Biochemistry (Moscow)

Hyperosmotic stimulation of endothelial cells often leads to its dysfunction accompanied, among other things, by proinflammatory response. The mechanisms of this phenomenon are not fully understood. It may arise due to increase in the plasma Na+ concentration, due to increase in the extracellular osmolarity, increase in the intracellular Na+i/K+i ratio, and/or change in the cell stiffness. In the present study we investigated the effects of short-term increase in osmolarity of extracellular medium on the mRNA content of some genes important for endothelial function (including Na+i/K+i-sensitive ones) and the equivalent elasticity constant of human umbilical vein endothelial cells membranes. Hyperosmotic stimulation of these cells with NaCl but not mannitol resulted in accumulation of Na+ ions inside the cells despite the Na,K-ATPase activation, and was also accompanied by the decrease in their equivalent elasticity constant. The amount of IL1α mRNA decreased with increasing osmolarity of the extracellular medium, whereas the amount of ATF3, PAR2, and PTGS2 mRNAs increased only in response to the increasing NaCl concentration. At the same time, under the conditions of our experiments, we did not detect changes in the expression of the osmoprotective transcription factor NFAT5. The obtained data indicate that the increase of extracellular Na+ concentration in the physiological range is an independent factor that affects intracellular Na+i/K+i ratio and regulates expression of some genes (in particular, ATF3, PAR2, PTGS2) in endothelial cells.

Several ferryl states of the catalytic heme a3-CuB center of the respiratory cytochrome c oxidases (CcOs) are observed during the reduction of O2 to H2O. One of the P-type ferryl forms, PM, is produced by the reaction of the two-electron reduced CcO with O2. In this state, the heme a3 iron is in the ferryl state and a free radical should be also present at the catalytic center. However, the energetics of the PM formation has not been experimentally established yet. Here, the generation of PM by the reaction of oxidized bovine CcO (O) with one molecule of H2O2 was investigated by the isothermal titration calorimetry and UV-Vis absorption spectroscopy. Two kinetic phases, corresponding to the formation of PM and its endogenous conversion back to O, were resolved by both methods. The ΔH of the entire process (–66 kcal/mol H2O2) was larger than the heat (–50.8 kcal/mol O2) liberated during O2 reduction by ferrocytochrome c (pH 8, 25°C). Interestingly, ΔH of the first phase (–32 kcal/mol ferryl state) far exceeds the enthalpy of the PM production. The data indicate that during the first phase, the radical in PM is quenched and spectrally similar second P-type ferryl form (PR) is produced. Additionally, it was shown that the entropy contribution to the Gibbs energy change (ΔG = –46 kcal/mol O2) during the catalytic reduction of O2 by ferrocytochrome c is negligible (–0.7 cal·mol–1·K–1).

Genomes of photoautotrophic organisms containing type I photosynthetic reaction center were searched for the rnf genes encoding Na+-translocating ferredoxin:NAD+ oxidoreductase (RNF). These genes were absent in heliobacteria, cyanobacteria, algae, and plants; however, genomes of many green sulfur bacteria (especially marine ones) were found to contain the full rnf operon. Analysis of RNA isolated from the marine green sulfur bacterium Chlorobium phaeovibrioides revealed a high level of rnf expression. It was found that the activity of Na+-dependent flavodoxin:NAD+ oxidoreductase detected in the membrane fraction of Chl. phaeovibrioides was absent in the membrane fraction of the freshwater green sulfur bacterium Chlorobaculum limnaeum, which is closely related to Chl. phaeovibrioides but whose genome lacks the rnf genes. Illumination of the membrane fraction of Chl. phaeovibrioides but not of Cba. limnaeum resulted in the light-induced NAD+ reduction. Based on the obtained data, we concluded that in some green sulfur bacteria, RNF may be involved in the NADH formation that should increase the efficiency of light energy conservation in these microorganisms and can serve as the first example of the use of Na+ energetics in photosynthetic electron transport chains.

This review discusses current knowledge on signal transduction pathways controlling chemotaxis of neutrophils and similar cells. Most neutrophil chemoattractants bind to seven-transmembrane-helix receptors. These receptors activate trimeric G proteins of the Gi class in neutrophils to initiate chemotaxis. Phospholipases Cβ, phosphoinositide 3-kinase γ, and PH domain-containing proteins play various roles in signaling further downstream. The actin cytoskeleton is crucial for cell motility, and is controlled by Rhofamily GTP-binding proteins. PIP 5-kinase, LIM kinase, myosin light chain kinase and phosphatase, or WASP-like proteins may be important links between Rho GTPases and actin during chemotaxis. Newly emerging ideas on the regulation of the “compass” of chemotaxing cells, which may involve Cdc42 and certain PH domain-containing proteins, are also presented.

The review discusses modern methods for the quantitative and semi-quantitative analysis of miRNAs, which are small non-coding RNAs affecting numerous biological processes such as development, differentiation, metabolism, and immune response. miRNAs are considered as promising biomarkers in the diagnosis of various diseases.

Cancer cells experience strong oxidative stress caused by disorders in cell metabolism and action of external factors. For survival, cancer cells have developed a highly efficient system of antioxidant defense, some of the most important elements of which are peroxiredoxins (Prxs). Prxs are an evolutionarily ancient family of selenium-independent peroxidases that reduce a wide range of organic and inorganic hydroperoxides in the cell and the extracellular space. In addition, some Prxs exhibit chaperone and phospholipase activities. Prxs play an important role in the maintenance of the cell redox homeostasis; they prevent oxidation and aggregation of regulatory proteins, thereby affecting many cell signaling pathways. Prxs are involved in the regulation of cell growth, differentiation, and apoptosis. Due to their versatility and wide representation in all tissues and organs, Prxs participate in the development/suppression of many pathological conditions, among which cancer occupies a special place. This review focuses on the role of Prxs in the development of various forms of cancer. Understanding molecular mechanisms of Prx involvement in these processes will allow to develop new approaches to the prevention and treatment of cancer.

The conversion of uridine diphosphate N-acetyl-D-glucosamine into uridine diphosphate N-acetyl-L-fucosamine was demonstrated with enzymes from cytoplasmic fraction of Salmonella arizonae O:59 cells in the presence of NAD+ (NADP+) and NADPH. The reaction product was identified by ion-pair, reverse-phase HPLC with the use of synthetic nucleoside diphosphate sugar standards under conditions specially developed for separation of uridine diphosphate 2-acetamido-2,6-dideoxyhexoses. L-Fucose dehydrogenase from porcine liver was shown to be applicable for determination of N-acetyl-L-fucosamine, this enzyme being used to confirm L-configuration of the amino sugar residue in the sugar nucleotide formed.