ASSOSIATION ANALYSYS OF 11 POLYMORPHISMS OF SNPS WITH ENDOTHELIUM DEPENDENT VASODILATATION IN CHILDREN WITH DIABETES MELLITUS TYPE 1.

We have studied the association with the level of the endothelium dependent vasodilatation (EDVD) among 11 single nucleotide polymorphisms (SNPs) of 10 genes in 45 children suffering from diabetes mellitus type 1. Following polymorphisms have been studied: G894→T of the eNOS exon 7 and Т-786→С of the eNOS promotor, А1266→G of the Eln exon 16, Т-381→C of the NPPB promotor, І\D of the ACE, Arg60→His of the LMP2, Met235→Thr of the AGT, A1166→C of the ATR1, C-1562→T of the MMP9, C-1306→T of the MMP2, and С-8→G of the PSMA6. It was shown that children with genotypes G/T by eNOS (G894→T), G/G by Eln (А1266→G), C/C by NPPB (Т-381→C) and І/D by ACE genes have lower EDVD (Р<0,05) than patients with others allelic variants of these genes, and this does not depend on duration of the disease, level of glicated hemoglobin and initial diameter of a humeral (brachial) artery. The combination of the above-stated genotypes influences most significantly on EDVD decrease (r=0,61; Р<0,01), comparing to each genotype separately.

Silencing of TERT decreases levels of miR-1, miR-21, miR-29a and miR-208a in cardiomyocytes.

To test the hypothesis that telomerase reverse transcriptase (TERT) as an RNA-dependent RNA polymerase could be involved in the amplification of microRNA (miRNA), we have determined the levels of immature and mature miRNA in cultured neonatal rat cardiomyocytes, during the silencing of TERT by siRNA. The silencing of the TERT gene led to the reduction of both telomerase activity and the TERT mRNA expression when compared with scrambled RNA. TERT gene silencing resulted in the decrement of three studied mature miRNAs levels: miRNA-21, miRNA-29a and miRNA-208a when compared with scrambled RNA; but miRNA-1, it was not changed significantly. At the same time, levels of immature miRNA-1 and miRNA-208a were not changed, although the levels of immature miRNA-29a and pri-miRNA-1 were decreased. The data obtained allow us to permit that TERT is a genome-independent source of mature miRNA, and the changes in telomerase activity can significantly influence the level of miRNA in cardiomyocytes.

[The expression of Akt kinase in the heart ventricles under hypoxic preconditioning and myocardial remodeling].

Activation of Akt-dependent mechanisms may play a significant role in the cellular response under hypoxic preconditioning and myocardial remodeling. The impact of hypoxic preconditioning, and remodeling on the expression of Akt kinase in the heart ventricles was investigated. Wistar male rats, the residents of plains or middle altitude (2100 m above sea level), were exposed to hypoxic preconditioning by "lifting" in the barochamber at the "height" of 5,600 m in 3 h. In the right and left ventricles of the heart, Akt protein expression was determined by Western blotting. It was shown, that hypoxic preconditioning causes the induction of Akt kinase in the ventricles during the period of delayed cardioprotection (1-3 days after preconditioning). Myocardial remodeling induced by chronic hypoxia in middle altitude was associated with elevated Akt expression in the myocardium, more pronounced in the left ventricle. Progression of hypoxic myocardial remodeling found in part of the animals was accompanied by a reduction of the cell hypoxic reactivity, including Akt induction in response to preconditioning. Thus, Akt kinase is involved in the mechanisms of hypoxia induced late preconditioning and myocardial remodeling in chronic hypoxia. Inhibitory regulatory mechanism was found to limit the induction of Akt in myocardium after remodeling.

[Hypoxic preconditioning prevents the induction and activation of 5-lipoxygenase during ischemia and reperfusion of rat heart].

Male Wistar rats were subjected to hypoxic preconditioning (10% O2 in nitrogen for 3 h). In 24 h heart were isolated and subjected to 30 min ischemia and 40 min reperfusion. Changes in expression of 5-lipoxygenase (5-LO) protein in rat heart ventricles, and in myocardial subcellular fractions were evaluated by Western blotting. It was found that hypoxic preconditioning attenuated reperfusion damage of cardiomyocytes with reducing the release of LDH by 27.6%. After ischemia and reperfusion, expression of 5-LO was 10.5-fold elevated in the left ventricle and 14.3-fold - in the right one. During ischemia and reperfusion occurred gradual translocation of 5-LO protein in nuclear subcellular compartment, more expressive in the left ventricle. Hypoxic preconditioning did not significant increase in 5-LO expression, but fully prevented its growth in the following ischemia-reperfusion, and partly reduced protein translocation at reperfusion in the left ventricle. Thus, hypoxic preconditioning limits proinflammatory effects ofischemia and reperfusion in myocardium, preventing the increase in expression of 5-LO, and reducing the alteration of cardiomyocytes.

[The frequency of polymorphism in promoter gene coding epoxygenase 2J2 in patents with acute coronary syndrome].

G(-50) --> T promoter polymorphism of gene encoding human epoxygenase 2J2 in 107 patients with acute coronary syndrome and in 104 practically healthy people was determined. It was shown that interrelation of genotypes G/G, G/T and T/T was 91%, 9% Ta 0% correspondingly (in control--92%, 7%, 1%; P > 0.05 by chi2-test). The data indicate that epoxygenase 2J2 gene polymorphism is not a risk factor of acute coronary syndrome in Ukrainian population.

[Allele polymorphism of genes coding proteasome subunits is associated with an enhanced risk for arterial hypertension in adolescents].

Large multifunctional protease LMP2 (Arg60-->His), LMP7 (Lys145-->Gln) and PSMA6 (C(-8)-->G) gene allelic polymorphisms in 147 young patients with essential hypertension and in 208 practically healthy people were determinated. It was shown that interrelation of genotypes Arg/Arg, Arg/His and His/His in LMP2 gene polymorphisms account 42.5 %, 46.4% and 11.1% correspondingly (in control--63.9%, 28.6%, 7.5%; P = 0.001 by c2-test). Allelic variants of PSMA6 dispense the next manner: C/C--76.2%, C/G--21.1%, G/G--2.7% in adolescents with EH (in control--69.8%, 29.7% and 0.5% correspondingly, P = 0,047). Analysis of LMP7 gene polymorphism showed identical frequency of different genotypes in patients (Lys/Lys--92.4%, Lys/Gln--7.6%, Gln/Gln--0%) and practically healthy people (97.3%, 2.7%, 0% correspondingly; P = 0.16). Obtained data suggest the LMP2 and PSMA6 gene polymorphisms significance as the risk factors of essential hypertension in adolescents.

[The role of endogenous bioregulators in formation of cardiogenic reflex effects on circulation].

The possible role of endogenous endothelium-derived bioactive substances in organization of cardiogenic depressor reflexes under cardiac receptor stimulation (by veratrine and bradykinin) was investigated in acute experiments on anesthetized rats. The results have shown that endothelium-derived bioactive substances take part in forming of the cardiogenic depressor reflex humoral components of nervous response or nervous modulators. These data contribute to understanding of the role of endogenous endothelium-derived bioactive substances (prostacyclin) and different NOS isoforms in mechanisms of depressor reflex development and species differences in their involvement in reflex vasomotor reactions.

Proteasome inhibitors eliminate protective effect of postconditioning in cultured neonatal cardiomyocytes.

A role of proteasomal proteolysis in the pathogenesis of ischemia-reperfusion is being actively studied. To evaluate the participation of the proteasome in postconditioning phenomenon, we used primary culture of neonatal cardiomyocytes. 30 minutes of anoxia followed by 60 minutes of reoxygenation was undergone. Postconditioning was modeled by 3 cycles of 1-minute reoxygenation followed by 1-minute anoxia, respectively. Clasto-lactacystin b-lactone, a specific proteasome inhibitor, in the dose that does not cause cell death (2.5 mM) was added to the culture medium just before the cycles of postconditioning. Percentages of living, necrotic, and apoptotic cells were determined by staining with bisBenzimide and propidium iodide. Autophagy was demonstrated by staining vacuolar structures with monodansyl cadaverine. Proteasomal activity was determined by cleavage intensity of specific fluorogenic substrates. Trypsin-like, chymotrypsin-like and peptidyl-glutamyl peptide-hydrolyzing (PGPH) activities were decreased after anoxia. Reoxygenation led to an increase in trypsin-like and chymotrypsin-like activities comparing to anoxia, but these parameters never reached the control levels. PGPH activity was restored up to the initial level. Postconditioning increased numbers of living cells and decreased that of necrotic, apoptotic and autophagic cells. Paradoxically, it was established, that proteasome inhibitors prevented the necrotic and apoptotic cell death of cardiomyocytes in anoxia-reoxygenation, but in the same concentration abolished the effects of postconditioning. The data obtained permit to suppose that proteasome inhibitors can be used for pharmacological postconditioning.

[The influence of quercetin on the activity of purified 20S, 26S proteasome and proteasomal activity in isolated cardiomyocytes].

For the clarification of the effect of quercetin on the proteasome experiments were performed using purified 20S proteasome, 26S proteasome from the proteasomal fraction II (PF II), as well as cardiomyocyte culture which underwent anoxia-reoxygenation. In the experiments with purified 20S proteasome it was shown, that quercetin in a dose-dependent manner inhibits all three peptidase activities of the proteasome, comparable to a specific proteasome inhibitor. The highest quercetin inhibition was observed in the case of chymotrypsin-like activity of proteasome. In the same way quercetin inhibited the activity of 26S proteasome from the PF II. Quercetin decreased trypsin-like (by 26%, p = 0.03), chymotrypsin-like (by 63.7%, p = 0.04) and peptidyl-glutamyl peptide-hydrolyzing (by 34.2%, p = 0.16) activities in the cardiomyocytes culture. It appears, that quercetin and its water-soluble analogue korvitin affect the cardiomyocytes in the same manner, as specific proteasome inhibitors clasto-lactacystin-beta-lactone. In the concentrations 5 and 10 mM quercetin and korvitin resulted in the decrease of the amount of living cardiomyocytes, increasing the amount of necrotic and apoptotic cells. In the concentration 2.5 mM quercetin and korvitin significantly abolished damaging effect of anoxia-reoxygenation, decreasing the amount of necrotic and apoptotic cells. These data suggest that the mechanisms of cardioprotective effect of quercetin connected with inhibition of proteasome.

[Role of nitric oxide in the mechanisms of formation of reflex vasomotor responses].

This review focuses on modem data concerning the role of nitric oxide (NO) in the mechanisms of vasomotor regulation. On the background of the literature data and own experimental results, we have discussed some questions of NO integration into transmission of impulses in central and autonomic nervous system under condition of realization of cardiogenic and sinocarotid pressor and depressor reflexes, reflectory vasomotor responses formation under acute myocardial ischemia. According to literature and own functional and morphological data we suggest species differences in NO participations in mechanisms of reflex self--regulation of circulation.

[Frequencies of allelic polymorphism of endothelial NO-synthase gene in patients with acute coronary syndrome in Ukrainian population].

Endothelial NO-synthase (eNOS) gene allelic polymorphism in 221 patients with acute coronary syndrome and in 83 practically healthy people was determined. It was shown that interrelations of normal homozygotes, heterozygotes and pathologic homozygotes in T/C promoter polymorphism analysis accout 48%, 36% and 16% correspondingly (in control--48%, 46%, 6%; P < 0.05 by chi2-test); in G894 --> T polymorphism ofexon 7 analysis--34%, 58%, 8% (in control--29%, 67%, 4%; P > 0.05), and in determination of 4a/4b polymorphism of intron 4--64.5%, 31% and 4.5% (in control--62.5%, 32.5%, 5%; P > 0.05). Obtained data show that eNOS C/C promoter variant is a risk factor of acute coronary syndrome in Ukrainian population.

Postconditioning prevents apoptotic necrotic and autophagic cardiomyocyte cell death in culture.

In the paper the data concerning the possibility of the reproduction of the postconditioning phenomena in the cardyomicyte culture are presented. Primary cultures of cardiomyocytes from neonatal rats underwent 30 minutes of anoxia followed by 60 minutes of reoxygenation. Three different models of postconditioning were used: 3 cycles of 1, 3, or 5 minutes of reoxygenation followed by 1, 3, or 5 minutes of anoxia, respectively. The percentage of living, necrotic, and apoptotic cells were determined by staining with Hoechst 33342 and propidium iodide. Autophagy was demonstrated by the staining of vacuolar structures in vivo by monodansyl cadaverine. After anoxia and reoxygenation the amount of living, necrotic and apoptotic cells were 79 +/- 1.5, 7.8 +/- 0.9 and 13 +/- 1.5 %, respectively (in unstimulated cell culture 90 +/- 0.8, 3.3 +/- 0.3, and 5.5 +/- 0.7, P < 0.0001 for all). Postconditioning with 1 min anoxia 3-fold increased the amount of living cells and decreased the number of necrotic and apoptotic cells (P = 0.002, P = 0.02 and P = 0.043 respectively). Postconditioning with cycles of 3 and 5 minutes had a gradually reduced effect compared to cycles of 1 minute. The percentage of autophagic cells in control cell culture was 4.3 +/- 0.3%. This number increased after anoxia-reoxygenation to 14 +/- 0.8%, and was reduced by postconditioning (P < 0.001). The data obtained indicate that postconditioning is one of the effective methods of cardioprotection and could effectively decrease the amount of cardiomyocytes with traits of programmed or non-programmed cell death.

[Effect of N-3-methyladenine on peptidase and ribonuclease activity of proteasome].

Using a culture of cardiomyocytes it has been shown, that a well-known inhibitor of autophagy, N-3-methyladenine causes a 1.4 fold increase (p = 0.023) of the chymotrypsin-like activity, a 1.5 fold increase (p = 0.09) of the peptidyl-glutamyl peptide-hydrolyzing activity and 1.5 fold decrease (p = 0.07) of the trypsin-like activity of the proteasome. N-3-methyladenine in a dose-dependent manner inhibits chymotrypsin-like and peptidyl-glutamyl peptide-hydrolyzing activities of the purified 20S proteasome, but activates it trypsin-like activity. Chymotrypsin-like and peptidyl-glutamyl peptide-hydrolyzing activities of the 26S proteasome from proteasome fraction II did change in the same way, as in the case of 20S proteasome, but trypsin-like activity decreased. Using the above method of determining ribonuclease activity, we have shown, that N-3-methyladenine and clasto-lactacystin b-lactone inhibit the RNase activity of the proteasome. Specific proteasome inhibitor exhibits more powerful action, almost completely preventing RNA of actin and myosin from degradation. These data show a multitarget action of N-3-methyladenine, resulting in changes of peptidase and ribonuclease activity of the proteasome.

[The frequency of allelic polymorphism of genes encoding immunoproteasome catalytic subunits in acute coronary syndrome patients].

Large multifunctional protease LMP2 (Arg60-->His) and LMP7 (Lys145-->Gln) gene allelic polymorphism in 200 patients with acute coronary syndrome and in 80 practically healthy people was determinated. It was shown that interrelation of genotypes Arg/Arg, Arg/His and His/His in LMP2 gene polymorphism is 52, 40.5 and 7.5 % correspondingly (in control group 53.8, 38.7, 7.5 %; P > 0.05 by chi2-test). Analysis of LMP7 gene polymorphism has shown that Lys/Lys - 89.5 %, Lys/Gln - 10.5 %, Gin/Gin - 0 % (in control group 93.8, 6.2, 0 % correspondingly; P > 0.05). The data show that LMP2 and LMP7 gene polymorphism is not a risk factor of acute coronary syndrome in Ukrainian population.

[Effect of proteasomal proteolysis on NO-synthase activity in isolated platelets].

In experiments with isolated platelets it was shown, that application of proteasomal fraction II (PF II) from rabbit's reticulocytes changes the activity of endothelial nitric oxide synthase (eNOS). During incubation of sonicated platelets with PF II eNOS activity increased by 24.6% (p = 0.02). Methylated ubiquitin and clasto-lactacystin beta-lacton significantly eliminated this effect. So, it is not eNOS that is subsequent to proteasomal degradation, but a certain negative regulator of its activity. eNOS activity in platelets, treated with H2O2 (1 mM), after incubation with PF II increased to a higer extent, and was 3.4 +/- 0.36 UF/min x 10(6) cells (for 51.3% more, than in control), but H2O2 did not affect the activity of enzyme in platelets under analogous condition without addition of PF II. It was established, that eNOS activity decreases after 60 min of incubation with 10 mM of clasto-lactacystin beta-lacton by 11.6%, and with 20 mM--by 28.6% (p < 0.05). Data obtained witnesses about participation of ubiquitin-dependent proteasomal proteolysis in regulation of eNOS activity and possibility of the effect upon intensity of NO production due to acceleration of degradation of intracellular regulators of this enzyme's activity.

[Proteolytic enzymes and apoptosis]. / Proteoliticheskie fermenty i apoptoz.

Information about basic mechanisms of programmed cell death (apoptosis) development with participation of proteolytic enzymes is given in the review. The basic mechanisms of apoptosis launching are conditionally subdivided into three groups, depending on the "points of application" of apoptosis development initiating factor: membrane (receptor-dependent), mitochondrial and nuclear. Attention is accentuated on ubiquitin-dependent proteasomal proteolysis as the key system regulating apoptosis. The possible disturbances of apoptotic program realization are specified under various pathological processes and diseases.

Arrhythmogenic peroxynitrite-induced alterations in mammalian heart contractility and its prevention with quercetin-filled liposomes.

The aim of the present study was to evaluate the effects of quercetin-filled phosphatidylcholine liposomes (PCLs) on peroxynitrite (ONOO-)-induced cardiac arrhythmias. Experiments were done using different experimental models, including isolated rat papillary muscle, Langendorff perfused rat hearts, and anesthetized animals. Being exogenously applied in a concentration greater than 50 microM, ONOO- caused inhibition of isometric twitch amplitude in isolated papillary muscles and led to an appearance of arrhythmias. Decomposed ONOO- had no similar effects and reversibly increased twitch amplitude. Authentic nitric oxide (NO, 100 microM) did not produce arrhythmias and had no significant effect on twitch amplitude. Verapamil and ruthenium red were with-out effect on ONOO- -induced arrhythmias, whereas tetrodotoxin and nicorandil effectively prevented arrhythmias development. Ouabain increased the arrhythmogenic effect of ONOO-. ONOO- significantly decreased coronary perfusion pressure (CPP) and mean left-ventricular pressure (MLVP) in the Langendorff perfused rat heart and produced severe arrhythmias. Authentic nitric oxide (NO) decreased CPP and MLVP insignificantly and resulted in a low incidence of arrhythmias. The NO donor SIN-1 in doses greater than 50 microM led to the appearance of low-incidence arrhythmias in anesthetized rats. Intraventricular injection of ONOO- promotes the appearance of a high incidence of arrhythmias in anesthetized rats and decreased MLVP. PCLs filled with the antioxidant quercetin restored normal cardiac contractility in both isolated tissues and anesthetizes animals. In conclusion, we hypothesized that ONOO-, but not its decomposed products, can initiate membrane lipid peroxidation and damage the phospholipid environment of ionic channels in myocardial cell plasma membranes inducing abnormal cardiac action potentials, arrhythmogenesis, and contractile dysfunction. Quercetin-filled PCL provide reliable protection against peroxynitrite-induced myocardial injury in isolated cardiac tissues and anesthetized animals primarily as a result of the decomposition of endogenously formed ONOO-.