Can summary nitrite+nitrate content serve as an indicator of NO synthesis intensity in body tissues?

Studies with the use of a highly specific enzymatic sensor demonstrated that, contrary to the common opinion, normally nitrate is in fact not present in the most important physiological fluids. NO metabolites in the amniotic fluid and semen are mainly presented by NO donor compounds. Therefore, the intensity of NO synthesis can be evaluated by the total content of all its metabolites, but not by the widely used summary nitrite+nitrate content.

[Antidiabetes drug metformin is a donor of nitric oxide: ESR measurement of efficiency].

It is shown that metformin, which is a drug used for treatment of type 2 diabetes mellitus, metabolizes itself in vivo in the intestine and liver of mice with the release of nitric oxide. Subsequently the released nitric oxide forms paramagnetic mono- and dinitrosyl iron complexes which can be registered by EPR method. It is suggested that nitric oxide is just responsible for multifarious therapeutic action of metformin such as lowering of blood glucose level, reduction of arterial hypertension and other biological effects.

[Accumulation of magnetic nanoparticles in plants grown on soils of Apsheron peninsula].

The disclosure of magnetic nanoparticles in five plant species growing in Apsheron peninsula have been detected by the EPR method. The EPR spectra of these nanoparticles proved to be similar to those of synthesized magnetic nanoparticles. The result demonstrated that plants are capable of absorbing magnetic nanoparticles from the soil. The accumulation of nanoparticles in plants is confirmed by the presence of a broad EPR signal whose maximum position of the low-field component changes from g = 2.38 and halfwidth of the signal of 32 mT at room temperature to g = 2.71 and 50-55 mT at 80 K. The intensity of the broad EPR signal for plants grown in radioactively contaminated areas (170-220 mkR per h) was substantially lower compared with plants grown on clean soil. The parameters of the broad EPR signal and its dependence on the temperature of recording were identical with those for synthetic magnetic nanoparticles. The photosynthetic activity and changes in the genome of irradiated plants by the analysis of PCR products were studied.

Effect of plastoquinone derivative 10-(6'-plastoquinonyl)decyltriphenylphosphonium (SkQ1) on contents of steroid hormones and NO level in rats.

Introduction of the plastoquinone derivative 10-(6'-plastoquinonyl)decyltriphenylphosphonium (SkQ1) into male Wistar rats once a day for two weeks in doses of 25 and 250 nmol/kg led to elevation of 17ß-estradiol level in blood serum by 33 and 41%, respectively. At the same time, nitrate and nitrite contents in the rat blood serum increased by 49 and 34%, respectively. ESR spectroscopy with diethyldithiocarbamateiron complex as a spin trap showed more than twofold increase in NO production in lungs, but not in blood, liver, and intestines, following the SkQ1 daily introduction at a dose of 25 nmol/kg.

[Effect of dinitrosyl iron complexes with thiol-containing ligands on the penile cavernosum bodies in rats].

A beneficial effect of dinitrosyl iron complexes (DNIC) with thiol-containing ligands on penile cavernus tissue was shown in rats subjected to penile denervation. Histological and histochemical investigations demonstrated that intracavernous injections of dinitrosyl iron complexes (2 times per one week during 6 months) blocked the reinforcement of endothelial cell proliferation in the tissue characteristic of the cavernous tissue when the penile nerve was removed. On the other hand, treatment with dinitrosyl iron complexes led to the preservation of mitotic activity of smooth myocytes and protected against the appearance in these cells of collagenase, an indicator of muscle transformation into fibrous tissue. It was shown that the process of fibrous transformation of myocytes correlates with a decrease in the mitotic activity of fibroblasts in the adventive part of cavernosa. The mitotic activity increased in cavernous tissue in the absence of dinitrosyl iron complexes. The efficiency of long-term action of dinitrosyl iron complexes on the erection in both intact animals and animals subjected to neuroectomy of cavernous tissue nerve was shown. The injection of low-molecular dinitrosyl iron complexes to the cavernous tissue resulted in the formation of protein-bound dinitrosyl iron complexes in the tissue, which were detected by the EPR technique. It is assumed that these dinitrosyl iron complexes function as a depot of nitric oxide, providing long-lasting penis erection.

[Dinitrosyl-iron complexes with cysteine or glutathione accelerate skin wound healing in animals].

The beneficial effect of NO-donors, dinitrosyl-iron complexes with cysteine or glutathione on the healing of skin wound in rats was demonstrated by hystological and hystochemical methods: dinitrosyl-iron complexes accelerated efficiently repair processes in wound tissue after a twofold injection of an aqueous solution of a dinitrosyl-iron complex into wound tissue at a total dose of 5 mmol on days 1 and 2 after skin wounding, and the granulocyte volume increased 3-4 times on the fourth day after wounding compared with the control. Higher doses of dinitrosyl-iron complex provoked an inflammation process in the wound. Similar experiments with of another NO donor S-nitrosoglutathione affected adversely the wound. S-Nitrosoglutathione was added to the wound at a total dose of 10 mmol, which ensured the administration of NO to the wound tissue in the amount equal to that introduced upon the injection of dinitrosyl-iron complex. The addition of dinitrosyl-iron complex with glutathione at a dose of 2.5 mmol was accompanied by the formation of protein-bound dinitrosyl-iron complex in wound tissue. The formation of dinitrosyl-iron complex was also observed after the injection of S-nitrosoglutathione. However, the amount of complexes was more than 25 times less than that after the administration of dinitrosyl-iron complex. The beneficial effect of dinitrosyl-iron complex on the wound was suggested to be due to the formation of a self-regulated chemical system in wound tissue, which is characterized by the mutual transformation of low-molecular dinitrosyl-iron complex and S-nitrosoglutathione. This system ensures a regulated delivery of NO to its intracellular targets without the formation of high amounts of peroxynitrite which could adversely affect the intracellular processes. It was assumed that the self-regulated system of dinitrosyl-iron complex and S-nitrosoglutathione is not formed after the addition of S-nitrosoglutathione to the wound, probably due to a low amount of intracellular iron which could provide the formation of dinitrosyl-iron complex. The rapid decomposition of S-nitrosoglutathione results in the appearance of high amounts of NO and hence peroxynitrite, which adversely affects the wound.

[Why does iron abrogate the cytotoxic effect of S-nitrosothiols on human and animal cultured cells?].

It was found that dinitrosyl iron complexes (DNIC) with thiol-containing ligands (cysteine or glutathione) of concentrations up to 1 mM produce no cytotoxic effect on cultured cells from human milk gland carcinoma (MCF-7). The cytotoxic action on MCF-7 cells was produced by S-nitrosocysteine: at a concentration of 1 mM, it induced the death of 50% cells. A more stable S-nitrosothiol, S-nitrosoglutathione, did not produce any cytotoxic effect at the same concentration. It is assumed that the negative action of nitrosocysteine is due to its rapid degradation, which results in the accumulation of large amounts of free NO molecules followed by their oxidation by superoxide ions to peroxynitrite, an efficient inhibitor of metabolic processes. These processes seem to be not characteristic of the more stable S-nitrosoglutathione. The cytotoxic effect of nitrosocysteine was completlly abrogated by the addition of 0.2 mM ferrous citrate complex to the medium. When S-nitrosoglutathione NO (0.5 mM) or S-nitrosoglutathione (0.5 mM) + Fe(2+)-citrate (0.2 mM) were added to the medium, protein-bound dinitrosyl iron complexes formed with the involvement of endogenous or exogenous iron were detected in cells. The amount of the complexes in the presence of exogenous iron increased four times, reaching the value of 1.6 nmole/5 x 10(6) cells. Therefore, it was proposed that the blockade of the cytotoxic action of S-nitrosoglutathione by iron complexes is due to Cys-NO transformation of S-nitrosocysteine into dinitrosyl iron complexes. The high stability of these complexes ensures only a gradual accumulation of nitric oxide in cells.

[Changes in the free-radical state and level of free iron during the formation of drug resistance in tumor cells].

The development of resistance of K562 human erythroleukemia cells to doxorubicin, a widely used antitumor antibiotic with the prooxidant action, leads to changes in the free-radical state of cells. It has been found that the formation of superoxide anion in resistant cells decreases. The introduction of doxorubicin to the culture medium induced a considerably lesser increase in the formation of O2*- in resistant cells compared to sensitive cells. At the same time, a strong decrease in the ESR signal of semiquinone type with a g-factor of 2.006 was observed in a culture of resistant cells grown in the absence of doxorubicin as compared with sensitive cells grown under similar conditions. At the same time, a decrease in the level of paramagnetic nitrosyl complexes of nonheme iron in resistant cells was recorded, indicating a decrease in the content of free nonheme iron as a result of the formation of drug resistance. In addition, a decrease in the level of mRNA of the transferrin receptor in resistant cells was found by the RT-PCR. These data indicate the development of a coodinated redox-dependent adaptive response, which makes itself evident as a suppression of free radical processes during the formation of resistance of K562 cells to doxorubicin.

Complexes of Fe2+ with diethyldithiocarbamate or N-methyl-D-glucamine dithiocarbamate as traps of nitric oxide in animal tissues: comparative investigations.

In EPR experiments on mice it was demonstrated that a hydrophobic complex Fe2+ with diethyldithiocarbamate (DETC) is a more efficient selective NO trap than a hydrophilic complex Fe2+ with N-methyl-D-glutamine dithiocarbamate (MGD). This difference can be due to the higher stability of paramagnetic nitrosyl iron complex with DETC (MNIC-DETC) formed by NO binding to Fe2+-DETC in animal tissues in vivo. The complex analogue MNIC-MGD is reversibly oxidized in animal blood to transform into the diamagnetic EPR-silent form. The latter is detectable also in urine of animals, especially of those treated with bacterial lipopolysaccharide which initiates the enhanced NO production in the organism. We suggest that NO2 or peroxynitrite formed from endogenous NO can serve as an agent reversibly oxidizing MNIC-MGD in these animals.

The source of non-heme iron that binds nitric oxide in cultivated macrophages.

In cultured macrophages (J 774 line) a decrease in iron-sulfur centers (ISC) was not observed after 5 min treatment with nitric oxide (NO) (10(-7) M NO/10(7) cells). The content of these centers was measured by electron spin resonance (ESR) spectroscopy at 16-60 K. However, the appearance of a characteristic ESR signal at g(av) = 2.03 indicated the formation of dinitrosyl iron complex (DNIC) in these cells. These findings suggest that loosely bound non-heme iron (free iron) but not iron from ISC is mainly involved in DNIC formation. ISC might release iron for DNIC formation after their destruction induced by the products of NO oxidation (NO2, N2O3, etc).

Comparison of physico-chemical characteristics of four laccases from different basidiomycetes.

New strains of basidiomycetes producing extracellular laccases (Trametes ochracea 92-78, and Trametes hirsuta 56) have been found by screening of isolates of Trametes fungi. The laccases from T. hirsuta 56 and T. ochracea 92-78 as well as two laccases from previously found and described strains of basidiomycetes, namely Cerrena maxima and Coriolopsis fulvocinerea, were purified to homogeneity. The standard redox potentials of type 1 copper in the enzymes were determined and found to be 780, 790, 750, and 780 mV, respectively. The spectral and biochemical studies showed that the enzymes had no significant differences between the structures of their active sites (T1, T2, and T3). In spite of this fact, the basic biochemical properties as well as the redox potentials of the T1 sites of the enzymes were found to be different. The molecular weights of the laccases range from 64 to 70 kDa, and their pI values range from 3.5 to 4.7. The pH-optima are in the range 3.5-5.2. The temperature optimum for activity is about 50 degrees C. The thermal stabilities of the enzymes were studied. The catalytic and Michaelis constants for catechol, guaiacol, hydroquinone, sinapinic acid, and K(4)Fe(CN)(6) were determined. Based on these results as well as results obtained by comparing with published properties of several laccases, it could be concluded that T. hirsuta and Cerrena maxima laccases have some superior characteristics such as high stability, high activity, and low carbohydrate content, making them attractive objects for further investigations as well as for application in different areas of biotechnology.

Radiation dosage accumulated by reindeer from Novaya Zemlya.

ESR spectrometry of tooth enamel revealed a significant difference in accumulated radiation dose between reindeer which lived on Novaya Zemlya when underground nuclear tests were performed there and those which lived there after the tests were stopped.

Relationships between ESR-evaluated doses estimated from enamel and activity of radionuclides in bone and teeth of reindeer.

Doses of radiation estimated from ESR analysis of tooth enamel were compared with activities of alpha- and beta-emitters in enamel and in bone tissue of mandibles of 77 reindeer from populations with different levels of radiation contamination. Contribution of the radionuclides incorporated into bone (or bone-seeking radionuclides in food) to ESR-evaluated doses was substantial and the contribution of the radionuclides incorporated into enamel itself proved to be relatively small.

Estimation of accumulated dose of radiation by the method of ESR-spectrometry of dental enamel of mammals.

ESR-spectrometry was used to investigate radiation-induced paramagnetic centers in enamel of mammals: carnivores (polar bear and fox), ungulates (reindeer, European bison, moose), and man. Values at half the microwave power saturation of the radiation signal, P1/2, evaluated at room temperature, was found to range from 16 to 26 mW for animals and man. A new approach to discrimination of the radiation induced signal from the total ESR spectrum of reindeer enamel is proposed. 'Dose-response' dependencies of enamel of different species mammals were measured within the dose range from 0.48 up to 10.08 Gy. Estimations of 'radiosensitivity' enamel of carnivores and ungulates showed good agreement with radiosensitivity enamel of man by ESR method.

[The dynamics of the radiation load on reindeer on Novaya Zemlya]. / Dinamika radiatsionnoi nagruzki na severnykh olenei Novoi Zemli.

The ESR analysis was carried out aiming at studying the activity of radionuclides in the dental enamel, dentin, and mandible bone tissues of reindeer of Novaya Zemlya. Doses of absorbed ionizing radiation were assessed. It was determined that the doses obtained by reindeer during underground nuclear tests were ten times higher and the activity of 90Sr was greater than those for reindeer living after the nuclear tests were suspended. Relatively high values of doses were observed for reindeer living before the nuclear weapons were invented. This fact is accounted for by an elevated level of natural radiation in the region.

[The effect of external factors on ATP synthesis during sudden pH increase in mitochondria]. / Vliianie vneshnikh faktorov na sintez ATF pri skachkoobraznom povyshenii pH v mitokhondriiakh.

Fast alkaline jump in aging uncoupled mitochondria results in the ATP synthesis. This process is completely inhibited with oligomycin and by 60-70% with FCCP. The NADH experiments have shown that mitochondrial membranes do not change their orientation during aging.

[New prospects in assessing the absorbed radiation dose by electron paramagnetic resonance]. / Novye vozmozhnosti v otsenke pogloshcheniia radiatsionnoi dozy metodom élektronnogo paramagnitnogo rezonansa.

Spin-lattice relaxation rates of radiation induced and background centers in dental enamel have been investigated by electron spin resonance techniques. It has been found that these centers differ in the value of the saturation half-power and the saturation process. Based on these experimental results a new approach to discrimination of the radiation induced signal from the total ESR spectrum of dental enamel is proposed. Comparison of the current methods of radiation dose estimation by the ESR analysis has been performed using the new approach.

[ATP synthesis in the membrane fragments of Staphylococcus aureus cells induced by a sudden increase in pH]. / Sintez ATF v membranno-stenochnykh fragmentakh kletok Staphylococcus aureus, indutsirovannyi skachkoobraznym povysheniem pH.

Membrane fragments of St. aureus are able to synthesize up to 7 nmol/mg protein of ATP after a jump-like increase of pH value. The presence of respiratory substrates is obligatory. The effect is realized only during the jump, if pH passes of 7.4 value. ADP phosphorylation induced by pH-jump is completely inhibited by DCCD.

[Hydrophobic and hydrophilic complexes of Fe2+ with dithiocarbamate derivatives as a nitric oxide trap in mice]. / Gidrofobnye i gidrofil'nye kompleksy Fe2+ s proizvodnymi ditiokarbamatakak lovushki oksida azota v organizme myshei.

It has been shown by using EPR method that hydrophobic complexes Fe(2+)-diethyldithiocarbamate (DETC) act more efficiently as a selective traps of nitric oxide (NO) in mice organisms than hydrophilic complexes Fe(2+)-N-methyl-D-glutamyldithiocarbamate (MGD). This difference seemed to be due to higher stability of paramagnetic mononitrosyl iron complexes with DETC (MNIC-DETC) formed in vivo in animal tissues in a result of NO binding with Fe(2+)-DETC complexes. Analogous complexes MNIC-MGD appeared a blood were oxidized to diamagnetic, EPR silent form. The latter was also detected in mouse urine especially for animals which were pretreated with bacterial lipopolysaccharide inducing increased NO generation in mice organisms. Nitrogen dioxide or peroxynitrite formed endogenous NO were suggested to be the agents which oxidized MNIC-MGD by reversible way in mice organisms.

[Formation of paramagnetic nitrosyl complexes of non heme iron in animals with participation of nitric oxide from exogenous and endogenous sources]. / Obrazovanie paramagnitnykh nitrozil'nykh kompleksov negemovogo zheleza v organizme zhivotnykh pri uchastii oksida azota iz ékzogennykh i éndogennykh istochnikov.

It was found that thiosulfate has a stabilizing effect on exogenous and endogenous dinitrosyl-iron complexes in mice treated with bacterial lipopolysaccharide. It was assumed that thiosulfate protects dinitrosyl-iron complexes from the destructive influence of superoxide and peroxinitrite whose enhanced synthesis, together with the synthesis of nitric oxide, is initiated in mice by the lipopolysaccharide. For the first time, the formation of dinitrosyl-iron complexes was demonstrated, which occurs with the participation of nitric oxide generated enzymatically via the L-arginine-dependent pathway. The injection of exogenous dinitrosyl-iron complexes with thiosulfate, which, together with diethyldithiocarbamate, provide the formation of exogenous mononitrosyl iron-diethyldithiocarbamate complexes, made it possible to use the ABC method, which markedly enhances the efficiency of scavenging of endogenous nitric oxide in mice treated with lipopolysaccharides.