F. Vanyushin (505-516).
Wheat core histones and various subfractions of histone H1 modulate differently the action of endonucleases WEN1 and WEN2 from wheat seedlings. The character of this modulation depends on the nature of the histone and the methylation status of the substrate DNA. The modulation of enzyme action occurs at different stages of processive DNA hydrolysis and is accompanied by changes in the site specificity of the enzyme action. It seems that endonuclease WEN1 prefers to bind with protein-free DNA stretches in histone H1-DNA complex. The endonuclease WEN1 does not compete with histone H1/6 for DNA binding sites, but it does compete with histone H1/1, probably for binding with methylated sites of DNA. Unlike histone H1, the core histone H2b binds with endonuclease WEN1 and significantly increases its action. This is associated with changes in the site specificity of the enzyme action that is manifested by a significant increase in the amount of low molecular weight oligonucleotides and mononucleotides produced as a result of hydrolysis of DNA fragments with 120–140-bp length. The WEN2 endonuclease binds with histone-DNA complexes only through histones. The action of WEN2 is increased or decreased depending on the nature of the histone. Histone H1/1 stimulated the exonuclease activity of WEN2. It is supposed that endonucleases WEN1 and WEN2, in addition to the catalytic domain, should have a regulatory domain that is involved in binding of histones. As histone H1 is mainly located in the linker chromatin areas, it is suggested that WEN2 should attack DNA just in the chromatin linker zones. As differentiated from WEN2, DNA hydrolysis with endonuclease WEN1 is increased in the presence of core histones and, in particular, of H2b. Endonuclease WEN1 initially attacks different DNA sites in chromatin than WEN2. Endonuclease WEN2 activity can be increased or diminished depending on presence of histone H1 subfractions. It seems that just different fractions of the histone H1 are responsible for regulation of the stepwise DNA degradation by endonuclease WEN2 during apoptosis. Modulation of the action of the endonucleases by histones can play a significant role in the epigenetic regulation of various genetic processes and functional activity of genes.
Keywords: endonucleases; H1; core histones; plants; wheat; modulation of action

Structural investigations of recombinant urokinase growth factor-like domain by I. B. Beloglazova; R. Sh. Beabealashvilli; Ya. G. Gursky; E. V. Bocharov; K. S. Mineev; E. V. Parfenova; V. A. Tkachuk (517-530).
Urokinase-type plasminogen activator (uPA) is a serine protease that converts the plasminogen zymogen into the enzymatically active plasmin. uPA is synthesized and secreted as the single-chain molecule (scuPA) composed of an N-terminal domain (GFD) and kringle (KD) and C-terminal proteolytic (PD) domains. Earlier, the structure of ATF (which consists of GFD and KD) was solved by NMR (A. P. Hansen et al. (1994) Biochemistry, 33, 4847–4864) and by X-ray crystallography alone and in a complex with the soluble form of the urokinase receptor (uPAR, CD87) lacking GPI (C. Barinka et al. (2006) J. Mol. Biol., 363, 482–495). According to these data, GFD contains two β-sheet regions oriented perpendicularly to each other. The area in the GFD responsible for binding to uPAR is localized in the flexible Ω-loop, which consists of seven amino acid residues connecting two strings of antiparallel β-sheet. It was shown by site-directed mutagenesis that shortening of the Ω-loop length by one amino acid residue leads to the inability of GFD to bind to uPAR (V. Magdolen et al. (1996) Eur. J. Biochem., 237, 743–751). Here we show that, in contrast to the above-mentioned studies, we found no sign of the β-sheet regions in GFD in our uPA preparations either free or in a complex with uPAR. The GFD seems to be a rather flexible and unstructured domain, demonstrating in spite of its apparent flexibility highly specific interaction with uPAR both in vitro and in cell culture experiments. Circular dichroism, tryptophan fluorescence during thermal denaturation of the protein, and heteronuclear NMR spectroscopy of 15N/13C-labeled ATF both free and in complex with urokinase receptor were used to judge the secondary structure of GFD of uPA.
Keywords: urokinase kurokinase receptor kNMR kΩ-loop

Two fish species, Cyprinion macrostomus macrostomus and Garra rufa obtuse, tolerate adverse conditions in the Kangal hot springs and cope with multiple stressors such as food deprivation, extreme temperature, toxins, protein degradation, hypoxia, and microbial damage. These fish have evolved strategies to counteract the stressors including the induction of heat shock proteins (Hsps). Hsps play an essential role in maintaining cellular homeostasis, and one of the key proteins in the mechanism is Hsp70. Hsp70 itself is exposed to the same stressors as all other proteins, and, hence, the stability of Hsp70 was investigated. For this purpose, Hsp70 ATPase activity was determined at different urea concentrations. It was found that the protein maintains considerable ATP hydrolysis activity at higher denaturant conditions. Temperature effects on the substrate peptide binding showed that Hsp70s bind prominently at elevated temperatures. Furthermore, temperature effects on Hsp70 aggregation indicated that the presence of nucleotides decreases the aggregation process. The present work has determined the stability and activity of cmHsp70 and grHsp70 themselves under extreme conditions. The stability of the Hsp70 proteins maintains substrate proteins in the native state, which may aid in the adaptation of the fish species to the hot spring environment.
Keywords: Hsp70; stability; aggregation

MGT, a macrolide UDP-glycosyltransferase from Streptomyces lividans, has been employed as a synthetic “tool kit” to synthesize a series of modified antibiotics owing to its broad substrate plasticity. Other than MGT, a number of UDP-glycosyltransferases with substrate promiscuity were also used to alter glycosylation patterns of secondary metabolites in an emerging method called “chemoenzymatic glycorandomization”. However, the structural basis of tolerating variant acceptors for these glycosyltransferases is still not clear. In this study, the relationship between the amino acid residues forming the acceptor binding pocket and the specificity of an MGT was investigated in evolutionary and structural aspects. Interestingly, alterations of the volume and hydrophobic environment of the binding pocket by replacing Ile127 or Val151 with a bulky Phe conferred on the MGT novel activities for glycosylating flavonoids that are not accepted by the wild-type MGT.
Keywords: glycosyltransferase; specificity; mutagenesis; macrolide; flavonoid

Role of oxidative stress and mitochondria in onset of urinary bladder dysfunction under acute urine retention by V. I. Kirpatovsky; E. Y. Plotnikov; I. S. Mudraya; S. A. Golovanov; V. V. Drozhzheva; R. A. Khromov; D. Y. Chernikov; V. P. Skulachev; D. B. Zorov (542-548).
Acute urine retention is a frequent complication in patients with benign hyperplasia of the prostate gland. According to studies made on experimental animals and people, it is accompanied by the deterioration of the bladder blood supply. This study attempts to explore the relationship of intramural blood flow, production of reactive oxygen species, and functional state of the bladder detrusor in modeling of acute urine retention in rats, as well as the impact of mitochondria-targeted antioxidants (which are supposed to alleviate the effects of oxidative stress induced by experimental ischemia) on these parameters. The study showed beneficial effects of mitochondria-targeted antioxidant SkQR1 in preventing damage of the bladder caused by acute urinary retention, which suggests the therapeutic use of this type of compounds for the treatment of ischemic abnormalities of the urinary bladder.
Keywords: ischemia; oxidative stress; urine retention; urinary bladder; mitochondria-targeted antioxidants; SkQ; mitochondria

Decrease in pool of T lymphocytes with surface phenotypes of effector and central memory cells under Influence of TCR transgenic β-chain expression by Yu. Yu. Silaeva; A. A. Kalinina; M. S. Vagida; L. M. Khromykh; A. V. Deikin; T. G. Ermolkevich; E. R. Sadchikova; I. L. Goldman; D. B. Kazansky (549-559).
Peripheral T lymphocytes can be subdivided into naive and antigen-experienced T cells. The latter, in turn, are represented by effector and central memory cells that are identified by different profiles of activation markers expression, such as CD44 and CD62L in mice. These markers determine different traffic of T lymphocytes in the organism, but hardly reproduce real antigenic experience of a T lymphocyte. Mechanisms of homeostasis maintenance of T lymphocytes with different activation phenotypes remain largely unknown. To investigate impact of T cell receptor (TCR) transgenic chains on formation of T lymphocytes, their peripheral survival and activation surface phenotypes, we have generated the transgenic mouse strain expressing transgenic β-chain of TCR 1D1 (belonging to the Vβ6 family) on the genetic background B10.D2(R101). Intrathymic development of T cells in these transgenic mice is not impaired. The repertoire of peripheral T lymphocytes in these mice contains 70–80% of T cells expressing transgenic β-chain and 20–30% of T cells expressing endogenous β-chains. The ratio of peripheral CD4+CD8 and CD4CD8+ T lymphocytes remained unchanged in the transgenic animals, but the percent of T lymphocytes with the “naive” phenotype CD44CD62L+ was significantly increased, whereas the levels of effector memory CD44+CD62L and central memory CD44+CD62L+ T lymphocytes were markedly decreased in both subpopulations. On the contrary, T lymphocytes expressing endogenous β-chains had surface phenotype of activated T cells CD44+. Thus, for the first time we have shown that the pool of T lymphocytes with different activation phenotypes depends on the structure of T cell receptors.
Keywords: T cell receptor; transgenic animals; lymphocyte; effector; memory cell