[Low-Molecular Thiols as a Factor Improving the Sensitivity of Escherichia coli Mutants with Impaired ADP-Heptose Synthesis to Antibiotics].

Low molecular-weight thiols as glutathione and cysteine are an important part of the cell's redox regulation system. Previously, we have shown that inactivation of ADP-heptose synthesis in Escherichia coli with a gmhA deletion induces the oxidative stress. It is accompanied by rearrangement of thiol homeostasis and increased sensitivity to antibiotics. In our study, we found that restriction of cysteine metabolism (ΔcysB and ΔcysE) and inhibition of glutathione synthesis (ΔgshAB) lead to a decrease in the sensitivity of the ΔgmhA mutant to antibiotics but not to its expected increase. At the same time, blocking of the export of cysteine (ΔeamA) or increasing import (Ptet-tcyP) into cells of the oxidized form of cysteine-cystine leads to an even greater increase in the sensitivity of gmhA-deleted cells to antibiotics. In addition, there is no correlation between the cytotoxic effect of antibiotics and the level of reactive oxygen species (ROS), the total pool of thiols, or the viability of the initial cell population. However, a correlation between the sensitivity to antibiotics and the level of oxidized glutathione in cells was found in our study. Apparently, a decrease in the content of low-molecular-weight thiols saves NADPH equivalents and limits the processes of protein redox modification. This leads to increasing of resistance of the ΔgmhA strain to antibiotics. An increase in low-molecular-weight thiols levels requires a greater expenditure of cell resources, leads to an increase in oxidized glutathione and induces to greater increase in sensitivity of the ΔgmhA strain to antibiotics.

[Metabolic Stress of Red Blood Cells Induces Hemoglobin Glutathionylation].

Metabolic stress caused by a lack of glucose significantly affects the state of red blood cells, where glycolysis is the main pathway for the production of ATP. Hypoglycemia can be both physiological (occurring during fasting and heavy physical exertion) and pathological (accompanying a number of diseases, such as diabetes mellitus). In this study, we have characterized the state of isolated erythrocytes under metabolic stress caused by the absence of glucose. It was established that 24 h of incubation of the erythrocytes in a glucose-free medium to simulate blood plasma led to a two-fold decrease in the ATP level into them. The cell size, as well as intracellular sodium concentration increased. These findings could be the result of a disruption in ion transporter functioning because of a decrease in the ATP level. The calcium level remained unchanged. With a lack of glucose in the medium of isolated erythrocytes, there was no increase in ROS and a significant change in the level of nitric oxide, while the level of the main low-molecular weight thiol of cells, glutathione (GSH) decreased by almost 2 times. It was found that the metabolic stress of isolated red blood cells induced hemoglobin glutathionylation despite the absence of ROS growth. The cause was the lack of ATP, which led to a decrease in the level of GSH because of the inhibition of its synthesis and, probably, due to a decrease in the NADPH level required for glutathione (GSSG) reduction and protein deglutathionylation. Thus, erythrocyte metabolic stress induced hemoglobin glutathionylation, which is not associated with an increase in ROS. This may have an important physiological significance, since glutathionylation of hemoglobin changes its affinity for oxygen.

[Combination of RNase Binase and AKT1/2 Kinase Inhibitor Blocks Two Alternative Survival Pathways in Kasumi-1 Cells].

Treatment of malignant neoplasms often requires the use of combinations of chemotherapeutic agents. However, in order to select combinations that are effective against specific tumor cells, it is necessary to understand the mechanisms of action of the drugs that make up the combination. Bacillus pumilus ribonuclease (binase) is considered as an adjuvant antitumor agent, and the sensitivity of malignant cells to the apoptogenic effect of binase depends on the presence of certain oncogenes. In the acute myelogenous leukemia cell line Kasumi-1, binase blocks the proliferation pathway mediated by the mutant tyrosine kinase KIT, which, as shown in our work, activates an alternative proliferation pathway through AKT kinase. In Kasumi-1 cells, binase in combination with an Akt1/2 inhibitor induces apoptosis, and their toxic effects add up: the Akt1/2 inhibitor blocks the binase-induced pathway after suppression of the KIT-dependent pathway. Thus, a combination of binase and AKT kinase inhibitors can effectively block various pathways of tumor cell proliferation and be used for their elimination.

[Blood-Brain Barrier Transwell Modeling].

In vitro blood-brain barrier (BBB) modeling with the use of the brain endothelial cells grown on a transwell membrane is widely used to investigate BBB disorders and factors intended to ameliorate these pathologies. Endothelial cells, due to tight junction proteins, ensure selective permeability for a number of substances. The low integrity (i.e., high permeability) of the BBB model, as compared to the physiological one, complicates evaluation of the effects caused by different agents. Thus, the selection of conditions to improve barrier integrity is an essential task. In this study, mouse brain endothelial cells bEnd.3 are used in experiments on transwell modeling. To determine which factors enhance BBB integrity, the effects of the cultivation medium, the number of cells during seeding, the state of the transwell membrane, and cultivation in the presence or in the absence of primary mouse neurons and matrigel as a matrix on the passage of a fluorescent label through the cell monolayer were assessed. The effect of fetal bovine serum on the tight junction protein claudin-5 was analyzed by immunocytochemistry. The obtained cultivation parameter data facilitate the solution to the problem of low integrity of the BBB transwell model and bring the model closer to the physiologically relevant indicators.

[Influence of the Donor of Hydrogen Sulfide GYY4137 on the Activation of Human Neutrophils by E. coli Lipopolysaccharides].

Lipopolysaccharides (LPS), components of the cell wall of gram-negative bacteria, activate neutrophils that trigger pathological processes, including gram-negative sepsis. LPS inhibit spontaneous apoptosis of neutrophils that leads to inflammation. In this work we tested the action of H2S donor (GYY4137) on the activation of human neutrophils by E. coli LPS. We estimated the changes in redox status (ROS level, intracellularglutathione, NO), apoptosis and mitochondrial potential of neutrophils under the LPS action in the presence and absence of GYY4137. GYY4137 reduces the ROS level, slightly reduces GSH, does not influence the NO level and has no apoptogenic effect. LPS induce the increasing of ROS level and inhibit spontaneous apoptosis of neutrophils. We found that GYY4137 prevents the growth of ROS caused by LPS and leads to a reduction of LPS-induced inhibition of neutrophil apoptosis. Thus the mechanism of GYY4137 protection against inflammation, triggered by bacterial infection, is concerned with the neutralization of LPS effect on neutrophils.

Glutathionylation of Na,K-ATPase Alpha-Subunit Alters Enzyme Conformation and Sensitivity to Trypsinolysis.

We found earlier that Na,K-ATPase is purified from duck salt glands in partially glutathionylated state (up to 13 of the 23 cysteine residues of the Na,K-ATPase catalytic α-subunit can be S-glutathionylated). To determine the effect of glutathionylation on the enzyme conformation, we have analyzed the products of trypsinolysis of Na,K-ATPase α-subunit in different conformations with different extent of glutathionylation. Incubation of the protein in the E1 conformation with trypsin produced a large fragment with a molecular mass (MM) of 80 kDa with the following formation of smaller fragments with MM 40, 35.5, and 23 kDa. Tryptic digestion of Na,K-ATPase in the E2 conformation also resulted in the generation of the fragments with MM 40, 35.5, and 23 kDa. Deglutathionylation of Na,K-ATPase α-subunit increases the rate of proteolysis of the enzyme in both E1 and E2 conformations. The pattern of tryptic digestion of the α-subunit in E2 conformation additionally glutathionylated with oxidized glutathione is similar to that of partially deglutathionylated Na,K-ATPase. The pattern of tryptic digestion of the additionally glutathionylated α-subunit in E1 conformation is similar to that of the native enzyme. The highest rate of trypsinolysis was observed for the α-subunit in complex with ouabain (E2-OBN conformation). Additional glutathionylation increased the content of high-molecular-weight fragments among the digestion products, as compared to the native and deglutathionylated enzymes. The data obtained were confirmed using molecular modeling that revealed that number of sites accessible for trypsinolysis is higher in the E2P-OBN conformation than in the E1- and E2-conformations and that glutathionylation decreases the number of sites accessible for trypsin. Therefore, glutathionylation affects enzyme conformation and its sensitivity to trypsinolysis. The mechanisms responsible for the changes in the Na,K-ATPase sensitivity to trypsinolysis depending on the level of enzyme glutathionylation and increase in the enzyme sensitivity to proteolysis upon its binding to ouabain, as well as physiological role of these phenomena are discussed.

[Enhancement of Na,K-ATPase Activity as a Result of Removal of Redox Modifications from Cysteine Residues of the al Subunit: the Effect of Reducing Agents].

Na,K-ATPase is a transmembrane enzyme that creates a gradient of sodium and potassium, which is necessary for the viability of animal cells. The activity of Na,K-ATPase depends on the redox status of the cell, decreasing with oxidative stress and hypoxia. Previously, we have shown that the key role in the redox sensitivity of Na,K-ATPase is played by the regulatory glutathionylation of cysteine residues of the catalytic alpha subunit, which leads to the inhibition of the enzyme. In this study, the effect of reducing agents (DTT, ME, TCEP) on the level of glutathionylation of the alpha subunit of Na,K-ATPase from rabbit kidneys and the enzyme activity has been evaluated. We have found that the reducing agents partially deglutathionylate the protein, which leads to its activation. It was impossible to completely remove glutathionylation from the native rabbit kidney protein. The treatment of a partially denatured protein on the PVDF membrane with reducing agents (TCEP, NaBH4) also does not lead to the complete deglutathionylation of the protein. The obtained data indicate that Na,K-ATPase isolated from rabbit kidneys has both regulatory and basal glutathionylation, which appears to play an important role in the redox regulation of the function of Na, K-ATPase in mammalian tissues.

[Changes in the receptor function of Na,K-ATPase during hypoxia and ischemia].

Na,K-ATPase maintains sodium and potassium homeostasis. It is the only known receptor for cardiotonic steroids such as ouabain. Binding of ouabain to Na,K-ATPase leads to the activation of Src kinase and the subsequent initiation of intracellular signaling pathways, including the induction of apoptosis. Changes in Na,K-ATPase activity is one of the earliest responses to hypoxia and is most critical for cell survival. However, it is not known how the hypoxia affects the functioning of Na,K-ATPase as a receptor. We have shown that, under the conditions of hypoxia and ischemia, ouabain is less toxic for murine fibroblast cells (SC-1 cell line) and ouabain does not cause an increase in the level of reactive oxygen species, which is typically observed at 20% pO2. Under hypoxia, the treatment of cells with ouabain also does not lead to the activation of Na,K-ATPase-associated Src kinase. Thus, at low oxygen content, the receptor function of Na,K-ATPase is altered, and cells become less sensitive to cardiotonic steroids. The decrease in sensitivity to cardiotonic steroids, which is evident at hypoxic conditions, should be taken into account in clinical practice. At the same time, in the presence of ouabain the cells are less sensitive to hypoxia, which indicates that cardiotonic steroids can be protective in acute ischemia.

Alzheimer's disease Aß42 peptide induces an increase in Na,K-ATPase glutathionylation.

We have shown that the inhibition of Na,K-ATPase during its long-term incubation with amyloid beta (Aß42), an Alzheimer's disease protein, is caused by the change in the thiol redox status of cells leading to induction of glutathionylation α-subunit of Na,K-ATPase. To restore the activity of Na,K-ATPase, it is proposed to use reducing agents, which promote normalization of the redox status of cells and deglutathionylation of the protein.

Identification of a Region of the Polypeptide Chain of Na,K-ATPase α-Subunit Interacting with 67-kDa Melittin-Like Protein.

It was shown earlier that a 67-kDa protein purified from mouse kidney using polyclonal antibodies against melittin (a peptide from bee venom) interacted with Na,K-ATPase from rabbit kidney. In this study, a 43-kDa proteolytic fragment of Na,K-ATPase α-subunit interacting with the 67-kDa melittin-like protein was found. The α-subunit was hydrolyzed by trypsin in the presence of 0.5 mM ouabain (E2-conformation of Na,K-ATPase). A proteolytic fragment interacting with the 67-kDa melittin-like protein that was identified by mass-spectrometry is a region of the cytoplasmic domain of Na,K-ATPase α-subunit located between amino acid residues 591 and 775. The fragment includes a conservative DPPRA motif that occurs in many P-type ATPases. It was shown earlier that this motif of H,K-ATPase from gastric mucosa binds to melittin. We suggest that namely this motif of P-type ATPases is able to interact with proteins containing melittin-like modules.

[Isomerization of Asp7 increases the toxic effects of amyloid ß and its phosphorylated form in SH-SY5Y neuroblastoma cells].

The generation of amyloid ß (Aß) toxic oligomers during the formation of senile plaques and amyloid fibrils is thought to play a central role in the onset and progression of Alzheimer's disease. Aß production is a physiological process, but the factors that trigger a transition to pathogenic Aß aggregation remain unknown. Posttranslational modifications of Aß could potentially induce the transition. The effects of Aß and its modified forms containing isomerized Asp7, phosphorylated Ser8, or both, were studied in SH-SY5Y human neuroblastoma cells. Asp7 isomerization of was shown to increase cytotoxicity of both the intact and phosphorylated Aß. An increase in cytotoxicity was not associated with an increased internalization of the isomerized Asp7-containing Aß or an influence on the function of mitochondria or reduced glutathione and reactive oxygen species levels. The nitric oxide (NO) level was identified as a determinant of the cytotoxic effect of isomerized Asp7-containing peptides, a decrease in NO level correlating with an increase in cytotoxicity.

[Ribonuclease binase induces death in T-cell acute lymphoblastic leukemia cells by apoptosis].

Bacterial ribonuclease binase is a potential anticancer agent. In the present study, we have determined the toxic effect of binase towards cell lines of T-cell acute lymphoblastic leukemia Jurkat and CEMss. We have shown that binase induces apoptosis in these cells. At the same time, binase does not cause toxic effects in leukocytes of healthy donors, which suggests that binase activity towards leukemic cells is selective. We have found that the treatment of cancer cells with binase leads to a reduction in reactive oxygen species and transcription factor NFκB levels, and demonstrated that these effects are a common feature of the action of RNases on cancer cells.

[The ability of cells to adjust to the low oxigen content associated with Na,K-ATPase glutationilation].

Decreasing the amount of oxygen in the tissues under hypoxic and ischemic conditions, observed at a number of pathologic processes, inevitably leads to their damage. One of the main causes of cell damage and death is a violation of the systems maintaining ionic balance. Na,K-ATPaseis a basic ion-transporting protein of animal cell plasma membrane and inhibition of the Na,K-ATPase activity at lower concentrations of oxygen is one of the earliest and most critical events for cell viability. Currently there is an active search for modulators of Na,K-ATPase activity. For this purpose traditionally used cardiac glycosides but the existence of serious adverse effects forced to look for alternative inhibitors of Na,K-ATPase. Previously we have found that the glutathionylation of Na,K-ATPase catalytic subunit leads to a complete-inhibition of the enzyme. In this paper it is shown that the agents which increase the level of Na,K-ATPase glutathionylation: ethyl glutathione (et-GSH), oxidized glutathione (GSSG) and N-acetyl cysteine (NAC), increase cell survival under oxygen deficiency conditions, prevent decline of ATP in the cells and normalize their redox status. Concentration range in which these substances have a maximum protective effect, and does not exhibit cytotoxic properties was defined: for et-GSH 0.2-0.5 mM, for GSSG 0.2-1 mM, for NAC 10 to 15 mM. The results show prospects for development of methods for tissues protection from damage caused by oxygen starvation by varying the degree of Na,K-ATPase glutathionylation.

Glutathionylation of the alpha-subunit of Na,K-ATPase from rat heart by oxidized glutathione inhibits the enzyme.

A partially purified Na,K-ATPase preparation from rat heart containing α1- and α2-isoforms of the enzyme was shown to include both subunits in S-glutathionylated state. Glutathionylation of the α1-subunit (but not of the α2-subunit) was partially removed when the preparation was isolated in the presence of dithiothreitol. The addition of oxidized glutathione irreversibly inhibited both isoforms. Inhibition of the enzyme containing the α1-subunit was biphasic, and the rate constants of the inhibition were 3745 ± 360 and 246 ± 18 M(-1)·min(-1). ATP, ADP, and AMP protected the Na,K-ATPase against inactivation by oxidized glutathione.

[Expression of FLT3-ITD oncogene confers mice progenitor B-cells BAF3 sensitivity to the ribonuclease binase cytotoxic action].

Acute myeloid leukemia is the most common acute leukemia affecting adults, and its incidence increases with age. Along with chromosomal translocations in leukemic cells mutations in the genes of receptor tyrosine kinases KIT and FLT3 were found with a high frequency. Here we show that transgenic progenitor of B-cells BAF3/FLT3-ITD are much more sensitive to the ribonuclease binase cytotoxic effects than the original BAF3 cells. The principal difference between BAF3/FLT3-ITD and the original BAF3 cells is the expression of FLT3-ITD oncogene, which leads to a change in the normal cell signaling pathways. Earlier, we described a similar effect for the cytotoxic action of binase on Kasumi-1 and FDC-P1-N822K cells, which express the activated KIT-N822K oncogene. Increased binase cytotoxicity toward the cells, expressing FLT3-ITD oncogene, suggests that, as in the case of FDC-P1 cells, transduced by KIT oncogene, the expression of an activated oncogene determines the sensitivity of cells to binase.

Neutral endopeptidase neprilysin is copurified with Na,K-ATPase from rabbit outer medulla and hydrolyzes its α-subunit.

Preparations of Na,K-ATPase from outer medulla of rabbit kidney purified in accordance with the method of P. L. Jorgensen were shown to contain as admixture a protease that moves with α-subunit (~100 kDa) as a single protein band during one-dimensional SDS-PAGE. The electro-elution of proteins of this band from polyacrylamide gel results in the appearance of two protein fragments (~67 and 55 kDa) that are stained with polyclonal antibodies against Na,K-ATPase α-subunit. Liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis showed that the neutral membrane-bound endopeptidase neprilysin is located in one protein band together with the Na,K-ATPase α-subunit. Addition of thiorphan, a specific inhibitor of neutral endopeptidase, eliminates proteolysis of the α-subunit. The data demonstrate that Na,K-ATPase α-subunit may be a natural target for neprilysin.

[Different domain organization of two main conformational states of Na+/K+-ATPase].

Na+/K+-ATPase generates an electrochemical gradient of Na+ and K+, which is necessary for the functioning of animal cells. During the catalytic act, the enzyme passes through two ground conformational states, E1 and E2. To characterize the domain organization of the protein in these conformations, the thermal denaturation of Na+/K+-ATPase from duck salt glands and rabbit kidneys has been studied in the presence of Na+ and K+, which induce the transition of the enzyme to the conformation E1 or E2. The melting curves for the apoforms of Na+/K+-ATPases have different shapes: the curve for the enzyme from the rabbit shows one transition at 56.1 degrees C, whereas the denaturation of Na+/K+-ATPase from the duck is characterized by two transitions, at 49.8 and 56.9 degrees C. Sodium and potassium ions abolish the difference in the domain organization of Na+/K+-ATPases. The melting curves for Na+/K+-ATPases in conformation E2 in both cases exhibit a single peak of thermal absorption at about 63 degrees C. The melting curves for the enzymes in conformation E1 show three peaks of thermal absorption, indicating the denaturation of three domains. The difference in the domain organization of Na+/K+-ATPase in conformations E1 and E2 may be of importance in different sensitivity of these conformations of the enzyme to temperature, proteolytic enzymes, and oxidative stress.

[Binase possesses a selective cytotoxic action on kit-transformed precursors of myeloid cells].

The effect of cationic microbial ribonuclease from Bacillus intermedius (binase) on normal precursors of myeloid cells of FDC-P1 mice and kit-transformed precursors expressing the receptor of the growth factor of stem cells has been studied by flow-through cytometry. Selective apoptogenic properties of binase toward kit-transformed cells were revealed. Viable kit-transformed cells responded to binase by an increase in the concentration of cytosolic calcium. The content of calcium in the cytosol of both cell types in which apoptosis was induced by binase decreased in a dose-dependent manner. The death of cells was not accompanied by a substantial decrease in the content of intracellular RNA. A possible mechanism of binase-induced effects, which involves changes in the expression of genes due to the interference of exogenous RNAse into the RNA interference, was considered.

[An experimental study of potentiated aqueous solutions].

A systematic study was undertaken of luminescent aqueous solutions of homeopathic preparation of sodium chloride at a dilution from D1 to D30, produced by "Weleda" company (Moscow) was carried out. It was shown that intensity of luminescence versus the degree of dilution is a non-monotonous function with several maxima, the main maximum corresponds to 13-14 decimal dilution. The dynamics of spectra was registered for several weeks. A systematic study of water samples (D1-D30) exposed to a similar procedure of potentization but without salt addition was also performed. The difference in the luminescence spectra of water of different stages of potentization was shown. The motility of infusoria Spirostoma ambiquum in solutions being examined was studied. A significant negative correlation between the infusoria motility and luminescence intensity was registered.

[Physico-chemical properties of aqueous solutions, prepared in a membrane electrolyzer]. / Fiziko-khimicheskie svoistva vodnykh rastvorov, poluchennykh v membrannom élektrolizere.

The physicochemical properties of aqueous solutions resulting from membrane electrolysis were studied. It was shown that the catholyte contains hydrogen peroxide at a concentration of 10(-7), which is formed during the reduction of soluble oxygen. It was found that the relaxation of the catholyte redox potential is caused by the transition of the reducing agent to the gaseous phase. The relaxation characteristics of the redox potentials of the catholyte and molecular hydrogen solution were compared. The similarity of the relaxation characteristics of the catholyte and the hydrogen solution as well as the fact that the catholyte, despite its low redox potential, does not reduce either potassium ferricyanide or 5-5'-dithiobis(2-nitrobenzoic acid) support the suggestion that the redox potential of the catholyte is due to molecular hydrogen. However, based on this suggestion, it is impossible to explain the increase in the relaxation time of the catholyte with increasing ionic strength and the fact that, as the redox potential of the catholyte decreases, the concentration of other gases dissolved in the catholyte remains unchanged. Thus, the question regarding the nature of the reducing agent remains open.