[Possible mechanism of antioxidant action of dinitrosyl iron complexes]. / Vozmozhnyi mekhanizm antioksidantnogo deistviia dinitrozil'nykh kompleksov zheleza.

The antioxidant effect of dinitrosyl iron complexes (DNICs) was studied in various model systems. DNICs with glutathione ligands effectively inhibited Cu2+-induced peroxidation of low density lipoproteins (LDL). The antioxidant effect of DNICs with phosphate ligands and free reduced glutathione (GSH) was less pronounced. In addition, DNICs with glutathione suppressed the formation of reactive oxygen species during co-oxidation of lecithin liposomes and glucose. Free radical oxidation in this system was induced with a lipophilic azo initiator and evaluated by luminol-dependent chemiluminescence. NO sharply stimulated chemiluminescence during co-oxidation of glucose and liposomes, thus suggesting the formation of potent oxidants under these conditions. Glutathione DNICs scavenge the superoxide radical anion generated in the xanthine-xanthine oxidase system. Superoxide production was assessed by lucigenin-dependent chemiluminescence and electron paramagnetic resonance (EPR) spectroscopy. Chemiluminescence revealed the dose-dependent character of antiradical effect of glutathione DNICs; moreover, these complexes turned out to be more efficient than GSH. EPR spectra of the adducts of the DEPMPO spin trap with free radicals suggest that the interaction of glutathione DNICs and superoxide does not result in the formation of the thiyl radical of glutathione. Here we propose a mechanism of the antioxidant action of glutathione DNICs, suggesting that unstable intermediate complexes are formed upon their interaction with superoxide or lipid radicals. Further, as a result of intramolecular rearrangement, these intermediates decompose without the free radical as the by-products.

Changes in Transcriptional Regulation of Postnatal Morphogenesis of the Adrenal Zona Fasciculata Caused by Endocrine Disruptor Dichlorodiphenyltrichloroethane.

We studied the expression of transcriptional factors regulating postnatal morphogenesis of the adrenal zona fasciculata in rats after developmental exposure to endocrine disruptor DDT. It was found that tissue reparation after trophic disorders and cell death triggered by prenatal and postnatal exposure to DDT was accompanied by an increase in the number of Oct4- and Shh-expressing cells forming a pool located outside the regeneration zones and involved in the maintenance of tissue homeostasis in the zona fasciculata. DDT exposure also disrupted the expression of antiproliferative factor Hhex. The data showed that proliferation of fasciculata cells after termination of adrenal cortex growth was downregulated by inhibition of the expression of Oct4 and Shh and suppression of canonical Wnt signaling, i.e. due to a decrease in the reserve cell pool essential for physiological regeneration, which can reduce the reactive potential of the zona fasciculata.

The Enhancement of Metal-Binding Properties in Hemoglobin: The Role of Mild Damaging Factors.

X-ray studies revealed the considerable enhancement of metal-binding properties in human hemoglobin under exposure to mild damaging factors (in the presence of 0.09 M urea or upon heating for 30 min at 50 °C). Changes in the element composition of the hemoglobin monolayer, formed on the water subphase in the Langmuir trough, have been monitored in real time by the total external reflection X-ray fluorescence measurements. X-ray absorption spectroscopy has been applied to study the local environment of zinc ions bound on hemoglobin molecules. According to these data, each zinc ion is coordinated by four ligands, two of which are cysteine and histidine. The oxidative stress has been found to accelerate extensively the enhancement of metal-binding ability in protein. A two-stage mechanism has been proposed as a possible explanation of the observed phenomenon: First, in the presence of the mild damaging agents, protein molecules can undergo a transition from the native conformation to a more labile intermediate state that increases the accessibility of amino acid residues (in particular cysteine). At the second stage, oxidation of cysteine and the subsequent activation of cysteine SH groups can affect markedly the protein-metal interaction. The presented investigations provide a deeper insight into the pathogenesis of metabolic disorders that excessive concentrations of the endogenic toxicants might trigger in an organism.

Electrophilic Signaling: The Role of Reactive Carbonyl Compounds.

Reactive carbonyl compounds (RCC) are a group of compounds with clearly pronounced electrophilic properties that facilitate their spontaneous reactions with numerous nucleophilic reaction sites in proteins, lipids, and nucleic acids. The biological functions of RCC are determined by their concentration and governed by the hormesis (biphasic reaction) principle. At low concentrations, RCC act as signaling molecules activating defense systems against xenobiotics and oxidizers, and at high concentrations, they exhibit the cytotoxic effect. RCC participate in the formation of cell adaptive response via intracellular signaling pathways involving regulation of gene expression and cytoplasmic mechanisms related to the structure-functional rearrangements of proteins. Special attention in this review is given to the functioning of electrophiles as mediators of cell general adaption syndrome manifested as the biphasic response. The hypothesis is proposed that electrophilic signaling can be a proto-signaling system.

Alternate and Additional Functions of Erythrocyte Hemoglobin.

The review discusses pleiotropic effects of erythrocytic hemoglobin (Hb) and their significance for human health. Hemoglobin is mostly known as an oxygen carrier, but its biochemical functions are not limited to this. The following aspects of Hb functioning are examined: (i) catalytic functions of the heme component (nitrite reductase, NO dioxygenase, monooxygenase, alkylhydroperoxidase) and of the apoprotein (esterase, lipoxygenase); (ii) participation in nitric oxide metabolism; (iii) formation of membrane-bound Hb and its role in the regulation of erythrocyte metabolism; (iv) physiological functions of Hb catabolic products (iron, CO, bilirubin, peptides). Special attention is given to Hb participation in signal transduction in erythrocytes. The relationships between various erythrocyte metabolic parameters, such as oxygen status, ATP formation, pH regulation, redox balance, and state of the cytoskeleton are discussed with regard to Hb. Hb polyfunctionality can be considered as a manifestation of the principle of biochemical economy.

Whole-genome sequencing targets drug-resistant bacterial infections.

During the past two decades, the technological progress of whole-genome sequencing (WGS) had changed the fields of Environmental Microbiology and Biotechnology, and, currently, is changing the underlying principles, approaches, and fundamentals of Public Health, Epidemiology, Health Economics, and national productivity. Today's WGS technologies are able to compete with conventional techniques in cost, speed, accuracy, and resolution for day-to-day control of infectious diseases and outbreaks in clinical laboratories and in long-term epidemiological investigations. WGS gives rise to an exciting future direction for personalized Genomic Epidemiology. One of the most vital and growing public health problems is the emerging and re-emerging of multidrug-resistant (MDR) bacterial infections in the communities and healthcare settings, reinforced by a decline in antimicrobial drug discovery. In recent years, retrospective analysis provided by WGS has had a great impact on the identification and tracking of MDR microorganisms in hospitals and communities. The obtained genomic data are also important for developing novel easy-to-use diagnostic assays for clinics, as well as for antibiotic and therapeutic development at both the personal and population levels. At present, this technology has been successfully applied as an addendum to the real-time diagnostic methods currently used in clinical laboratories. However, the significance of WGS for public health may increase if: (a) unified and user-friendly bioinformatics toolsets for easy data interpretation and management are established, and (b) standards for data validation and verification are developed. Herein, we review the current and future impact of this technology on diagnosis, prevention, treatment, and control of MDR infectious bacteria in clinics and on the global scale.

Carbonyl Stress in Bacteria: Causes and Consequences.

Pathways of synthesis of the α-reactive carbonyl compound methylglyoxal (MG) in prokaryotes are described in this review. Accumulation of MG leads to development of carbonyl stress. Some pathways of MG formation are similar for both pro- and eukaryotes, but there are reactions specific for prokaryotes, e.g. the methylglyoxal synthase reaction. This reaction and the glyoxalase system constitute an alternative pathway of glucose catabolism - the MG shunt not associated with the synthesis of ATP. In violation of the regulation of metabolism, the cell uses MG shunt as well as other glycolysis shunting pathways and futile cycles enabling stabilization of its energetic status. MG was first examined as a biologically active metabolic factor participating in the formation of phenotypic polymorphism and hyperpersistent potential of bacterial populations. The study of carbonyl stress is interesting for evolutionary biology and can be useful for constructing highly effective producer strains.

[Formation of a new type of dinitrosyl iron complexes bound to cysteine modified with methylglyoxal].

It has been shown that interaction of cysteine dinitrosyl iron complexes with methylglyoxal leads to the formation of a new type of dinitrosyl iron complexes., EPR spectrum of these complexes essentially differs from spectra of dinitrosyl iron complexes containing unmodified thiol. The products of the cysteine reaction with methylglyoxal are hemithioacetals, Schiff bases and thiazolidines, which most likely serve as ligands for the new type of dinitrosyl iron complexes. It has been shown that the new type of dinitrosyl iron complexes as cysteine dinitrosyl iron complexes, which are physiological donors of nitric oxide, exert a vasodilator effect. It has also been found that the oxidative destruction of the new type of dinitrosyl iron complexes occurs at normal oxygen partial pressure, but these dinitrosyl iron complexes remain rather stable under hypoxia modeling. An assumption that the destruction of the new type of dinitrosyl iron complexes is caused by the formation of a bound peroxynitrite-containing intermediate is made.

Measurement of plasma hemoglobin peroxidase activity.

We described a spectrophotometric method for measuring hemoglobin peroxidase activity in human plasma using o-dianisidine (o-DA) as the substrate and myeloperoxidase specific inhibitor 4-aminobensoic acid hydrazide (ruling out the probable contribution of myeloperoxidase to the measured parameter value). The optimal conditions (pH 5.5; 2 mM H2O2) have been determined, at which hemoglobin makes the main contribution to plasma oxidation of o-DA. A significant positive correlation between hemoglobin peroxidase activity measured by the spectrophotometric method and hemoglobin level measured by the pyridine hemochromogenic method has been detected (r=0.624; p<0.01) in plasma specimens from 16 donors. Plasma hemoglobin peroxidase activities were measured in healthy individuals and patients with type 2 diabetes mellitus and coronary heart disease. High plasma hemoglobin peroxidase activities in both groups of patients indicates disorders in the mechanisms of clearance of hemoglobin and its highly reactive derivatives and can serve as specific markers of diseases associated with oxidative stress.

Genetic Diversity of Bacillus thuringiensis from Different Geo-Ecological Regions of Ukraine by Analyzing the 16S rRNA and gyrB Genes and by AP-PCR and saAFLP.

The Bacillus cereus group consists of closely related species of bacteria and is of interest to researchers due to its importance in industry and medicine. However, it remains difficult to distinguish these bacteria at the intra- and inter-species level. Bacillus thuringiensis (Bt) is a member of the B. cereus group. In this work, we studied the inter-species structure of five entomopathogenic strains and 20 isolates of Bt, which were collected from different geo-ecological regions of Ukraine, using various methods: physiological and biochemical analyses, analysis of the nucleotide sequences of the 16S rRNA and gyrB genes, by AP-PCR (BOX and ERIC), and by saAFLP. The analysis of the 16S rRNA and gyrB genes revealed the existence of six subgroups within theB.cereus group: B anthracis, B. cereus I and II, Bt I and II, and Bt III, and confirmed that these isolates belong to the genus Bacillus. All strains were subdivided into 3 groups. Seventeen strains belong to the group Bt II of commercial, industrial strains. The AP-PCR (BOX and ERIC) and saAFLP results were in good agreement and with the results obtained for the 16S rRNA and gyrB genes. Based on the derived patterns, all strains were reliably combined into 5 groups. Interestingly, a specific pattern was revealed by the saAFLP analysis for the industrial strain Bt 0376 р.о., which is used to produce the entomopathogenic preparation "STAR-t".

[Formation of glycated recombinant leghemoglobin in Escherichia coli cells].

A nonenzymatic glycation of the recombinant leghemoglobin expressed in Escherichia coli cells was demonstrated for the first time. This process involved the heme pocket and gave low-spin leghemoglobin species. A correlation between the degree of E. coli protein glycation and synthesis of poly-beta-hydroxybutyric acid was found, suggesting that the accumulation of reserve carbon sources and nonenzymatic glycation could be alternative processes.

[Formation of dinitrosyl iron complexes in cardiac mitochondria].

It has been established that, in the presence of S-nitrosothiols, cysteine, and mitochondria, dinitrosyl iron complexes (DNIC) coupled to low-molecular-weight ligands and proteins are formed. The concentration of DNIC depended on oxygen partial pressure. It was shown that, under the conditions of hypoxia, the kinetics of the formation of low-molecular DNIC was diphasic. After the replacement of anaerobic conditions of incubation to aerobic ones, the level of DNIC came down; in this case, protein dinitrosyl complexes became more stable. We proposed that iron- and sulfur-containing proteins and low-molecular-weight iron complexes are the sources of iron for DNIC formation in mitochondrial suspensions. It was shown that a combination of DNIC and S-nitrosothiols inhibited effectively the respiration of cardiomyocytes.

[Hemoglobins: diversity of structures and functions].

This review briefs the modem concepts of the diversity of hemoglobin functions. The hemoglobins discovered in the representatives of all kingdoms of living nature are described. Specific structural features of various groups of these proteins, including flavohemoglobins and truncated hemoglobins, are discussed. The transport, catalytic, and sensory functions of these proteins are described as well as their roles in oxidative, nitrosative, and carbonyl stresses and the changes in their functions caused by modifications of the molecule. The issues of hemoglobin origin and evolution are discussed.

[Regulatory genes of garden pea (Pisum sativum L.) controlling the development of nitrogen-fixing nodules and arbuscular mycorrhiza: a review of basic and applied aspects].

The review sums up the long experience of the authors and other researchers in studying the genetic system of garden pea (Pisum sativum L.), which controls sthe development of nitrogen-fixing symbiosis and arbuscular mycorrhiza. A justified phenotypic classification of pea mutants is presented. Progress in identifying and cloning symbiotic genes is adequately reflected. The feasibility of using double inoculation as a means of increasing the plant productivity is demonstrated, in which the potential of a tripartite symbiotic system (pea plants-root nodule bacteria-arbuscular mycorrhiza) is mobilized.

[Interaction between albumin-bound dinitrosyl iron complexes and reactive oxygen species].

It has been established that albumin-bound dinitrosyl iron complexes can be destroyed by superoxide radicals generated in a xanthine-xanthine oxidase system. It was shown that peroxynitrite also effectively destroyed albumin-bound dinitrosyl iron complexes. At the same time, hydrogen peroxide and tert-butyl hydroperoxide did not stimulate the destruction of albumin-bound dinitrosyl iron complexes up to concentrations one order higher than the content of NO. The data have been obtained indicating that dinitrosyl iron complexes possess the vasodilatory activity. It has been proposed that peroxynitrite and superoxide radical, by causing the destruction of albumin-bound dinitrosyl iron complexes, affect the physiological properties of nitric oxide.

[Method of determination of the content of hemoglobin-like proteins in heterogenic mixtures].

A spectrophotometric method of quantification of hemoglobin-like proteins, which makes it possible to determine their concentration in mixtures of various composition, was proposed. This method is based on comparing the optical density of different redox forms of proteins when their spectra are superposed at the isosbestic point. The coefficients used in calculations were determined by us. The proposed technique is simple and does not require special procedures. It was tested using myoglobin solutions of various concentrations, myoglobin solutions containing other proteins, and solutions of leghemoglobin (a myoglobin-like protein from legumes).

[Biochemical aspects of growth-stimulating effects of steroid phytohormones on lupine plants].

Presowing treatment of seeds of lupine of various species and cultivars with brassinosteroids--homobrassinolide and epibrassinolide--caused an increase in protein content and a change in the proportion of some amino acids. Both hormones increased heterogeneity of high- and medium-molecular-weight nonhistone chromatin proteins but had no effect on the polypeptide profile of histones. Using epibrassinolide as an example, we showed that the brassinosteroid-induced increase in protein content in lupine seeds was due primarily to accumulation of the low-molecular-weight components of beta-conglutin. The content of some amino acids in both alpha- and beta-conglutins changed. These changes in protein metabolism correlated with an increase in the content of indoleacetic acid and a decrease in the content of abscisic acid.

[Interaction between dinitrosyl iron complexes and intermediate products of oxidative stress].

The interaction between glutathione-containing dinitrosyl iron complexes and superoxide radicals has been studied under the conditions of superoxide radical generation in mitochondria and in a model system xanthine-xanthine oxidase. It has been shown that both superoxide radical and hydroxyl radical are involved in the destruction of dinitrosyl iron complexes. At the same time, iron contained in dinitrosyl iron complex, apparently, does not catalyze the decomposition of hydrogen peroxide with the formation of hydroxyl radical. It has been found that dinitrosyl iron complexes with different anion ligands inhibit effectively the formation of phenoxyl probucol radical in a hemin-H2O2 a system. In this process, different components of the dinitrosyl iron complexes take part in the antioxidant action of these complexes.

Interaction of oxoferrylmyoglobin and dinitrosyl-iron complexes.

It is shown that dinitrosyl-iron complexes (DNIC) with glutathione can reduce oxoferrylmyoglobin forming on interaction of tert-butyl hydroperoxide and metmyoglobin. A rapid decrease in the DNIC concentration was observed under the conditions of production of tert-butyl free radicals; however, destruction of DNIC in the presence of oxoferrylmyoglobin alone was negligible. It is demonstrated that DNIC reduces oxoferrylmyoglobin more than an order more efficiently than S-nitrosoglutathione and glutathione. DNIC also inhibits formation of the thiyl radicals of glutathione in a medium containing metmyoglobin and tert-butyl hydroperoxide. A mechanism of the antioxidant action of DNIC based on regeneration of the nitrosyl complexes from the products of their interaction with oxoferrylheme is proposed.

[Isolation of multiple forms of metleghemoglobin reductase and leghemoglobin components from lupin nodules]. / Metod polucheniia mnozhestvennykh form metlegoglobinreduktazy i komponentov legoglobina iz kluben'kov liupina.

Method for simultaneous obtaining in homogeneous state of two main forms of metleghemoglobin reductase and main leghemoglobin components from lupine nodules was worked out. The method included steps of saturation with ammonium sulphate (40-80%), gel-filtration on Ultrogel AcA 44, isoelectric focusing and repeated isoelectric focusing. As result the forms of metleghemoglobin reductase with molecular weights 62 and 66 kDa were obtained purified 725 and 402 times respectively and obtained in homogeneous state. The total yield of activity was 37%. The form with 62 kDa molecular weight was more active.