[MicroRNA Biogenesis in Cell Senescence Induced by Chronic Endoplasmic Reticulum Stress].

MicroRNAs are small noncoding RNAs that regulate gene expression; stabilize the cell phenotype; and play an important role in cell differentiation, development, and apoptosis. A canonical microRNA biogenesis pathway includes several posttranscriptional steps of processing and transport and ends with cytoplasmic cleavage of pre-miRNA by type III ribonuclease DICER to form a mature duplex, which is included in RISC. MicroRNA biogenesis and role in cell stress are still poorly understood. Using flow cytometry and high-throughput analysis of gene expression, we have shown that chronic endoplasmic reticulum (ER) stress, which is associated with improper protein folding in the ER, induce a cellular senescence phenotype in fibroblast-like FRSN cells. While acute ER stress can reduce miRNA biogenesis, chronic stress does not cause a significant drop in global microRNA expression and is accompanied by only a slight decrease in DICER1 mRNA expression. Heterogeneity with respect to lysosomal ß-galactosidase activity was found to increase in the cell population exposed to ER stress. We do not exclude induced cell heterogeneity regarding expression of components of the microRNA biogenesis pathway.

[Protein Homeostasis Dysregulation in Pathogenesis of Neurodegenerative Diseases].

The formation and accumulation of unfolded, misfolded, or damaged cellular proteins leads to development of endoplasmic reticulum stress (ER stress). A series of protective reactions is initiated in response to ER stress. These reactions are aimed at restoring the balance between protein synthesis and degradation, which is key to maintaining protein homeostasis (proteostasis). The main protective mechanisms are the attenuation of protein synthesis, increase of chaperone levels, and activation of protein degradation systems. Insufficiency or malfunction of these mechanisms induce apoptosis. Proteostasis dysregulation accompanied by protein aggregation and subsequent cell death in specific regions of the nervous system is a common pathogenetic hallmark of most neurodegenerative diseases. We discuss targeted regulation of the ER stress signaling pathways as a potential therapeutic strategy that can slow or even halt the disease progression.

Detection of autoantibodies to potentially amyloidogenic protein, gamma-synuclein, in the serum of patients with amyotrophic lateral sclerosis and cerebral circulatory disorders.

In this study, we analyzed serum for the presence of antibodies to gamma-synuclein in patients with amyotrophic lateral sclerosis (ALS) compared to the control group of patients with other neurological diseases and healthy control donors. As a result, antibodies against gamma-synuclein are not an ALS-specific feature and have been identified in patients with ALS as well as in the control group patients. Patients with the impaired cerebral circulation showed increased incidence of autoantibodies to gamma-synuclein, yet the difference lacks statistical representativeness due to limited sample size.

[The Effects of Inhibitors of Rho- and tyrosine c-Src-kinases on serotonin-induced constrictions of the aorta and mesenteric artery in rats].

We found that the inhibitor of Rho-kinase fasudil selectively inhibited constriction of isolated rings of the aorta and mesenteric artery in rats in response to application of the agonists of 5HT2A-(DOI and TBC-2) and 5HT1A-receptors (8-OH-DPAT) and did not influence vasoconstriction induced by serotonin. We demonstrate for the first time that application of the agonists of 5HT2C-receptors (MK 212 and SCH 23390) did not influence the tone of "intact" vessels. The marked vasoconstrictory effect of the agonists of 5HT2C-receptors was observed in the vessels preconstricted due to angiotensin II or vasopressin. We found that the inhibitor of Rho-kinase did not influence negatively on MK 212 or SCH 23390-induced constriction of isolated rings of the aorta and mesenteric artery in rats. We suppose.that, in the presence of fasudil, serotonin induces constriction of vessels through the interaction with 5HT2C-receptors and signal transduction from these receptors does not involve Rho-kinase activity. We found that fasudil attenuated vasoconstriction induced by norepinephrine and vasopressin by 40%. We.demonstrated that tyrosine c-Src-kinase plays the most important role in signal transduction from 5HT-receptors because its effects are specific with relation to these receptors.

[Agonists of 5HT2C-receptors SCH 23390 and MK 212 incresase the force of rat aorta contraction in the presence of vasopressin and angiotensin II].

We investigated the role of 5HT2C receptors in regulation of blood vessel contractility. We determined expression of 5HT2C receptors in smooth muscle cell line A7r5 as well as on isolated rat aorta. It was shown that strong vasoconstriction effect of 5HT2C receptor agonists - SCH 23390 and MK 212 appeared on blood vessels after preliminary activation of angiotensin ATIA- and vasopressin V1A-receptors. Biphasic contraction (a rhythmic alternation of contraction and subsequent relaxation phases of aoitic rings) and tonic contraction were observed in 75% and 25% of the cases after 5HT2C receptor activation, respectively. Periodic high amplitude constrictions of isolated rat aorta, induced by SCH 23390 and MK 212 agonists, were persisted for a long time (>1 hour). It was revealed that calmodulin and c-Src kinase play a central role in the mechanisms of signal transduction from 5HT2C receptors. Trifluoperazine and PP2, the inhibitors of calmodulin and c-Src kinase, respectively, abolished vasoconstriction reaction of isolated aortic rings in response to SCH 23390 and MK 212 but did not affect the strength gain of the vasoconstriction caused by fluoroaluminate, a G-protein activator. Taken together, these date suggest that 5HT2C receptors are in a latent state in blood vessels (<> receptors) and activation of these receptors is dependent on the functional state of the receptors of other endogenous vasoconstrictors.

[Molecular logic of the endoplasmic reticulum stress signal pathways: the system of unfolded protein response].

Endoplasmic reticulum (ER) is the central organelle of a eukaryotic cell, it provides the synthesis and maturation of the majority of secretory and transmembrane proteins. The intensity of ER-related protein synthesis and ER loading varies in different cell types and depends on the cell microenvironment, physiological state of the cells, the stage of cellular differentiation. Quality control of transmembrane and secretory proteins in ER is a high precision process. The proteins with non-native conformations which are difficult or energetically disadvantageous to refold undergo ubiquitin-dependent proteolytic degradation. Homeostatic control of protein maturation in ER is mediated by a system of interconnected signaling pathways represented sensors located in the lumen of the ER, and effectors, that transmit information to other cell compartments. This system of intracellular signaling pathways play an important role in the endoplasmic reticulum stress and initiates a complex cellular response to the proteins with non-native conformations (Unfolded Protein Response, UPR). However, if homeostasis is not restored, cell death is triggered via apoptosis, which is a supracellular level adaptation mechanism that protects the tissues and the whole organism from dysfunctional and potentially immunogenic unfolded proteins. Malfunctions of the UPR, as well as ER-associated protein degradation (ERAD) process contribute to the development of many diseases: cardiovascular, neurodegenerative, endocrine diseases, autoimmune. The list of factors and mechanisms involved in ER stress is constantly updated, which is a result of significant attention to ER stress as a typical pathophysiological process that forms the basis of many diseases.

[Cluster analysis in biomedical researches].

Cluster analysis is one of the most popular methods for the analysis of multi-parameter data. The cluster analysis reveals the internal structure of the data, group the separate observations on the degree of their similarity. The review provides a definition of the basic concepts of cluster analysis, and discusses the most popular clustering algorithms: k-means, hierarchical algorithms, Kohonen networks algorithms. Examples are the use of these algorithms in biomedical research.

[Total aminothioles of rat plasma under intraperitoneal and subcutaneous introduction of homocysteine].

In the presented work a variation of total aminothiols (cysteine, glutathione, cysteinylglycine and homocysteine) in blood plasma have been shown at modelling hyperhomocysteinemia by daily intraperitoneal (0.6 mkmol/g body weight) and subcutaneous (0.12 mkmol/g body weight) introduction of homocysteine. During two weeks of the intraperitoneal introduction a significant concentration growth (from -40 to 180 mkM) of cysteine was observed. We also observed a moderate change of concentration levels for glutathione (from 10-15 to 30 mkM) and cysteinylglycine (from 1,5 to 4,5 mkM). The homocysteine level has decreased from 300 to 200-250 mkM at second week of experiments. Experimental results with subcutaneous introduction were similar. In this case a stable homocysteine level (-70 mkM) and increase of cysteine level (to 60 mkM) was observed at second week. These data reflect dose-depended processes of organism adaptation to hyperhomocysteinemia, i.e. reinforced capability for homocysteine metabolism and at the same time retention low glutathione level which correlates with hyperhomocysteinemia degree and duration.

[Deposition of von Willebrand factor in human endothelial cells HUVEC in the endoplasmic reticulum stress induced by an excess of homocysteine in vitro].

Von Willebrand factor (vWF) is an adhesive glycoprotein synthesized and secreted by endothelial cells and megakaryocytes. Violation of vWF secretion by endothelial cells is a characteristic feature of endothelial dysfunction in hyperhomocysteinemia. In our study we examined to clarify the concentration-dependent effect of homocysteine (Hcy) on the expression of vWF. Our studies have shown that homocysteine excess induces changes in the intracellular deposition of von Willebrand factor in cultured human endothelial cells in vitro. Primary cultures of human umbilical vein endothelial cells (HUVEC) were incubated with the various concentrations of D,L-homocysteine (0.025 - 5 mM). Homocysteine at a concentration of 0.025 and 0.25 mM after 18 h incubation caused an increase in the intracellular fraction of vWF in HUVEC cells. High concentrations of homocysteine induced a dose-dependent decrease in the intracellular fraction of vWF. These dose-dependent variations may indicate the modulation by homocysteine of different mechanisms of the deposition, the constitutive secretion and the degradation of vWF in human endothelial cells. We proposed that Endoplasmic reticulum stress, in HUVEC cells by the action of an excess of homocysteine associated with increased intracellular levels of vWF at a relatively low concentration of the inducer. We found decline in intracellular vWF at the same duration but higher concentrations of inducer, which may be due to the ER-associated protein degradation.

[Induction of the endoplasmic reticulum stress in conditions of acid-base imbalance in human cells of T-lymphoblastic leukemia].

Endoplasmic reticulum stress - typical molecular pathophysiological process that underlies many cardiovascular, endocrine and other diseases. Violations of the protein conformational maturation processes in the endoplasmic reticulum can cause proteotoxic stress. Compensatory mechanisms are activated in response to ER stress include increased expression of enzymes involved in the formation of disulfide bonds in proteins. The sulfhydryl groups oxidation in the electron transport chain (PDI-ERO1-O2) is associated with reactive oxygen species (ROS) generation. Increased activity of oxidoreductase ERO1 could be one of the mechanisms of oxidative stress - however, a direct relationship of ER stress with the overproduction of ROS remains a subject of debate. In this study we have shown that induced by dithiothreitol (DTT) violation of the redox balance with low ROS production leads to the endoplasmic reticulum stress in Jurkat cells. ER-stress in these cells is not associated with increased ROS production, DTT treatment leads to induction of apoptosis. Modulation of intracellular levels of ROS can influence the apoptosis-inducing effects of ER-stress. Given the possible involvement of ROS in the generation of disulfide bonds, the role of ROS in ER stress may be a modulation of disulfide proteome including client proteins.

[Determination of reduced homocysteine fraction in blood plasma with HPLC-MS].

Homocysteine is a bioactive compound involved in many impotant processes of clinical significance. High sensitivity methods associated with preparation of oxidation-resistant derivatives are of demand for determination of different homocysteine fractions. In this work a derivatization agent N-ethylmaleinimide (NEM) was proposed by us for detection of reduced homocysteine fraction with HPLC-MS. Linearity, detection limit, and precision were 25-1500 nM, 10 nM, and 95-105% respectively, with reproducibility within 12%.

[Nonviral gene transfer in vivo in gene therapy]. / Nevirusnyi perenos genov in vivo v gennoi terapii.

Recent findings connected with in vivo use of artificial macromolecular complexes (genosomes) for functional gene transfer and delivery are discussed in the paper. Non-viral methods are the most safe for the purpose of human gene delivery. The cationic liposomes containing cholesterol are the most suitable for this purpose, because they possess high biodegradability and stability in blood stream. The DNA-liposome complexes should: (i) contain DNA in the condition at most protected from environmental influence, (ii) be rather homogeneous and of small size (40-80 nm). Injections of complexes into blood are the most effective; in a respect of organospecifity may be achieved by appropriate ligand selection. It is the most perspectively to increase the expression level by combining liposomes with viral peptides.