NADPH phagocyte oxidase knockout mice control Trypanosoma cruzi proliferation, but develop circulatory collapse and succumb to infection

^•NO is considered to be a key macrophage-derived cytotoxic effector during Trypanosoma cruzi infection. On the other hand, the microbicidal properties of reactive oxygen species (ROS) are well recognized, but little importance has been attributed to them during in vivo infection with T. cruzi. In order to investigate the role of ROS in T. cruzi infection, mice deficient in NADPH phagocyte oxidase (gp91^{phox −/−} or phox KO) were infected with Y strain of T. cruzi and the course of infection was followed. phox KO mice had similar parasitemia, similar tissue parasitism and similar levels of IFN-γ and TNF in serum and spleen cell culture supernatants, when compared to wild-type controls. However, all phox KO mice succumbed to infection between day 15 and 21 after inoculation with the parasite, while 60% of wild-type mice were alive 50 days after infection. Further investigation demonstrated increased serum levels of nitrite and nitrate (NOx) at day 15 of infection in phox KO animals, associated with a drop in blood pressure. Treatment with a NOS2 inhibitor corrected the blood pressure, implicating NOS2 in this phenomenon. We postulate that superoxide reacts with ^•NO in vivo, preventing blood pressure drops in wild type mice. Hence, whilst superoxide from phagocytes did not play a critical role in parasite control in the phox KO animals, its production would have an important protective effect against blood pressure decline during infection with T. cruzi.     

A review and discussion of platelet nitric oxide and nitric oxide synthase: do blood platelets produce nitric oxide from <Emphasis Type="SmallCaps">l</Emphasis>-arginine or nitrite?

The NO/sGC/cGMP/PKG system is one of the most powerful mechanisms responsible for platelet inhibition. In numerous publications, expression of functional NO synthase (NOS) in human and mouse platelets has been reported. Constitutive and inducible NOS isoforms convert l-arginine to NO and l-citrulline. The importance of this pathway in platelets and in endothelial cells for the regulation of platelet function is discussed since decades. However, there are serious doubts in the literature concerning both expression and functionality of NOS in platelets. In this review, we aim to present and critically evaluate recent data concerning NOS expression and function in platelets, and to especially emphasise potential pitfalls of detection of NOS proteins and measurement of NOS activity. Prevailing analytical problems are probably the main sources of contradictory data on occurrence, activity and function of NOS in platelets. In this review we also address issues of how these problems can be resolved. NO donors including organic nitrites (RONO) and organic nitrate (RONO₂) are inhibitors of platelet activation. Endogenous inorganic nitrite (NO₂ ^?), the product of NO autoxidation, and exogenous inorganic nitrite are increasingly investigated as NO donors in the circulation. The role of platelets in the generation of NO from nitrite is also discussed.     

The hemerythrin-like diiron protein from Mycobacterium kansasii is a nitric oxide peroxidase

The hemerythrin-like protein from Mycobacterium kansasii (Mka HLP) is a member of a distinct class of oxo-bridged diiron proteins that are found only in mycobacterial species that cause respiratory disorders in humans. Because it had been shown to exhibit weak catalase activity and a change in absorbance on exposure to nitric oxide (NO), the reactivity of Mka HLP toward NO was examined under a variety of conditions. Under anaerobic conditions, we found that NO was converted to nitrite (NO₂⁻) via an intermediate, which absorbed light at 520 nm. Under aerobic conditions NO was converted to nitrate (NO₃⁻). In each of these two cases, the maximum amount of nitrite or nitrate formed was at best stoichiometric with the concentration of Mka HLP. When incubated with NO and H₂O₂, we observed NO peroxidase activity yielding nitrite and water as reaction products. Steady-state kinetic analysis of NO consumption during this reaction yielded a K_m for NO of 0.44 μM and a k_cat/K_m of 2.3 × 10⁵ M⁻¹s⁻¹. This high affinity for NO is consistent with a physiological role for Mka HLP in deterring nitrosative stress. This is the first example of a peroxidase that uses an oxo-bridged diiron center and a rare example of a peroxidase utilizing NO as an electron donor and cosubstrate. This activity provides a mechanism by which the infectious Mycobacterium may combat against the cocktail of NO and superoxide (O₂^•−) generated by macrophages to defend against bacteria, as well as to produce NO₂⁻ to adapt to hypoxic conditions.     

Nitrate Reductase Activity in Shoots and Roots of Maize Seedlings as Affected by the Form of Nitrogen Nutrition and the pH of the Nutrient Solution

The effect of nitrogen form (NH₄-N, NH₄-N + NO₃⁻, NO₃⁻) on nitrate reductase activity in roots and shoots of maize (Zea mays L. cv INRA 508) seedlings was studied. Nitrate reductase activity in leaves was consistent with the well known fact that NO₃⁻ increases, and NH₄⁺ and amide-N decrease, nitrate reductase activity. Nitrate reductase activity in the roots, however, could not be explained by the root content of NO₃⁻, NH₄-N, and amide-N. In roots, nitrate reductase activity in vitro was correlated with the rate of nitrate reduction in vivo. Inasmuch as nitrate reduction results in the production of OH⁻ and stimulates the synthesis of organic anions, it was postulated that nitrate reductase activity of roots is stimulated by the released OH⁻ or by the synthesized organic anions rather than by nitrate itself. Addition of HCO₃⁻ to nutrient solution of maize seedlings resulted in a significant increase of the nitrate reductase activity in the roots. As HCO₃⁻, like OH⁻, increases pH and promotes the synthesis of organic anions, this provides circumstantial evidence that alkaline conditions and/or organic anions have a more direct impact on nitrate reductase activity than do NO₃⁻, NH₄-N, and amide-N.     

Platelet Content of Nitric Oxide Synthase 3 Phosphorylated At Serine1177 Is Associated with the Functional Response of Platelets to Aspirin

Objective

To analyse if platelet responsiveness to aspirin (ASA) may be associated with a different ability of platelets to generate nitric oxide (NO).

Patients/Methods

Platelets were obtained from 50 patients with stable coronary ischemia and were divided into ASA-sensitive (n = 26) and ASA-resistant (n = 24) using a platelet functionality test (PFA-100).

Results

ASA-sensitive platelets tended to release more NO (determined as nitrite + nitrate) than ASA-resistant platelets but it did not reach statistical significance. Protein expression of nitric oxide synthase 3 (NOS3) was higher in ASA-sensitive than in ASA-resistant platelets but there were no differences in the platelet expression of nitric oxide synthase 2 (NOS2) isoform. The highest NOS3 expression in ASA-sensitive platelets was independent of the presence of T-to-C mutation at nucleotide position −786 (T⁻⁷⁸⁶→C) in the NOS3-coding gene. However, platelet content of phosphorylated NOS3 at Serine (Ser)¹¹⁷⁷, an active form of NOS3, was higher in ASA-sensitive than in ASA-resistant platelets. The level of platelet NOS3 Ser¹¹⁷⁷ phosphorylation was positively associated with the closure time in the PFA-100 test. In vitro, collagen failed to stimulate the aggregation of ASA-sensitive platelets, determined by lumiaggregometry, and it was associated with a significant increase (p = 0.018) of NOS3 phosphorylation at Ser¹¹⁷⁷. On the contrary, collagen stimulated the aggregation of ASA-resistant platelets but did not significantly modify the platelet content of phosphorylated NOS3 Ser¹¹⁷⁷. During collagen stimulation the release of NO from ASA-sensitive platelets was significantly enhanced but it was not modified in ASA-resistant platelets.

Conclusions

Functional platelet responsiveness to ASA was associated with the platelet content of phosphorylated NOS3 at Ser¹¹⁷⁷.     

Studies of the Uptake of Nitrate in Barley : IV. Electrophysiology

Transmembrane electrical potential differences (Δψ) of epidermal and cortical cells were measured in intact roots of barley (Hordeum vulgare L. cv Klondike). The effects of exogenous NO₃⁻ on Δψ (in the concentration range from 100 micromolar to 20 millimolar) were investigated to probe the mechanisms of nitrate uptake by the high-affinity (HATS) and low-affinity (LATS) transport systems for NO₃⁻ uptake. Both transport systems caused depolarization of Δψ, demonstrating that the LATS (like the HATS) for NO₃⁻ uptake is probably mediated by an electrogenic cation (H⁺?) cotransport system. Membrane depolarization by the HATS was “inducible” by NO₃⁻, and saturable with respect to exogenous [NO₃⁻]. By contrast, depolarization by the LATS was constitutive, and first-order in response to external [NO₃⁻]. H⁺ fluxes, measured in 200 micromolar and in 5 millimolar Ca(NO₃)₂ solutions, failed to alkalinize external media as anticipated for a 2 H⁺:1 NO₃⁻ symport. However, switching from K₂SO₄ solutions (which were strongly acidifying) to KNO₃ solutions at the same K⁺ concentration caused marked reductions in H⁺ efflux. These observations are consistent with NO₃⁻ uptake by the HATS and the LATS via 2 H⁺:1 NO₃⁻ symports. These observations establish that the HATS for nitrate uptake by barley roots is essentially similar to those reported for Lemna and Zea mays by earlier workers. There are, nevertheless, distinct differences between barley and corn in their quantitative responses to external NO₃⁻.     

Dietary nitrite restores NO homeostasis and is cardioprotective in endothelial nitric oxide synthase-deficient mice

Endothelial production of nitric oxide (NO) is critical for vascular homeostasis. Nitrite and nitrate are formed endogenously by the stepwise oxidation of NO and have, for years, been regarded as inactive degradation products. As a result, both anions are routinely used as surrogate markers of NO production, with nitrite as a more sensitive marker. However, both nitrite and nitrate are derived from dietary sources. We sought to determine how exogenous nitrite affects steady-state concentrations of NO metabolites thought to originate from nitric oxide synthase (NOS)-derived NO as well as blood pressure and myocardial ischemia-reperfusion (I/R) injury. Mice deficient in endothelial nitric oxide synthase (eNOS-/-) demonstrated decreased blood and tissue nitrite, nitrate, and nitroso proteins, which were further reduced by low-nitrite (NOx) diet for 1 week. Nitrite supplementation (50 mg/L) in the drinking water for 1 week restored NO homeostasis in eNOS-/- mice and protected against I/R injury. Nitrite failed to alter heart rate or mean arterial blood pressure at the protective dose. These data demonstrate the significant influence of dietary nitrite intake on the maintenance of steady-state NO levels. Dietary nitrite and nitrate may serve as essential nutrients for optimal cardiovascular health and may provide a novel prevention/treatment modality for disease associated with NO insufficiency.     

Nitrite oxidation in the Namibian oxygen minimum zone

Nitrite oxidation is the second step of nitrification. It is the primary source of oceanic nitrate, the predominant form of bioavailable nitrogen in the ocean. Despite its obvious importance, nitrite oxidation has rarely been investigated in marine settings. We determined nitrite oxidation rates directly in ¹⁵N-incubation experiments and compared the rates with those of nitrate reduction to nitrite, ammonia oxidation, anammox, denitrification, as well as dissimilatory nitrate/nitrite reduction to ammonium in the Namibian oxygen minimum zone (OMZ). Nitrite oxidation (⩽372 nM NO₂⁻ d⁻¹) was detected throughout the OMZ even when in situ oxygen concentrations were low to non-detectable. Nitrite oxidation rates often exceeded ammonia oxidation rates, whereas nitrate reduction served as an alternative and significant source of nitrite. Nitrite oxidation and anammox co-occurred in these oxygen-deficient waters, suggesting that nitrite-oxidizing bacteria (NOB) likely compete with anammox bacteria for nitrite when substrate availability became low. Among all of the known NOB genera targeted via catalyzed reporter deposition fluorescence in situ hybridization, only Nitrospina and Nitrococcus were detectable in the Namibian OMZ samples investigated. These NOB were abundant throughout the OMZ and contributed up to ∼9% of total microbial community. Our combined results reveal that a considerable fraction of the recently recycled nitrogen or reduced NO₃⁻ was re-oxidized back to NO₃⁻ via nitrite oxidation, instead of being lost from the system through the anammox or denitrification pathways.     

The Effect of Lipopolysaccharide from <Emphasis Type="Italic">Proteus mirabilis</Emphasis> on the Level of the Stable End Metabolic Products of Nitric Oxide in Blood Platelets

Nitric oxide (^•NO) plays an important role in a number of physiologic processes. Evidence exists that ^•NO, which stimulates soluble guanylate cyclase and enhances cyclic guanosine monophosphate (cGMP) levels, may inhibit platelet activation. In contrast, during platelet activation induced by different agonists, synthesis of ^•NO in platelets occurs. In these studies, production of the stable end-products of ^•NO-nitrite and nitrate (NO_x) in human platelets, stimulated by different doses of lipopolysaccharide from Proteus mirabilis (LPS; endotoxin), has been evaluated. LPS is a weak platelet agonist that may activate various steps of platelet activation with the generation of reactive oxygen species. The mechanism of platelet activation induced by the endotoxin is not known. The aim of the present study was to measure the level of nitrite and NO_x in blood platelets treated with LPS and to examine the level of nitrotyrosine in platelet proteins caused by LPS. Our results show that LPS at a low concentration (6.8 ng/ml) caused a decrease (approximately 80%) in the NO_x level, whereas at higher concentrations (13.6 and 25 ng/ml) it induced an increase in the NO_x level (approximately 210% and 260%, respectively). Our results indicate that LPS, like other agonists (thrombin, platelet-activating factor), can stimulate ^•NO production in platelets. After incubating platelets with LPS, we also observed a distinct increase in platelet protein nitration (3-nitrotyrosine).     

ReviewMethods of quantitative analysis of the nitric oxide metabolites nitrite and nitrate in human biological fluids

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.     

Nitrate enrichment alters a Daphnia–microparasite interaction through multiple pathways

Nutrient pollution has the potential to alter many ecological interactions, including host–parasite relationships. One of the largest sources of nutrient pollution comes from anthropogenic alteration of the nitrogen (N) cycle, specifically the increased rate of nitrate (NO₃-N) deposition to aquatic environments, potentially altering host–parasite relationships. This study aimed to assess the mechanisms through which nitrate may impact host–pathogen relationships using a fungal pathogen (Metschnikowia bicuspidata) parasitic to crustacean zooplankton (Daphnia dentifera) as a tractable model system. First, the influence of nitrate on host population dynamics was assessed along a gradient of nitrate concentrations. Nitrate decreased host population size and increased infection prevalence. Second, the influence of nitrate on host reproduction, mortality, and infection intensity was assessed at the individual host level by examining the relationship between pathogen dose and infection prevalence at ambient (0.4 mg NO₃-N*L⁻¹) and intermediate (12 mg NO₃-N*L⁻¹) levels of nitrate. Host fecundity and infection intensity both decreased with increasing pathogen dose, but increased nitrate levels corresponded to greater infection intensities. Nitrate had no effect on host growth rate, suggesting that hosts do not alter feeding behavior in nitrate-treated media compared with ambient conditions. This study suggests that nutrient enrichment may enhance disease through increased transmission and infection intensity, but that high levels of nitrate may result in smaller epidemics through reduced transmission caused by smaller population sizes and increased pathogen mortality.     

Inducible nitric oxide synthase in heart tissue and nitric oxide in serum of Trypanosoma cruzi-infected rhesus monkeys: association with heart injury

Background

The factors contributing to chronic Chagas'' heart disease remain unknown. High nitric oxide (NO) levels have been shown to be associated with cardiomyopathy severity in patients. Further, NO produced via inducible nitric oxide synthase (iNOS/NOS2) is proposed to play a role in Trypanosoma cruzi control. However, the participation of iNOS/NOS2 and NO in T. cruzi control and heart injury has been questioned. Here, using chronically infected rhesus monkeys and iNOS/NOS2-deficient (Nos2 ^−/−) mice we explored the participation of iNOS/NOS2-derived NO in heart injury in T. cruzi infection.

Methodology

Rhesus monkeys and C57BL/6 and Nos2 ^−/− mice were infected with the Colombian T. cruzi strain. Parasite DNA was detected by polymerase chain reaction, T. cruzi antigens and iNOS/NOS2⁺ cells were immunohistochemically detected in heart sections and NO levels in serum were determined by Griess reagent. Heart injury was assessed by electrocardiogram (ECG), echocardiogram (ECHO), creatine kinase heart isoenzyme (CK-MB) activity levels in serum and connexin 43 (Cx43) expression in the cardiac tissue.

Results

Chronically infected monkeys presented conduction abnormalities, cardiac inflammation and fibrosis, which resembled the spectrum of human chronic chagasic cardiomyopathy (CCC). Importantly, chronic myocarditis was associated with parasite persistence. Moreover, Cx43 loss and increased CK-MB activity levels were primarily correlated with iNOS/NOS2⁺ cells infiltrating the cardiac tissue and NO levels in serum. Studies in Nos2 ^−/− mice reinforced that the iNOS/NOS2-NO pathway plays a pivotal role in T. cruzi-elicited cardiomyocyte injury and in conduction abnormalities that were associated with Cx43 loss in the cardiac tissue.

Conclusion

T. cruzi-infected rhesus monkeys reproduce features of CCC. Moreover, our data support that in T. cruzi infection persistent parasite-triggered iNOS/NOS2 in the cardiac tissue and NO overproduction might contribute to CCC severity, mainly disturbing of the molecular pathway involved in electrical synchrony. These findings open a new avenue for therapeutic tools in Chagas'' heart disease.     

Visualization of NO3?/NO2? Dynamics in Living Cells by Fluorescence Resonance Energy Transfer (FRET) Imaging Employing a Rhizobial Two-component Regulatory System

Nitrate (NO₃⁻) and nitrite (NO₂⁻) are the physiological sources of nitric oxide (NO), a key biological messenger molecule. NO₃⁻/NO₂⁻ exerts a beneficial impact on NO homeostasis and its related cardiovascular functions. To visualize the physiological dynamics of NO₃⁻/NO₂⁻ for assessing the precise roles of these anions, we developed a genetically encoded intermolecular fluorescence resonance energy transfer (FRET)-based indicator, named sNOOOpy (sensor for NO₃⁻/NO₂⁻ in physiology), by employing NO₃⁻/NO₂⁻-induced dissociation of NasST involved in the denitrification system of rhizobia. The in vitro use of sNOOOpy shows high specificity for NO₃⁻ and NO₂⁻, and its FRET signal is changed in response to NO₃⁻/NO₂⁻ in the micromolar range. Furthermore, both an increase and decrease in cellular NO₃⁻ concentration can be detected. sNOOOpy is very simple and potentially applicable to a wide variety of living cells and is expected to provide insights into NO₃⁻/NO₂⁻ dynamics in various organisms, including plants and animals.     

Diel changes in nitrogen and carbon resource status and use for growth in young plants of tomato (Solanum lycopersicum)

Background and Aims

Modellers often define growth as the development of plant structures from endogenous resources, thus making a distinction between structural (W_S) and total (W) dry biomass, the latter being the sum of W_S and the weight of storage compounds. In this study, short-term C and N reserves were characterized experimentally (forms, organ distribution, time changes) in relation to light and nutrition signals, and organ structural growth in response to reserve levels was evaluated.

Methods

Tomato plants (Solanum lycopersicum) were grown hydroponically in a growth room with a 12-h photoperiod and an adequate supply of NO₃⁻ (3 mol m⁻³). Three experiments were carried out 18 d after sowing: [NO₃⁻] was either maintained at 3 mol m⁻³, changed to 0·02 mol m⁻³ or to 0 mol m⁻³. Plants were sampled periodically throughout the light/dark cycles over 24–48 h. Organ W_S was calculated from W together with the amount of different compounds that act as C and N resources, i.e. non-structural carbohydrates and carboxylates, nitrate and free amino acids.

Key Results

With adequate nutrition, carbohydrates accumulated in leaves during light periods, when photosynthesis exceeded growth needs, but decreased at night when these sugars are the main source of C for growth. At the end of the night, carbohydrates were still high enough to fuel full-rate growth, as W_S increased at a near constant rate throughout the light/dark cycle. When nitrate levels were restricted, C reserves increased, but [NO₃⁻] decreased progressively in stems, which contain most of the plant N reserves, and rapidly in leaves and roots. This resulted in a rapid restriction of structural growth.

Conclusions

Periodic darkness did not restrict growth because sufficient carbohydrate reserves accumulated during the light period. Structural growth, however, was very responsive to NO₃⁻ nutrition, because N reserves were mostly located in stems, which have limited nitrate reduction capacity.Key words: Solanum lycopersicum, tomato, nitrogen, carbon, structural growth, reserves, nitrate, amino acids, carbohydrate, carboxylate     

Effective charge measurements reveal selective and preferential accumulation of anions, but not cations, at the protein surface in dilute salt solutions

Specific-ion effects are ubiquitous in nature; however, their underlying mechanisms remain elusive. Although Hofmeister-ion effects on proteins are observed at higher (>0.3M) salt concentrations, in dilute (<0.1M) salt solutions nonspecific electrostatic screening is considered to be dominant. Here, using effective charge (Q*) measurements of hen-egg white lysozyme (HEWL) as a direct and differential measure of ion-association, we experimentally show that anions selectively and preferentially accumulate at the protein surface even at low (<100 mM) salt concentrations. At a given ion normality (50 mN), the HEWL Q* was dependent on anion, but not cation (Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺, GdnH⁺, and Ca²⁺), identity. The Q* decreased in the order F⁻ > Cl⁻ > Br⁻ > NO₃⁻ ∼ I⁻ > SCN⁻ > ClO₄⁻ ≫ SO₄²⁻, demonstrating progressively greater binding of the monovalent anions to HEWL and also show that the SO₄²⁻ anion, despite being strongly hydrated, interacts directly with the HEWL surface. Under our experimental conditions, we observe a remarkable asymmetry between anions and cations in their interactions with the HEWL surface.     

Nitric oxide exerts protective effects against bleomycin-induced pulmonary fibrosis in mice

Background

Increased expression of nitric oxide synthase (NOS) and an increase in plasma nitrite plus nitrate (NOx) have been reported in patients with pulmonary fibrosis, suggesting that nitric oxide (NO) plays an important role in its development. However, the roles of the entire NO and NOS system in the pathogenesis of pulmonary fibrosis still remain to be fully elucidated. The aim of the present study is to clarify the roles of NO and the NOS system in pulmonary fibrosis by using the mice lacking all three NOS isoforms.

Methods

Wild-type, single NOS knockout and triple NOS knockout (n/i/eNOS^−/−) mice were administered bleomycin (BLM) intraperitoneally at a dose of 8.0 mg/kg/day for 10 consecutive days. Two weeks after the end of the procedure, the fibrotic and inflammatory changes of the lung were evaluated. In addition, we evaluated the effects of long-term treatment with isosorbide dinitrate, a NO donor, on the n/i/eNOS^−/− mice with BLM-induced pulmonary fibrosis.

Results

The histopathological findings, collagen content and the total cell number in bronchoalveolar lavage fluid were the most severe/highest in the n/i/eNOS^−/− mice. Long-term treatment with the supplemental NO donor in n/i/eNOS^−/− mice significantly prevented the progression of the histopathological findings and the increase of the collagen content in the lungs.

Conclusions

These results provide the first direct evidence that a lack of all three NOS isoforms led to a deterioration of pulmonary fibrosis in a BLM-treated murine model. We speculate that the entire endogenous NO and NOS system plays an important protective role in the pathogenesis of pulmonary fibrosis.     

Intracellular Conversion of Environmental Nitrate and Nitrite to Nitric Oxide with Resulting Developmental Toxicity to the Crustacean Daphnia magna

Background

Nitrate and nitrite (jointly referred to herein as NO_x) are ubiquitous environmental contaminants to which aquatic organisms are at particularly high risk of exposure. We tested the hypothesis that NO_x undergo intracellular conversion to the potent signaling molecule nitric oxide resulting in the disruption of endocrine-regulated processes.

Methodology/Principal Findings

These experiments were performed with insect cells (Drosophila S2) and whole organisms Daphnia magna. We first evaluated the ability of cells to convert nitrate (NO₃ ⁻) and nitrite (NO₂ ⁻) to nitric oxide using amperometric real-time nitric oxide detection. Both NO₃ ⁻ and NO₂ ⁻ were converted to nitric oxide in a substrate concentration-dependent manner. Further, nitric oxide trapping and fluorescent visualization studies revealed that perinatal daphnids readily convert NO₂ ⁻ to nitric oxide. Next, daphnids were continuously exposed to concentrations of the nitric oxide-donor sodium nitroprusside (positive control) and to concentrations of NO₃ ⁻ and NO₂ ⁻. All three compounds interfered with normal embryo development and reduced daphnid fecundity. Developmental abnormalities were characteristic of those elicited by compounds that interfere with ecdysteroid signaling. However, no compelling evidence was generated to indicate that nitric oxide reduced ecdysteroid titers.

Conclusions/Significance

Results demonstrate that nitrite elicits developmental and reproductive toxicity at environmentally relevant concentrations due likely to its intracellular conversion to nitric oxide.     

Influence of Belonolaimus longicaudatus on Nitrate Leaching in Turf

Experiments were conducted to quantify the effects of the sting nematode (Belonolaimus longicaudatus) on root reductions and quantity of nitrate (NO₃ ⁻) leached from ‘Tifdwarf’ bermudagrass in lysimeters. Forty lysimeters were planted with ‘Tifdwarf’ bermudagrass, of which 20 were inoculated with B. longicaudatus and 20 were noninoculated. Root length was compared between treatments at six, 12, and 18 weeks after initiation of the experiments. Turf was fertilized every three weeks, and leaching events were simulated at 21 and 42-day intervals in trial one and trial two, respectively. Leachate was collected, and the quantity of NO₃ ⁻ leached was compared between treatments. Root reductions were observed in lysimeters inoculated with B. longicaudatus at all evaluation dates. Quantity of NO₃ ⁻ leached was greater in inoculated lysimeters at the 18-week evaluation during both trials. This study indicates that nematode damage to turf roots limits root vigor and N uptake, thereby increasing nitrate leaching, adding to water quality concerns.     

Nitrite and Nitric Oxide as Inhibitors of Nitrogenase from Soybean Bacteroids

Nitrite was able to strongly inhibit C₂H₂ reduction by nitrogenase from soybean bacteroids, whereas H₂ evolution was unaffected under the same conditions. NO inhibited both C₂H₂ reduction and H₂ evolution; during C₂H₂ reduction, sensitivity of nitrogenase to NO was higher than to NO₂⁻, and the K_i values were, respectively, 0.056 and 0.52 mM. Production of NO resulting from a reduction of NO₂⁻ by dithionite in nitrogenase incubations was observed. However, the characteristics of inhibitions and the low level of NO generated by nitrite reduction ruled out the suggestion concerning a direct role of NO to explain the inhibitory effect of NO₂⁻ on nitrogenase.     

An integrated network analysis reveals that nitric oxide reductase prevents metabolic cycling of nitric oxide by Pseudomonas aeruginosa

Nitric oxide (NO) is a chemical weapon within the arsenal of immune cells, but is also generated endogenously by different bacteria. Pseudomonas aeruginosa are pathogens that contain an NO-generating nitrite (NO₂⁻) reductase (NirS), and NO has been shown to influence their virulence. Interestingly, P. aeruginosa also contain NO dioxygenase (Fhp) and nitrate (NO₃⁻) reductases, which together with NirS provide the potential for NO to be metabolically cycled (NO→NO₃⁻→NO₂⁻→NO). Deeper understanding of NO metabolism in P. aeruginosa will increase knowledge of its pathogenesis, and computational models have proven to be useful tools for the quantitative dissection of NO biochemical networks. Here we developed such a model for P. aeruginosa and confirmed its predictive accuracy with measurements of NO, O₂, NO₂⁻, and NO₃⁻ in mutant cultures devoid of Fhp or NorCB (NO reductase) activity. Using the model, we assessed whether NO was metabolically cycled in aerobic P. aeruginosa cultures. Calculated fluxes indicated a bottleneck at NO₃⁻, which was relieved upon O₂ depletion. As cell growth depleted dissolved O₂ levels, NO₃⁻ was converted to NO₂⁻ at near-stoichiometric levels, whereas NO₂⁻ consumption did not coincide with NO or NO₃⁻ accumulation. Assimilatory NO₂⁻ reductase (NirBD) or NorCB activity could have prevented NO cycling, and experiments with ΔnirB, ΔnirS, and ΔnorC showed that NorCB was responsible for loss of flux from the cycle. Collectively, this work provides a computational tool to analyze NO metabolism in P. aeruginosa, and establishes that P. aeruginosa use NorCB to prevent metabolic cycling of NO.