Blood Level of Nitric Oxide Metabolites and Expression of Genes Regulating NO Synthesis in Early Forms of Non-Alcoholic Fatty Liver Disease.

The levels of NO metabolites in the plasma and mRNA of the NOS3, ATG9B, and NOS2 genes in peripheral blood leukocytes of healthy people and patients with early forms of non-alcoholic fatty liver disease (steatosis and weak activity non-alcoholic steatohepatitis) were studied. In patients with steatohepatitis, the concentration of NO metabolites in the blood and the level of mRNA of the NOS2 gene were higher than in patients with steatosis and healthy people. These differences can be of diagnostic value for distinguishing between steatosis and weak activity steatohepatitis in non-alcoholic fatty liver disease. A correlation between the levels of NO metabolites and the expression of the NOS2 gene in weak activity steatohepatitis was established, which indicates activation of NO synthesis in non-alcoholic steatohepatitis due to the expression of the inducible NO synthase gene. The level of the NOS2 gene mRNA in peripheral blood leukocytes of patients with weak activity steatohepatitis correlated with the level of TNFα and IL-6 cytokines. An increase in the level of NO in the blood in weak activity steatohepatitis correlated with the level of MDA, an indicator of oxidative stress.

Quantitative Assay of SARS-CoV-2 RNA and Level of Proinflammatory Protein Gene Transcripts in Peripheral Blood Leukocytes after a Novel Coronavirus Infection.

The possibility of finding persistent SARS-CoV-2 viral particles in human peripheral blood leukocytes after a novel coronavirus infection was shown. The results of droplet digital PCR showed that 19 of 24 examined subjects had from 4 to 555 copies of the Nsp4 SARS-CoV-2 gene in 5-6 months after infection. The presence of this transcript in peripheral blood leukocytes was associated with reduced expression of FOXP3 gene and increased level of RORγ gene mRNA. The copy number of the Nsp4 gene negatively correlated with the level of FOXP3 gene mRNA (r=-0.45; p=0.028), but showed a positive correlation with the DANCR long non-coding RNA (r=0.94; p<0.001). In SARS-CoV-2-positive healthy individuals, the level of TLR2, NLRP3, and IL1B gene transcripts was higher than in SARS-CoV-2-negative donors. The presence of SARS-CoV-2 in a persistent form is probably associated with impaired immunosuppression and the development of chronic inflammation in apparently healthy volunteers after a new coronavirus infection.

The Role of Inducible NOS2 Gene Polymorphism in the Development of Essential Arterial Hypertension.

The risk of essential arterial hypertension was assessed in carriers of the NOS2 gene variants (rs1800482 (-954G>C), rs3730017 (C>T)). In subjects carrying C allele (rs1800482), the risk for essential arterial hypertension developing was higher by 1.7 times (OR=1.712, 95%CI 1.07-2.74), while the presence of T-allele (rs3730017) had a protective effect (OR=0.304, 95%CI 0.192-0.482). In patients with essential arterial hypertension, the presence of the C allele (rs1800482) was associated with a higher content of NO metabolites in the blood plasma. A positive correlation was found between the plasma content of nitrites and nitrates and the level of transcripts of VCAM1, ICAM1 genes in peripheral blood leukocytes. We found the influence of the C allele carriership on the expression VCAM1 and ICAM1 genes in patients with essential hypertension. It was hypothesized that this polymorphic site in the NOS2 gene can be involved in the development of endothelial dysfunction and essential arterial hypertension through modulation of NO level under condition of inflammation.

Analysis of the Association of TNF -238G>A Gene Polymorphism with the Risk of Rheumatoid Arthritis Development in Russian Population in the Republic of Karelia.

We studied association of the TNF gene -238G>A polymorphism (rs361525) with the risk of rheumatoid arthritis development in the Russian population living in the Republic of Karelia. The influence of rs361525 on the development of rheumatoid arthritis was revealed: genetic predisposition to this disease is associated with the presence of GG genotype. The effect of the genotype on the polymorphic locus of -238G>A on TNF mRNA content was revealed. Increased content of transcripts of this gene is associated with the presence of A allele.

DICER and DROSHA gene expression in peripheral mononuclear blood cells from rheumatoid arthritis patients under methotrexate therapy.

AIM: The aim of this research was studying the level of DROSHA and DICER genes expression in peripheral mononuclear blood cells (PBMC) in rheumatoid arthritis (RA) patients under methotrexate therapy. MATERIALS AND METHODS: 82 people (from 45 to 70 years) enrolled in this study were divided into 3 groups (the first one - healthy donors (n=33 median age 49.93±1.87); the second group - rheumatoid arthritis patients without any therapy (n=15; median age 57.28±15.18) and the third group (n=34; median age 60.88±9.02) - rheumatoid arthritis patients who treated at least 4 weeks with methotrexate therapy (10-20 mg/week). The DICER and DROSHA genes expression level was determined by real-time PCR. RESULTS: The number of DICER gene transcripts in PBMC in rheumatoid arthritis patients without therapy as in RA patients treated with methotrexate was reduced in comparison with the healthy donors (p<0.001 and p>0.05 respectively). The level of DROSHA gene expression in PBMC was not significantly different in all groups enrolled in this study (p>0.05). CONCLUSION: Our findings suggest that that the DICER gene expression level in perifheral mononuclear blood cells decreased with the development of rheumatoid arthritis. Methotrexate doesn't influence on the mRNA level of this gene.

Expression of DROSHA and DICER genes in peripheral blood leukocytes in lung sarcoidosis.

AIM: To study the expression level of the genes DROSHA and DICER in peripheral blood leukocytes (PBL) of patients with sarcoidosis of the lungs. MATERIALS AND METHODS: The study included 32 patients diagnosed with persistent lung sarcoidosis (mean age 41.56±1.27 years) and 36 healthy donors (control; mean age 42.79±1.95 years). The level of expression of messenger RNA (mRNA) of the genes DROSHA and DICER were determined in PBL of healthy donors and patients with sarcoidosis of the lung by polymerase chain reaction in real time. RESULTS: As a result of the conducted researches it is established that the level of drosha gene expression in PBL patients with sarcoidosis of lungs is significantly reduced in comparison with the control (p<0.01). We also found a significant decrease in the number of Dicker gene transcripts in the PBL of the study group of patients (p<0.01). CONCLUSION: According to the results of the conducted studies, a significant decrease in the number of DROSHA and DICER transcripts in PBL patients with the development of lung sarcoidosis has been found, which can contribute to the pathogenesis of this disease.

[Analysis of an association of TNF -308G>A polymorphism with a risk for pulmonary sarcoidosis in the Russian population of the Republic of Karelia]. / Analiz assotsiatsii polimorfizma -308G>A gena TNF s riskom razvitiya sarkoidoza legkikh u russkogo naseleniya Respubliki Kareliya.

AIM: To analyze an association of TNF -308G>A polymorphism with a risk for pulmonary sarcoidosis (PS) in the Russian population of the Republic of Kareli. SUBJECTS AND METHODS: 84 patients with persistent PS and 96 donors without clinical manifestations of this disease (a control group) were examined. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was used to identify alleles and genotypes by the marker of TNF -308G>A polymorphism. The level of transcripts of the above gene in the peripheral blood leukocytes of healthy and sick people was determined by real-time PCR. RESULTS: There were no significant differences in the distribution of allelic and genotypic frequencies by the marker of TNF -308G>A polymorphism between the control and PS patient groups. There was a significant increase in the number of TNF gene transcripts in the peripheral blood leukocytes of patients with PS compared to the controls. At the same time, there were no marked differences in mRNA expression levels in the above gene in the carriers of different genotypes by the marker of TNF -308G>A polymorphism in all the examined groups. CONCLUSION: The marker of TNF -308G>A polymorphism is unassociated with the risk of PS in the Russian population of the Republic of Karelia. No differences in TNF mRNA levels in the carriers of different genotypes by the above marker may suggest that the found elevated level of transcripts in the above gene in patients with diagnosed with PS is due to the development of the body's inflammatory responses in this disease.

[Association of FOXP3 gene -3279 C>A polymorphism with the risk of pulmonary sarcoidosis]. / Assotsiatsiia polimorfizma -3279 C>A gena FOXP3 s riskom razvitiia sarkoidoza legkikh.

AIM: To investigate the association of the polymorphic marker -3279 C>A of the FOXP3 gene with the risk of pulmonary sarcoidosis (PS) and to estimate the transcription level of this gene in the carriers of different genotypes of this polymorphic marker. SUBJECTS AND METHODS: The investigation included 99 patients of Russian ethnicity (mean age, 45.41±1.31 years) living in the Republic of Karelia, who were diagnosed with persistent PS, and 116 healthy donors (mean age, 42.06±1.30 years) in the control group. The alleles and genotypes of the polymorphic marker -3279 C>A of the FOXP3 gene were identified using polymerase chain reaction (PCR)-restriction fragment length polymorphism. The number of transcripts of the studied gene in the peripheral blood leukocytes of healthy donors and PS patients was determined with real-time PCR. RESULTS: The control group and the PS patient one had no statistically significant differences in the distribution of the frequencies of alleles and genotypes by the polymorphic marker -308G>A of the FOXP3 gene (p > 0.05). The number of FOXP3 gene transcripts was not statistically significantly different in the peripheral blood leukocytes of patients with PS and control individuals. No statistically significant differences were observed in the mRNA expression levels in the above-mentioned gene in the carriers of different genotypes by the polymorphic marker -3279 C>A of the FOXP3 gene in all examined groups. CONCLUSION: The polymorphic marker -3279 C>A of the FOXP3 gene is unassociated with the risk of PS.

The level of cytokines and expression of caspase genes in rheumatoid arthritis.

The level of TNFα and IL6 in the blood plasma of patients with rheumatoid arthritis (RA) who received antiinflammatory therapy with methotrexate (MT) was significantly lower than in the patients without MT treatment. The level of caspase 6 and 9 gene transcripts in peripheral blood lymphocytes in patients with rheumatoid arthritic diagnosed for the first time and in patients with MT treatment were not significantly different. At the same time, the level of caspase 3 mRNA expression was significantly higher in the cells of the RA patients with MT therapy compared to the patients without MT therapy.

[Genetic diversity of insular natural populations of Festuca pratensis Huds.: RAPD analysis].

In natural populations of Festuca pratensis Huds. from the islands of Onega Lake, the level of genetic diversity was evaluated. In three populations variability of 64 RAPD loci was tested. The level of genetic diversity (P95% = 30.2; Hexp = 0.093) was low for a cross-pollinating plant species. Furthermore, genetic similarity between the plants from insular populations was found to be high (IN = 0.887). It was demonstrated that genetic variation among the population accounted for at most 5.3% of total genetic diversity, which, however, was higher than the FST values for continental populations (FST = 0.022). It was suggested that specific features of the genetic structure of insular population, i.e., low gene diversity within the populations along with high differentiation among the populations, were caused by the gene flow attenuation, as a result of isolation, and intensification of inbreeding. These features had negative effect on total population adaptation.

[Cellular mechanisms of induction of apoptosis in human erythroleukaemic K562 cells line treated by quinoline-N-oxide derivatives].

We have studied the influence of 2-(4'-nitrostyryl)-quinoline-1-oxide (2-NSQO) and 4-(4'-nitrostyryl)-quinoline-1-oxide (4-NSQO) on modulation activity of microsomal NADPH-oxidoreductases, concentration of nicotinamide enzymes and induction of apoptosis in human erythroleukaemic K562 cells. It was shown, that activity of microsomal NADPH-cytochrome c-reductases in cancer cells was inhibited by 10 microM 4-NSQO (15%), and 10 microM 2-NSQO (50%). Treatment of cells with these reagents for two days, was accompanied by caspases-9 and -3 activation, rise of EtBr and DAPI fluorescence related to DNA binding and induction of apoptosis. Apoptosis was preceded by the decrease of concentration of nicotinamide enzymes. Thus 4-NSQO is a promising compound for further experimental trials as an anticancer drug with low toxic action on the tissues of organism.

[Phorbol 12-myristate 13-acetate inhibits apoptosis in erythroleukemia K562 cells induced by some nucleosides].

We studied the ability of phorbol 12-myristate 13-acetat to prevent erythroid differentiation and apoptosis in erythroleukemic K562 cells induced by cytidine, thymidine, and guanosine. The exposure of cancer cells to combinations of phorbol 12-myrsitate 13-acetate (100 nM) nucleosides for two days led to a loss of hemoglobin production (marker of erythroid differentiation) in cells and increased expression of monocyte-macrophage lineage associated surface antigen CD14. The treatment of K562 cells with nucleosides only was accompanied by the activation of caspase-3 and caspase-9, rather than caspase-6, increased fluorescence of ethidium bromide and DAPI upon binding to DNA, and apoptosis. Intracellular activation of caspase-6, inhibition of caspase-9, a markedly decreased activity of caspase-3 and of fluorescence of DNA-binding dyes, and inhibition of apoptosis were observed when the cells were treated with phorbol 12-myeristet 13-acetate combined with nucleosides.

[Comparative analysis of differentiating and apoptogenic effects of cytidine, thymidine, and guanosine on cells of human erythromyeloleukemic cell line K562]. / Sravnitel'nyi analiz differentsiruiushchego i apoptogennogo deistvii tsitidina, timidina i guanozina na kletki éritromieloleikoznoi linii cheloveka K562.

The antiproliferative, differentiating and apoptogenic effects of purinic (cytidine and thymidine) and pyrimidinic (guanosine) nucleosides on K562 cells were investigated. Induction of erythroid differentiation (haemoglobin synthesis) in cancer cells was observed after 2 and 4 days incubation with 2 mM thymidine and 1 mM guanosine. Increase of haemoglobin synthesis in K 562 cells incubated with 2 mM cytidine was detected at 4 day. Analysis of the DNA-degrading effect (induction of apoptosis) of the reagents used on K 562 cells by fluorescent dyes and modulation activity of caspase 9 and also caspases 3,7,10 is induced with thymidine and guanosine but not with cytidine.