Production of Nitric Oxide and Nitrous Oxide During Denitrification by Corynebacterium nephridii

Resting cells of Corynebacterium nephridii reduce nitrate, nitrite, and nitric oxide to nitrous oxide under anaerobic conditions. Nitrous oxide production from nitrite was optimal from pH 7.0 to 7.4. The stoichiometry of nitrous oxide production from nitrite was 99% of the theoretical-two moles of nitrite was used for each mole of nitrous oxide detected. Hydroxylamine increases gas evolution from nitrite but inhibits the reduction of nitric oxide to nitrous oxide. Hydroxylamine is converted to nitrogenous gas(es) by resting cells only in the presence of nitrite. Under certain conditions nitric oxide, as well as nitrous oxide, was detected.     

Effect of Temperature on Consecutive Denitrification Reactions in Brookston Clay and Fox Sandy Loam

The kinetics of several steps in the microbial denitrification process in Brookston clay and Fox sandy loam, two soils common to Southwestern Ontario, were studied in the temperature range of 5 to 25°C. The extent of chemical denitrification was also determined in otherwise identical but sterilized soils at temperatures up to 80°C. A gas flow system was used in which soil gases were continuously removed from anaerobic soil columns by argon carrier gas. Net steady-state rates of NO and N₂O production, rates of loss of NO₃⁻, and production and loss of NO₂⁻ were measured over periods of up to 5 days. Arrhenius activation energies for the zero-order process NO₃⁻ → NO₂⁻ were calculated to be 50 ± 9 kJ mol⁻¹ for Brookston clay and 55 ± 13 kJ mol⁻¹ for Fox sandy loam. The overall reaction, NO₂⁻ → NO (chemodenitrification), in both sterile soils was accurately first order with respect to NO₂⁻; the activation energy was 70 ± 2.8 kJ mol⁻¹ in Brookston clay and 79 ± 1.2 kJ mol⁻¹ in the sandy loam, and the preexponential factors were (2.3 ± 1.2) × 10⁹ and (5.7 ± 1.2) × 10⁹ min⁻¹, respectively.     

Production of Nitric Oxide in Loam Under Aerobic and Anaerobic Conditions

Measurements of gas flow through soil columns of loam from Kjettslinge, Uppland, Sweden, gave average NO production rates of 0.06 ± 0.01 ng of NO N g of soil⁻¹ min⁻¹ in aerobic conditions and 3.7 ± 0.6 ng of NO N g of soil⁻¹ min⁻¹ in anaerobic conditions at 25°C. Approximately 30% of the NO₃⁻ loss in anaerobic conditions was as NO. In aerobic conditions an equilibrium concentration for NO was found. Above this concentration there was uptake of NO. Autoclaved samples indicated that less than 10% of the NO production was abiological, and there was no abiological NO uptake. The NO production reached anaerobic rates at soil O₂ levels between 0.5 and 0.05%.     

Terpenoids from Rhizomes of Alpinia japonica Inhibiting Nitric Oxide Production

A new sesquiterpenoid, 1 , and three new diterpenoids, 3 – 5 , along with five known compounds, 2 and 6 – 9 , were isolated from rhizomes of Alpinia japonica. The structures of the new compounds were determined as (1R,4R,6S,7S,9S)‐4α‐hydroxy‐1,9‐peroxybisabola‐2,10‐diene ( 1 ), methyl (12E)‐16‐oxolabda‐8(17),12‐dien‐15‐oate ( 3 ), (12R)‐15‐ethoxy‐12‐hydroxylabda‐8(17),13(14)‐dien‐16,15‐olide ( 4 ), and methyl (11E)‐14,15,16‐trinorlabda‐8(17),11‐dien‐13‐oate ( 5 ) by means of spectroscopic data. The absolute configurations at C(4) in 1 and C(12) in 4 were deduced from the circular dichroism (CD) data of the in situ‐formed [Rh₂(CF₃COO)₄] complexes. Inhibitory effects of the isolates on NO production in lipopolysaccharide‐induced RAW264.7 macrophages were evaluated, and 2 – 4, 6 , and 7 were found to exhibit inhibitory activities with IC₅₀ values between 14.6 and 34.3 μM .     

Inhibition of Anaerobic Phosphate Release by Nitric Oxide in Activated Sludge

Activated sludge not containing significant numbers of denitrifying, polyphosphate [poly(P)]-accumulating bacteria was grown in a fill-and-draw system and exposed to alternating anaerobic and aerobic periods. During the aerobic period, poly(P) accumulated up to 100 mg of P · g of (dry) weight. When portions of the sludge were incubated anaerobically in the presence of acetate, 80 to 90% of the intracellular poly(P) was degraded and released as orthophosphate. Degradation of poly(P) was mainly catalyzed by the concerted action of polyphosphate:AMP phosphotransferase and adenylate kinase, resulting in ATP formation. In the presence of 0.3 mM nitric oxide (NO) in the liquid-phase release of phosphate, uptake of acetate, formation of poly-β-hydroxybutyrate, utilization of glycogen, and formation of ATP were severely inhibited or completely abolished. In cell extracts of the sludge, adenylate kinase activity was completely inhibited by 0.15 mM NO. The nature of this inhibition was probably noncompetitive, similar to that with hog adenylate kinase. Activated sludge polyphosphate glucokinase was also completely inhibited by 0.15 mM NO. It is concluded that the inhibitory effect of NO on acetate-mediated phosphate release by the sludge used in this study is due to the inhibition of adenylate kinase in the phosphate-releasing organisms. The inhibitory effect of nitrate and nitrite on phosphate release is probably due to their conversion to NO. The lack of any inhibitory effect of NO on adenylate kinase of the poly(P)-accumulating Acinetobacter johnsonii 210A suggests that this type of organism is not involved in the enhanced biological phosphate removal by the sludges used.     

Kinetic Explanation for Accumulation of Nitrite, Nitric Oxide, and Nitrous Oxide During Bacterial Denitrification

The kinetics of denitrification and the causes of nitrite and nitrous oxide accumulation were examined in resting cell suspensions of three denitrifiers. An Alcaligenes species and a Pseudomonas fluorescens isolate characteristically accumulated nitrite when reducing nitrate; a Flavobacterium isolate did not. Nitrate did not inhibit nitrite reduction in cultures grown with tungstate to prevent formation of an active nitrate reductase; rather, accumulation of nitrite seemed to depend on the relative rates of nitrate and nitrite reduction. Each isolate rapidly reduced nitrous oxide even when nitrate or nitrite had been included in the incubation mixture. Nitrate also did not inhibit nitrous oxide reduction in Alcaligenes odorans, an organism incapable of nitrate reduction. Thus, added nitrate or nitrite does not always cause nitrous oxide accumulation, as has often been reported for denitrifying soils. All strains produced small amounts of nitric oxide during denitrification in a pattern suggesting that nitric oxide was also under kinetic control similar to that of nitrite and nitrous oxide. Apparent K_m values for nitrate and nitrite reduction were 15 μM or less for each isolate. The K_m value for nitrous oxide reduction by Flavobacterium sp. was 0.5 μM. Numerical solutions to a mathematical model of denitrification based on Michaelis-Menten kinetics showed that differences in reduction rates of the nitrogenous compounds were sufficient to account for the observed patterns of nitrite, nitric oxide, and nitrous oxide accumulation. Addition of oxygen inhibited gas production from ¹³NO₃⁻ by Alcaligenes sp. and P. fluorescens, but it did not reduce gas production by Flavobacterium sp. However, all three isolates produced higher ratios of nitrous oxide to dinitrogen as the oxygen tension increased. Inclusion of oxygen in the model as a nonspecific inhibitor of each step in denitrification resulted in decreased gas production but increased ratios of nitrous oxide to dinitrogen, as observed experimentally. The simplicity of this kinetic model of denitrification and its ability to unify disparate observations should make the model a useful guide in research on the physiology of denitrifier response to environmental effectors.     

Nitric Oxide Production in Plants: Facts and Fictions

There is now general agreement that nitric oxide (NO) is an important and almost universal signal in plants. Nevertheless, there are still many controversial observations and opinions on the importance and function of NO in plants. Partly, this may be due to the difficulties in detecting and even more in quantifying NO. Here, we summarize major pathways of NO production in plants, and briefly discuss some methodical problems.Key Words: chemiluminescence, DAF-fluorescence, mitochondria, nitrate reductase, nitric oxide, nitric oxide synthase, NO detection, NO signaling     

Distinguishing Nitrous Oxide Production from Nitrification and Denitrification on the Basis of Isotopomer Abundances

The intramolecular distribution of nitrogen isotopes in N₂O is an emerging tool for defining the relative importance of microbial sources of this greenhouse gas. The application of intramolecular isotopic distributions to evaluate the origins of N₂O, however, requires a foundation in laboratory experiments in which individual production pathways can be isolated. Here we evaluate the site preferences of N₂O produced during hydroxylamine oxidation by ammonia oxidizers and by a methanotroph, ammonia oxidation by a nitrifier, nitrite reduction during nitrifier denitrification, and nitrate and nitrite reduction by denitrifiers. The site preferences produced during hydroxylamine oxidation were 33.5 ± 1.2‰, 32.5 ± 0.6‰, and 35.6 ± 1.4‰ for Nitrosomonas europaea, Nitrosospira multiformis, and Methylosinus trichosporium, respectively, indicating similar site preferences for methane and ammonia oxidizers. The site preference of N₂O from ammonia oxidation by N. europaea (31.4 ± 4.2‰) was similar to that produced during hydroxylamine oxidation (33.5 ± 1.2‰) and distinct from that produced during nitrifier denitrification by N. multiformis (0.1 ± 1.7‰), indicating that isotopomers differentiate between nitrification and nitrifier denitrification. The site preferences of N₂O produced during nitrite reduction by the denitrifiers Pseudomonas chlororaphis and Pseudomonas aureofaciens (−0.6 ± 1.9‰ and −0.5 ± 1.9‰, respectively) were similar to those during nitrate reduction (−0.5 ± 1.9‰ and −0.5 ± 0.6‰, respectively), indicating no influence of either substrate on site preference. Site preferences of ~33‰ and ~0‰ are characteristic of nitrification and denitrification, respectively, and provide a basis to quantitatively apportion N₂O.     

Glycoinositolphospholipids from Trypanosomatids Subvert Nitric Oxide Production in Rhodnius prolixus Salivary Glands

Background

Rhodnius prolixus is a blood-sucking bug vector of Trypanosoma cruzi and T. rangeli. T. cruzi is transmitted by vector feces deposited close to the wound produced by insect mouthparts, whereas T. rangeli invades salivary glands and is inoculated into the host skin. Bug saliva contains a set of nitric oxide-binding proteins, called nitrophorins, which deliver NO to host vessels and ensure vasodilation and blood feeding. NO is generated by nitric oxide synthases (NOS) present in the epithelium of bug salivary glands. Thus, T. rangeli is in close contact with NO while in the salivary glands.

Methodology/Principal Findings

Here we show by immunohistochemical, biochemical and molecular techniques that inositolphosphate-containing glycolipids from trypanosomatids downregulate NO synthesis in the salivary glands of R. prolixus. Injecting insects with T. rangeli-derived glycoinositolphospholipids (Tr GIPL) or T. cruzi-derived glycoinositolphospholipids (Tc GIPL) specifically decreased NO production. Salivary gland treatment with Tc GIPL blocks NO production without greatly affecting NOS mRNA levels. NOS protein is virtually absent from either Tr GIPL- or Tc GIPL-treated salivary glands. Evaluation of NO synthesis by using a fluorescent NO probe showed that T. rangeli-infected or Tc GIPL-treated glands do not show extensive labeling. The same effect is readily obtained by treatment of salivary glands with the classical protein tyrosine phosphatase (PTP) inhibitor, sodium orthovanadate (SO). This suggests that parasite GIPLs induce the inhibition of a salivary gland PTP. GIPLs specifically suppressed NO production and did not affect other anti-hemostatic properties of saliva, such as the anti-clotting and anti-platelet activities.

Conclusions/Significance

Taken together, these data suggest that trypanosomatids have overcome NO generation using their surface GIPLs. Therefore, these molecules ensure parasite survival and may ultimately enhance parasite transmission.     

Withanolides from Physalin angulata and Their Inhibitory Effects on Nitric Oxide Production

Six new withanolides, angulasteroidins A−F ( 1 – 6 ), along with twelve known analogs ( 7-18 ) were isolated from the whole plants of Physalis angulata. Their structures were elucidated by analysis of 1D and 2D NMR, ECD and IR spectra, HR-ESI-MS data, and ECD calculation. Compounds 1 and 6 were rare 1–10 seco withanolides. Compounds 2 – 4 , 7 – 9 , and 15 exhibited significant inhibitory activity on the production of nitric oxide in the LPS-activated RAW 264.7 mouse macrophage cell lines with IC₅₀ values ranging from 0.23 to 9.06 μM.     

Nitric Oxide Production in Striatum and Pallidum of Cirrhotic Rats

Ammonium and manganese are neurotoxic agents related to brain metabolic disturbances observed after prolonged liver damage. The aim of this study was to assess the production of nitric oxide (NO) in the brain of cirrhotic rats exposed to manganese. We induced cirrhosis by bile duct ligation for 4 weeks in rats. From brain, striatum and globus pallidus were dissected out, and NO synthase activity and the content of nitrites plus nitrates (NOx) were determined. In pallidum we found a diminished constitutive NO synthase activity from cirrhotic rats, independently of manganese exposure. This result was confirmed by low levels of NOx in the same brain area (P<0.05, two-way ANOVA). This finding was not related to protein expression of NO synthase since no differences were observed in immunoblot signals between cirrhotic and sham-operated animals. Results from present study suggest that the production of NO is reduced in basal ganglia during cirrhosis.     

Leptin and Nitric Oxide Production in Normotensive and Hypertensive Men

Objective: Recent findings have shown that leptin, the product of the obesity gene, may actively participate in the regulation of blood pressure and other cardiovascular functions through the nitric oxide (NO)‐dependent mechanism. Research Methods and Procedures: In this study, to test the hypothesis that leptin regulation of NO metabolism is impaired in hypertension, we examined the possible relationship between circulating leptin and plasma NO metabolite level in normotensive (NT) and hypertensive (HT) men. Results: There were significant correlations between circulating leptin and BMI in both the NT and HT groups (NT: r = 0.64, n = 26, p < 0.01; HT: r = 0.59, n = 22, p < 0.01). The concentration of circulating leptin was similar between the NT and HT men, although the plasma NO metabolite level (nitrite and nitrate) was significantly reduced in the HT men compared with the NT men (NT: 51.0 ± 4.9 μM, n = 26; HT: 37.1 ± 2.5 μM, n = 22, p < 0.05). The circlating leptin was significantly correlated with the plasma NO metabolite level in the overall analysis of the NT and HT men (r = 0.35, n = 48, p < 0.05). When the analysis of the correlation for the NT and HT men was performed separately, there was a significant correlation between circulating leptin and plasma NO metabolites in the NT men (r = 0.45, n = 26, p < 0.05) but not in the HT men (r = 0.15, n = 22). The results of this study are consistent with the hypothesis that leptin‐related metabolism of NO might be altered in HT men.     

Undescribed Phenolic Glycosides from Syzygium attopeuense and Their Inhibition of Nitric Oxide Production

Four undescribed phenolic glycosides including three stilbene derivatives ( 1 and 3 ) and sodium salt of 3 ( 2 ), and a chalcone glycoside ( 4 ), together with thirteen known compounds ( 5 – 17 ) were isolated from the leaves of Syzygium attopeuense (Gagnep.) Merr. & L.M.Perry. Their chemical structures were elucidated to be (Z)-gaylussacin ( 1 ), 6′′-O-galloylgaylussacin sodium salt ( 2 ), 6′′-O-galloylgaylussacin ( 3 ), 4′-O-[β-D-glucopyranosyl-(1→6)-glucopyranosyl]oxy-2′-hydroxy-6′-methoxydihydrochalcone ( 4 ), gaylussacin ( 5 ), pinosilvin 3-O-β-D-glucopyranoside ( 6 ), myricetin-3-O-(2′′-O-galloyl)-α-L-rhamnopyranoside ( 7 ), myricetin-3-O-(3′′-O-galloyl)-α-L-rhamnopyranoside ( 8 ), myricetin-3-O-α-L-rhamnopyranoside ( 9 ), quercitrin ( 10 ), myricetin-3-O-β-D-glucopyranoside ( 11 ), myricetin-3-O-β-D-galactopyranoside ( 12 ), quercetin 3-O-α-L-arabinopyranoside ( 13 ), myricetin-3-O-2′′-O-galloyl)-α-L-arabinopyranoside ( 14 ), (+)-gallocatechin ( 15 ), (−)-epigallocatechin ( 16 ), and 3,3’,4’-trimethoxyellagic acid 4-O-β-D-glucopyranoside ( 17 ) by the analysis of HR-ESI-MS, 1D and 2D NMR spectra in comparison with the previously reported data. Compounds 1–3 , 5 , and 6 significant inhibition of NO production in LPS-activated RAW264.7 cells, with IC₅₀ values ranging from 18.37±1.38 to 35.12±2.53 μM, compared to a positive control (dexamethasone) with an IC₅₀ value of 15.37±1.42 μM.     

Increased Nitric Oxide Production in Patients with Systemic Sclerosis

Nitric oxide (NO, nitrogen monoxide) is a messenger molecule whose synthesis can be induced by proinflammatory cytokines. Increased production of NO has been reported in various inflammatory and autoimmune diseases. We studied serum nitrite and citrulline as surrogate markers for NO production in patients with systemic sclerosis (SSc) and looked for correlation with extent of disease, disease duration, age, and systemic involvement. Thirty-four patients were studied against 20 controls. The nitrite levels were significantly higher in the disease group (1588.4 +/- 998.2 nmol/ml compared to 327.8 +/- 137.7 nmol/ml; P < 0.001). The citrulline levels of the disease group were also significantly higher (5490.1 +/- 2518.3 nmol/ml compared to 3264.5 +/- 2509.7 nmol/ml in the controls; P = 0.005). There was no significant difference among limited and diffuse subgroups. There was no significant difference in patients with or without arthritis or interstitial lung disease or with other systemic involvement. On multivariate analysis there was a trend toward a rising level of nitrite with worsening lung functions (P = 0.07). Hence, there is evidence of increased NO production in patients with SSc. There is no difference between NO levels in disease subgroups or those with systemic involvement.     

Characterization of the Magnitude and Mechanism of Aldehyde Oxidase-mediated Nitric Oxide Production from Nitrite

Aldehyde oxidase (AO) is a cytosolic enzyme with an important role in drug and xenobiotic metabolism. Although AO has structural similarity to bacterial nitrite reductases, it is unknown whether AO-catalyzed nitrite reduction can be an important source of NO. The mechanism, magnitude, and quantitative importance of AO-mediated nitrite reduction in tissues have not been reported. To investigate this pathway and its quantitative importance, EPR spectroscopy, chemiluminescence NO analyzer, and immunoassays of cGMP formation were performed. The kinetics and magnitude of AO-dependent NO formation were characterized. In the presence of typical aldehyde substrates or NADH, AO reduced nitrite to NO. Kinetics of AO-catalyzed nitrite reduction followed Michaelis-Menten kinetics under anaerobic conditions. Under physiological conditions, nitrite levels are far below its measured K_m value in the presence of either the flavin site electron donor NADH or molybdenum site aldehyde electron donors. Under aerobic conditions with the FAD site-binding substrate, NADH, AO-mediated NO production was largely maintained, although with aldehyde substrates oxygen-dependent inhibition was seen. Oxygen tension, substrate, and pH levels were important regulators of AO-catalyzed NO generation. From kinetic data, it was determined that during ischemia hepatic, pulmonary, or myocardial AO and nitrite levels were sufficient to result in NO generation comparable to or exceeding maximal production by constitutive NO synthases. Thus, AO-catalyzed nitrite reduction can be an important source of NO generation, and its NO production will be further increased by therapeutic administration of nitrite.