[Effect of quercetin on proteasome activity in the aorta and heart tissues of spontaneously hypertensive rats].

To determine the role of proteasome proteolysis in the pathogenesis of hypertension, we have studied the proteolytic activity of the proteasome in the aorta and heart tissues of rats with spontaneous hypertension (line SHR), and used quercetin, the drug that can inhibit the activity of this multicatalytic complex. In the aorta of SHR, the activities of the proteasome were not significantly different from that observed in Wistar rats. At the same time, in the heart tissues the trypsin-like (at 40%, P > 0.05), and chymotrypsin-like (by 1.7 times, P < 0.03) activities were significantly less in SHR. Significant morphological changes (fibrosis of the left ventricle was 4.7%, aorta intima width was increased and heart weight index was higher by 21.6% (3.7 +/- 0.6 mg/g) compared with Wistar rats (2.9 +/- 0,4 mg/g, P < 0.004) were observed in these animals functional disorders (reduced stroke volume by 3 times (P < 0.0001), ejection fraction by 2.5 times (P < 0.0001), increased end diastolic pressure by 6.5 times (P < 0.005), end systolic pressure by 15% (P < 0.004)) were revealed. Pharmacological drug "Qvercetin" effectively inhibited trypsin-like and chymotrypsin-like proteasome activities in the aorta (2.7-fold (P < 0.005) and 2-fold (P < 0.003), correspondingly) and trypsin-like, and peptidyl-glutamyl peptide-hydrolyzing-like activities (2.4-fold, P > 0.05 and 9.3-fold, P < 0.02, correspondingly) activities in the heart, leading to a significant improvement of morphological and functional parameters of the heart. Whereas the drug "Qvercetin" that is widely used in clinical practice (especially in therapy of acute myocardial infarction) it could be recommended for the use in prevention of cardiac remodeling with high level of blood pressure.

[The changes in the activity of tripeptidyl peptidase II in experimental atherosclerosis and hypertension].

The changes in the activity of intracellular proteolytic systems are important mechanisms in the damage of blood vessels walls and arterial hypertension. Tripeptidyl peptidase II (TPP II) is one of the giant intracellular protease that is still poorly known. It fulfils hydrolysis of peptides, coming from proteasomal proteolysis. Modeling of cholesterol atherosclerosis in rabbits (1% of cholesterol in diet for 2 month) results in the significant decrease of TPP II activity in aorta tissues. This diet in spontaneously hypertensive rats (SHR) leads to a decrease of TPP II activity in aorta tissues (on 50%, P < 0.05) but has no influence on the activity of TPP II in Wistar rats. Application of Quercetin prevents the inhibition of TPP II activity in aorta tissues of rabbits and SHR at experimental hypercholesterolemia. The data received show that changes in the activity of TPP II play an important role in pathogenesis of blood vessels wall in atherosclerosis and arterial hypertension.

[Frequency of allele polymorphism of immune proteasome catalytic subunits in patients with ischemic stroke].

Frequency of allelic variants of proteasome subunits genes LMP2 (Arg60 --> His) and PSMA6 were determined in patients with ischemic stroke using real-time PCR. Allelic variants of PSMA6 were disposed in the next manner: C/C - 80.2%, C/G - 19.8%, G/G--were not (in control) and C/C - 75.5%, C/G - 21.4%, G/G - 3.1% (P = 0.22) in patients with IS. It was shown that distribution of LMP2 allelic variants was the following: Arg/Arg - 53.3%, Arg/His - 43.5%, His/His - 6.7% in control and Arg/Arg - 55.9%, Arg/His - 34.3%, His/His - 9.8% in IS group (P > 0.05). The data show that LMP2 and PSMA6 gene polymorphism is not a risk factor of ischemic stroke in Ukrainian population.

[T(-786) --> C-polymorphism of the endothelial nitric oxide synthase promoter gene (eNOS) and exercise performance in sport].

Given the significant impact of the T(-786) --> C-polymorphism of the eNOS gene in the process of adaptation to physical stress, we aimed to investigate the effect of this polymorphism on physical performance in sportsmen and establish the possibility of its use as a marker of predisposition to the sport. DNA of 516 people, of which 195 qualified athletes and 321 people who had no experience of regular exercise was investigated. The frequency of genotypes and alleles of the T(-786) --> C-polymorphism of the eNOS gene in groups of athletes of different sports, the distribution of genotypes and alleles among athletes and those who are not involved in sports were studied. T allele frequency in a group of athletes on 6.4% (r(chi)2 = 0.03) than in control group. The association of the T allele of the T(-786) --> C-polymorphism of the eNOS gene with a predisposition for speed and power was established. In the group of athletes in speed and power sports, the T-allele frequency was higher than that in the control group by 12% (r(chi)2 = 0.002) and than in group endurance sports by 10% (r(chi)2 = 0.004). We found that the T(-786) --> C-polymorphism of the eNOS gene influence the power and efficiency ofthe functioning of the cardiorespiratory system of athletes during exercise.

[RNA interference of proteasome subunit of PSMbeta7 gene restricts proteasome subunit PSMbeta1 and PSMbeta5 mRNA expression and peptidyl-glutamyl peptide-hydrolyzing proteasome activity in neonatal cardiomyocytes].

Using small interfering RNA (siRNA) transfection of neonatal cardiomyocytes to inhibit expression of nonproteolytic proteasome beta7 subunit, we observed a significant decrease in beta1 proteolytic subunit mRNA expression. Proteasome peptidyl-glutamyl peptide-hydrolyzing activity decreased to 28% (0.48 +/- 0.2 nM AMC/min) compared to control (1.7 +/- 0.5 nM AMC/min) (P < 0.05). Beta5 Subunit mRNA expression decreased 21 times (P < 0.05) with no changes in its chymotrypsin-like activity. Proteasome trypsin-like activity and activity of another proteolytic enzyme tripeptidyl-peptidase II remained unchanged.

[The frequency of allelic polymorphism of matrix Gla-protein gene in acute coronary syndrome patients].

Allelic polymorphisms of matrix Gla protein (MGP) gene were determinated in 115 patients with acute coronary syndrome (ACS) and in 110 practically healthy people. It was shown that interrelation of major homozygotes, heterozygotes and minor homozygotes by T-138-->C MGP promoter polymorphism in patients with ACS were 59.8%, 32.7%, 7.5% and in control 58.7%, 36.7%, 4.6% (P>0.05 by X2-test); by G-7-->A polymorphism: 42.1%, 45.6%, 12.3% and 41.8%, 54.5%, 3.6% correspondingly (P<0.05); and by Thr83Ala exon 4 polymorphism: 42.6%, 43.5%, 13.9% compared to 43.9%, 45.9%, 10.2% in control (P>0.05). The obtained data show that MGP A/A variant of MGP promoter G-7-->A polymorphism is a risk factor of ACS in Ukrainian population. These data indicate also that the intensity of calcification influences atherosclerotic injury of coronary artery.

[Interrelation between cardiac pump function disturbances and cardiac contractility after beta-adrenergic hyperstimulation of the heart in rats].

The complex of structural and functional changes of myocardium was investigated in experiments with rats with chronic beta-adrenergic activation for 1 month. We observed substantial attenuation of myocardial pump function, particularly reduction of stroke volume by 38.50% (P < 0.01), cardiac output by 42.38% (P < 0.01), and ejection fraction by 35.61% (P < 0.01). Furthermore, 2-fold increase of end-diastolic left ventricular pressure (P < 0.01) and rise of active relaxation constant Tau by 12.91% (P < 0.05) were observed. This indicates on an impaired diastolic function of the heart that is associated with accumulation of connective tissue elements in myocardium and increase of its end-diastolic stiffness that finally leads to cardiac pump function disturbances. Surprisingly, myocardial contractility was considerably augmented not only after the treatment with beta-adrenergic agonist but also on the 26th day after drug cessation. This phenomenon is associated with elevation of dP/dt(max) by 49.9% (P < 0.01), 2.5-fold increase of end-systolic elastance (P < 0.01) as well as maximal myocardial elastance by 42.53% (P < 0.05). It can be explained by compensatory influence of increased contractility that nevertheless failed to maintain adequate cardiac pump function and furthermore it may result in depletion of cardiac energy resource.

[Correction of lipid peroxidation and antioxidant system disorders by bioflavonoids during modeling of cholesterol atherosclerosis in rabbits].

We have studied the influence of bioflavonoids (quercetin, corvitin) on lipid peroxidation and antioxidant enzymes in the modeling of cholesterol atherosclerosis in rabbits. It has been shown that simultaneous administration of the quercetin derivative corvitin suppressed lipid peroxidation. We showed that under hypercholesterolemia, the concentration of malone dialdehyde in myocardial tissue in rabbits is significantly increased, while administration of bioflavonoids decreased the concentration of malone dialdehyde by 38.3%. Furthermore, corvitin caused activating effects on antioxidant enzymes superoxide dismutase and catalase in cardiac tissue. Our data suggest that bioflavonoids are able to suppress lipid peroxidation and prevent the decrease ofantioxidant enzymes activity in rabbits with cholesterol-rich diet induced atherosclerosis.

[Increased expression of voltage-dependent anion channel and adenine nucleotide translocase and the sensitivity of calcium-induced mitochondrial permeability transition opening pore in the old rat heart].

We investigated mRNA and protein expression of voltage-dependent anion channel (VDAC), mRNA adenine nucleotide translocase (ANT) as well as the sensitivity of the mitochondrial permeability transition pore opening (MPTP) to Ca2+ in the adult and old rat heart. It was shown that in the old rats hearts VDAC mRNA expression increased by 1.7 (p < 0.05) times and mRNA ANT expression increased by 1.8 (p < 0.05) times in comparison with adult animals. The Western Blot analysis showed that the level of VDAC protein expression in the old rat hearts also significantly increased compared with adult animals. In the hearts of old rats, the sensitivity of MPTP opening to calcium (10(-7)-10(-4) mol/l) determined by mitochondria swelling, increased two-fold (p < 0.05). Therefore, an increased VDAC and ANT expression, as the main structural functional components of the MPTP, and an increased sensitivity of MPTP opening to Ca2+ caused an increase in the permeability of mitochondrial membranes in aging. Each of these factors may contribute to alterations in mitochondrial barrier properties and lead to mitochondrial dysfunction.

[Effects of ubiquitin gene silencing in anoxia-reoxygenation of cultured cardiomyocytes].

Ubiquitin-dependent proteasomal proteolysis is crucialin the turnover of cardiomyocytes functional proteins (actin, myosin, ion channels at. al.), therefore, investigation of cell death after ubiquitin (UBB) gene silencing using RNA interference and anoxia-reoxygenation (AR) modeling appears to be attractive. Cardiomyocytes were transfected by siRNA to ubiquitin gene using electroporation procedure, and then primary culture was treated by 30 min of anoxia and 60 min of reoxygenation. The number of living, necrotic and apoptotic cardiomyocytes was determined by fluorescence microscopy. The level of UBB and proteasome subunits beta5 (PSMB5) and beta9 (PSMB9) mRNA expression was estimated by real-time PCR. It was shown that UBB mRNA expression was increased by 2.1 times after AR modelling (P < 0.05). Small interference RNA injection in cell culture decreased ubiquitin, PSMB5 and PSMB9 expression by 2.4 (P < 0.05), 1.3 (P > 0.05) and 1.6 (P < 0.05) times, respectively, compared with control (scrambled siRNA introduction). At the same time, the number of living cardiomyocytes decreased to 70.26 +/- 1.54%, P<0.05, and the level of necrotic cells, apoptotic cells and cells with signs of autophagy augmented by 25.92 +/- 1.52%, (P = 0.38), 4.32 +/- 0.53% (P = 0.15) and 38.2 +/- 3.81% (P = 0.001), respectively. Ubiquitin silencing after AR (30 min/l h) increased the number of living cells by 3.7% and decreased the number of necrotic cells by 4.7% and did not alter the apoptotic and autophagic cells populations. The data obtained indicate that ubiquitin gene silencing, mRNA expression of which augmented during AR, induces necrotic and autophagic death of intact neonatal cardiomyocytes in culture, but enhances the AR resistance of these cells to some extent.

[Expression of proteasome subunits PSMB5 and PSMB9 mRNA in hippocampal neurons in experimental diabetes mellitus: link with apoptosis and necrosis].

There is a huge body of evidence showing that long-termed diabetes mellitus is followed with hippocampal dysfunction. The goal of this work was to investigate the expression of proteasome subunits PSMB5 and PSMB9 mRNA in CA1, CA2 and CA3 areas of hippocampus in parallel with processes of cell death (apoptosis and necrosis) in development dynamics of streptozotocine-induced diabetes. We have studied hippocampal neurons using chromatine dye Hoechst-33342 and immunohistochemical detection of apoptotic cell death marker caspase-3. At day 3 and 7 after injection of streptozotocine we have performed visualization of caspase-3-immunopositive neurons showing signs of neurodegeneration in hippocampal sections using confocal microscope Olympus FV1000. The rate of proteasome subunits PSMB5 and PSMB9 mRNA expression was determined with RT-PCR. The results indicated elevation of PSMB9 mRNA content (from 4807 +/- 0.392 arbU up to 20,023 +/- 4949 arbU on day 3 and up to 20,253 +/- 5141 arbU on day 7). A maximal number of cells with signs of chromatin condensation was observed at day 3 and day 7 in CA2 and CA3 area (11.51% and 12.49% respectively). That indicates an intensification of proapoptotic processes. Summarizing the results presented above we can conclude that during the first week of diabetes mellitus development, hippocampal cells undergo the process of impairment and degeneration.

[Omega-3 polyunsaturated fatty acids stimulate the expression of PPAR target genes].

In experiments on isolated neonatal cardiomyocytes and hearts of adult rats we investigated the influence of polyunsaturated fatty acids (PUFA) on expression of two peroxisome proliferator-activated receptor (PPAR) target genes--fatty acid transport protein (FATP) and interleukin-1 receptor antagonist (IL-1ra). We determined that cell membranes of adult rats heart and cardiomyocytes of neonatal rats which were born by females fed with omega-3 PUFA for 4 weeks, contain more omega-3 PUFA (alpha-linolenic and docosahexaenoic acids) in comparison with the control. It was shown that omega-3 PUFA elevated FATP and IL-1ra mRNA expression in 2.25 (P = 0.07) and 8.4 (P < 0.00001) folds correspondingly in adult rats tissues. In isolated cardiomyocytes, the mRNA expression of FATP and IL-1ra were augmented 2.5 folds (P = 0.22) and 8.1 folds (P < 0.00001), accordingly. Our data show that omega-3 PUFA stimulate PPAR target genes expression and allow to permit genetic mechanisms in cardioprotective effects of omega-3 PUFA.

[Second anoxia-reoxygenation does not cause the apoptotic cell death of cultured neonatal cardiomyocytes].

The cells death and genes expression in neonatal cardiomyocytes culture at two anoxia-reoxygenation modeling were investigated. The primary culture of neonatal cardiomyocytes was undergone 30 min of anoxia followed by 24 h (A-R1) and the second anoxia-reoxygenation--30 min and 60 min respectively (A-R2). The percentages of living, necrotic, apoptotic and autophagic cells were determined by staining with bis-benzimide, propidium iodide and monodansylcadaverine. Anoxia-reoxygenation significantly influenced the ratio of living, necrotic, apoptotic and autophagic cells both at its first A-RI and second A-R2 episodes. It was shown that the main mechanism of cell death after the both periods of anoxia-reoxygenation is necrosis. The changes of mRNA levels of genes of heat shock proteins HSP70 and HSP90, antiapoptotic protein Bcl2 and key regulator of autophagy FRAP in cardiomyocytes culture were established. The data obtained allow to make suggestion that in 24 h after the first episode of anoxia-reoxygenation A-R1 the overexpression of heat shock proteins starts the cascade of reactions that causes the necrotic cell death prevalent and the blocking of apoptotic program at second anoxia-reoxygenation A-R2.

[Functional and molecular changes of large-conductance calcium-dependent potassium in rat aortic smooth muscle cells after ionizing irradiation].

The effects of whole body gamma-irradiation on large conductance Ca(2+)-activated K+ channels (BK(Ca)) function and mRNA expression in rat thoracic aorta smooth muscle cells (SMCs) were studied using combined patch-clamp technique in whole-cell modification and RT-PCR analysis. The stimulation of control SMCs by increasingly depolarized voltage steps showed clearly expressed outward K+ currents in control SMCs. Outward currents in SMCs obtained from irradiated animals on the 9th and 30th days post-irradiation demonstrated a significant decrease of K current density amplitudes. Paxillin was without effect on irradiated cells on 30th day post-irradiation indicating the absence of conductance through BK(Ca) channels. The results of RT-PCR analysis showed that expression both alpha-subunit and beta1-subunit of BK(Ca) channels appears to be considerably diminished on 30th day post-irradiation. It is likely that radiation-induced malfunction functional activity of channels is related with insufficient expression of BK(Ca) structural elements in SMCs. In conclusion, the data obtained clearly demonstrate that decreased of the BK(Ca) channels alpha- and beta1-subunit expression in SMCs is a key factor of abnormality in BK(Ca) channels activity. This abnormality may contribute to vasorelaxing force depression following non-fatal whole-body gamma-irradiation.

[Ultrastructural changes in isolated cardiomyocytes in modeling of endoplasmic reticulum stress].

At modeling of endoplasmatic reticulum (ER) stress by it classic inducer thapsigargin, anoxia-reoxygenation and simultaneous inhibition ofproteasomal proteolysis, autophagy and apoptosis a diversity of ultrastructural peculiarities was shown. Their comparison allows to make a conclusion that changes in these groups of experiments are similar and typical for ER stress. Thapsigargin application was shown to result in accumulation of giant mitochondria in perinuclear zone of cardiomyocytes. Some of these mitochondria had destroyed and high condensed matrix. The structure of ER was normal but in some regions of cells the dilation of ER cisterns occurred that, to our opinion, is an essential sign of ER stress. In another group of cells thapsigargin caused dehydratation and osmiophilia of cytoplasm, significant dilation of ER cisterns, partial or complete degranulation of these organelles that often formed vacuoles with high electron density material. Also, the significant decrease of the number and size of mitochondria that had partially destroyed and condensed matrix was observed in these cells. The accumulation of lipofuscin and myophilament destruction at preservation of sarcoplasmic membrane integrity was detected. However, in conditions of simultaneous inhibition ofproteasomal proteolysis, aytophagy and apoptosis the loss of membrane integrity was shown, and we propose that it unconditionally should cause necrotic cell death. That was confirmed by use of fluorogenic dyes to detect necrosis and apoptosis. Our data indicate the important role of ER stress in processes of cardiomyocytes death at anoxia-reoxygenation and inhibition of proteasomal and autophagic proteolysis.

[Expression of genes encoding vanilloid receptors type 1 and type 2 in the cultured hippocampal neurons].

Expression of mRNA of 1 and 2 type (TRPVI and TRPV2) vanilloid receptors in the cultured hippocampal neurons was determined. With the use of reverse transcription polymerase chain reaction (RT-PCR) of single neurons (single-cell RT-PCR), was shown the TRPV1 and TRPV2 receptors expression in both neurons and glia cell. With use of real-time (real-time PCR) we determined that the level of these genes expression in different neurons is identical. It was also shown that the level of mTRPVI in GABAergic neurons was considerably higher, than the level of mTRPV2, while expression of genes in the glutamatergic neurons did not differ. Findings about expression of TRPV 1 and TRPV2 allow to suppose that vanilloid receptors can play an important functional role in the hippocampal neurons.

[Proteasome activity changes in the aorta, heart tissues, and blood leucocytes in modelling of cholesterol atherosclerosis].

In experiments on modelling of cholesterol atherosclerosis in rabbits (1% cholesterol-rich diet during 2 month) it was determined changes of trypsin-like (TL), chymotrypsin-like (CHTL) and peptydilglutamilpeptidase (PGPG) proteasomal activity in tissues of aorta, heart and isolated blood leukocytes. It was shown that cholesterol-rich diet caused significant increase of TL (3.2 fold, P=0.003), PGPG (1.8 fold, P=0.003) proteasomal activity in aorta tissues, and PGPG activity (1.8-times, P=0.003) in myocardium. In isolated blood monocytes, the CHTL and PGPG activities were significantly increased (1.9 fold, P=0.05 and 11.6 fold, P=0.0001, respectively) and in PMN leucocytes the PGPG activity of proteasome was also significantly increased (1.8 fold, P=0.031). Proteasomal activity in lymphocytes during cholesterol atherosclerosis modelling had no significant changes. The data obtained indicate that alimentary hypercholesterolemia induces considerable changes of proteasomal activity in cardio-vascular system and blood cells that take part in atherogenesis.

[Proteasomal degradation of ribonucleic acids of different NO-synthase isoforms].

Using a developed method of determination of the RNase activity of the proteasome in vitro with the application of reverse transcription followed by subsequent polymerase chain reaction it was shown that 26S proteasome from the proteasomal fraction II effectively cleaves RNA, encoding actin, myosin and all isoforms of NO synthase. The intensity of RNA degradation by proteasome and specific RNases is similar. It was also shown that clasto-lactacystin beta-lactone, a specific proteasome inhibitor significantly depresses RNase activity of the proteasome. Thus, proteasome is capable to degrade certain eukariotic RNA in vitro and the proposed method can be used in order to discover specific substances such as inhibitors of RNase activity of the proteasome.

[Identification of E-4031-sensitive potassium current component in murine P19 embryonic carcinoma cell line differentiated in cardiomyocytes].

Pluripotent mouse P19 embryonic carcinoma cells represent a convenient in vitro model for studying various aspects of cardiac differentiation. Here by using whole-cell patch-clamp recording we have identified the rapid delayed rectifier K+ current, I(Kr) in P19 cell induced to differentiate into cardiac phenotype by DMSO (1%). Cardiac differentiation was confirmed by the appearance of spontaneously beating cells, their morphological features, ultrastructural clusterization of mitochondria around contraction elements, expression of cardiac actin mRNAs and MLC2v, and by the presence of inward sodium and calcium currents. I(Kr) was isolated based on the sensitivity to the specific blocker, E-4031, which at concentration of 1 MM blocked more than 50% of the total outward K+ current. However, in contrast to I(Kr) in native cardiac myocytes and in heterologous systems expressing I(Kr)-carrying ERG1 potassium channel, E-4031-sensitive K+ current in cardiac-like P19 cells lacked characteristic inward rectification, suggesting specific regulation and/or subunit composition of endogenous ERG -based channel in these cells. Establishing the reason(s) for this phenomenon will advance the understanding of the mechanisms of I(Kr)-channel rectification. Cardiac-differentiated P19 cells might also be useful for studying pharmacological modulation of I(Kr), which is recognized target for cardiotoxic side effects of numerous drugs.

[Characteristics of changes in respiratory function of humans after a long stay in Antarctic region].

Fifteen participants of the Antarctic expedition (men, 27-51 years old) have been investigated after their return from a one-year stay there. All subjects have signs of latent hypoxia. Compensation of hypoxic reactions depended on the initial state of organism oxygen regimen as well as on the features of a genotype. It was supposed that, after a long stay by a person in the coastal Antarctic conditions, the latent form of hypoxia could develop. The latter was accompanied by reorganizations of oxygen regimen and determined by specific features of a genotype.