Increased nitric oxide formation followed by increased arginase activity induces relative lack of arginine at the wound site and alters whole nutritional status in rats almost within the early healing period

Nitric oxide (NO) production and free amino acid fluxes at the wound side during the first 3 days following cutaneous wound were investigated. Experiments were performed on Albino Oxford rats (n = 18) underwent cutaneous implantation of polyvinyl sponges. Intact animals (n = 6) were controls. Nitrites, nitrates, free amino acids and urea were measured both in plasma and wound fluids. Inducible nitric oxide synthase (iNOS) gene expressions at wound site were analyzed, too. The highest levels of both iNOS gene expression and its activity (increased wound fluid citrulline and nitrites) were at the first day. Wound fluid nitrates were significantly above plasma levels throughout the whole period, while molar nitrate to nitrite ratio steadily increased. It was associated with gradual increase of both ornithine and urea as well as steadily decreases of arginine and increases of phenylalanine at the wound site. Gradual decrease in glycine to branched-chain molar ratio was observed both in plasma and wound fluids. In conclusion, an early locally induced alterations in Arg metabolism, due to increased NO formation followed by increased arginase activity, produces relative lack of Arg at the wound site and disturbs nutritional status of the whole body almost within early healing period following cutaneous wound in rats. It is likely that NO autoxidation at the wound side is influenced by substrate availability.     

Precursors of carcinogenic N-nitroso compounds in Lake Peipsi

The concentration of precursors of carcinogenic N-nitroso compounds, nitrates and nitrites as well as ammonia, in the surface water of Lake Peipsi and its tributaries has been determined during the period 1985–1988. The nitrate and nitrite content was also analysed in bottom sediments and fish from the lake.The nitrate concentration in the water of Lake Peipsi varied from 0.01 to 2.33 mg NO₃&z.sbnd;N l^–1, the average value from 0.27 to 1.60 mg NO₃&z.sbnd;N l^–1, with the lowest concentrations in summer. The variations may be caused by different pollution loads, meteorological conditions, and assimilation of nitrates by plants and algae.The nitrate content in the water of rivers was on an average somewhat higher in comparison with its concentrations in the lake. The concentrations of nitrites were, as a rule, about an order of magnitude lower than those of nitrates. The amount of ammonia varied from 0.15 to 0.36 mg NH₄&z.sbnd;N l^–1.At present the concentrations of the studied nitrogen compounds are not essential and do not prevent from using the lake for recreation and drinking water supply.     

Relationship between synovial fluid and plasma manganese,arginase, and nitric oxide in patients with rheumatoid arthritis

Nitric oxide (NO) participates in the pathogenesis of inflammatory reactions in many autoimmune diseases such as rheumatoid arthritis (RA). There is a reciprocal pathway between arginase and nitric oxide synthese (NOS) for NO production, and Mn is required for arginase activity and stability. To investigate whether NO production related with the arginine-nitric oxide pathway in patients with RA, we measured synovial fluid and plasma nitrite (NO_x) levels, arginase activities, and its cofactor manganese (Mn) concentrations in 21 RA patients and 13 healthy control subjects. Plasma albumin levels were measured as an index of nutritional status. NO_x levels were determined after the reduction of nitrates to nitrites using the Griess reaction. Whereas, synovial fluid arginase activities and Mn levels were found to be significantly lower (p<0.001, p<0.001, respectively), plasma arginase activities and Mn levels were similar in patients with RA when compared to the control subjects. Plasma and synovial fluid NO levels were similar in patients with RA and in healthy subjects (p>0.05, p>0.05, respectively). There were significantly positive correlations between synovial fluid and plasma arginase activities vs Mn content (r=0.543, p=0.011; r=0.516, p=0.017, respectively) and significantly negative correlations between synovial fluid and plasma NO levels vs arginase activities (r=−0.497, p=0.022; r=−0.508, p=0.019 respectively) in the patients group. Our results indicate that the lower concentration of synovial fluid Mn could cause lower arginase activity and this could also upregulate NO production by increasing L-arginine content in patients with RA.     

Nitric oxide regulates steroid synthesis by bovine antral granulosa cells in a chemically defined medium

Nitric oxide (NO) in bovine ovary has been characterized as one of the controllers of granulosa cells’ (GC) steroidogenesis and apoptosis. One of the pathways used by NO to have these effects is cGMP. The objectives of the present study were to verify the effect of sodium nitroprusside (SNP), a NO donor, on steroidogenesis, cell viability (mitochondrial activity) and GC cell cycle distribution and if this effect occurs by the NO-cGMP signaling pathway with the addition of SNP with or without 1H-[1,2,3] oxadiaziolo[4,3a]quinoxaline-1-one (ODQ), a selective soluble guanylate cyclase inhibitor. The antral GC from 3 to 5 mm diameter cattle follicles was cultured without treatment (control), with ODQ (10⁻⁴ M) and 10⁻⁵, 10⁻³ and 10⁻¹ M SNP with or without ODQ for 24 h. Nitrate/nitrite (NO₃⁻/N0₂⁻) concentrations were evaluated by Griess method, progesterone (P₄) and 17β-estradiol (E₂) concentrations by chemiluminescence, viability and cell cycle stage by MTT method (3-[4,5-dimethylthiazol-2yl]-2,3 dipheniltetrazolium bromide) and flow cytometry, respectively. Nitrate/nitrite concentration in culture medium increased (P < 0.05) in a dose-dependent manner according to SNP concentration added to the culture medium. The GC cultured without treatment, with ODQ and with 10⁻⁵ M SNP in the presence or absence of ODQ developed into cell aggregates and did not vary in cell viability (P > 0.05), while GC cultured with 10⁻³ and 10⁻¹ M SNP with or without ODQ presented disorganized GC aggregates or did not develop into cell aggregates and also had substantially decreased cell viability (mitochondrial activity inhibition) and steroids synthesis (P < 0.05), and effects were not reversed with us of ODQ. Most GC cultured without treatment (control) or with ODQ, 10⁻⁵ and 10⁻³ M SNP with or without ODQ were in the G0/G1 (80–75%) stage and in a lesser proportion (20–25%) in the S + G2/M stage of the cell cycle, while the 10⁻¹ M SNP treatment resulted in GC in G1 phase arrest. The treatment with 10⁻⁵ M SNP increased (P < 0.05) E₂ synthesis and inhibited (P < 0.05) progesterone synthesis. The addition of ODQ reversed (P < 0.05) the stimulatory effect of 10⁻⁵ M SNP treatment on E₂, but not on P₄ synthesis (P > 0.05). These results demonstrated that E₂ synthesis by antral GC from small follicles is modulated by lesser NO concentrations via the cGMP pathway, but not P₄ while steroids inhibition cGMP pathway independent, mitochondrial damage and the interference on cell cycle progression caused by greater NO concentration can lead to cell death.     

Nitrate and ammonium absorption by plants growing at a sufficient or insufficient level of phosphorus in nutrient solutions

Summary Absorption of nitrate and ammonium was studied in water culture experiments with 4 to 6 weeks old plants of barley (Hordeum vulgare L.), buckwheat (Fagopyrum esculentum L. Moench) and rape (Brassica napus L.). The plants were grown in a complete nutrient solution with nitrate (5.7±0.2 mM) or nitrate (5.6±0.2 mM) + ammonium (0.04±0.02 mM). The pH of the nutrient solution was kept at 5.0 using a pH-stat. It was found that phosphorus deficiency reduced the rate of nitrate uptake by 58±3% when nitrate was the sole N source and by 83±1% when both nitrate and ammonium were present. The reduction occurred even before growth was significantly impeded by P deficiency. The inhibition of the uptake of ammonium was less,i.e. ammonium constituted 10±1% of the total N uptake in the P sufficient plants and 30±5% in the P deficient plants. The reduction of nitrate absorption greatly decreased the difference between the uptake of anions and cations. It is suggested that P deficiency reduced the assimilation of NO ₃ ^– into the proteins, which might cause a negative feedback on NO ₃ ^– influx and/or stimulate NO ₃ ^– efflux.     

Nitrate Reduction to Nitrite,Nitric Oxide and Ammonia by Gut Bacteria under Physiological Conditions

The biological nitrogen cycle involves step-wise reduction of nitrogen oxides to ammonium salts and oxidation of ammonia back to nitrites and nitrates by plants and bacteria. Neither process has been thought to have relevance to mammalian physiology; however in recent years the salivary bacterial reduction of nitrate to nitrite has been recognized as an important metabolic conversion in humans. Several enteric bacteria have also shown the ability of catalytic reduction of nitrate to ammonia via nitrite during dissimilatory respiration; however, the importance of this pathway in bacterial species colonizing the human intestine has been little studied. We measured nitrite, nitric oxide (NO) and ammonia formation in cultures of Escherichia coli, Lactobacillus and Bifidobacterium species grown at different sodium nitrate concentrations and oxygen levels. We found that the presence of 5 mM nitrate provided a growth benefit and induced both nitrite and ammonia generation in E.coli and L.plantarum bacteria grown at oxygen concentrations compatible with the content in the gastrointestinal tract. Nitrite and ammonia accumulated in the growth medium when at least 2.5 mM nitrate was present. Time-course curves suggest that nitrate is first converted to nitrite and subsequently to ammonia. Strains of L.rhamnosus, L.acidophilus and B.longum infantis grown with nitrate produced minor changes in nitrite or ammonia levels in the cultures. However, when supplied with exogenous nitrite, NO gas was readily produced independently of added nitrate. Bacterial production of lactic acid causes medium acidification that in turn generates NO by non-enzymatic nitrite reduction. In contrast, nitrite was converted to NO by E.coli cultures even at neutral pH. We suggest that the bacterial nitrate reduction to ammonia, as well as the related NO formation in the gut, could be an important aspect of the overall mammalian nitrate/nitrite/NO metabolism and is yet another way in which the microbiome links diet and health.     

The relationship of nitrogen source and in vivo nitrate reductase actiity to root formation in Euphorbia esula cell suspension cultures

Root formation and in vivo nitrate reductase (NR) activity were determined in leafy spurge cell suspensions. Cells grown in B5 media with 1 mg L^–1 2,4-D were transferred to B5 media without 2,4-D, but containing either high (92:8) or low (15:85) ratios of nitrogen as NO ₃ ^– -N:NH ₄ ⁺ -N. In older cell lines root formation occurred only in the low NO ₃ ^– medium with =<30 roots per flask. In younger cell lines root numbers were greatest in the high NO ₃ ^– medium (1000 to 3000 per flask). Cells grown in low NO ₃ ^– medium were about one-third the final dry weight as those in high NO ₃ ^– medium. Root length was consistently greater for cell lines of all ages in the low NO ₃ ^– medium. Developmental profiles of NR activity were similar in cell lines of all ages, whether or not roots were formed. NR activity was lower, however, in cultures grown in low NO ₃ ^– medium compared to high NO ₃ ^– medium. There was no consistent relationship between NR activity and root initiation. Therefore, nitrate reductase does not appear to be a primary target for regulation of leafy spurge growth by chemical application.     

Effect of seabuckthorn leaf extracts on circulating energy fuels, lipid peroxidation and antioxidant parameters in rats during exposure to cold, hypoxia and restraint (C–H–R) stress and post stress recovery

The present study was carried out to study mechanism of adaptogenic activity of seabuckthorn leaf extract, administered orally in rats both in single and five doses at a dose of 100 mg/kg body weight 30 min prior to C–H–R exposure. The efficacy of the extract was studied on circulating energy fuels, lipid peroxidation and anti-oxidant parameters in rats on attaining the T_rec 23 °C during C–H–R exposure and after recovery (T_rec 37 °C) from C–H–R induced hypothermia. Single dose treatment in rats restricted rise in blood malondialdehyde (MDA) levels and decrease in glutathione (GSH) and catalase (CAT) levels. Both single and five doses also restricted the rise in serum free fatty acids (FFA) and lactate dehydrogenase (LDH) levels on attaining T_rec 23 °C during C–H–R exposure, suggesting more efficient utilization of FFA for energy production and better maintained cell membrane permeability. This suggested that the adaptogenic activity of the extract might be due to its anti-oxidative activity, maintained blood glucose levels, better utilization of FFA and improved cell membrane permeability.     

Growth of dinoflagellates, Ceratium furca and Ceratium fusus in Sagami Bay, Japan: The role of nutrients

In order to study the influence of nutrients on the growth characteristics of the dominant dinoflagellates, Ceratium furca and Ceratium fusus, in the temperate coastal area of Sagami Bay, Japan, we conducted field monitoring from January 2000 to December 2005 and performed laboratory culture experiments. In the field study, population densities of C. furca and C. fusus were high, even in low nutrient concentrations (N: 1.58 μM, P: 0.17 μM). Both species were more abundant in the surface and sub-surface layers than in the bottom layers during the stratification periods. In the laboratory study, the specific growth rates of C. furca and C. fusus increased gradually along with increasing nutrients up to the T₅ (N: 5 μM, P: 0.5 μM) and T₁₀ (N: 10 μM, P: 1 μM) concentration levels, after which the growth rate plateaued at the T₅₀ (N: 50 μM, P: 5 μM) concentration level. In contrast, the nutrient uptake rates of both species continuously increased, indicating “luxury consumption”, i.e., excessive cellular storage not related to growth rate. The half-saturation constants (K_s) of C. furca for nitrate (0.49 μM) and phosphate (0.05 μM) were slightly higher than C. fusus (0.32 and 0.03 μM, respectively). We offer two reasons why the two Ceratium population densities were maintained at high levels in low nutrient conditions. First, these two species have a competitive advantage over other algal species because of low K_s values and specific characteristics for nutrient uptake such as luxury consumption. Their ability to obtain nutrients through alternative methods, such as phagotrophy, might contribute to bloom formation and population persistence. Second, the cell densities of both Ceratium species increased along with nitrate concentrations in the media even when phosphorus was held constant. In particular, the growth of C. furca was directly supported by various nitrogen sources such as nitrate, ammonium, and urea, although the highest growth rates were observed only in the nitrate-enriched cultures. Our field and laboratory results revealed that the growth rates of the two Ceratium species increased readily in high N:P nutrient conditions (i.e., conditions of P limitation) indicating an advantage over other algal species in phosphorus-limited environments such as Sagami Bay.     

Nitrate and nitrite uptake and reduction by intact sunflower plants

Nitrogen-starved sunflower plants (Helianthus annuus L. cv. Peredovic) cannot absorb NO ₃ ^– or NO ₂ ^– upon initial exposure to these anions. Ability of the plants to take up NO ₃ ^– and NO ₂ ^– at high rates from the beginning was induced by a pretreatment with NO ₃ ^– . Nitrite also acted as inducer of the NO ₂ ^– -uptake system. The presence of cycloheximide during NO ₃ ^– -pretreatment prevented the subsequent uptake of NO ₃ ^– and NO ₂ ^– , indicating that both uptake systems are synthesized de novo when plants are exposed to NO ₃ ^– . Cycloheximide also suppressed nitrate-reductase (EC 1.6.6.1) and nitrite-reductase (EC 1.7.7.1) activities in the roots. The sulfhydryl-group reagent N-ethylmaleimide greatly inhibited the uptake of NO ₃ ^– and NO ₂ ^– . Likewise, N-ethylmaleimide promoted in vivo the inactivation of nitrate reductase without affecting nitrite-reductase activity. Rates of NO ₃ ^– and NO ₂ ^– uptake as a function of external anion concentration exhibited saturation kinetics. The calculated K_m values for NO ₃ ^– and NO ₂ ^– uptake were 45 and 23 M, respectively. Rates of NO ₃ ^– uptake were four to six times higher than NO ₃ ^– -reduction rates in roots. In contrast, NO ₂ ^– -uptake rates, found to be very similar to NO ₃ ^– -uptake rates, were much lower (about 30 times) than NO ₂ ^– -reduction rates. Removal of oxygen from the external solution drastically suppressed NO ₃ ^– and NO ₂ ^– uptake without affecting their reduction. Uptake and reduction were also differentially affected by pH. The results demonstrate that uptake of NO ₃ ^– and NO ₂ ^– into sunflower plants is mediated by energy-dependent inducible-transport systems distinguishable from the respective enzymatic reducing systems.Abbreviations CHI cycloheximide - NEM N-ethylmaleimide - NiR nitrite reductase - NR nitrate reductase - pHME p-hydroxymercuribenzoate This research was supported by grant PB86-0232 from the Dirección General de Investigatión Científica y Técnica (Spain). One of us (E.A.) thanks the Consejeria de Educación y Ciencia de la Junta de Andalucia for the tenure of a fellowship. We thank Miss G. Alcalá and Miss C. Santos for their valuable technical and secretarial assistance.     

Nitrate induces enzymes of the mannitol cycle and suppresses versicolorin synthesis inAspergillus parasiticus

Addition of sodium nitrate to growing cultures ofAspergillus parasiticus (ATCC 36537) induces the synthesis of enzymes involved in nitrate assimilation (NO ₃ ^– reductase), of enzymes in the pentose pathway (glucose-6-phosphate dehydrogenase), and of enzymes in the mannitol cycle (mannitol- and mannitol-1-phosphate dehydrogenases). Addition of NO ₃ ^– also causes a dose-dependent suppression of synthesis of the polyketide secondary metabolite, versicolorin A. We suggest that in the presence of NO ₃ ^– plus peptone, the cytoplasmic NADPH/NADP ratio may be elevated, resulting in increased conversion of malonyl coenzyme A to fatty acid rather than to polyketide.     

Nitrate and nitrite reductase negative mutants of N2-fixingAzospirillum spp.

Chlorate resistant spontaneous mutants ofAzospirillum spp. (syn.Spirillum lipoferum) were selected in oxygen limited, deep agar tubes with chlorate. Among 20 mutants fromA. brasilense and 13 fromA. lipoferum all retained their functional nitrogenase and 11 from each species were nitrate reductase negative (nr^–). Most of the mutants were also nitrite reductase negative (nir^–), only 3 remaining nir⁺. Two mutants from nr⁺ nir⁺ parent strains lost only nir and became like the nr⁺ nir^– parent strain ofA. brasilense. No parent strain or nr⁺ mutant showed any nitrogenase activity with 10 mM NO ₃ ^– . In all nr^– mutants, nitrogenase was unaffected by 10 mM NO ₃ ^– . Nitrite inhibited nitrogenase activity of all parent strains and mutants including those which were nir^–. It seems therefore, that inhibition of nitrogenase by nitrate is dependent on nitrate reduction. Under aerobic conditions, where nitrogenase activity is inhibited by oxygen, nitrate could be used as sole nitrogen source for growth of the parent strains and one mutant (nr^– nir^–) and nitritite of the parent strains and 10 mutants (all types). This indicates the loss of both assimilatory and dissimilatory nitrate reduction but only dissimilatory nitrite reduction in the mutants selected with chlorate.     

Genotype-Dependent Effect of Exogenous Nitric Oxide on Cd-induced Changes in Antioxidative Metabolism, Ultrastructure, and Photosynthetic Performance in Barley Seedlings (Hordeum vulgare)

A greenhouse hydroponic experiment was performed using Cd-sensitive (cv. Dong 17) and Cd-tolerant (Weisuobuzhi) barley seedlings to evaluate how different genotypes responded to cadmium (Cd) toxicity in the presence of sodium nitroprusside (SNP), a nitric oxide (NO) donor. Results showed that 5 μM Cd increased the accumulation of O₂^•−, H₂O₂, and malondialdehyde (MDA) but reduced plant height, chlorophyll content, net photosynthetic rate (P _n), and biomass, with a much more severe response in the Cd-sensitive genotype. Antioxidant enzyme activities increased significantly under Cd stress in the roots of the tolerant genotype, whereas in leaves of the sensitive genotype, superoxide dismutase (SOD) and ascorbate peroxide (APX), especially cytosol ascorbate peroxidase (cAPX), decreased after 5–15 days Cd exposure. Moreover, Cd induces NO synthesis by stimulating nitrate reductase and nitric oxide synthetase-like enzymes in roots/leaves. A Cd-induced NO transient increase in roots of the Cd-tolerant genotype might partly contribute to its Cd tolerance. Exogenous NO dramatically alleviated Cd toxicity, markedly diminished Cd-induced reactive oxygen species (ROS) and MDA accumulation, ameliorated Cd-induced damage to leaf/root ultrastructure, and increased chlorophyll content and P _n. External NO counteracted the pattern of alterations in certain antioxidant enzymes induced by Cd; for example, it significantly elevated the depressed SOD, APX, and catalase (CAT) activities in the Cd-sensitive genotype after 10- and 15-day treatments. Furthermore, NO significantly increased stromal APX and Mn-SOD activities in both genotypes and upregulated Cd-induced decrease in cAPX activity and gene expression of root/leaf cAPX and leaf CAT1 in the Cd-sensitive genotype. These data suggest that under Cd stress, NO, as a potent antioxidant, protects barley seedlings against oxidative damage by directly and indirectly scavenging ROS and helps to maintain stability and integrity of the subcellular structure.     

Role of arginine in superficial wound healing in man

Arginine supplementation has been identified as advantageous in experimental wound healing. However, the mechanisms underlying this beneficial effect in tissue repair remain unresolved. Animal studies suggest that the beneficial role of arginine supplementation is mediated, at least in part through NO. The latter component mediates processes involved in tissue repair, including angiogenesis, epithelialization and collagen formation. This prospective study is performed to investigate arginine metabolism in acute surgical wounds in man. Expression of enzymes, known to be involved in arginine metabolism, was studied in donor sites of skin grafts of 10 hospitalized patients undergoing skin transplantation. Plasma and wound fluid levels of arginine metabolites (ornithine, citrulline, nitrate and nitrite = NOx) were measured using High Performance Liquid Chromatography. Expression of iNOS, eNOS, arginase-1 and arginase-2 was studied by immunohistochemistry in paraffin sections of skin tissue. Arginase-1 concentration was measured in plasma and wound fluid using ELISA. Arginase-2 was determined using Western blot analysis. We observed increased levels of citrulline, ornithine, NOx and arginase-1 in wound fluid when compared with plasma. Arginase-2 was expressed in both plasma and wound fluid and seemed higher in plasma. iNOS was expressed by neutrophils, macrophages, fibroblasts, keratinocytes and endothelial cells upon wounding, whereas eNOS reactivity was observed in endothelial cells and fibroblasts. Arginase-1 was expressed in neutrophils post-wounding, while arginase-2 staining was observed in endothelial cells, keratinocytes, fibroblasts, macrophages and neutrophils. For the first time, human data support previous animal studies suggesting arginine metabolism for an NO- as well as arginase-mediated reparation of injured skin.     

N-acetyl cysteine enhances imatinib-induced apoptosis of Bcr-Abl+ cells by endothelial nitric oxide synthase-mediated production of nitric oxide

Introduction  Imatinib, a small-molecule inhibitor of the Bcr-Abl kinase, is a successful drug for treating chronic myeloid leukemia (CML). Bcr-Abl kinase stimulates the production of H₂O₂, which in turn activates Abl kinase. We therefore evaluated whether N-acetyl cysteine (NAC), a ROS scavenger improves imatinib efficacy. Materials and methods  Effects of imatinib and NAC either alone or in combination were assessed on Bcr-Abl⁺ cells to measure apoptosis. Role of nitric oxide (NO) in NAC-induced enhanced cytotoxicity was assessed using pharmacological inhibitors and siRNAs of nitric oxide synthase isoforms. We report that imatinib-induced apoptosis of imatinib-resistant and imatinib-sensitive Bcr-Abl⁺ CML cell lines and primary cells from CML patients is significantly enhanced by co-treatment with NAC compared to imatinib treatment alone. In contrast, another ROS scavenger glutathione reversed imatinib-mediated killing. NAC-mediated enhanced killing correlated with cleavage of caspases, PARP and up-regulation and down regulation of pro- and anti-apoptotic family of proteins, respectively. Co-treatment with NAC leads to enhanced production of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS). Involvement of eNOS dependent NO in NAC-mediated enhancement of imatinib-induced cell death was confirmed by nitric oxide synthase (NOS) specific pharmacological inhibitors and siRNAs. Indeed, NO donor sodium nitroprusside (SNP) also enhanced imatinib-mediated apoptosis of Bcr-Abl⁺ cells. Conclusion  NAC enhances imatinib-induced apoptosis of Bcr-Abl⁺ cells by endothelial nitric oxide synthase-mediated production of nitric oxide.     

Reaction of nitric oxide with the oxidized di-heme and heme–copper oxygen-reducing centers of terminal oxidases: Different reaction pathways and end-products

Inhibition of terminal oxidases by nitric oxide (NO) has been extensively investigated as it plays a role in regulation of cellular respiration and pathophysiology. Cytochrome bd is a tri-heme (b₅₅₈, b₅₉₅, d) bacterial oxidase containing no copper that couples electron transfer from quinol to O₂ (to produce H₂O) with generation of a transmembrane protonmotive force. In this work, we investigated by stopped-flow absorption spectroscopy the reaction of NO with Escherichia coli cytochrome bd in the fully oxidized (O) state. We show that under anaerobic conditions, the O state of the enzyme binds NO at heme d with second-order rate constant k_on = 1.5 ± 0.2 × 10² M⁻¹ s⁻¹, yielding a nitrosyl adduct (d³⁺–NO or d²⁺–NO⁺) with characteristic optical features (an absorption increase at 639 nm and a red shift of the Soret band). The reaction mechanism is remarkably different from that of O cytochrome c oxidase in which the heme–copper binuclear center reacts with NO approximately three orders of magnitude faster, forming nitrite. The data allow us to conclude that in the reaction of NO with terminal oxidases in the O state, Cu_B is indispensable for rapid oxidation of NO into nitrite.     

Influence of bed media characteristics on ammonia and nitrate removal in shallow horizontal subsurface flow constructed wetlands

Two bed media were tested (gravel and Filtralite) in shallow horizontal subsurface flow (HSSF) constructed wetlands in order to evaluate the removal of ammonia and nitrate for different types of wastewater (acetate-based and domestic wastewater) and different COD/N ratios. The use of Filtralite allowed both higher mass removal rates (1.1 g NH₄–N m⁻² d⁻¹ and 3 g NO₃–N m⁻² d⁻¹) and removal efficiencies (>62% for ammonia, 90–100% for nitrate), in less than 2 weeks, when compared to the ones observed with gravel. The COD/N ratio seems to have no significant influence on nitrate removal and the removal of both ammonia and nitrate seems to have involved not only the conventional pathways of nitrification–denitrification. The nitrogen loading rate of both ammonia (0.8–2.4 g NH₄–N m⁻² d⁻¹) and nitrate (0.6–3.2 g NO₃–N m⁻² d⁻¹) seem to have influenced the respective removal rates.     

Characterization of a symbiotic,heterocystous, N2-fixing cyanobacterium fromCycas coralloid roots

A symbiotic, heterocystous, N₂-fixing blue-green alga, isolated from the coralloid roots of a xerophytic plant,Cycas revoluta, grew best in liquid medium supplemented with 4 mM NO ₃ ^– . Morphologically, the isolated alga was identical to that of the natural endophyte but the cell size had decreased markedly. The alga was heterotrophic. Intact coralloid roots had nearly 4 to 5 times more nitrogenase activity compared with natural- and laboratory-grown agla but nitrate reductase was inducible in both the forms. Plasmid(s) were found in both algal forms.     

Role of nitric oxide on quality of freshly ejaculated bull spermatozoa during heparin-induced in vitro capacitation

The objective of this study was to assess the effects of nitric oxide (NO) on heparin-induced capacitation in vitro of fresh bull sperm, through the addition of Nω-nitro-l-arginine methyl ester (L-NAME, a NO-synthesis inhibitor) and l-arginine (L-Arg, a NO-synthesis precursor) to the capacitation medium. In Experiment 1, different concentrations of L-NAME (0.1, 1, 10 mM) and of L-Arg (10 mM) were added to the capacitation medium. Sperm motility and vigor were subjectively appraised using direct light microscopy; sperm membrane integrity was examined using a 2% Trypan blue solution while the concentration of nitrate/nitrite (NO₃⁻/NO₂⁻) was determined by using the Griess method over a 5 h capacitation period. The addition of 10 mM L-NAME has inhibited NO synthesis, sperm progressive motility, sperm vigor and sperm membrane integrity (P < 0.05) as compared to control. The addition of 10 mM L-Arg to the capacitation medium increased all variables evaluated in comparison to the control (P < 0.05). In Experiment 2, mitochondrial activity was assessed through the MTT test (3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), and sperm capacitation was assessed through the test of penetration in homologous oocytes after addition of the 10 mM L-NAME, and of the 10 mM L-Arg. The addition of 10 mM L-NAME caused mitochondrial activity (40%) and the percentage of oocytes penetrated (77%) to decrease in relation to the control (P < 0.05). After addition of 0.6 mM L-Arg + 10 mM L-NAME, partial reversal of mitochondrial activity did occur (only 20%). The addition of 10 mM L-Arg increased the percentage of oocytes penetrated as compared to control (21%) (P < 0.05). These results indicate that: (1) NO is involved in control of progressive sperm motility, vigor, membrane integrity, and mitochondrial activity along the period of heparin-induced capacitation of fresh bovine sperm via NOS/NO; (2) adequate L-Arg/NO concentrations into the capacitation medium can potentiate heparin action or act independently for increasing the number or the quality of capacitated sperm.     

Nitrate use by tobacco cells in response to N-stress and ammonium nutrition

Summary Characterization of NO ₃ ^– use by suspension cultured tobacco cells during a culture cycle is needed to take advantage of cell cultures for further study of the biochemical regulation of NO ₃ ^– uptake induction and decay processes. Tobacco (Nicotiana tabacum L., cv. Ky14) cells were cultured with media containing different N sources. Cells cultured with a mixture of NO ₃ ^– and NH ₄ ⁺ (40 mM NO ₃ ^– plus 20 mM NH ₄ ⁺ , in Murashige and Skoog media) initially grew slightly faster but attained the same maximum cell culture density as those cultured with 40 mM NO ₃ ^– only. Cells subcultured with N-free media grew at a similar rate for the first 3 d as those cells grown with N, then ceased further growth. The cessation of growth of cells subcultured with N-free media coincided with depletion of cell NO ₃ ^– . The NO ₃ ^– influx of cells subcultured with N-free media increased eleven-fold and those grown with N increased four- to five-fold before declining. Maximal NO ₃ ^– influx rates occurred at the onset of the stationary growth phase for N-stressed cells, while cells grown with N reached maximums prior to the stationary phase of cell growth. Cells grown with a mixture of NO ₃ ^– and NH ₄ ⁺ had lower NO ₃ ^– reductase (NR) activity and higher cell NO ₃ ^– levels than those of cells grown with NO ₃ ^– only. The NR activity of cells subcultured with N-free media peaked within 1 d after subculture before declining to a constitutive level when cell NO ₃ ^– was depleted. The level of cell NO ₃ ^– plays a critical role in the expression of the NO ₃ ^– uptake and reduction processes. The transitions in the expression of NO ₃ ^– uptake and reduction activities of tobacco cell suspension cultures should prove valuable for further study of the biochemical and molecular basis for the regulation of these processes.Abbreviations DTT DL-dithiothreitol - EDTA ethylenediamine tetraacetate - FW fresh weight - MS media Murashige & Skoog media - NADH ß-nicotinamide adenine dinucleotide reduced form - PMSF phenylmethyl-sulfonyl fluoride