Dependence of the content of individual molecules in agranucocytes of whole blood at coronary heart disease from the level of phosphorylation of protein kinase r 38 in terms of low intensity microwave radiation

Molecular indicators reflecting the states of stress-limiting systems of mononuclear leucocytes in the blood, as well as the effects of low-intensive microwave radiation in patients with coronary artery disease were studied. The work it was evaluated the content in mononuclear leucocytes whole blood of components PI3P/AKT/mTOR/p70S6K1 of signaling pathway, heat shock proteins (HSP27, HSP70, HSP90), the concentra-tion of antioxidants and peroxides depending on the level of phosphorylation of the terminal protein kinase MAPK/SAPK of signaling pathway – p38. The results of the study. It was revealed the dependence of a level of studied factors from the degree of phosphorylation p38 in patients with coronary heart disease. It was defined the 38 p sensitivity to the effects of low-intensity microwave radiation, it is manifested by increased level of phosphorylation in the irradiated cul-tures. This study revealed the sensitivity to low-intensity microwave irradiation of content in the mononuclear cells phosphorylated forms of the protein kinases AMPK, AKT1, p70S6K1, as well as the antioxidant status and protein of p53-dependent initial content in the cell phosphorylated form p38. It was shown a possibility of microwave radiation to reduce the content in the cells of the protein kinase and p70 ACT more pronounced at high levels of phosphorylation p38.

POLYMORPHISM OF GENES OF IMMUNOSUPRESSIVE CYTOKINE IL-10 AND TGF-β AT TUBERCULOSIS INFECTION

The aim of the work was the study of connection of allelic polymorphism of IL10 and TGFВgenes with changes in the basal and BCG-induced production of immunosuppressive cytokines IL-10 and TGF-β by mononuclear leukocytes in vitro in patients with the first diagnosed pulmonary tuberculosis (TB), depending on the clinical form of the disease. The evaluation of the cytokines production was conducted by measuring its concentration in culture supernatants by ELISA. The allele-specific amplification of specific stretches of the genome was used for the study of polymorphic genes of cytokines. The DNA and supernatants of culture suspensions of blood mononuclear leucocytes in healthy volunteers and patients with TB were the material of the research. It was shown in the research conducted that the basal and BCG-induced over-production of IL-10 in vitro occurs in patients with TB, regardless of the genotype of the locus of C-592AIL10 gene. In addition, genotype AA of polymorphism of IL10gene in patients with infiltrative and disseminated TB is associated with the maximum production of IL-10 in vitroand genotype CC – with the minimum production of this cytokine in vitro. Analysis of the production of TGF-β in vitro in patients with TB showed its increase only in case of carriage of allele T (C-509T) of TGFB gene. In patients with disseminated TB and homosygotic genotype TT the increase in both basal and BCG-induced production of TGF-β was determined, and in patients with infiltrative TB – only after induction of cells by BCG-antigen.Thus, the over-production of cytokines with inhibiting activity in patients with TB is genetically determined and promotes the formation of suppressive mode of immune-regulation. The increase in the secretion of cytokines IL-10 and TGF-β in vitro in patients with TB are associated with carriage of allele A and genotype AA (C-592A) of IL10gene and allele T and genotype TT (C-509T) of TGFB gene.

Antidepressant-induced differential ubiquitination of β-arrestins 1 and 2 in mononuclear leucocytes of patients with depression

β-Arrestins 1 and 2, cytosolic proteins known to mediate receptor desensitization, endocytosis and G protein-independent signalling, are post-translationally modified by ubiquitination regulating their ability to serve as adaptors and scaffolds. β-Arrestins were suggested to play a role in the pathophysiology of depression and in antidepressant mechanism of action. To determine whether a depressive episode or antidepressant treatment induce significant selective differences in β-arrestin 1 and 2 levels or their ubiquitination patterns in leucocytes of patients with depression, 46 outpatients diagnosed with a depressive episode were examined before and after 4-wk antidepressant treatment compared with age- and gender-matched control subjects. β-Arrestin levels were measured by immunoblotting using anti-arrestin antibodies. Ubiquitination of β-arrestins was measured using anti-ubiquitin antibodies followed by an immunoprecipitation step and immunoblotting using anti-arrestin antibodies. Antidepressants induced selective alterations in leucocyte β-arrestin 1 and 2 levels and ubiquitination. The levels of β-arrestin 1 and 2 and their ubiquitinated forms in leucocytes of yet untreated patients with depression were significantly decreased in a symptom severity correlated manner compared to control subjects. Antidepressants normalized β-arrestin 1 and 2 levels and uncovered novel differences between the two isoforms: (a) while antidepressants normalized ubiquitination of β-arrestin 1, ubiquination of β-arrestin 2 was unaffected; (b) while under antidepressants ubiquitination extent of β-arrestin 1 positively correlated with its level, an inverse picture of negative correlation was found between ubiquitination extent of β-arrestin 2 and its level. We conclude that antidepressants may serve as a tool to detect functional differences between the two β-arrestin isoforms and that through these differential effects antidepressants can induce specific alterations in alternative cellular signalling.

The role of intracellular gaseous transmitters hydrogen sulfide and nitric oxide in apoptosis regulation of normal and cancer cells

Investigation of influence of gases nitric oxide and hydrogen sulfide on apoptotic cell death of Jurlat cells and mononuclear leucocytes of healthy donors was conducted. It was shown that 100 mmol sodium nitroprussidi increased the apoptosis of T lymphoblast leukemia cells after 15’ incubation. 10 and 100 mmol donor of hydrogen sulfide caused apoptotic death of Jurkat cells after 15’ incubation. 15’ exposure of nitric oxide and hydrogen sulfide donors did not lead to the changes of cell death of mononuclear leucocytes. Gaseous transmitters NO and H2S increased necrosis of Jurkat cells and mononuclear leucocytes after 24 h incubation with the appropriate gase’s donor.

Role of protein kinases JNK and p38 in regulation of mononuclear leucocytes apoptosis in oxidative stress

Programmed cell death of peripheral blood mononuclear leucocytes taken from healthy donors and cultivated with various concentration of Н2О2, selective inhibitors of JNK (SP600125), 38 (ML3403) and in case of pneumonia was investigated. Intensification of intracellular production of reactive oxy р МАРК - gen species was accompanied by the increase in number of apoptotic and TNFR1-presented cells and mononuclears with reduced value of mitochondrial transmembrane potential in a case of oxidative stress induction with 1 mM hydrogen peroxide and in blood taken from patients with pneumonia. Action of inhibitors SP600125 and ML3403 in vitro in oxidative stress conditions prevents the increase in number of annexin- positive mononuclear cells, that confirms the participation of JNK and 38 -kinases in mechanisms of oxidative stress-mediated apoptosis dysregulation.