Enhanced enzymatic production of cholesteryl 6ʹ-acylglucoside impairs lysosomal degradation for the intracellular survival of Helicobacter pylori

Background During autophagy defense against invading microbes, certain lipid types are indispensable for generating specialized membrane-bound organelles. The lipid composition of autophagosomes remains obscure, as does the issue of how specific lipids and lipid-associated enzymes participate in autophagosome formation and maturation. Helicobacter pylori is auxotrophic for cholesterol and converts cholesterol to cholesteryl glucoside derivatives, including cholesteryl 6ʹ-O-acyl-α-d-glucoside (CAG). We investigated how CAG and its biosynthetic acyltransferase assist H. pylori to escape host-cell autophagy. Methods We applied a metabolite-tagging method to obtain fluorophore-containing cholesteryl glucosides that were utilized to understand their intracellular locations. H. pylori 26695 and a cholesteryl glucosyltransferase (CGT)-deletion mutant (ΔCGT) were used as the standard strain and the negative control that contains no cholesterol-derived metabolites, respectively. Bacterial internalization and several autophagy-related assays were conducted to unravel the possible mechanism that H. pylori develops to hijack the host-cell autophagy response. Subcellular fractions of H. pylori-infected AGS cells were obtained and measured for the acyltransferase activity. Results The imaging studies of fluorophore-labeled cholesteryl glucosides pinpointed their intracellular localization in AGS cells. The result indicated that CAG enhances the internalization of H. pylori in AGS cells. Particularly, CAG, instead of CG and CPG, is able to augment the autophagy response induced by H. pylori. How CAG participates in the autophagy process is multifaceted. CAG was found to intervene in the degradation of autophagosomes and reduce lysosomal biogenesis, supporting the idea that intracellular H. pylori is harbored by autophago-lysosomes in favor of the bacterial survival. Furthermore, we performed the enzyme activity assay of subcellular fractions of H. pylori-infected AGS cells. The analysis showed that the acyltransferase is mainly distributed in autophago-lysosomal compartments. Conclusions Our results support the idea that the acyltransferase is mainly distributed in the subcellular compartment consisting of autophagosomes, late endosomes, and lysosomes, in which the acidic environment is beneficial for the maximal acyltransferase activity. The resulting elevated level of CAG can facilitate bacterial internalization, interfere with the autophagy flux, and causes reduced lysosomal biogenesis.

CONTENT OF PRIMARY AND SECONDARY LIPID PEROXIDATION PRODUCTS IN SUBCELLULAR FRACTIONS OF CARDIOMYOCYTES DURING MYOCARDIAL INFARCTION IN RATS IN AN EXPERIMENT AND THEIR CORRECTION BY TRANSPLANTATION OF MESENCHYMAL STEM CELLS

Experimental modeling of myocardial infarction in rats was carried out by ligation of the anterior intergastric artery after the first division. There were 3 groups of 20 animals each: control group I — to verify normal parameters, group II — a model of myocardial infarction, and group III — animals which, after modeling myocardial infarction, underwent transplantation of mesenchymal stem cells. The level of lipid peroxidation products — diene conjugates and malondialdehyde — was studied by spectrophotometry. Comparison of the content and their ratio in the cytoplasm and mitochondria of myocardiocytes was carried out. It turned out that transplantation of mesenchymal stem cells significantly levels the activation of lipid peroxidation processes in subcellular fractions of cardiomyocytes, which is accompanied by a decrease in the primary and secondary products of oxidative stress. The ratio of malondialdehyde to diene conjugates both in the cytoplasm and in the mitochondria of cardiomyocytes after transplantation returned to control values. This indicates the normalization of physiological processes with underlying ischemic heart damage. The results indicate the cytoprotective effect of mesenchymal stem cell transplantation and the preservation of a larger number of cell pools, compared with the control group of animals that did not receive any treatment.

The effect of prenatal stress on antioxidant glutathion-assosiated enzymes activity in subcellular fractions of rat's liver

The activity of glutathione-associated antioxidant enzymes in subcellular fractions (cytosolic, mitochondrial, and cell nucleus fractions) was investigated in the liver of adult male Wistar rats born after prenatal stress was. Two groups of animals were studied in the experiment: (1) control group included — animals was born by intact mothers, and (2) prenatal stress group included animals whose mothers were subjected to immobilization stress in high-light conditions from the 15th to the 19th day of pregnancy. The activity of glutathione peroxidase (EC 1.11.1.9) in prenatally stressed animals decreased in the fractions of nuclei and mitochondria compared to the control group, while the activity of glutathione reductase (EC 1.8.1.7.) increased in the same subcellular fractions. The activity of glutathione transferase (EC 2.5.1.18) in prenatally stressed rats reduced in the cytosol and mitochondrial fractions as compared to control group. Redistribution of the antioxidant enzyme activity in the cytosol, the fraction of nuclei and the mitochondrial fraction of liver tissue may contribute to the formation of the pathological phenotype of prenatally stressed offspring.

Oxidative carbonilation of liver tissue proteins under the influence of pesticide based on glyphosate in a subchronic experiment

Introduction. Currently, the pathogenetic mechanisms of the action on the body of widely used glyphosate-based herbicides and the search for the markers of the health status that changes under the influence of these herbicides remain open for study and discussion. Carbonyl derivatives of proteins can be used as the markers of oxidative stress caused by the herbicidal composition of the isopropylamine salt of glyphosate. The purpose of this study was to assess the severity of oxidative carbonylation of liver tissue proteins under the influence of a glyphosate-based pesticide in a subchronic experiment. Material and methods. The material for the study was subcellular fractions of liver tissue homogenates obtained from 90 Wistar rats, which were administered solutions of glyphosate isopropylamine salt at doses of 280 mg/kg and 100 mg/kg per os for 3 months. The severity of oxidative carbonylation of proteins was determined by the method of R.L. Levine modified by E.E. Dubinina. Results. Statistically significant changes in the content of products of oxidative damage to proteins at various stages of the experiment in the study groups were obtained compared with the control group due to a significant increase in the products of oxidative damage to proteins in subcellular fractions of liver tissue. Conclusion. The changes in carbonyl stress indices and the decrease in the reserve-adaptive potential indicate the depletion of antioxidant protection in the liver cells. A statistically significant increase in secondary markers of carbonyl stress after 1 month from the start of seeding of animals compared with the control with a slight increase in this index after 3 months may indicate the launch of adaptation mechanisms, including the induction of proteolytic utilization of oxidized proteins or additional protein synthesis.