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Nitrosyl iron complexes are remarkably multifactorial pharmacological agents. These compounds have been proven to be particularly effective in treating cardiovascular and oncological diseases. We evaluated and compared the antioxidant activity of tetranitrosyl iron complexes (TNICs) with thiosulfate ligands and dinitrosyl iron complexes (DNICs) with glutathione (DNIC-GS) or phosphate (DNIC-PO 4 - ) ligands in hemoglobin-containing systems. The studied effects included the production of free radical intermediates during hemoglobin (Hb) oxidation by tert -butyl hydroperoxide, oxidative modification of Hb, and antioxidant properties of nitrosyl iron complexes. Measuring luminol chemiluminescence revealed that the antioxidant effect of TNICs was higher compared to DNIC-PO 4 - . DNIC-GS either did not exhibit antioxidant activity or exerted prooxidant effects at certain concentrations, which might have resulted from thiyl radical formation. TNICs and DNIC-PO 4 - efficiently protected the Hb heme group from decomposition by organic hydroperoxides. DNIC-GS did not exert any protective effects on the heme group; however, it abolished oxoferrylHb generation. TNICs inhibited the formation of Hb multimeric forms more efficiently than DNICs. Thus, TNICs had more pronounced antioxidant activity than DNICs in Hb-containing systems.

P-glycoprotein (Pgp, ABCB1, MDR1 ) is an efflux transporter protein that removes molecules from the cells (outflow) into the extracellular space. Pgp plays an important role in pharmacokinetics, ensuring the absorption, distribution, and excretion of drugs and its substrates, as well as in the transport of endogenous molecules (steroid and thyroid hormones). It also contributes to tumor cell resistance to chemotherapy. In this review, we summarize the mechanisms of Pgp regulation during oxidative stress. The currently available data suggest that Pgp has a complex variety of regulatory mechanisms under oxidative stress, involving many transcription factors, the main ones being Nrf2 and Nf-kB. These factors often overlap, and some can be activated under certain conditions, such as the deposition of oxidation products, depending on the severity of oxidative stress. In most cases, the expression of Pgp increases due to increased transcription and translation, but under severe oxidative stress, it can also decrease due to the oxidation of amino acids in its molecule. At the same time, Pgp acts as a protector against oxidative stress, eliminating the causative factors and removing its by-products, as well as participating in signaling pathways.

In many proteins, supplementary metal-binding centers appear under stress conditions. They are known as aberrant or atypical sites. Physico-chemical properties of proteins are significantly changed after such metal binding, and very stable protein aggregates are formed, in which metals act as "cross-linking" agents. Supplementary metal-binding centers in proteins often arise as a result of posttranslational modifications caused by reactive oxygen and nitrogen species and reactive carbonyl compounds. New chemical groups formed as a result of these modifications can act as ligands for binding metal ions. Special attention is paid to the role of cysteine SH-groups in the formation of supplementary metal-binding centers, since these groups are the main target for the action of reactive species. Supplementary metal binding centers may also appear due to unmasking of amino acid residues when protein conformation changing. Appearance of such centers is usually considered as a pathological process. Such unilateral approach does not allow to obtain an integral view of the phenomenon, ignoring cases when formation of metal complexes with altered proteins is a way to adjust protein properties, activity, and stability under the changed redox conditions. The role of metals in protein aggregation is being studied actively, since it leads to formation of non-membranous organelles, liquid condensates, and solid conglomerates. Some proteins found in such aggregates are typical for various diseases, such as Alzheimer's and Huntington's diseases, amyotrophic lateral sclerosis, and some types of cancer.

Dinitrosyl iron complexes (DNICs) are important physiological derivatives of nitric oxide. These complexes have a wide range of biological activities, with antioxidant and antiradical ones being of particular interest and importance. We studied the interaction between DNICs associated with the dipeptide L-carnosine or serum albumin and prooxidants under conditions mimicking oxidative stress. The ligands of these DNICs were histidine residues of carnosine or His39 and Cys34 in bovine serum albumin. Carnosine-bound DNICs reduced the level of piperazine free radicals in the reaction system containing tert -butyl hydroperoxide ( t -BOOH), bivalent iron ions, a nitroxyl anion donor (Angeli's salt), and HEPES buffer. The ability of carnosine DNICs to intercept organic free radicals produced from t -BOOH decay could lead to this effect. In addition, carnosine DNICs reacted with the superoxide anion radical (O 2 •- ) formed in the xanthine/xanthine oxidase enzymatic system. They also reduced the oxoferryl form of the heme group formed in the reaction of myoglobin with t -BOOH. DNICs associated with serum albumin were found to be rapidly destroyed in a model system containing metmyoglobin and t-BOOH. At the same time, these protein DNICs inhibited the t -BOOH-induced oxidative degradation of coenzymes Q 9 and Q 10 in rat myocardial homogenate. The possible mechanisms of the antioxidant and antiradical action of the DNICs studied and their role in the metabolism of reactive oxygen and nitrogen species are discussed.

Carbonyl stress occurs when reactive carbonyl compounds (RCC), such as reducing sugars, dicarbonyls etc., accumulate in the organism. The interaction of RCC carbonyl groups with amino groups of molecules is called the Maillard reaction. One of the most active RCCs is α-dicarbonyl methylglyoxal (MG) that modifies biomolecules forming non-enzymatic glycation products. Organic free radicals are formed in the reaction between MG and lysine or Nα-acetyllysine. S-nitrosothiols and nitric oxide ( • NO) donor PAPA NONOate increased the yield of organic free radical intermediates, while other • NO-derived metabolites, namely, nitroxyl anion and dinitrosyl iron complexes (DNICs) decreased it. At the late stages of the Maillard reaction, S-nitrosoglutathione (GSNO) also inhibited the formation of glycation end products (AGEs). The formation of a new type of DNICs, bound with Maillard reaction products, was found. The results obtained were used to explain the glycation features of legume hemoglobin-leghemoglobin (Lb), which is a lysine-rich protein. In Lb, lysine residues can form fluorescent cross-linked AGEs, and • NO-derived metabolites slow down their formation. The knowledge of these processes can be used to increase the stability of Lb. It can help in better understanding the impact of stress factors on legume plants and contribute to the production of recombinant Lb for biotechnology.

Donors of nitroxyl and nitroxyl anion (HNO/NO - ) are considered to be promising pharmacological treatments with a wide range of applications. Remarkable chemical properties allow nitroxyl to function as a classic antioxidant. We assume that HNO/NO - can level down the non-enzymatic glycation of biomolecules. Since erythrocyte hemoglobin (Hb) is highly susceptible to non-enzymatic glycation, we studied the effect of a nitroxyl donor, Angeli's salt, on Hb modification with methylglyoxal (MG) and organic peroxide- tert -butyl hydroperoxide ( t -BOOH). Nitroxyl dose-dependently decreased the amount of protein carbonyls and advanced glycation end products (AGEs) that were formed in the case of Hb incubation with MG. Likewise, nitroxyl effectively protected Hb against oxidative modification with t -BOOH. It slowed down the destruction of heme, formation of carbonyl derivatives and inter-subunit cross-linking. The protective effect of nitroxyl on Hb in this system is primarily associated with nitrosylation of oxidized Hb and reduction of its ferryl form, which lowers the yield of free radical products. We suppose that the dual (antioxidant and antiglycation) effect of nitroxyl makes its application possible as part of an additional treatment strategy for oxidative and carbonyl stress-associated diseases.

Dinitrosyl iron complexes (DNICs) are a physiological form of nitric oxide ( • NO) in an organism. They are able not only to deposit and transport • NO, but are also to act as antioxidant and antiradical agents. However, the mechanics of hemoglobin-bound DNICs (Hb-DNICs) protecting Hb against peroxynitrite-caused, mediated oxidative modification have not yet been scrutinized. Through EPR spectroscopy we show that Hb-DNICs are destroyed under the peroxynitrite action in a dose-dependent manner. At the same time, DNICs inhibit the oxidation of tryptophan and tyrosine residues and formation of carbonyl derivatives. They also prevent the formation of covalent crosslinks between Hb subunits and degradation of a heme group. These effects can arise from the oxoferryl heme form being reduced, and they can be connected with the ability of DNICs to directly intercept peroxynitrite and free radicals, which emerge due to its homolysis. These data show that DNICs may ensure protection from myocardial ischemia.

Leghemoglobin (Lb) is an oxygen-binding plant hemoglobin of legume nodules, which participates in the symbiotic nitrogen fixation process. Another way to obtain Lb is its expression in bacteria, yeasts, or other organisms. This is promising for both obtaining Lb in the necessary quantity and scrutinizing it in model systems, e.g., its interaction with reactive oxygen (ROS) and nitrogen (RNS) species. The main goal of the work was to study how Lb expression affected the ability of Escherichia coli cells to tolerate oxidative and nitrosative stress. The bacterium E. coli with the embedded gene of soybean leghemoglobin a contains this protein in an active oxygenated state. The interaction of the expressed Lb with oxidative and nitrosative stress inducers (nitrosoglutathione, tert -butyl hydroperoxide, and benzylviologen) was studied by enzymatic methods and spectrophotometry. Lb formed NO complexes with heme-nitrosylLb or nonheme iron-dinitrosyl iron complexes (DNICs). The formation of Lb-bound DNICs was also detected by low-temperature electron paramagnetic resonance spectroscopy. Lb displayed peroxidase activity and catalyzed the reduction of organic peroxides. Despite this, E. coli -synthesized Lb were more sensitive to stress inducers. This might be due to the energy demand required by the Lb synthesis, as an alien protein consumes bacterial resources and thereby decreases adaptive potential of E. coli .

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.

The paper overviews the peculiarities of carbonyl stress in nucleus-free mammal red blood cells (RBCs). Some functional features of RBCs make them exceptionally susceptible to reactive carbonyl compounds (RCC) from both blood plasma and the intracellular environment. In the first case, these compounds arise from the increased concentrations of glucose or ketone bodies in blood plasma, and in the second-from a misbalance in the glycolysis regulation. RBCs are normally exposed to RCC-methylglyoxal (MG), triglycerides-in blood plasma of diabetes patients. MG modifies lipoproteins and membrane proteins of RBCs and endothelial cells both on its own and with reactive oxygen species (ROS). Together, these phenomena may lead to arterial hypertension, atherosclerosis, hemolytic anemia, vascular occlusion, local ischemia, and hypercoagulation phenotype formation. ROS, reactive nitrogen species (RNS), and RCC might also damage hemoglobin (Hb), the most common protein in the RBC cytoplasm. It was Hb with which non-enzymatic glycation was first shown in living systems under physiological conditions. Glycated HbA1c is used as a very reliable and useful diagnostic marker. Studying the impacts of MG, ROS, and RNS on the physiological state of RBCs and Hb is of undisputed importance for basic and applied science.

X-ray absorption near edge structure (XANES) spectra for protein layers adsorbed at liquid interfaces in a Langmuir trough have been recorded for the first time. We studied the parkin protein (so-called E3 ubiquitin ligase), which plays an important role in pathogenesis of Parkinson disease. Parkin contains eight Zn binding sites, consisting of cysteine and histidine residues in a tetracoordinated geometry. Zn K-edge XANES spectra were collected in the following two series: under mild radiation condition of measurements (short exposition time) and with high X-ray radiation load. XANES fingerprint analysis was applied to obtain information on ligand environments around zinc ions. Two types of zinc coordination geometry were identified depending on X-ray radiation load. We found that, under mild conditions, local zinc environment in our parkin preparations was very similar to that identified in hemoglobin, treated with a solution of ZnCl 2 salt. Under high X-ray radiation load, considerable changes in the zinc site structure were observed; local zinc environment appeared to be almost identical to that defined in Zn-containing enzyme alkaline phosphatase. The formation of a similar metal site in unrelated protein molecules, observed in our experiments, highlights the significance of metal binding templates as essential structural modules in protein macromolecules.

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.

Lupinus angustifolius L. is a legume culture known as a source of valuable feed protein and the N 2 -fixator for improving soil fertility. However, its low ecological resistance does not allow for a stable yield of the crop. Earlier, we have shown that steroid phytohormone 24-epibrassinolide (EBR) increases the tolerance of lupine to chlorine ions by activating the protective proteins in ripening seeds (such as proteinase inhibitors that prevent protein breakdown) and lectins. Here we investigated the effect of EBR on the functional status of the N 2 -fixing system in root nodules, protein synthesis in ripening seeds and the resistance of lupine plants to various pathogens. It was found that EBR enhanced the nodulation process, N 2 -fixing activity of nitrogenase and the accumulation of poly-β-hydroxybutirate in the bacteroides, increased the leghemoglobin content in the nodules as well as the metabolic activity of bacteroides. According to data on the inclusion of 14 C-leucine in maturing seed proteins, EBR increased the accumulation of protein in them against the background of a short-term decrease in protein synthesis and its subsequent regeneration to the control level. Gradual inhibition of protein synthesis, characteristic of other legumes, was observed in control variants of lupine. EBR increased lupine resistance to phytopathogenic fungi of Colletotrichum genus and insects of Noctuidae and Scarabaeidae families. We concluded that a more complete implementation of the potential productivity and sustainability of lupine under the action of EBR was achieved due to the anabolic/anti-catabolic effect on the N 2 fixation system in root nodules, as well as on protein synthesis in ripening seeds., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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.

Hypochlorous acid (HOCl), one of the major precursors of free radicals in body cells and tissues, is endowed with strong prooxidant activity. In living systems, dinitrosyl iron complexes (DNIC) with glutathione ligands play the role of nitric oxide donors and possess a broad range of biological activities. At micromolar concentrations, DNIC effectively inhibit HOCl-induced lysis of red blood cells (RBCs) and manifest an ability to scavenge alkoxyl and alkylperoxyl radicals generated in the reaction of HOCl with tert -butyl hydroperoxide. DNIC proved to be more effective cytoprotective agents and organic free radical scavengers in comparison with reduced glutathione (GSH). At the same time, the kinetics of HOCl-induced oxidation of glutathione ligands in DNIC is slower than in the case of GSH. HOCl-induced oxidative conversions of thiolate ligands cause modification of DNIC, which manifests itself in inclusion of other ligands. It is suggested that the strong inhibiting effect of DNIC with glutathione on HOCl-induced lysis of RBCs is determined by their antioxidant and regulatory properties.

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.

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.

Dinitrosyl iron complexes (DNICs) are physiological NO derivatives and account for many NO functions in biology. Polyfunctional dipeptide carnosine (beta-alanyl-L-histidine) is considered to be a very promising pharmacological agent. It was shown that in the system containing carnosine, iron ions and Angeli's salt, a new type of DNICs bound with carnosine as ligand {(carnosine) 2 -Fe-(NO) 2 }, was formed. We studied how the carbonyl compound methylglyoxal influenced this process. Carnosine-bound DNICs appear to be one of the cell's adaptation mechanisms when the amount of reactive carbonyl compounds increases at hyperglycemia. These complexes can also participate in signal and regulatory ways of NO and can act as protectors at oxidative and carbonyl stress conditions.

This review considers dinitrosyl iron complexes (DNICs) and some other metabolites of nitric oxide (NO) in plants. Nitric oxide is vital for all living organisms, although its role in plants has been studied insufficiently compared with that in animals. We presume that the spectrum of its functions in plants is even wider than in animals. The main NO metabolites could be S-nitrosothiols, DNICs and peroxynitrite. Of particular interest are pro- and antioxidant properties of these compounds. DNICs function and their potential biosynthetic role in plants are practically unknown and brought to the limelight in this review. Since the process of NO biosynthesis in plants is still under discussion, we also specially examine this problem.

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.

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.

Background: Nitric oxide (NO) and its metabolites can nitrosylate hemoglobin (Hb) through the heme iron. Nitrihemoglobin (nitriHb) can be formed as result of porphyrin vinyl group modification with nitrite. However, in those with diabetes the non-enzymatic glycation of Hb amino acids residues (the Maillard reaction) can take place. The objectives of this study were to investigate effects of the Maillard reaction on the interaction of methemoglobin (metHb) with S-nitrosoglutathione (GSNO) and nitrite., Methods: Nitrosylhemoglobin production was registered using increasing optical density at 572 nm and compared with 592 nm, and with EPR spectroscopy. Formation of nitriHb was determined using an absorbance band of reduced hemochromogen (582 nm) in the alkaline pyridine solution. Accumulation of fluorescent advanced glycation end-products of Hb was measured through increasing of fluorescence at 385-395 nm (excitation λ=320 nm)., Results: We determined that NO metabolites such as GSNO and nitrite at physiological pH values and aerobic conditions caused modification of metHb porphyrin vinyl groups with nitriHb formation. It was ascertained that this formation was inhibited by superoxide dismutase. In microaerobic conditions metHb was nitrosylated under the action of GSNO or GSNO with methylglyoxal. Nitrite nitrosylated metHb only in the presence of methylglyoxal. It was shown that GSNO inhibited accumulation of fluorescent products which formed during Hb glycation with methylglyoxal., Conclusions: The assumption was made that intermediates of the Hb glycation reaction play an important role both in vinyl group nitration and in heme iron nitrosylation. Oxygen content in reaction medium is an important factor influencing these processes. These effects can play an important role in pathogenesis of the diseases connected with carbonyl, oxidative and nitrosative stresses.

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.

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.

The Maillard reaction is the key process in protein modification during pathologies connected with carbonyl stress. It was shown in system modeling that Maillard reaction interaction of L-lysine (L-lys) with methylglyoxal (MG) led to the formation of compounds reducing methemoglobin (metHb). Under the above conditions and in the presence of S-nitrosoglutathione (GSNO), metHb nitrosylation took place. Processes of metHb reduction and nitrosylation had the lag phase that was dependent on the presence of oxygen (O2) in the reaction mixture. Oxygen interacting with organic free radicals of the Maillard reaction inhibited hemoglobin (Hb) reduction and hence Hb nitrosylation during the first minutes of the reaction. It was also shown that the yield of organic free-radical intermediates of the L-lys with MG was increased in the presence of GSNO and metHb. All effects described could be a result of the formation of active red-ox GSNO derivates in the Maillard reaction. These derivates are probably mediators of one-electron oxidation of dialkylimine by MG. Anion radicals of S-nitrosothiols can function as such mediators.

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".

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.

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.

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.

Destructive effect of superoxide anions O2- derived from KO(2) or xanthine-xanthine oxidase system on dinitrosyl-iron complexes bound with bovine albumin or methemoglobin (DNIC-BSA or DNIC-MetHb) was demonstrated. The sensitivity of DNIC-BSA synthesized by the addition of DNIC with cysteine, thiosulfate or phosphate (DNIC-BSA-1, DNIC-BSA-2 or DNIC-BSA-3, respectively) to destructive action of O2- decreased in row: DNIC-BSA-1>DNIC-BSA-3>DNIC-BSA-2. The estimated rate constant for the reaction between O2- and DNIC-BSA-3 was equal to approximately 10(7)M(-1)s(-1). However, hydrogen peroxide and tert-butyl hydrogenperoxide (t-BOOH) did not induce any noticeable degradation of DNIC-BSA-3 even when used at concentrations exceeding by one order of magnitude those of the complex. As to their action on DNIC-MetHb both hydrogen peroxide and t-BOOH-induced rapid degradation of the complex. Both agents could induce the process due to the effect of alkylperoxyl or protein-derived free radicals formed at the interaction of the agents with ferri-heme groups of MetHb. Peroxynitrite (ONOO(-)) could also initiate protein-bound DNIC degradation more efficiently in the reaction with DNIC-BSA-3. Higher resistance of DNIC-MetHb to peroxynitrite was most probably due to the protective action of heme groups on ONOO(-). However, the analysis allows to suggest that the interaction of protein-bound DNICs with O2- is the only factor responsible for the degradation of the complexes in cells and tissues.

Prooxidant and antioxidant properties of nitric oxide (NO) during oxidative stress are mostly dependent on its interaction with reactive oxygen species, Fe ions, and hemoproteins. One form of NO storage and transportation in cells and tissues is dinitrosyl iron complexes (DNIC), which can bind with both low-molecular-weight thiols and proteins, including hemoglobin. It was shown that dinitrosyl iron complexes bound with hemoglobin (Hb-DNIC) were formed in rabbit erythrocytes after bringing low-molecular-weight DNIC with thiosulfate into blood. It was ascertained that Hb-DNIC intercepted free radicals reacting with hemoglobin SH-groups and prevented oxidative modification of this protein caused by hydrogen peroxide. Destruction of Hb-DNIC can take place in the presence of both hydrogen peroxide and tert-butyl hydroperoxide. Hb-DNIC can also be destroyed at the enzymatic generation of superoxide-anion radical in the xanthine-xanthine oxidase system. If aeration in this system was absent, formation of the nitrosyl R-form of hemoglobin could be seen during the process of Hb-DNIC destruction. Study of Hb-DNIC interaction with reactive oxygen metabolites is important for understanding NO and Hb roles in pathological processes that could result from oxidative stress.

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.

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).

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.

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.

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.

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.

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.

An electrochemical cell for simultaneous potentiometric and spectrophotometric measurements of solutions of various substances is described. A schematic diagram of the cell, its advantages, method of use, and possible areas of application are discussed. The cell is suggested to be used for electrochemical potentiometric redox titration of leghemoglobin and electrochemical reduction of organic reductants commonly used in biochemical research.

To assess the role of O2 in the regulation of nodule metabolism following a decrease or an increase in temperature, the fractional oxygenation of leghemoglobin (FOL) was measured in soybean (Glycine max L. Merr.) nodules during rapid and gradual changes in temperature from 20[deg]C to either 15 or 25[deg]C. The affinity of leghemoglobin for O2 was also measured at each temperature and the values were used to calculate the infected cell O2 concentration (Oi). After nodules were transferred to 15[deg]C, FOL and Oi increased and adenylate energy charge (AEC = [ATP + 0.5ADP]/[ATP + ADP + AMP]) increased from 0.70 to 0.78. The temperature increase was associated with a decrease in FOL and Oi. We concluded that changes in nodule temperature alter the respiratory demand of the nodules for O2, resulting in a change in Oi and a shift in the balance between ATP consumption and ATP production within the nodule tissue.

The purification and properties of metlegoglobin reductase from lupine (Lupinus luteus L.) nodules are described. The purification procedure results in a 1056-fold purification of the enzyme with a total yield of 21%. The enzyme possesses the NADH-diaphorase activity. Metlegoglobin reductase is heterogenous during electrophoresis and isoelectric focusing. Electrophoresis produces two vicinal active bands, while isoelectrofocusing results in four active fractions. The fraction possessing the highest activity has a pI of 4.4. The enzyme is a flavoprotein, in which all flavins are represented by FAD. The molecular weight of the enzyme is 30 000. In some properties metlegoglobin reductase from lupine nodules is similar to methemoglobin reductase from erythrocytes and metmyoglobin reductase from muscles.

The interaction between metlegoglobin reductase from lupin root nodules cytosol and some substrates and inhibitors was studied. The Km values for electron acceptors: dichlorophenol indophenol, potassium ferricyanide, methylene blue and cytochrome c were 5.7 x 10(-5), 2.1 x 10(-5), 1.75 x 10(-4) and 2.5 x 10(-5) M, respectively. The Km value for electron donor NADH was 2.4 x 10(-5) M. Hydroxymercurybenzoate and ethylmaleimide inhibited the metlegoglobin reductase activity; the enzyme activity was also inhibited by NAD. Metlegoglobin reductase was inhibited by quinacrine, which confirmed the flavoproteid nature of the enzyme earlier discovered by the authors. Cytochrome b5 from rabbit liver microsomes can be an electron intermediate during cytochrome c reduction by metlegoglobin reductase. The temperature optimum of metlegoglobin reductase is 40 degrees. The enzyme is comparatively thermostable; and was inactivated by 85% only after 5 min heating at 100 degrees.