Changes of the echocardiographic parameters in chronic heart failure patients with Ile337val, Glu23lys, and Ser1369ala polymorphisms of genes encoding the ATP-sensitive potassium channels subunits in the Ukrainian population.

Different allelic variants of genes that encode ATP-sensitive potassium (KATP ) channels' subunits may contribute to the development of heart failure. The purpose of the work to investigate SNPs in genes that encode KATP channels in relation to echocardiographic parameters in chronic heart failure (CHF) patients. Ninety-nine people with CHF of ischemic origin with left ventricular systolic dysfunction were examined. The control group is represented by 108 clinically healthy subjects. KCNJ11 polymorphisms Ile337Val and Glu23Lys, and ABCC8 polymorphism Ser1369Ala were genotyped using polymerase chain reaction. In CHF patients, the frequency of the Ile337Val genotype was: Ile/Ile, 40.4%; Ile/Val, 45.5%; and Val/Val, 14.1%. The patients with the Val/Val genotype had left ventricular (LV) mass that was 334.15 g, which was 27.3% (P < 0.05) lower versus Ile/Val patients (425.48 g). The index of this parameter was also significantly lower (28.4%, P < 0.05). In CHF patients, the frequency of Glu23Lys and Ser1369Ala was: Glu/Glu and Ser/Ser, 43.4%; heterozygote, 44.4%; Lys/Lys and Ala/Ala, 12.2%. The patients with the Lys/Lys and Ala/Ala genotypes had a significantly lower LV mass index and LV end-diastolic volume (22.9% and 26.8%, P < 0.05) versus heterozygotes. Thus, the greatest LV mass and LV end-diastolic volume values are associated with heterozygotes, while the smallest are associated with minor homozygotes.

Establishment of transgenic lettuce plants producing potentially anti-hypertensive shRNA sequencing data.

Development of RNAi-based therapeutics is a fast growing field of pharmaceutical industry. Using plants for production of pharmaceutically valuable siRNAs may have significant advantages of cost-effectiveness, scalability and low risk of contamination with human pathogens. If edible plant species are genetically engineered to synthesize siRNAs, the costly stage of target product purification may be omitted. We describe the establishment of transgenic lettuce plants producing shRNA targeting delta isoform of protein kinase C (PKC-delta), an effective target for RNAi-based treatment of arterial hypertension. Transgenic lettuce plants were obtained by Agrobacterium-mediated transformation with genetic constructs harboring antiPKC and scrambled (control) shRNA genes. The presence of transgenes was proved by PCR analysis, and the accumulation of antiPKC shRNA was estimated using RT-qPCR technique. Six transgenic lettuce lines showed varying levels of antiPKC shRNA expression with the highest value reaching 14 ± 9 % of highly abundant endogenous lettuce micro RNA (miR156a), or 12.7 fmol/g dry weight. Plants carrying either antiPKC or scrambled shRNA genes flowered normally, but did not produce seeds. The described transgenic lettuce plants accumulating antiPKC siRNA are the subject for animal testing and can be considered as a raw material for the development of novel antihypertensive drugs.

PROGESTERONE RECEPTOR GENE POLYMORPHISMS AS GENETIC RISK FACTOR OF THREATENED ABORTION.

Determination of the progesterone receptor gene polymor- phisms rs590688 C/G and rs500760 A/G was provided using PCR method. To investigate the genetical precursors of threatened abortion the next groups were included: 67 patients with threatened abortion and 93 healthy persons. These allelic variants have the significantly different at rs590688 study: C/C - 23,9%, C/G - 44,8%, GIG - 31,3%, and C/C - 27,2%, C/G - 58,7%, GIG - 14,1% in the control group (P<0,05 by x2-test). The allelic variants of the rs500760 polymorphism did not differ statistically in the study group A/A 53,7%, A/G - 40,3%, GIG - 6% in the control group A/A- 52,2%, A/G - 44,6%, GIG - 3,3% (P>0,05 by x2-test). Distribution of minor homozygote GIG polymorphism rs590688 progesterone receptor gene in patients with threatened abortion in our study almost in 10 times higher in comparing with women of Taiwanese Han population, who had idiopathic recurrent pregnancy loss. The obtained data indicate significant ethnic differences in PGR and show polymorphism rs590688 clinical significance.

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.

[ALLELIC VARIANT FREQUENCY OF PROMOTER (G(-47)-->A) γ-CRYSTALLIN GENE AFFECTS THE LEVEL OF ITS EXPRESSION IN PLATELETS].

To investigate the genetical precursors of cataract development the next groups were included: patients suffering from cataract (96) and 96 healthy persons. The determination of γ-crystallin polymorphism (G(-47)-->A) (rs2289917) was provided using PCR method and further analyses of restriction fragment length polymorphism. These allelic variants have the significant different: G/G--35.37%, G/A--53.66%, A/A--10.98%, and G/G--55.06%, G/A--35.96%, A/A--8.99% comparing with the control group (P = 0.03, by χ2-test). While investigating the level of expression of γ-crystallin gene (CRYGB) in platelets, showed that the quantity of mRNA in homozygotes G/G in 3.9 times (P < 0.05) higher than in carriers of A allele (genotype G/A and A/A). This paper shows the significant difference in distribution of CRYGB promoter (G(-47)-->A) genotypes in patients with cataract compared to the control group. Furthermore, here we provide the data concerning its functional meaning: level of mRNA expression of crystallin is different in carriers of various CRYGB promoter (G(-47)-->A) genotypes.

[SINGLE NUCLEOTIDE POLYMORPHISMS IN HUMAN PROGESTERONE RECEPTOR GENE AND ITS VALUE IN MISCARRIAGE OR PRETERM DELIVERY].

This article presents the current understanding of progesterone and its role in female reproductive function, described the structure and functionality of the progesterone receptor. The attention is focused on the meaning of the terms miscarriage and preterm delivery. Was described the today known causes of the miscarriage and preterm delivery, shown the value of single nucleotide polymorphisms of the progesterone receptor in miscarriage and preterm delivery by the analysis of publications in recent years in order to find connections between different polymorphisms of the progesterone receptor gene and a spontaneous abortion, premature labor, recurrent miscarriage and its impact on progesterone therapy.

[Association of allelic polymorphisms of genes matrix Gla-protein system with ischemic atherothrombotic stroke].

There are results of the determination of 10 polymorphisms of matrix Gla-protein system (gene MGP-T(-138)-->C (rs1800802), G(-7)-->A (rs1800801), Thr83-->Ala (rs4236), gene VDR-FokI (rs2228570), BsmI (rs1544410), ApaI (rs7975232), TaqI (rs731236), gene GGCX-Arg325-->Gln (rs699664), gene VKORS1-T(2255)-->C (rs2359612), gene BMP-2-Ser37-->Ala (rs2273073)) into 170 patients with ischemic atherothrombotic stroke (IATS) and 124 healthy individual is (control group). It is established that there is a connection between the IATS and polymorphic variants of genes MGP (G(-7)-->A) and VKORC1 (T(2255)-->C). The risk of IATS in carriers of minor allele A/A (G(-7)-->A polymorphism) in 2.6 times higher than in carriers of the major allele (G/A + G/G), and C/C genotype (T(2255)-->C polymorphism) in 2.2 times higher than the homozygotes of major allele. The coincidence of patients T/C and G/G, C/C and G/A genotypes, and A/A genotype (G(-7)-->A polymorphism) with any genotype T(2255)-->C polymorphism are increases the risk of IATS.

Proteasomal activity in brain tissue following ischemic stroke in Wistar rats.

Functional as well as structural reorganization of brain tissues takes place in the surrounding and remotes brain areas after focal ischemic lesions. In particular, reactive or regenerative processes have been described to occur in the infarction areas and the contralateral hemisphere. Experiments were performed on 63 rats, divided into 3 groups (each consisted of 21 animals): sham operated, short-term occlusion of the right middle cerebral artery (MCAO) group, and long-term MCAO group. We have studied changes in proteasome proteolysis during transient occlusion of the middle cerebral artery using method of Koizumi J., duration 2 and 60 min and made the comparison between changes in different types of proteasome activity and severity of ischemic injury and showed three types of decrease inproteolytic activity (trypsin-, chymotrypsin-like, peptidylglutamyl peptide-hydrolyzing) in the brain tissues. Chymotrypsin-like activity of ischemic areas of the brain for short-term MCAO decreased 4.1 times compared with controls (P > 0.05), for long-term MCAO decreased 5.8 times compared with controls (P < 0.05). Trypsin-like activity of ischemic areas of brain for short-term MCAO decreased 7.1 times compared with controls (P > 0.05), for long-term MCAO decreased 12.5 times compared with controls (P < 0.05). PGPH activity of ischemic areas for short-term MCAO decreased 8 times compared with controls (P > 0.05), for long-term MCAO decreased 2.8 times compared with controls (P < 0.05). The similar dynamics was observed also in the penumbra and the core zone of the brain at 6 h of reperfusion, in the long run there is no significant difference between the core and contralateral zones. Our results suggest that proteasome activity may play also a role in contralateral cortical plasticity occurring after focal cerebral ischemia.

Hippocampus remodeling by chronic stress accompanied by GR, proteasome and caspase-3 overexpression.

Chronic stress is a threat to homeostasis for many brain regions. While hippocampal formation is one of the most stress-sensitive areas of the cortex, molecular changes occurring as a result of increased glucocorticoid neurotoxicity in hippocampus are largely unknown. The aim of these studies was to investigate mRNA expression of mineralocorticoid and glucocorticoid receptors (MR, GR), proteasome subunits ß5 (constitutive subunit) and ß1i (inducible immunoproteasome subunit), mTOR (mammalian target of rapamycin), bcl-2; as well as caspase-3 immunoreactivity (confocal microscopy) in adult Wistar rat hippocampus following 10-day restraint stress (plastic restrainers, 6h daily). Chronic restraint led to a significant reduction in number of neuronal and astroglial cells in hippocampal regions CA1-3. This reaction was combined with substantial increase in GR and decrease in MR mRNA levels with the greatest response - 1.5-fold amplitude increase - observed in dentate gyrus and CA3 correspondingly. Stress did not change the expression of constitutive ß5 subunit but dramatically enhanced expression of inducible ß1i subunit and increased mTOR, and bcl-2 mRNA expression. Multiple scattered cells demonstrating caspase-3(+) profile were found in hippocampus of stressed animals. The study demonstrates that hippocampal remodeling induced by chronic restraint stress is associated with GR, immunoproteasome, mTOR, caspase-3 and bcl-2 overexpression in hippocampus.

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.

[Experimental therapy of cardiac remodeling with quercetin-containing drugs].

It was shown that continuous beta-adrenergic hyperstimulation resulted in cardiac function disturbances and fibrosis of cardiac tissue. Treatment with quercetin-containing drugs, particularly, water-soluble corvitin and tableted quertin exerted favourable effect on cardiac hemodynamics, normalized systolic and diastolic function in cardiac remodeling, induced by sustained beta-adrenergic stimulation. It was estimated that conducted experimental therapy limited cardiac fibrosis area almost three-fold, that could be associated with first and foremost improved cardiac distensibility, characteristics of diastolic and also pump function in cardiac remodeling.

Genetic predisposition to essential hypertension in children: analysis of 17 single nucleotide polymorphisms.

Study of 17 single nucleotide polymorphisms has been performed to determine the factors of genetic predisposition to essential hypertension. Polymerase chain reaction (PCR) with subsequent analysis of restriction fragment length, allele specific PCR or real-time PCR was used for genotyping of 17 single nucleotide polymorphisms in 14 genes in 145 children with essential hypertension and 144 healthy persons with following complex multivariate statistical analysis. Two single nucleotide polymorphisms--MMP9 (C(-1562) --> T) and NOS3 (Glu298 --> Asp)--rs3918242 and rs1799983--were shown to represent the main independent effects with the highest predictive potential (77.1% as indicated by binary logistic regression and 74.6% testing accuracy shown by Multifactorial Dimensionality Reduction). MMP9 (C(-1562 --> T) and NOS3 (Glu298 --> Asp) potentially may be used to create predictive algorithm for determination of predisposition to arterial hypertension in children.

The association of gene polymorphisms with athlete status in ukrainians.

UNLABELLED: Athletic performance is a polygenic trait influenced by both environmental and genetic factors. OBJECTIVE: To investigate individually and in combination the association of common gene polymorphisms with athlete status in Ukrainians. METHODS: A total of 210 elite Ukrainian athletes (100 endurance-oriented and 110 power-orientated athletes) and 326 controls were genotyped for ACE I/D, HIF1A Pro582Ser, NOS3 -786 T/C, PPARA intron 7 G/C, PPARG Pro12Ala and PPARGC1B Ala203Pro gene polymorphisms, most of which were previously reported to be associated with athlete status or related intermediate phenotypes in different populations. RESULTS: Power-oriented athletes exhibited an increased frequency of the HIF1A Ser (16.1 vs. 9.4%, P = 0.034) and NOS3 T alleles (78.3 vs. 66.2%, P = 0.0019) in comparison with controls. Additionally, we found that the frequency of the PPARG Ala allele was significantly higher in power-oriented athletes compared with the endurance-oriented athletes (24.7 vs. 13.5%; P = 0.0076). Next, we determined the total genotype score (TGS, from the accumulated combination of the three polymorphisms, with a maximum value of 100 for the theoretically optimal polygenic score) in athletes and controls. The mean TGS was significantly higher in power-oriented athletes (39.1 ± 2.3 vs. 32.6 ± 1.5; P = 0.0142) than in controls. CONCLUSIONS: We found that the HIF1A Ser, NOS3 T and PPARG Ala alleles were associated with power athlete status in Ukrainians.

Expression of TNF-alpha mRNA, but not of TNF-alpha receptors mRNA, is detected in single murine oocyte and decreases during oocyte meiotic maturation: single-cell RT-PCR data.

BACKGROUND: The present study was conducted to investigate the expression of TNF-alpha and its receptors (types I and II) in both oocytes with germinal vesicle and the first polar body in mice. METHODS: Oocytes with intact germinal vesicle were isolated from mouse ovaries and subjected to in vitro maturation to obtain oocytes forming the first polar body. A reverse transcription polymerase chain reaction (RT-PCR) was used to examine the expression of TNF-alpha and its receptors at mRNA level. RESULTS: mRNA TNF-alpha was expressed in single oocytes and its level was decreasing during transition from germinal vesicle to the first polar body stage. At the same time the expression of TNF-receptors was not observed in single oocyte. CONCLUSIONS: These data are the important link in understanding of the molecular mechanisms regulating oocyte maturation as well as follicle development.

[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.

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.

[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.