[Changes of the genome-wide DNA methylation level in various regions of the rat brain with incomplete cerebral ischemia]. / Izmenenie urovnya polnogenomnogo metilirovaniya DNK v razlichnykh oblastyakh golovnogo mozga krys pri nepolnoi tserebral'noi ishemii.

OBJECTIVE: To quantify genome-wide DNA methylation in the olfactory bulbs, fronto-parietal and occipital regions, and cerebellum in normal male Wistar rats and in modeling incomplete cerebral ischemia caused by permanent bilateral occlusion of the common carotid arteries. MATERIAL AND METHODS: The study was performed on 23 male Wistar rats divided into groups: «Sham operation¼ and «Cerebral ischemia¼. The level of genome-wide methylation of CCGG sites was determined by methyl-sensitive restriction using MspI/HpaII endonucleases followed by densitometric analysis of electrophoregrams by ImageJ software. RESULTS: Incomplete cerebral ischemia on the 7th day leads to 56.3% (95% CI: 33.2-76.90) mortality. In the surviving rats of the «Cerebral ischemia¼ group, compared with the animals of the «Sham operation¼ group, a pronounced neurological deficit was observed, which was accompanied by changes in the level of whole-genome DNA methylation in the nervous tissue of brain structures (p<0.05). Incomplete cerebral ischemia in male Wistar rats was characterized by interhemispheric asymmetry in the severity and direction of the epigenomic reaction of the nervous tissue in both ischemic and non-ischemic areas of the brain. CONCLUSION: It is likely that it is precisely this dynamics of changes in the status of genome-wide DNA methylation in the nervous tissue that imparts plasticity to neuronal function during ischemic damage.

[DNA methylation in experimental ischemic brain injury]. / Metilirovanie DNK pri eksperimental'nom ishemicheskom povrezhdenii golovnogo mozga.

The enrichment of angioneurology with fundamental advances leads to the understanding of new important facets in the pathogenesis of cerebral ischemia. The knowledge of epigenetic mechanisms in the development of stroke, in particular, DNA methylation, which makes a significant contribution to the development and formation of cerebral damage, is becoming more and more relevant. This review reflects an analysis of animal studies proving the relationship of DNA methylation with cerebral ischemia. As a result of the search work, 282 articles from the PubMed database were selected for keywords that corresponded to this topic. Of these publications, 8 studies were devoted to genome-wide DNA methylation, and 6 published the results of DNA methylation of candidate genes in experimental cerebral ischemia. The results have demonstrated that brain DNA methylation in animals is associated with the development of ischemic stroke and may play a role in several pathogenetic mechanisms. In two studies, a decrease in the level of DNA methylation in 2 genes in ischemic brain tissues of laboratory animals was found, at the same time, in four studies, 8 genes, in which methylation increased after ischemic stroke, were reported. These data suggest that the assessment of the level of DNA methylation in stroke is a promising biomarker for the search and improvement of pharmacological and non-pharmacological methods for limiting brain damage in ischemic and reperfusion injury at the stages of preclinical and clinical studies.

[CHANGE IN BCL-2 PROTEIN EXPRESSION IN NEURONS OF THE HIPPOCAMPAL AREAS AFTER THE APPLICATION OF ISCHEMIC CEREBRAL POSTCONDITIONING].

Bcl-2 protein expression was studied in hippocampal CA1, CA2, CA3 and CA4 pyramidal neurons in Mongolian gerbils (Meriones unguiculatus), of the in in the early (Day 2) and late (Day 7) reperfusion period after a 7-minute forebrain ischemia and following ischemic postconditioning (IPostC), as well as in sham-operated animals (n=60). In the latter, the highest level, of Bcl-2-expression was found in CA4 neurons, while the lowest--in-CA1 neurons (P<0.01). Reversible ischemic brain damage led to the increasing deficit of morphologically unchanged hippocampal neurons with the increasing duration of reperfusion period. This was accompanied by a significant decrease in expression of Bcl-2 in the early reperfusion period, but in the late reperfu- sion period this decrease largely disappeared. IPostC, applied as three episodes of ischemia-reperfusion lasting 15/15 seconds, contributed to significant increase in the number of morphologically unchanged CA1 and CA3 neurons in the early reperfusion period, while the expression of Bel-2 was increased in morphologically unchanged neurons in all the hippocampal areas. In the late reperfusion period after IPostC, the number of unchanged neurons was increased in hippocampal areas CA1, CA3 and CA4 (P<0.05), while a significant increase in Bcl-2 expression (by 12.7%, P<0.01) was detected only in CA1 neurons. The results suggest that the cytoprotective effect of IPostC in hippocampal CA1 area is realized through a mechanism leading to increased expression of Bcl-2 protein, i.e., by blocking apoptosis.

[Morpho-functional changes of the pyramidal neurons in various hippocampal areas after the ischemic postconditioning].

The aim of this study was to determine the effect of ischemic postconditioning (IP) on the viability of neurons in various hippocampal areas as well as on cytoplasmic activity of succinatedehydrogenase (SDH) in these cells in 30 male Mongolian gerbils (Meriones unguiculatus). Ischemic brain injury was induced by bilateral common carotid artery occlusion for 7 min. IP protocol comprised 3 cycles of 15 s of reperfusion/15 s of ischemia. After reperfusion for 48 h, the morphometric analysis was conducted, and SDH cytoplasmic activity was assessed using quantitative histochemistry in the pyramidal neurons of the hippocampal areas CA1, CA2, CA3, CA4. The experiment has demonstrated that 7-minute-long ischemia resulted in a significant decrease in the number of viable neurons in CA1 area (up to 24%) and in the CA3 (to 56%) of the hippocampus; besides, it lead to the elevation of SDH activity in the cytoplasm of the neurons in all the hippocampal areas as compared to that in sham-operated animals. The application of IP significantly increased the number of viable neurons in CA1 (up to 52.9%, P<0,01) and in CA3 areas of the hippocampus(up to 88%, P<0,05), and it was accompanied by reduction of SDH activity in surviving neurons in all the hippocampal areas.

Lox-dependent gene expression in transgenic plants obtained via Agrobacterium-mediated transformation.

Lox sites of the Cre/lox recombination system from bacteriophage P1 were analyzed for their ability to affect on transgene expression when inserted upstream from a gene coding sequence adjacent to the right border (RB) of T-DNA. Wild and mutated types of lox sites were tested for their effect upon bar gene expression in plants obtained via Agrobacterium-mediated and biolistic transformation methods. Lox-mediated expression of bar gene, recognized by resistance of transgenic plants to PPT, occurred only in plants obtained via Agrobacterium-mediated transformation. RT-PCR analysis confirms that PPT-resistant phenotype of transgenic plants obtained via Agrobacterium-mediated transformation was caused by activation of bar gene. The plasmid with promoterless gus gene together with the lox site adjacent to the RB was constructed and transferred to Nicotiana tabacum as well. Transgenic plants exhibited GUS activity and expression of gus gene was detected in plant leaves. Expression of bar gene from the vectors containing lox site near RB allowed recovery of numerous PPT-resistant transformants of such important crops as Beta vulgaris, Brassica napus, Lactuca sativa and Solanum tuberosum. Our results demonstrate that the lox site sequence adjacent to the RB can be used to control bar gene expression in transgenic plants.

Activity of succinate dehydrogenase in the neocortex and hippocampus of Mongolian gerbils with ischemic and reperfusion brain injury.

We analyzed changes in activity of SDH, one of the most important enzymes of the Krebs cycle, in the cytoplasm of hippocampal and cortical neurons of Mongolian gerbils (Meriones unguiculatus) at the early and delayed reperfusion period after global brain ischemia. The data indicate that SDH activity in pyramidal neurons of various hippocampal areas and in neurons of II, III and V layers of cerebral cortex after 7-min forebrain ischemia depends on both the localization of these neurons and duration of the postischemic reperfusion. SDH activity in neurons significantly increased on days 2 and 7 after reperfusion.

[The impact of early and late ischemic preconditioning on brain damage and degree of neurological deficiency in rats].

The present study was aimed to investigate neuroprotective effects of early and late ischemic preconditioning in the acute phase of ischemic brain damage in rats. It was found that a single five-minute ischemic episode of early ischemic preconditioning did not lead to significant neuroprotective effects in comparison with control group, while three five-minute ischemic episode early ischemic preconditioning accompanied by a significant increase in neurological deficit and growing damage rate in CA1 hippocampus neurons. In contrast, later ischemic preconditioning in form of single five-minute episode 24 hours before ischemia modeling, provided a significant neuroprotective effect, manifesting reduced neurological deficit and maintaining viability of CA1 hippocampus neurons.

[Molecular mechanisms of development of cerebral tolerance to ischemia (Review. Part 2)].

In the 2nd part the authors describe in details the main aspects of protective effect of preconditioning of the brain: inhibition of programmed cell death, weakening of phenomenon of excitotoxicity, activation of endogenous antioxidant systems, anti-inflammatory effects, modulation of glial cell function, changes in regional blood flow and vascular reactivity. In addition, data analysis on the impact of preconditioning on brain neurogenesis, the state of the blood-brain barrier, ion homeostasis and metabolism of neurons is presented. Review emphasizes the role of microRNAs in mechanisms of ischemic tolerance of brain. Profound understanding of molecular mechanisms of increased tolerance of brain to ischemic and reperfusion injury requires the implementation of this phenomenon in clinical practice.

[Molecular mechanisms of development of cerebral tolerance to ischemia. Part 1].

In the first part of this review molecular mechanisms of ischemic tolerance emerging as a result ofpreconditioning of the brain are discussed. Data on inductors, sensors, transducers and effectors of early and delayed ischemic tolerance are presented.

A new rat model of reversible global cerebral ischemia.

A new rat model of global cerebral ischemia-reperfusion was proposed via reversible occlusion of the major vessels originating from the aortic arch and supplying the brain. This technique can be used for the search and study of exogenous (pharmacological) and endogenous methods of brain protection from ischemia-reperfusion injury.

[Activity of lactate dehydrogenase in the brain cortex and hippocampus of Mongolian gerbils after global ischemia and reperfusion injuries].

Cerebral ischemia results in severe derangements of energy metabolism in the nervous tissue including activation of glycolytic pathway. Activity of cytosolic lactate dehydrogenase (LDH) in the specific brain structures remains unclear. The recent study was aimed at investigation into the LDH activity in the cytoplasm of both hippocampal and cortical neurons in Mongolian gerbils (Meriones unguiculatus) at different durations of reperfusion after global ischemia. Analysis showed that the activity of LDH in pyramidal neurons of various hippocampal areas and neurons of II, III and V cortical layers after 7-minute forebrain ischemia depended on both localization of the neurons and duration ofreperfusion. In general, the changes in postischemic cytosolic LDH activity include significant decrease in the LDH activity 2 days after reperfusion with varying degree of recovery on day 7 of reperfusion.

[Morpho-functional changes of hippocampal CA1 area in Mongolian gerbils after ischemic postconditioning].

The aim of this study was to determine the effect of ischemic postconditioning on hippocampal CA1 neuronal survival and cytoplasmic activity of lactate dehydrogenase (LDH) in the gerbil model of cerebral ischemia-reperfusion injury. Ischemia was induced by bilateral common carotid artery occlusion (for 7 min) in male Mongolian gerbils (Meriones unguiculatus). Ischemic postconditioning protocol comprised 3 cycles of 15 s reperfusion/15 s ischemia. After 48 h of reperfusion, CA1 neuronal death was detected by Nissl staining and the cytoplasmic LDH was demonstrated histochemically in CA1 area of the hippocampus with a quantitative cytophotometric assessment of the enzyme activity. The results have shown that 7 min ischemia resulted in a significant decrease in the number of viable neurons (up to 24%) in the CA1 area of hippocampus; in addition, it reduced the activity of LDH in these neurons (from 0.260 +/- 0.009 to 0.190 +/- 0.006 relative units). The application of ischemic postconditioning significantly increased the number of viable neurons (up to 52.9%, P < 0.01) in the CA1 area of hippocampus, and it was accompanied by an increase in the activity of LDH (0.240 +/- 0.008 relative units, P < 0.001).

Expression of genes for thioredoxin 1 and thioredoxin 2 in multidrug resistance ovarian carcinoma cells SKVLB.

The expression of genes for thioredoxin isoforms Trx1 and Trx2 was studied in sensitive SKOV-3 and resistant SKVLB human ovarian carcinoma cells. The development of doxorubicin resistance was accompanied by a significant increase in the expression of TRX1 gene and less pronounced increase in TRX2 gene expression.

Expression of genes for redox-dependent glutathione S-transferase isoforms GSTP1-1 and GSTA4-4 in tumor cell during the development doxorubicin resistance.

Expression of genes for redox-dependent glutathione S-transferase isoforms GSTP1-1 and GSTA4-4 in tumor cells K562, MCF-7, and SKOV-3 was studied during the development of resistance to doxorubicin. It was found that the development of resistance was accompanied by predominant increase in the expression of hGSTP1 gene in MCF-7 cells, and hGSTA4 gene in resistant K562/DOX and SKVLB cells.

[A comparative study of induction regulation in cytochromes family 1 P450 in cell cultures at different stages of tumor transformation].

Lipophilic xenobiotics, including some carcinogenic agents and cytostatics, are metabolized by cytochrome P450 isoforms (CYP). In tumours expression of CYP genes and their inducibility are lower than in a homologous normal tissue. This phenomenon determines the known higher cytostatic stability of tumour cells. To clarify, at which particular stage of tumour transformation the level of family 1 CYP may change, we compared mRNA expression of CYP1A1, CYP1B1 and also of proteins regulated CYP expression: Ah receptor, ARNT and AHRR. For this aim we studied embryonic and fibroblast-like cells, in addition to cells of the same types but immortalized by the Rausher virus, or spontaneously after crisis. Besides, we investigated transformed clones obtained by means of benzo/a/pyrene action on Rausher virus-immortalized cells. Constitutive expression of genes studied in all cell cultures was shown. Benzo/a/anthracene induction increases the mRNA expression of all inducible genes (CYP1A1, CYP1B1, AHRR) in the original embryonic cells, in Rausher virus-immortalized cells, and in transformed clone K2. In both spontaneously immortalized cells and transformed clone K1 only CYP1B1 was induced. In transformed clone K8 no inducible gene was induced. In summary, we have shown that: (1) the ability of immortalized cells to CYP induction is determined not only by their capacity for a non-limited persistence, but also by the nature of immortalization; (2) despite their common genesis, the transformed clones differ in their ability to induce CYP. In addition to Ah receptor and ARNT, some other, yet unknown factors may also take part in CYP induction.

[Changes in the free-radical state and level of free iron during the formation of drug resistance in tumor cells].

The development of resistance of K562 human erythroleukemia cells to doxorubicin, a widely used antitumor antibiotic with the prooxidant action, leads to changes in the free-radical state of cells. It has been found that the formation of superoxide anion in resistant cells decreases. The introduction of doxorubicin to the culture medium induced a considerably lesser increase in the formation of O2*- in resistant cells compared to sensitive cells. At the same time, a strong decrease in the ESR signal of semiquinone type with a g-factor of 2.006 was observed in a culture of resistant cells grown in the absence of doxorubicin as compared with sensitive cells grown under similar conditions. At the same time, a decrease in the level of paramagnetic nitrosyl complexes of nonheme iron in resistant cells was recorded, indicating a decrease in the content of free nonheme iron as a result of the formation of drug resistance. In addition, a decrease in the level of mRNA of the transferrin receptor in resistant cells was found by the RT-PCR. These data indicate the development of a coodinated redox-dependent adaptive response, which makes itself evident as a suppression of free radical processes during the formation of resistance of K562 cells to doxorubicin.

[Effect of lox-sites of the Cre lox recombination system on promoterless bar gene expression in transgenic plants].

We demonstrate that localization of lox site between the right border of T-DNA and promoterless bar gene (RB-lox-bar-) led to its highly efficient expression in transgenic plants of Nicotiana tabacum and N. africana. Plasmid vectors used in gene integration experiments contained neomycin phosphotransferase II (npt II) gene under nos promoter as well. Transgenic plants were selected according to their capacity to grow on the medium with kanamycin and then they were tested on the selective medium containing phosphinothricin. 80% of transgenic plants expressed bar gene at the level similar to that in plants transformed with the bar gene under widely used constitutive promoter. Transformation of plants with the plasmid vector containing only promoterless bar gene near T-DNA right border (RB-bar-) and with the vector containing lox site and promoterless bar gene in the middle of the construction (-lox-bar-) led to obtaining no more than 4.5% of transgenic plants resistant to phosphinothricin. PCR analyses confirmed both the absence of tandem repeats and of plasmid recombination resulting in transference of bar gene under promoter in plasmid vector. Nos-terminator situated between the lox site and the right border of T-DNA did not decrease bar gene expression.

[In vitro regeneration of Nicotiana africana plants from explants of different type and mesophyll protoplasts]. / Regeneratsiia in vitro rastenii Nicotiana africana iz éksplantov raznogo tipa i mezofil'nykh protoplastov.

Methods of in vitro cultivation of an African endemic species Nicotiana africana (Solanaceae) possessing some valuable traits have been elaborated. Influence of different concentrations of auxin (alpha-naphtaleneacetic acid) and cytokinins (6-benzylaminopurine, zeatin, thidiazuron) on morphogenesis and plant regeneration has been analysed using leaves and internodes as explants. The optimum method of regeneration of N. africana shoots in leaf explants is cultivation in the presence of 0.1 mg/l NAA and 1 mg/l BA; in internode explants--cultivation on the medium containing 0.5 mg/l NAA and 1 mg/l zeatin. The use of thidiazuron was the most effective at the concentration of 0.4 mg/l with subsequent transfer of explants to hormone-free medium. Method of isolation and culture of mesophyll protoplasts enabling to regenerate N. africana plants during 3-4 months has been proposed.

Cytochrome P-450 family 1 in rat embryo cell culture immortalized by Rausher leukemia virus.

We studied comparative expression and activity of cytochrome P450 family 1 (CYP1) isoforms in rat embryo cells, both primary and immortalized by Rausher leukemia virus (RLV). In RLV-infected embryonal cells compared with the initial ones the expression levels of CYP1A1 and 1B1 mRNAs and benzo[a]pyrene (BP) hydroxylase activity were higher, regardless of their treatment with the CYP1 inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin. The sensitivity to BP and 7,12-dimethylbenzo[a]anthracene was higher in the cells immortalized with RLV. The expression level of mRNAs of induction-mediating proteins aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator was the same in both cell cultures tested. Higher sensitivity of cells immortalized with RLV compared with the initial embryo cells to transforming effect of BP, which was described previously, is possibly associated with elevated expression of CYP1 isoforms.

[Genetic transformation of Nicotiana africana Merxm. with plasmids containing lox recombination]. / Geneticheskaia transformatsiia rastenii Nicotiana africana Merxm. plazmidami, soderzhashchimi saity recombinatsii lox.

An efficient genetic transformation method for african tobacco Nicotiana africana Merxm. has been established. African tobacco is a valuable source for cytoplasmic male sterility (CMS) and nuclear encoded resistance to potato virus Y (PVY). N. africana transgenic plants have been obtained using both Agrobacterium-mediated and direct transformation of leaf explants with gold particle bombardment using particle inflow gun. Plasmid vectors containing phosphinothricin resistance gene (bar gene) coding region without promoter and independent 35S promoter between lox sites (lox-bar-35S-lox) and nptII gene were used. Transgenic plants were selected according to growth capacity on the selective medium containing 50 mg/l kanamycin. PCR analyses of kanamycin-resistant plants confirmed the presence of nptII and bar genes in their genome. Agrobacterium-mediated transformation of root explants has proved to be the most efficient transformation method for N. africana.