Source: https://chemweb.com/articles/SV10541/0007900002
Timestamp: 2019-04-22 02:28:36+00:00

Document:
Expression of exogenous DNA methyltransferases: Application in molecular and cell biology by O. V. Dyachenko; S. V. Tarlachkov; D. V. Marinitch; T. V. Shevchuk; Y. I. Buryanov (77-87).
DNA methyltransferases might be used as powerful tools for studies in molecular and cell biology due to their ability to recognize and modify nitrogen bases in specific sequences of the genome. Methylation of the eukaryotic genome using exogenous DNA methyltransferases appears to be a promising approach for studies on chromatin structure. Currently, the development of new methods for targeted methylation of specific genetic loci using DNA methyltransferases fused with DNA-binding proteins is especially interesting. In the present review, expression of exogenous DNA methyltransferase for purposes of in vivo analysis of the functional chromatin structure along with investigation of the functional role of DNA methylation in cell processes are discussed, as well as future prospects for application of DNA methyltransferases in epigenetic therapy and in plant selection.
Kinase mTOR: Regulation and role in maintenance of cellular homeostasis, tumor development, and aging by A. A. Parkhitko; O. O. Favorova; D. I. Khabibullin; V. N. Anisimov; E. P. Henske (88-101).
Serine/threonine protein kinase mTOR regulates the maintenance of cellular homeostasis by coordinating transcription, translation, metabolism, and autophagy with availability of amino acids, growth factors, ATP, and oxygen. The mTOR kinase is a component of two protein complexes, mTORC1 and mTORC2, which are different in their composition and regulate different cellular processes. An uncontrolled activation of the mTOR kinase is observed in cells of the majority of tumors, as well as in diabetes and neurodegenerative and some other diseases. At present, inhibitors of the kinase complex mTORC1 are undergoing clinical trials. This review focuses on different aspects of the regulation of the mTORC1 and mTORC2 complexes, on their role in the regulation of protein synthesis, metabolism, and autophagy, as well as on using mTOR inhibitors for treatment of tumors and slowing of aging.
Pectin methylesterase-generated methanol may be involved in tobacco leaf growth by T. V. Komarova; D. V. Pozdyshev; I. V. Petrunia; E. V. Sheshukova; Y. L. Dorokhov (102-110).
Plant leaves undergo a sink-source modification of intercellular macromolecular transport during the transition from carbon import to carbon export. After assessing the role of metabolite signaling in gene regulation in Nicotiana tabacum sink and source leaves, we observed increased pectin methylesterase (PME)-mediated methanol generation in immature leaves. Using suppression subtractive hybridization (SSH), we identified a number of genes whose activity changes from sink to source leaves. The most abundant SSH-identified genes appeared to be sensitive to methanol. We hypothesize that tobacco leaf maturation and the sink-source transition are accompanied by a change in mRNA levels of genes that function in methanol-dependent cell signaling.
Hepatitis B virus can be inhibited by DNA methyltransferase 3a via specific zinc-finger-induced methylation of the X promoter by L. Xirong; L. Rui; Y. Xiaoli; H. Qiuyan; T. Bikui; Z. Sibo; Z. Naishuo (111-123).
In this work we explored whether DNA methyltransferase 3a (Dnmt3a) targeted to the HBV X promoter (XP) causes epigenetic suppression of hepatitis B virus (HBV). The C-terminus of Dnmt3a (Dnmt3aC) was fused to a six-zinc-finger peptide specific to XP to form a fused DNA methyltransferase (XPDnmt3aC). The binding and methyl-modifying specificity of XPDnmt3aC were verified with an electrophoretic mobility shift assay and methylation-specific PCR, respectively. XP activity and HBV expression were clearly downregulated in HepG2 cells transfected with plasmid pXPDnmt3aC. The injection of XPDnmt3aC into HBV transgenic (TgHBV) mice also showed significant inhibition, leading to low serum HBV surface protein (HBsAg) levels and a reduced viral load. Thus, XPDnmt3aC specifically silenced HBV via site-selective DNA methylation delivered by zinc-finger peptides. This study establishes the foundation of an epigenetic way of controlling HBV-related diseases.
Mitochondria-targeted antioxidants prevent TNFα-induced endothelial cell damage by I. I. Galkin; O. Yu. Pletjushkina; R. A. Zinovkin; V. V. Zakharova; I. S. Birjukov; B. V. Chernyak; E. N. Popova (124-130).
Increased serum level of tumor necrosis factor α (TNFα) causes endothelial dysfunction and leads to serious vascular pathologies. TNFα signaling is known to involve reactive oxygen species (ROS). Using mitochondria-targeted antioxidant SkQR1, we studied the role of mitochondrial ROS in TNFα-induced apoptosis of human endothelial cell line EAhy926. We found that 0.2 nM SkQR1 prevents TNFα-induced apoptosis. SkQR1 has no influence on TNFα-dependent proteolytic activation of caspase-8 and Bid, but it inhibits cytochrome c release from mitochondria and cleavage of caspase-3 and its substrate PARP. SkQ analogs lacking the antioxidant moieties do not prevent TNFα-induced apoptosis. The antiapoptotic action of SkQR1 may be related to other observations made in these experiments, namely SkQR1-induced increase in Bcl-2 and corresponding decrease in Bax as well as p53. These results indicate that mitochondrial ROS production is involved in TNFα-initiated endothelial cell death, and they suggest the potential of mitochondria-targeted antioxidants as vasoprotectors.
Search for ligand of N-acetylglucosaminyl-N-acetylmuramyl dipeptide using its peptide mimetic by G. V. Savinov; A. O. Shepelyakovskaya; Kh. M. Boziev; F. A. Brovko; A. G. Laman (131-138).
The method for searching for ligands exerting an adjuvant effect is described. The method involves isolation of polysomes using an immobilized peptide mimetic of N-acetylglucosaminyl-N-acetylmuramyl dipeptide (GMDP) — RN-peptide. After the affinity chromatography and washing, RN-peptide complexes with the target sequences were dissociated with guanidine hydrochloride. The obtained mRNA was used for cDNA synthesis and subsequent cloning in an expression vector. Further studies showed the effectiveness of this method. Clones interacting with the peptide were selected using biotinylated RN-peptide. It was found that all clones encode a sequence identical to the protein YB-1. Recombinant antibodies against protein YB-1 were selected from a phage display human scFv library. Using these antibodies, we determined the binding constant of RN-peptide to protein YB-1. Competitive analysis showed that RN-peptide and GMDP compete for the same portion of YB-1 at molar ratio 1: 12.
Inhibition of oxidative hemolysis in erythrocytes by mitochondria-targeted antioxidants of SkQ series by E. O. Omarova; Y. N. Antonenko (139-145).
In the present work we studied the effect of antioxidants of the SkQ1 family (10-(6′-plastoquinonyl)decyltriphenylphosphonium) on the oxidative hemolysis of erythrocytes induced by a lipophilic free radical initiator 2,2′-azobis(2,4-dimethylvaleronitrile) (AMVN) and a water-soluble free radical initiator 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH). SkQ1 was found to protect erythrocytes from hemolysis, 2 μM being the optimal concentration. Both the oxidized and reduced SkQ1 forms exhibited protective properties. Both forms of SkQ1 also inhibited lipid peroxidation in erythrocytes induced by the lipophilic free radical initiator AMVN as detected by accumulation of malondialdehyde. However, in the case of induction of erythrocyte oxidation by AAPH, the accumulation of malondialdehyde was not inhibited by SkQ1. In the case of AAPH-induced hemolysis, the rhodamine-containing analog SkQR1 exerted a comparable protective effect at the concentration of 0.2 μM. At higher SkQ1 and SkQR1 concentrations, the protective effect was smaller, which was attributed to the ability of these compounds to facilitate hemolysis in the absence of oxidative stress. We found that plastoquinone in the oxidized form of SkQ1 could be reduced by erythrocytes, which apparently accounted for its protective action. Thus, the protective effect of SkQ in erythrocytes, which lack mitochondria, proceeded at concentrations that are two to three orders of magnitude higher than those that were active in isolated mitochondria.
Study on ATP concentration changes in cytosol of individual cultured neurons during glutamate-induced deregulation of calcium homeostasis by A. M. Surin; L. R. Gorbacheva; I. G. Savinkova; R. R. Sharipov; B. I. Khodorov; V. G. Pinelis (146-157).
For the first time, simultaneous monitoring of changes in the concentration of cytosolic ATP ([ATP]c), pH (pHc), and intracellular free Ca2+ concentration ([Ca2+]i) of the individual neurons challenged with toxic glutamate (Glu) concentrations was performed. To this end, the ATP-sensor AT1.03, which binds to ATP and therefore enhances the efficiency of resonance energy transfer between blue fluorescent protein (energy donor) and yellow-green fluorescent protein (energy acceptor), was expressed in cultured hippocampal neurons isolated from 1–2-day-old rat pups. Excitation of fluorescence in the acceptor protein allowed monitoring changes in pHc. Cells were loaded with fluorescent low-affinity Ca2+ indicators Fura-FF or X-rhod-FF to register [Ca2+]i. It was shown that Glu (20 μM, glycine 10 μM, Mg2+-free) produced a rapid acidification of the cytosol and decrease in [ATP]c. An approximately linear relationship (r 2 = 0.56) between the rate of [ATP]c decline and latency of glutamate-induced delayed calcium deregulation (DCD) was observed: higher rate of [ATP]c decrease corresponded to shorter DCD latency period. DCD began with a decrease in [ATP]c of as much as 15.9%. In the phase of high [Ca2+]i, the plateau of [ATP]c dropped to 10.4% compared to [ATP]c in resting neurons (100%). In the presence of the Na+/K+-ATPase inhibitor ouabain (0.5 mM), glutamate-induced reduction in [ATP]c in the phase of the high [Ca2+]i plateau was only 36.6%. Changes in [ATP]c, [Ca2+]i, mitochondrial potential, and pHc in calcium-free or sodium-free buffers, as well as in the presence of the inhibitor of Na+/K+-ATPase ouabain, led us to suggest that in addition to increase in proton conductivity and decline in [ATP]c, one of the triggering factors of DCD might be a reversion of the neuronal plasma membrane Na+/Ca2+ exchange.
Glutathionylation of the alpha-subunit of Na,K-ATPase from rat heart by oxidized glutathione inhibits the enzyme by Meng Xianyu; I. Yu. Petrushanko; E. A. Klimanova; E. A. Dergousova; O. D. Lopina (158-164).
A partially purified Na,K-ATPase preparation from rat heart containing α1- and α2-isoforms of the enzyme was shown to include both subunits in S-glutathionylated state. Glutathionylation of the α1-subunit (but not of the α2-subunit) was partially removed when the preparation was isolated in the presence of dithiothreitol. The addition of oxidized glutathione irreversibly inhibited both isoforms. Inhibition of the enzyme containing the α1-subunit was biphasic, and the rate constants of the inhibition were 3745 ± 360 and 246 ± 18 M−1·min−1. ATP, ADP, and AMP protected the Na,K-ATPase against inactivation by oxidized glutathione.

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