The effect of different concentrations of the glucocorticoid (GC) methylprednisolone (MP) on CD4+CD95+HLA-DR+ T-cells and their ability to produce proinflammatory mediators in cultures of TCR-stimulated CD3+CD45RO+ T-lymphocytes in the in vitro system was investigated. T cells were obtained from healthy donors and patients with rheumatoid arthritis (RA).Under conditions of TCR-activation, MP increased the number of CD4+HLA-DR+CD95+ cells in CD3+CD45RO+ cultures obtained from RA patients and did not change their content in the control group. In general, MP decreased production of proinflammatory factors (IFN-, IL-2, IL-17, IL-21 and TNF-) by TCR-activated CD3+CD45RO+ cells from healthy donors and RA, consistent with the overall immunosuppressive mechanism of GC action. The correlation between CD4+CD45RO+HLA-DR+CD95+ T-cell contents and parameters reflecting production of proinflammatory mediators (IL-17, IL-21 and TNF-) in RA patients indicates maintenance of the pro-inflammatory potential of this T-cell population exposed to GC action. We suggest that relative resistance of CD4+CD45RO+CD95+HLA-DR+ T-cells of RA patients to the suppressor effect of GC leads to maintenance and even enhancement in the functional capacities of autoreactive cells in the pathogenesis of RA.

Hyperhomocysteinemia is a risk factor for many human diseases, including pulmonary pathologies. In this context much interest attracts secondary mitochondrial dysfunction, which is an important link in pathogenesis of diseases associated with hyperhomocysteinemia. The study was conducted using male Wistar rats. It was found that under conditions of severe hyperhomocysteinemia caused by administration of methionine, homocysteine was accumulated in lung mitochondria thus suggesting a direct toxic effect on these organelles. However, we have not observed any significant changes in the activity of mitochondrial enzymes involved in tissue respiration (succinate dehydrogenase) and oxidative phosphorylation (H+-ATPase) and of cytoplasmic lactate dehydrogenase. Also there was no accumulation of lactic acid in the cytoplasm. Animals with severe hyperhomocysteinemia had higher levels of lung mitochondrial protein carbonylation, decreased reserve-adaptive capacity, and increased superoxide dismutase activity. These results indicate that severe hyperhomocysteinemia causes development of oxidative stress in lung mitochondria, which is compensated by activation of antioxidant protection. These changes were accompanied by a decrease in the concentration of mitochondrial nitric oxide metabolites. Introduction to animals a nonselective NO-synthase inhibitor L-NAME caused similar enhancement of mitochondrial protein carbonylation. It demonstrates importance of reducing bioavailability of nitric oxide, which is an antioxidant in physiological concentrations, in the development of oxidative stress in lung mitochondria during hyperhomocysteinemia. Key words: hyperhomocysteinemia, nitric oxide, lung, oxidative stress, mitochondria.

Hepatitis C virus (HCV) induces the expression of the genes of proinflammatory cytokines, the excessive production of which may cause cell death, and contribute to development of liver fibrosis and hepatocarcinoma. The relationship between cytokine production and metabolic disorders in HCV-infected cells remains obscure. The levels of biogenic polyamines, spermine, spermidine, and their precursor putrescine, may be a potential regulator of these processes. The purpose of the present work was to study the effects of the compounds which modulate biogenic polyamines metabolism on cytokine production and HCV proteins expression. Human hepatocarcinoma Huh7.5 cells have been transfected with the plasmids that encode HCV proteins and further incubated with the following low-molecular compounds that affect different stages of polyamine metabolism: (1) difluoromethylornithine (DFMO), the inhibitor of ornithine decarboxylase, the enzyme that catalyzes the biosynthesis of polyamines; (2) N,N'-bis(2,3-butane dienyl)-1,4-diaminobutane (MDL72.527), the inhibitor of proteins involved in polyamine degradation; and (3) synthetic polyamine analog N^(I),N^(II)-diethylnorspermine (DENSpm), an inducer of polyamine degradation enzyme. The intracellular accumulation and secretion of cytokines (IL-6, IL-1β, TNF-α, and TGF-β) was assessed by immunocytochemistry and in the immunoenzyme assay, while the cytokine gene expression was studied using reverse transcription and PCR. The effects of the compounds under analysis on the expression of HCV proteins were analyzed using the indirect immunofluorescence with anti-HCV monoclonal antibodies. It has been demonstrated that, in cells transfected with HCV genes, DFMO reduces the production of three out of four tested cytokines, namely, TNF-α and TGF-β in cells that express HCV core, Е1Е2, NS3, NS5A, and NS5B proteins, and IL-1β in the cells that express HCV core, Е1Е2, and NS3 proteins. MDL72527 and DENSpm decreased cytokine production to a lesser extent. Incubation with DFMO led to a 28-32% decrease in the number of cells expressing NS5B or NS5A, both of which are key components of the HCV replication complex. The results obtained in the work indicate that a further detailed study of the antiviral activity of DFMO is required in order to assess its potential as an anti-hepatitis C therapeutic agent.

In murine bone-marrow stromal microenvironment cells and in human multipotent mesenchymal stromal cells (MMSCs), proinflammatory cytokine interleukin-1 beta (IL-1β) serves as a growth factor. In murine bone tissue, IL-1β expression increases in vivo after irradiation. Here, we have presented our evaluation of the effects of exogenous IL-1β on the expression of NF-kB transcription factors in human MMSCs and stromal layer cells of murine long-term bone marrow cultures (LTBMCs). The cytokine signaling pathway was also activated in murine LTBMC by braking electron radiation in doses of 3-12 Gy. The level of expression of genes that code for IL-1β, IL-1β type-I receptor and NF-kB and IKK protein families have been studied at different time points post exposure. In both human and murine stromal cells, exogenous IL-1β led to an increase in the level of expression of its own gene, while levels of expression of NF-kB and IKK gene families were not substantially changed. Nevertheless, in human cells, a significant correlation between levels of expression of IL-1β and all NF-kB family genes was detected. It points to a similarity in IL-1β signal pathways in mesenchymal and hematopoietic cells, where the posttranslational modifications of NF-kB transcription factors play a major role. The irradiation of murine LTBMC resulted in a transient increase in the expression of genes that code NF-kB transcription factors and IL-1β. These results indicate an important role of Rel, Rela, Relb, and Nfkb2 genes in the induction of IL-1β signal pathway in murine stromal cells. An increase in IL-1β expression after the irradiation of stromal cells may be related to both the induction of inflammation due to massive cell death and to a profound stimulation of the expression of this proinflammatory cytokine expression.

to study changes in the expression of angio- and vasculogenesis markers in colorectal adenocarcinoma metastases to the liver during combined cytotoxic and targeted anti-VEGF therapy versus cytotoxic monotherapy.

Alternative splicing of telomerase catalytic subunit hTERT pre-mRNA (human Telomerase Reverse Transcriptase) regulates telomerase activity. Increased expression of non-active splice variant hTERT results in inhibition of telomerase. Apoptotic endonuclease EndoG is known to participate in hTERT alternative splicing. Expression of EndoG can be induced in response to DNA damages. The aim of this study was to determine the ability of a DNA-damaging compound, cisplatin, to induce EndoG and its influence on alternative splicing of hTERT and telomerase activity in human CD4+ Т lymphocytes. Overexpression of EndoG in CD4+ T cells downregulated the expression of active full-length hTERT variant and upregulated its non-active spliced variant. Reduction of full-length hTERT caused downregulation of telomerase activity, shortening of telomeres length during cell divisions, converting cells to the replicative senescence state, activation of apoptosis and finally cell death. Few cells survived and underwent malignant transformation. Transformed cells have increased telomerase activity and proliferative potential compare to initial CD4+ T cells. These cells have phenotype of T lymphoblastic leukemic cells and are able to form tumors and cause death in experimental mice.

The effects of the Nothrombel on the formation of platelet-leukocyte complexes (PLCs) induced by thrombin was studied. It was shown, that Nothrombel dose-dependently inhibited the formation of PLCs. Its activity is higher than the activity of the comparison compounds Aspirin. The half maximal effective concentration (EC50) for Nothrombel is 1.75 mMol/mL, for Aspirin is much more than 2.5 mMol/mL. The inhibition mechanism of the PLCs formation by Nothrombel caused by the ability of this drug to inhibit the P-selectin translocation on the platelet membrane, the expression of membrane complex GPIb-IX-V, the mobilization of cytoplasmic calcium in platelets, as well as, apparently, its inhibitory effect on platelet P2Y12 purine receptors.

The effect of melatonin on the correlation between the intensity of the accumulation of the oxidative-modified protein content, activity of the antioxidant enzymes and the state of proteoly-sis in the basal nuclei (the nucleus caudatus, globus pallidus, nucleus accumbens, amigdaloid complex) of the brain under the conditions of acute hypoxia has been studied. Under the conditions of acute hypoxia in the basal nuclei an intensification of the protein peroxidation processes is observed, the activity of the antioxidant enzymes decreases, the intensity of the proteolysis increases. The injection of melatonin in a dose of 1 mg per kg of the body mass before the modeling of acute hypoxia results in the decreasing of protein peroxidation, increasing of the antioxidant enzyme activity and normalization of the parameters of proteolysis.

In order to explore the importance of the transformer (tra) gene in reproductive mode switching in Daphnia pulex, we studied the effect of silencing of this gene using RNA interference (RNAi). We obtained Dptra dsRNA by constructing and using a dsRNA expression vector and transcription method in vitro. D. pulex individuals in different reproductive modes were treated by soaking in a solution of Dptra dsRNA. We then assayed the expression of the endogenous Dptra mRNA after RNAi treatment using RT-PCR and obtained the suppression ratio. Expression of the tra gene in the RNAi groups was down-regulated compared with the controls after 16 h (p < 0.05). We also analyzed the effect of RNAi on the expression of the TRA protein using Western blot, which showed that the expression level of the TRA protein was reduced after RNAi treatment. Our experimental results showed that soaking of D. pulex adults in tra-specific dsRNA transcribed in vitro can specifically reduce the level of tra mRNA and also reduce the expression of the TRA protein, demonstrating effective in vivo silencing of the tra gene.

Breast cancer is one of the most common cancers in women worldwide. Tamoxifen (TAM) provided a successful treatment for ER-positive (ER+) breast cancer for many years. However, ER+ patients with metastatic diseases respond poorly to TAM therapy and many initial responders eventually relapse. Emerging evidence indicates that long non-coding RNAs (lncRNAs) may have a critical role in the regulation of cellular processes such as cancer progression and metastasis, though the function of lncRNAs in TAM-resistance is still unclear. To investigate the role of CCAT2 in the development of resistance to TAM treatment of breast cancer, we established TAM-resistant models in MCF-7 and T47D cells. The present study demonstrates that TAM-resistant cells show a higher level of CCAT2 expression compared with TAM-sensitive cells. Biologically, CCAT2 knockdown could inhibit proliferation and induce apoptosis in TAM-resistant cells exposed to TAM. Furthermore, knockdown of CCAT2 improves the sensitivity to TAM in TAM-resistant cells. Overall, the study results provide a novel therapeutic approach for TAM-resistant patients through depleting CCAT2 expression.

We investigated the role of genes of hevein-like antimicrobial peptides of the WAMP family in the protection of wheat plants against biotic and abiotic stress. The semiquantitative RT-PCR method was used to examine the expression of wamp genes in wheat seedlings in response to infection by pathogens and exposure to phytohormones and ions of a heavy metal ion—cadmium. We discovered that wheat germ contamination by harmful fungi significantly increases expression of genes of the wamp family, and the primary transcript is wamp-2. We determined that salicylic acid, rather than methyl jasmonate, induces expression of genes of the wamp family. We showed that abiotic stress induced by cadmium ions inhibits expression of wamp genes in the roots with no effect on their expression in shoots. The results support the protective role of wamp genes in the response of wheat plants to infections by pathogens. In turn, the resistance to abiotic stress induced by cadmium ions does not appear to be associated with expression of genes of the wamp family.

The dependence of expression of miRNAs and their precursors (pre-miRNAs) on the DNA methylation level in HeLa cells 8 days after mitomycin C treatment was studied. A massive parallel DNA sequencing method was applied to analyze miRNA expression. 5-Azacytidine (DNA methylation inhibitor) was added to the medium 6 days after mutagenic agent exposure. The results indicated that the change in expression for some mature miRNAs (39 of 61) was accompanied by the change in the expression of their pre-miRNAs, while there were no significant changes in the expression of pre-miRNA for other mature miRNAs (22 of 61). The aberrant expression was maintained by 8 of 61 mature miRNAs and 6 of 55 pre-miRNAs in the induced HeLa cells after 5-azacytidine treatment. In addition, the expression of more than 90% of miRNAs, which indicated a significant change in expression after mitomycin C treatment, does not depend or depends slightly on the DNA methylation level in HeLa cells without mitomycin C treatment. The results suggest that mitomycin C induces aberrant DNA methylation which affects maintenance of changes in the miRNA expression in cell generations after mutagen treatment.

The response of Triticum aestivum L. to infection by Septoria nodorum Berk, a pathogen causing speckled leaf blotch, was studied. The effect of salicylic acid (SA) and jasmonic acid (JA) signal molecules, as well as chitooligosaccharides (COSs) with different acetylation degrees (ADs), on the accumulation of hydrogen peroxide (Н2О2) in wheat leaves and the pathogenesis-related (PR) proteins of oxalate oxidase (AJ556991.1), peroxidase (TC 151917), and proteinase inhibitor (EU293132.1) was investigated. Treatment with the signal molecules inhibited S. nodorum growth and stimulated Н2О2 accumulation, as well as PR gene expression. SA and COS with 65% AD are found to be more efficient in Н2О2 induction and elevation of the transcriptional level of the oxalate oxidase and peroxidase genes. At the same time, JA and COS with 30% AD stimulated transcription of the proteinase inhibitor gene. The results suggest the existence of differential means of defense response induction by signal molecules with more prospects for the regulation of plant immunity.

Staphylococcus aureus and Pseudomonas aeruginosa are foregroundpathogens of bacteriemia and sepsis. They produce large spectrum of such factors of pathogenicity permitting them to proliferate and survive in bloodstream as hydrolytic enzymes, adenosine diphosphate-ribosylarginine toxins, plasmocoagulase, etc. The occurrence of alteration of growth and expression of virulence factors of S. aureus and P. aeruginosa at fermentation in LB-broth depending on iron concentration was demonstrated previously. The conditions in vivo significantly differ the laboratory conditions. The activity of growth and expression of pathogenicity factors of S. aureus and P. aeruginosa at fermentation in blood serum of donors with different alternative of iron homeostasis was analyzed. The study established expression of genes of hemolytic phospholipase C (plcH), alginate (algD), exotoxin A (exoA) for P. aeruginosa and genes of protein A (spA), virulence global regulator (RNAIII) for S. aureus. The iron-deficient serum and serum with normal iron homeostasis decreased activity of growth of S. aureus and P. aeruginosa. The growth rate and expression level of all analyzed genes turned out higher at fermentation of S. aureus and P. aeruginosa in serums containing excess of iron (more than 30 mkM). The fermentation of P. aeruginosa in iron-deficient serum resulted in decreasing of expression level of genes plcH, algD, exoA. The expression of RNA III and spaA S.aureus in iron-deficient serum increased towards normal content of iron in blood. The example of S. aureus and P. aeruginosa demonstrated that expression of many virulence factors of opportunistic microorganisms depends on iron homeostasis of host organism. The survival and proliferation of microorganisms in blood depend on both immune status of host organism and biological characteristics of pathogen.

The RARa is a transcription factor playing important role in such processes as proliferation, differentiation and apoptosis of cells in norm and in tumor. At the same time, it is little known about significance of expression of two major products of transcription of gene RARa - isoforms RARa and RARa - in pathogenesis of solid and non-solid tumors, including multiple myeloma. The actual data testify ambiguity of input made by isoforms RARa and RARa into processes of tumor development and progression of malignant tumors.

Effect of mast cell degranulation blockade on the inflammatory response and character of the lung tissue structure-functional changes were evaluated in the chronic obstructive pulmonary disease model produced in rats by 60-day intermittent exposure to nitrogen dioxide. The membrane stabilizer sodium cromoglicate was used to blockade of mast cell degranulation. Lung tissue sections were stained with toluidine blue to identify mast cells. Bronchoalveolar lavage fluid (BALF) cytogram was determined. The levels of mast cell tryptase and chymase, proinflammatory cytokine TNF-α, surfactant protein B were measured in BALF. Suppression of mast cell degranulation prevented the release of proteases in the bronchoalveolar space and reduced activity of the inflammatory process. The influx of inflammatory cells and TNF-α concentration decreased. There was no interstitial inflammatory infiltration. Bronchoalveolar epithelium structure was recovered that is the basis of its functional usefulness. The results confirm the active involvement of mast cells in the development of the inflammatory process in obstructive pulmonary diseases and allow us to consider them as a possible therapeutic target.

The effects of the salt stress (200 mM NaCl) and exogenous jasmonic acid (JA) on levels of osmolytes and flavonoids in leaves of four-week-old Arabidopsis thaliana L. plants of the wild-type (WT) Columbia-0 (Col-0) and the mutant jin1 (jasmonate insensitive 1) with impaired jasmonate signaling were studied. The increase in proline content caused by the salt stress was higher in the Col-0 plants than in the mutant jin1. This difference was especially marked if the plants had been pretreated with exogenous 0.1 µM JA. The sugar content increased in response to the salt stress in the JA-treated WT plants but decreased in the jin1 mutant. Leaf treatment with JA of the WT plants but not mutant defective in jasmonate signaling also enhanced the levels of anthocyanins and flavonoids absorbed in UV-B range. The presence of JA increased salinity resistance of the Col-0 plants, since the accumulation of lipid peroxidation products and growth inhibition caused by NaCl were less pronounced. Under salt stress, JA almost did not render a positive effect on the jin1 plants. It is concluded that the protein JIN1/MYC2 is involved in control of protective systems under salt stress.

In order to investigate the mechanism of apoptosis in rat intestinal epithelial cells (IEC-6) induced by hydrogen peroxide (H(2)O(2)), IEC-6 cells were subjected to 20 μmol/L H(2)O(2) and cell proliferation activity was determined using 3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide. Cell morphology was observed by microscopy and cell apoptosis was detected by acridine orange and ethidium bromide staining and the portion of apoptotic cells was measured by flow cytometry. Genes and proteins related to cell apoptosis were detected by RT-PCR and Western blotting, and the mitochondrial membrane potential was evaluated by fluorescence probes.

Tryptophan hydroxylase 2 (Tph-2) is the key enzyme in serotonin biosynthesis. Serotonin is one of the main neurotransmitters involved in the regulation of various physiological functions and behavior patterns. The influence of chronic ethanol consumption on the expression of the Bdnf, Bax, Bcl-xL, and CASP3 genes was studied in the brain structures of B6-1473C (C/C) and B6-1473G (G/G) mice that had been obtained on the base of the C57BL/6 strain. The strains differed in the genotype for the C1473G single nucleotide polymorphism in the Tph-2 gene and in Tph-2 enzyme activity. It was found that chronic alcohol treatment led to a significant increase in the expression of the Bdnf gene in the midbrain of B6-1473G mice, but not in B6-1473С. Chronic alcohol treatment considerably decreased the expression of the ultimate brain apoptosis effector, caspase 3, in the frontal cortex, but increased it in the hippocampus of B6-1473G mice. At the same time, chronic ethanol administration reduced the level of the antiapoptotic Bcl-xL mRNA in the midbrain of B6-1473C mice. Thus, the C1473G polymorphism in the Tph-2 gene considerably influenced the changes in the expression patterns of genes involved in the regulation of neurogenesis and neural apoptosis induced by chronic ethanol treatment.

The ratio of the expression levels of the immediate early genes c-jun and c-fos that encode components of the AP-1 transcription complex determines the direction of changes in the expression of genes controlled by the complex, including changes induced by glucocorticoids. The aim of the present work was to assess the levels of mRNA encoded by genes c-jun and c-fos and the ratio of expression levels of these genes in various regions of the neonatal rat brain after the administration of dexamethasone, a selective ligand of the glucocorticoid receptor. The level of mRNA encoded by the immediate early gene c-fos in the hippocampus and prefrontal cortex of 3-day-old rat pups was elevated at 30, 60, and 120 min after dexamethasone administration. The basal level of c-fos gene expression in the brainstem was higher than in the cortex and hippocampus, and administration of the hormone was followed by a reduction in the amount of transcript detectable in the brainstem after 2 h. As a result, the ratio of c-jun to c-fos transcript levels in the brainstem of neonatal rats was doubled after dexamethasone administration. The dexamethasone-induced shift of the ratio of c-jun to c-fos transcript levels in the brainstem of neonatal rats towards a predominance of c-jun reported for the first time in the present work may induce the expression of genes that contain AP-1 response elements in the promoters, since the glucocorticoid receptor can be involved in protein-protein interactions with the Jun/Jun homodimer of the AP-1 complex.