The aim of our work was to study Na -dependent ATPase activity in cellfree extracts of the sulfate-reducing bacteria Desulfovibrio piger Vib-7 and Desulfomicrobium sp. Rod-9 isolated from the human large intestine, and to carry out the kinetic analysis of the enzyme reaction. The maximum ATPase activity for both bacterial strains at +35 ºC was determined. The highest activities of the studied enzyme in the cell-free extracts of D. piger Vib-7 at pH 7.0 and Desulfomicrobium sp. Rod-9 at pH 6.5 were measured. Based on experimental data, the analysis of kinetic properties of the ATP-hydrolase reaction by the studied bacteria was carried out. The enzyme activity, initial (instantaneous) reaction rate (V 0 ) and maximum rate of the ATPase reaction (V max ) was significantly higher in D. piger Vib-7 cells than in Desulfomicrobium sp. Rod-9. Michaelis constants (K m ) of the enzyme reaction for both bacterial strains were determined. ATPase activity, hydrogen sulfide, intestinal microbiocenosis, bowel diseases, ulcerative colitisSulfate-reducing bacteria carry out the dissimilatory sulfate reduction during anaerobic respiration (Barton and Hamilton 2007). The final product of the sulfate reduction in the human intestine is hydrogen sulfide which is carcinogenic to its cells, and can cause inhibition of cytochrome oxidase, colonocytes oxidation of butyrate, destruction of epithelial cells, development of ulcers, and inflammation with subsequent development of colon cancer (Pitcher and Cummings 1996;Gibson et al. 1991;Cummings et al. 2003). The transport of sulfate ions and organic compounds in the cytoplasm of the bacterial cells occurs through active transport using ATP energy (Barton and Hamilton 2007). In this regard, it is very important to study the mechanisms of sulfate ions transport, enzymatic activity and kinetic properties of other ATP-dependent enzymes of sulfatereducing bacteria from human intestine.Plentiful data are available on the functions of biological membranes including the integral membrane protein ATP-dependent systems of transport ions (Lodish et al. 2000;Yuan et al. 2005;Tian et al. 2006). However, the ATPase activity of the sulfate-reducing bacteria Desulfovibrio piger and Desulfomicrobium sp. isolated from the human large intestine has not been studied yet. A comprehensive study of the functioning and role of Na + /K + -pump as a system of energy-dependent transport of different ions in the regulation of the dissimilatory sulfate reduction and accumulation of hydrogen sulfide will enable to form a holistic view on the participation of these systems in maintaining ion homeostasis of the sulfate-reducing bacteria cells.The aim of our work was to study Na-dependent ATPase activity in cell-free extracts of the sulfate-reducing bacteria Desulfovibrio piger Vib-7 and Desulfomicrobium sp. Rod-9 isolated from the human large intestine, and to carry out the kinetic analysis of the enzyme reaction.
The level of the products of lipid peroxidation measured as the thiobarbituric acid reactive substances (TBARS) and activity of enzymes of glutathione antioxidant protection system in spermatozoa were studied. It was shown that mean levels of TBARS in sperm cells of infertile men with oligozoo-, astenozoo-, oligoastenozoo-and leucocytospermia were 1.6-, 2.0-, 2.0-and 2.7-fold higher, respectively, compared with such levels in the fertile men. The glutathione peroxidase activity was decreased 2.2-fold in patients with oligozoospermia, 2.8-fold in patients with astenozoo-and oligoastenozoospermia and 3.8-fold in patients with leucocytospermia. It was found that glutathione reductase activity in patients with oligozoo-, astenozoo-, oligoastenozoo-and leucocytospermia were 2.3-, 1.6-, 1.7-and 3.3-fold lower than that in normozoospermic men, respectively. In addition, the glutathione transferase activity was decreased 1.8-2.1-fold in patients with oligozoo-, astenozoo-and oligoastenozoospermia and 5.0-fold in patients with leuco cytospermia in comparison with healthy donors. The most significant changes were observed in the infertile men with leucocytospermia. This manifestation could be explained by the fact that white blood cells stimulate the formation of reactive oxygen species, induction and development of oxidative stress in sperm cells.
SummaryBackgroundInfertility is an important worldwide problem which affects 10–15% of couples globally. Altered NO production has also been implicated in the pathogenesis of the male infertility. The present study was designed to evaluate the changes in the activity of NO-synthase (NOS) and arginase in spermatozoa of patients with infertility.MethodsThe total NOS, Ca2+-dependent constitutive (cNOS) and Ca2+-independent inducible (iNOS) activity and arginase activity were assessed in sperm cells of patients with different forms of pathospermia.ResultsWe found a significant increase in iNOS activity, but significantly decreased cNOS and arginase activity in sperm cells of infertile men vs fertile, normozoospermic men (p<0.001). The arginase/NOS ratio significantly decreased compared to control group. The iNOS/cNOS ratio was drastically increased in patients with decreased fertility potential indicating predominance of iNOS. Men with leuko cytospermia were distinguished to have the most express iNOS activity.ConclusionsThese observations provide evidence for a disturbed balance between the L-arginine metabolic pathways in sperm cells of infertile men. This imbalance includes the considerable activation of the inducible isoform of NO-synthase accompanied by significant inhibition of its constitutive isoform which indicates disturbances in NO production. In patients with decreased fertility potential the arginase/NOS was shifted towards predominance of iNOS-derived NO production.
Background: Inflammatory bowel diseases (IBDs) are multifactorial illnesses of the intestine, to which microorganisms are contributing. Among the contributing microorganisms, sulfate-reducing bacteria (SRB) are suggested to be involved in the process of bowel inflammation due to the production of hydrogen sulfide (H2S) by dissimilatory sulfate reduction. The aims of our research were to physiologically examine SRB in fecal samples of patients with IBD and a control group, their identification, the study of the process of dissimilatory sulfate reduction (sulfate consumption and H2S production) and biomass accumulation. Determination of biogenic elements of the SRB and evaluation of obtained parameters by using statistical methods were also included in the research. The material for the research consisted of 14 fecal samples, which was obtained from patients and control subjects. Methods: Microscopic techniques, microbiological, biochemical, biophysical methods and statistical analysis were included. Results: Colonies of SRB were isolated from all the fecal samples, and subsequently, 35 strains were obtained. Vibrio-shaped cells stained Gram-negative were dominant in all purified studied strains. All strains had a high percentage of similarity by the 16S rRNA gene with deposited sequences in GenBank of Desulfovibrio vulgaris. Cluster analysis of sulfate reduction parameters allowed the grouping of SRB strains. Significant (p < 0.05) differences were not observed between healthy individuals and patients with IBD with regard to sulfate reduction parameters (sulfate consumption, H2S and biomass accumulation). Moreover, we found that manganese and iron contents in the cell extracts are higher among healthy individuals in comparison to unhealthy individuals that have an intestinal bowel disease, especially ulcerative colitis. Conclusions: The observations obtained from studying SRB emphasize differences in the intestinal microbial processes of healthy and unhealthy people.
Background: Oxidative stress is considered as one of the causes of male subfertility or infertility. Among antioxidant enzymes, the crucial role belongs to glutathione S -transferases (GSTs). Data on the biological role of GSTs in the defense mechanisms of sperm cells in fertile and infertile men are limited. Aim: The aim of this study was to demonstrate the functional role of GSTs in sperm cells on the model of H 2 O 2 -induced stress on human ejaculated spermatozoa obtained from both normospermic and pathospermic patients. Subjects and Methods: We used a H 2 O 2 -induced stress on human ejaculated spermatozoa obtained from both normospermic and pathospermic patients. Results: Here, we report the effect of GST inhibitor ethacrynic acid on sperm motility and viability. Pharmacological inhibition of sperm GSTs activity leads to spermal membrane damage and rejected in the loss of motility and decrease of viability. For similar treatment conditions, thiobarbituric acid reactive substance (TBARS) levels increased significantly leading to decrease in sperm motility and viability. It is suggested that these functional impairments are related to the intensification of lipid peroxidation as expressed by TBARS levels in spermal membranes after GST inhibitor treatment. Conclusion: This study provides evidence that sperm GSTs are important in the defense mechanism against oxidative stress. Evaluation of GSTs activity in sperm cells of infertile men can be helpful in fertility assessment and for the evaluation of treatment by antioxidants.
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