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1.
Bisht NK Abbruzzetti S Uppal S Bruno S Spyrakis F Mozzarelli A Viappiani C Kundu S 《Biochimica et biophysica acta》2011,1814(8):1042-1053
Type 1 non-symbiotic rice hemoglobin (rHb1) shows bis-histidyl heme hexacoordination and is capable of binding diatomic ligands reversibly. The biological function is as yet unclear, but the high oxygen affinity makes it unlikely to be involved in oxygen transport. In order to gain insight into possible physiological roles, we have studied CO rebinding kinetics after laser flash photolysis of rHb1 in solution and encapsulated in silica gel. CO rebinding to wt rHb1 in solution occurs through a fast geminate phase with no sign of rebinding from internal docking sites. Encapsulation in silica gel enhances migration to internal cavities. Site-directed mutagenesis of FB10, a residue known to have a key role in the regulation of hexacoordination and ligand affinity, resulted in substantial effects on the rebinding kinetics, partly inhibiting ligand exit to the solvent, enhancing geminate rebinding and enabling ligand migration within the internal cavities. The mutation of HE7, one of the histidyl residues involved in the hexacoordination, prevents hexacoordination, as expected, but also exposes ligand migration through a complex system of cavities. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches. 相似文献
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Shimoda Y Nagata M Suzuki A Abe M Sato S Kato T Tabata S Higashi S Uchiumi T 《Plant & cell physiology》2005,46(1):99-107
We characterized the expression profiles of LjHb1 and LjHb2, non-symbiotic hemoglobin (non-sym-Hb) genes of Lotus japonicus. Although LjHb1 and LjHb2 showed 77% homology in their cDNA sequences, LjHb2 is located in a unique position in the phylogenetic tree of plant Hbs. The 5'-upstream regions of both genes contain the motif AAAGGG at a position similar to that in promoters of other non-sym-Hb genes. Expression profiles obtained by using quantitative RT-PCR showed that LjHb1 and LjHb2 were expressed in all tissues of mature plants, and expression was enhanced in mature root nodules. LjHb1 was strongly induced under both hypoxic and cold conditions, and by the application of nitric oxide (NO) donor, whereas LjHb2 was induced only by the application of sucrose. LjHb1 was also induced transiently by the inoculation with the symbiotic rhizobium Mesorhizobium loti MAFF303099. Observations using fluorescence microscopy revealed the induction of LjHb1 expression corresponded to the generation of NO. These results suggest that non-sym-Hb and NO have important roles in stress adaptation and in the early stage of legume-rhizobium symbiosis. 相似文献
3.
T. Sobko C.I. Reinders E.. Jansson E. Norin T. Midtvedt J.O. Lundberg 《Nitric oxide》2005,13(4):262-278
Denitrifying bacteria in soil generate nitric oxide (NO) from nitrite as a part of the nitrogen cycle, but little is known about NO production by commensal bacteria. We used a chemiluminescence assay to explore if human faeces and different representative gut bacteria are able to generate NO. Bacteria were incubated anaerobically in gas-tight bags, with or without nitrate or nitrite in the growth medium. In addition, luminal NO levels were measured in vivo in the intestines in germ-free and conventional rats, and in rats mono-associated with lactobacilli. We show that human faeces can generate NO after nitrate or nitrite supplementation. Lactobacilli and bifidobacteria generated much NO from nitrite, but only a few of the tested strains produced NO from nitrate and at much lower levels. In contrast, Escherichia coli, Bacteroides thetaiotaomicron, and Clostridium difficile did not produce significant amounts of NO either with nitrate or nitrite. NO generation in the gut lumen was also observed in vivo in conventional rats but not in germ-free rats or in rats mono-associated with lactobacilli. We conclude that NO can be generated by the anaerobic gut flora in the presence of nitrate or nitrite. Future studies will reveal its biological significance in regulation of gastrointestinal integrity. 相似文献
4.
Induction of inducible nitric oxide synthase by ginsenosides in cultured porcine endothelial cells 总被引:1,自引:0,他引:1
Mechanism of Nitric oxide (NO) production by ginsenosides was investigated in cultured porcine endothelial cells. Beta-nicotinamide adenine dinucleotide phosphate (beta-NADPH) staining showed that the NO production was significantly enhanced by the presence of 40 microg/ml ginsenosides with 10 microM L-arginine after 12 h incubation. NO production was suppressed by addition of 0.5 microM Nomega-Nitro-L-arginine (L-NNA), an inhibitor of NO synthases (NOSs), to the incubation medium. In addition, the immunoreactive signals of inducible NOS (iNOS) were appeared in endothelial cells after 12-h incubation of ginsenosides, whereas the signals were not observed in non-treated cells. Our findings suggest that ginsenosides can enhance NO production by induction of iNOS in addition to its direct effect on endothelial cells by increasing intracellular Ca2+ concentration. 相似文献
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Nitric oxide (NO) plays a fundamental role in maintaining normal vasomotor tone. Recent data implicate a critical function for hemoglobin and the erythrocyte in regulating the activity of NO in the vascular compartment. Intravascular hemolysis releases hemoglobin from the red blood cell into plasma (cell-free plasma hemoglobin), which is then able to scavenge endothelium-derived NO 600-fold faster than erythrocytic hemoglobin, thereby disrupting NO homeostasis. This may lead to vasoconstriction, decreased blood flow, platelet activation, increased endothelin-1 expression (ET-1), and end-organ injury, thus suggesting a novel mechanism of disease for hereditary and acquired hemolytic conditions such as sickle cell disease and cardiopulmonary bypass. Furthermore, therapy with NO gas inhalation or infusion of sodium nitrite during hemolysis may attenuate this disruption in vasomotor balance by oxidizing plasma cell-free hemoglobin, thereby preventing the consumption of endogenous NO and the associated pathophysiological changes. In addition to providing an NO scavenging role in the physiological regulation of NO-dependent vasodilation, hemoglobin and the erythrocyte may deliver NO as the hemoglobin deoxygenates. While this process has previously been ascribed to S-nitrosated hemoglobin, recent data from our laboratories suggest that deoxygenated hemoglobin reduces nitrite to NO and vasodilates the human circulation along the physiological oxygen gradient. This newly described role of hemoglobin as a nitrite reductase is discussed in the context of blood flow regulation, oxygen sensing, and nitrite-based therapeutics. 相似文献
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Induction by ouabain of hemoglobin synthesis in cultured friend erythroleukemic cells 总被引:1,自引:0,他引:1
Induction of erythroid differentiation in ouabain-resistant murine erythroleukemia cells by ouabain is reported. Ouabain induction results in the appearance of hemoglobin-containing cells 12–24 hr earlier than induction of the same clone by dimethyl sulfoxide. The levels of globin mRNA after ouabain induction are similar in amount to the globin mRNA levels observed after induction by dimethyl sulfoxide. The concentration of ouabain required to induce hemoglobin synthesis depends upon the K+ ion levels in the culture medium. Lowering the extracellular K+ ion concentration 2–4 fold reduced by 10–40 fold the ouabain concentration necessary for the induction of hemoglobin synthesis. In low K+ medium (1.8 mM), ouabain is an effective inducer of hemoglobin synthesis at a concentration of 0.02 mM. This K+ effect is specific for ouabain induction, since induction by other inducers, such as dimethyl sulfoxide and dimethyl acetamide, does not exhibit this marked sensitivity to the levels of K+ ions in the culture medium. These results suggest that the binding of ouabain to the plasma membrane enzyme, ATPase, is required for the induction of erythroid differentiation by ouabain. A small but significant proportion of wild-type, ouabain-sensitive cells also can be induced by ouabain, below ouabain concentrations that are toxic to these cells. The observation that the binding of ouabain to the ATPase induces hemoglobin synthesis suggests that changes in the intracellular concentration of K+ ions may be involved in the control of erythroid differentiation in Friend erythroleukemic cells. 相似文献
10.
Jansson EA Huang L Malkey R Govoni M Nihlén C Olsson A Stensdotter M Petersson J Holm L Weitzberg E Lundberg JO 《Nature chemical biology》2008,4(7):411-417
Inorganic nitrite (NO(2)(-)) is emerging as a regulator of physiological functions and tissue responses to ischemia, whereas the more stable nitrate anion (NO(3)(-)) is generally considered to be biologically inert. Bacteria express nitrate reductases that produce nitrite, but mammals lack these specific enzymes. Here we report on nitrate reductase activity in rodent and human tissues that results in formation of nitrite and nitric oxide (NO) and is attenuated by the xanthine oxidoreductase inhibitor allopurinol. Nitrate administration to normoxic rats resulted in elevated levels of circulating nitrite that were again attenuated by allopurinol. Similar effects of nitrate were seen in endothelial NO synthase-deficient and germ-free mice, thereby excluding vascular NO synthase activation and bacteria as the source of nitrite. Nitrate pretreatment attenuated the increase in systemic blood pressure caused by NO synthase inhibition and enhanced blood flow during post-ischemic reperfusion. Our findings suggest a role for mammalian nitrate reduction in regulation of nitrite and NO homeostasis. 相似文献
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Macrophage oxidation of L-arginine to nitrite and nitrate: nitric oxide is an intermediate 总被引:117,自引:0,他引:117
Previous studies have shown that murine macrophages immunostimulated with interferon gamma and Escherichia coli lipopolysaccharide synthesize NO2-, NO3-, and citrulline from L-arginine by oxidation of one of the two chemically equivalent guanido nitrogens. The enzymatic activity for this very unusual reaction was found in the 100,000g supernatant isolated from activated RAW 264.7 cells and was totally absent in unstimulated cells. This activity requires NADPH and L-arginine and is enhanced by Mg2+. When the subcellular fraction containing the enzyme activity was incubated with L-arginine, NADPH, and Mg2+, the formation of nitric oxide was observed. Nitric oxide formation was dependent on the presence of L-arginine and NADPH and was inhibited by the NO2-/NO3- synthesis inhibitor NG-monomethyl-L-arginine. Furthermore, when incubated with L-[guanido-15N2]arginine, the nitric oxide was 15N-labeled. The results show that nitric oxide is an intermediate in the L-arginine to NO2-, NO3-, and citrulline pathway. L-Arginine is required for the activation of macrophages to the bactericidal/tumoricidal state and suggests that nitric oxide is serving as an intracellular signal for this activation process in a manner similar to that very recently observed in endothelial cells, where nitric oxide leads to vascular smooth muscle relaxation [Palmer, R. M. J., Ashton, D. S., & Moncada, S. (1988) Nature (London) 333, 664-666]. 相似文献
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Background
Type 1 Diabetes Mellitus results from an autoimmune destruction of the pancreatic beta cells, which produce insulin. The lack of insulin leads to chronic hyperglycemia and secondary complications, such as cardiovascular disease. The currently approved clinical treatments for diabetes mellitus often fail to achieve sustained and optimal glycemic control. Therefore, there is a great interest in the development of surrogate beta cells as a treatment for type 1 diabetes. Normally, pancreatic beta cells produce and secrete insulin only in response to increased blood glucose levels. However in many cases, insulin secretion from non-beta cells engineered to produce insulin occurs in a glucose-independent manner. In the present study we engineered liver cells to produce and secrete insulin and insulin secretion can be stimulated via the nitric oxide pathway. 相似文献13.
Identification of nitric oxide and nitrous oxide as products of nitrite reduction by Pseudomonas cytochrome oxidase (nitrate reductase) 总被引:5,自引:0,他引:5
The cytosol fraction of rat adrenocortical tissue contains comparatively high levels of two prostaglandin metabolizing enzymes. The first, prostaglandin-9-ketoreductase, utilizes NADPH more effectively than NADH as cofactor, is inhibited by NADP, and exhibits an apparent Km of 304 μM for PGE1. 15-hydroxyprostaglandin dehydrogenase, tentatively identified as the type II NADP-dependent isozyme, is inhibited by NADPH but not NADH, and exhibits an apparent Km of 157 μM when PGE1 is used as substrate. Changes in specific activities of the two enzymes following ACTH, hypophysectomy, or dexamethasone treatment are inconclusive in defining a chronic regulatory role for adrenocorticotropin. 相似文献
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Role of nitrate and nitrite for production and consumption of nitric oxide during denitrification in soil 总被引:1,自引:0,他引:1
Abstract Anaerobic production and consumption of NO was measured in a calcic cambisol (KBE; pH 7.3) and a forest luvisol (PBE; pH 4.4) which were incubated at 80% water-holding capacity and continuously flushed with N2 . Both NO production and NO consumption were negligibly low when nitrate and nitrite concentrations in the soil were exhausted. Addition of glucose alone had no effect, but addition of nitrate ± glucose greatly stimulated both NO production and NO consumption. NO consumption followed an apparent first-order reaction at low NO mixing ratios (1–3 ppmv), but a higher NO mixing ratios it followed Michaelis-Menten kinetics. In PBE the apparent K m was 980 ppbv NO (1.92 nM in soil water). During reduction of nitrate, nitrite intermediately accumulated and simultaneously, production rates of NO and N2 O were at the maximum. Production rates of NO plus N2 O amounted to 20% and 34% of the nitrate reduction rate in KBE and PBE, respectively. NO production was hyperbolically related to the nitrite concentration, indicating an apparent Km of 1.6 μg nitrite-N g−1 d.w. soil (equivalent to 172 μM nitrite in soil solution) for the reduction of nitrite to NO in KBE. Under nitrate and nitrite-limiting conditions, 62–76% and 93–97% of the consumed NO-N were recovered as N2 O-N in KBE and PBE, respectively. Gassing of nitrate plus nitrite-depretsu KBE with increasing mixing ratios of NO2 resulted in increasing rates of NO2 uptake and presumably in the formation of low concentrations of nitrite and nitrate. This NO2 uptake resulted in increasing rates of both NO production and NO consumption indicating that nitrite or nitrate was limiting for both reactions. 相似文献
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Induction of nitrate and nitrite reductases in Anabaena cylindrica(FOGG strain) was investigated. At various stages of algal growthin the presence of nitrate or nitrite, the levels of these enzymeswere determined using cell-free preparations. Nitrate and nitritereductases were induced by the respective substrates. Nitratedid not act either as an inducer or as a repressor of nitritereductase.
1This work was supported by grant No. 8814 from the Ministryof Education
2Department of Biology, Faculty of Science, Tokyo MetropolitanUniversitySetagaya-ku, Tokyo, Japan (Received June 18, 1970; ) 相似文献
17.
Effects of simulated microgravity on arterial nitric oxide synthase and nitrate and nitrite content. 总被引:5,自引:0,他引:5
Jin Ma Chadi I Kahwaji Zhenmin Ni Nosratola D Vaziri Ralph E Purdy 《Journal of applied physiology》2003,94(1):83-92
The aim of the present work was to investigate the alterations in nitric oxide synthase (NOS) expression and nitrate and nitrite (NOx) content of different arteries from simulated microgravity rats. Male Wistar rats were randomly assigned to either a control group or simulated microgravity group. For simulating microgravity, animals were subjected to hindlimb unweighting (HU) for 20 days. Different arterial tissues were removed for determination of NOS expression and NOx. Western blotting was used to measure endothelial NOS (eNOS) and inducible NOS (iNOS) protein content. Total concentrations of NOx, stable metabolites of nitric oxide, were determined by the chemiluminescence method. Compared with controls, isolated vessels from simulated microgravity rats showed a significant increase in both eNOS and iNOS expression in carotid arteries and thoracic aorta and a significant decrease in eNOS and iNOS expression of mesenteric arteries. The eNOS and iNOS content of cerebral arteries, as well as that of femoral arteries, showed no differences between the two groups. Concerning NOx, vessels from HU rats showed an increase in cerebral arteries, a decrease in mesenteric arteries, and no change in carotid artery, femoral artery and thoracic aorta. These data indicated that there were differential alterations in NOS expression and NOx of different arteries after hindlimb unweighting. We suggest that these changes might represent both localized adaptations to differential body fluid redistribution and other factors independent of hemodynamic shifts during simulated microgravity. 相似文献
18.
Tsikas D 《Free radical research》2005,39(8):797-815
In human organism, the gaseous radical molecule nitric oxide (NO) is produced in various cells from L-arginine by the catalytic action of NO synthases (NOS). The metabolic fate of NO includes oxidation to nitrate by oxyhaemoglobin in red blood cells and autoxidation in haemoglobin-free media to nitrite. Nitrate and nitrite circulate in blood and are excreted in urine. The concentration of these NO metabolites in the circulation and in the urine can be used to measure NO synthesis in vivo under standardized low-nitrate diet. Circulating nitrite reflects constitutive endothelial NOS activity, whereas excretory nitrate indicates systemic NO production. Today, nitrite and nitrate can be measured in plasma, serum and urine of humans by various analytical methods based on different analytical principles, such as colorimetry, spectrophotometry, fluorescence, chemiluminescence, gas and liquid chromatography, electrophoresis and mass spectrometry. The aim of the present article is to give an overview of the most significant currently used quantitative methods of analysis of nitrite and nitrate in human biological fluids, namely plasma and urine. With minor exception, measurement of nitrite and nitrate by these methods requires method-dependent chemical conversion of these anions. Therefore, the underlying mechanisms and principles of these methods are also discussed. Despite the chemical simplicity of nitrite and nitrate, accurate and interference-free quantification of nitrite and nitrate in biological fluids as indicators of NO synthesis may be difficult. Thus, problems associated with dietary and laboratory ubiquity of these anions and other preanalytical and analytical factors are addressed. Eventually, the important issue of quality control, the use of commercially available assay kits, and the value of the mass spectrometry methodology in this area are outlined. 相似文献
19.
Dimitrios Tsikas 《Free radical research》2013,47(8):797-815
In human organism, the gaseous radical molecule nitric oxide (NO) is produced in various cells from l-arginine by the catalytic action of NO synthases (NOS). The metabolic fate of NO includes oxidation to nitrate by oxyhaemoglobin in red blood cells and autoxidation in haemoglobin-free media to nitrite. Nitrate and nitrite circulate in blood and are excreted in urine. The concentration of these NO metabolites in the circulation and in the urine can be used to measure NO synthesis in vivo under standardized low-nitrate diet. Circulating nitrite reflects consitutive endothelial NOS activity, whereas excretory nitrate indicates systemic NO production. Today, nitrite and nitrate can be measured in plasma, serum and urine of humans by various analytical methods based on different analytical principles, such as colorimetry, spectrophotometry, fluorescence, chemiluminescence, gas and liquid chromatography, electrophoresis and mass spectrometry. The aim of the present article is to give an overview of the most significant currently used quantitative methods of analysis of nitrite and nitrate in human biological fluids, namely plasma and urine. With minor exception, measurement of nitrite and nitrate by these methods requires method-dependent chemical conversion of these anions. Therefore, the underlying mechanisms and principles of these methods are also discussed. Despite the chemical simplicity of nitrite and nitrate, accurate and interference-free quantification of nitrite and nitrate in biological fluids as indicators of NO synthesis may be difficult. Thus, problems associated with dietary and laboratory ubiquity of these anions and other preanalytical and analytical factors are addressed. Eventually, the important issue of quality control, the use of commercially available assay kits, and the value of the mass spectrometry methodology in this area are outlined. 相似文献
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Macrophage oxidation of L-arginine to nitric oxide, nitrite, and nitrate. Tetrahydrobiopterin is required as a cofactor 总被引:27,自引:0,他引:27
A new metabolic pathway characterized recently that is expressed in activated macrophages involves the formation of nitric oxide ('N = O) as an intermediate. The 'N = O formed decomposes to nitrite (NO2-) and nitrate (NO3-). The substrate for the reaction is the amino acid arginine which is oxidized at the guanido nitrogen to yield citrulline as the other product of the reaction. The studies reported here show that the activity for this unusual oxidation reaction which is contained in the 100,000 x g supernatant was lost after desalting on a Sephadex G-25 column. A small molecule cofactor was found to be required for the restoration of activity. The addition of (6R)-tetrahydrobiopterin (BH4) and NADPH led to complete recovery of activity in this desalted protein. Analysis of macrophage cell extracts, using high performance liquid chromatography with electrochemical detection, showed that BH4 was present at 17 pmol/10(6) cells or 2.1 microM in macrophage supernatant. Only the (6R)-isomer was present. With the addition of BH4 and NADPH, there was loss of arginine that was equal to the NO2-, NO3-, and citrulline formed. With substoichiometric levels of NADPH relative to BH4, the loss of arginine was greater than the formation of the end products of the reaction. A scheme for the reaction pathway consistent with the results involves N-hydroxylation of arginine as the initial step. The participation of BH4 in this type of oxidative chemistry is consistent with previous characterizations of this co-factor. 相似文献