Nmol NO Research Articles

Determination of 17O Anomaly in Atmospheric Aerosol Nitrate

通过改造稳定同位素比质谱仪外设设备、优化系统参数、优化细菌反硝化方法等方法，建立了一种高灵敏度高精度的大气气溶胶硝酸盐氧同位素异常值快速测定方法。本方法的检出限为0.4 μg N，在最佳进样量为0.8 μg N时，测定精度可达到0.19‰。 A novel method for rapid determination of oxygen isotope anomaly (Δ 17 O) in atmospheric aerosol samples was developed. With this method, the measurement was completed in 15 min with a relatively high analytical precision and a low determination limit. To achieve an analytical precision of 0.6‰, this method required a minimum nitrate content of 29 nmol (0.4 μg N). The analytical precision was optimum with a nitrate content of 57 nmol (0.8 μg N). The nitrate in atmospheric aerosol samples was converted to nitrous oxide by optimized bacterial denitrification process within 4 hs. After that, a thermal decomposition of nitrous oxide in sample gas was carried out in a gold tube at 800 °C after separation and purification, followed by gas chromatographic separation and isotope analysis of oxygen. The experiment results showed that the analytical precision of Δ 17 O in this method could reach 0.19‰ with a sample content of 57 nmol NO 3 − , and the analytical precision of δ 15 N could reach 0.15‰. This method was not only suitable for oxygen isotope anomaly determination in atmospheric aerosol samples, but also for the other atmospheric environmental samples such as rain and snow samples with low concentrations. This research establishes a rapid determination method which can determinate the 17 O anomaly in atmospheric aerosol samples by transforming the peripheral equipment of stable isotope ratio mass spectrometer, optimizing the system parameters and the bacterial anti-nitrification method. This method requires a minimum of 0.4 μg N, and analytical precision could reach 0.19 ‰ at a sample content of 0.8 μg N.

<p>A Comparative Study of Inhaled Nitric Oxide and an Intravenously Administered Nitric Oxide Donor in Acute Pulmonary Hypertension</p>

PurposeInhaled nitric oxide (iNO) selectively vasodilates the pulmonary circulation but the effects are sometimes insufficient. Available intravenous (iv) substances are non-selective and cause systemic side effects. The pulmonary and systemic effects of iNO and an iv mono-organic nitrite (PDNO) were compared in porcine models of acute pulmonary hypertension.MethodsIn anesthetized piglets, dose–response experiments of iv PDNO at normal pulmonary arterial pressure (n=10) were executed. Dose–response experiments of iv PDNO (n=6) and iNO (n=7) were performed during pharmacologically induced pulmonary hypertension (U46619 iv). The effects of iv PDNO and iNO were also explored in 5 mins of hypoxia-induced increase in pulmonary pressure (n=2-4).ResultsPDNO (15, 30, 45 and 60 nmol NO kg−1 min−1 iv) and iNO (5, 10, 20 and 40 ppm which corresponded to 56, 112, 227, 449 nmol NO kg−1 min−1, respectively) significantly decreased the U46619-increased mean pulmonary arterial pressure (MPAP) and pulmonary vascular resistance (PVR) to a similar degree without significant decreases in mean arterial pressure (MAP) or systemic vascular resistance (SVR). iNO caused increased levels of methemoglobin. At an equivalent delivered NO quantity (iNO 5 ppm and PDNO 45 nmol kg−1 min−1 iv), PDNO decreased PVR and SVR significantly more than iNO. Both drugs counteracted hypoxia-induced pulmonary vasoconstriction and they decreased the ratio of PVR and SVR in both settings.ConclusionIntravenous PDNO was a more potent pulmonary vasodilator than iNO in pulmonary hypertension, with no severe side effects. Hence, this study supports the potential of iv PDNO in the treatment of acute pulmonary hypertension.

Mitochondrial nitric oxide production supported by reverse electron transfer

Heart phosphorylating electron transfer particles (ETPH) produced NO at 1.2 ± 0.1 nmol NO. min−1 mg protein−1 by the mtNOS catalyzed reaction. These particles showed a NAD+ reductase activity of 64 ± 3 nmol min−1 mg protein−1 sustained by reverse electron transfer (RET) at expenses of ATP and succinate. The same particles, without NADPH and in conditions of RET produced 0.97 ± 0.07 nmol NO. min−1 mg protein−1. Rotenone inhibited NO production supported by RET measured in ETPH and in coupled mitochondria, but did not reduce the activity of recombinant nNOS, indicating that the inhibitory effect of rotenone on NO production is due to an electron flow inhibition and not to a direct action on mtNOS structure. NO production sustained by RET corresponds to 20% of the total amount of NO released from heart coupled mitochondria. A mitochondrial fraction enriched in complex I produced 1.7 ± 0.2 nmol NO. min−1 mg protein−1 and reacted with anti-75 kDa complex I subunit and anti-nNOS antibodies, suggesting that complex I and mtNOS are located contiguously. These data show that mitochondrial NO production can be supported by RET, and suggest that mtNOS is next to complex I, reaffirming the idea of a functional association between these proteins.

The impaired synthesis of insulin and its inability to inhibit platelet aggregation in cerebrovascular accident.

Both ischemic stroke (IS) and hemorrhagic stroke (HS) are reported to occur due to thrombosis on the arteries of the brain. As diabetes mellitus is a risk factor for strokes and insulin is reported to prevent thrombosis, the role of insulin in IS and HS was investigated. Forty eight stroke victims (IS=22, HS=26) and equal number of aged and sex matched normal volunteers participated in the study. Nitric oxide was determined by methemoglobin method. Insulin and Dermcidin isoform-2 (DCN2) level was determined by ELISA by using insulin and dermcidin antibody. Insulin binding to the platelet membrane was analyzed by scat chard plot. Treatment of normal platelet rich plasma (10(8)platelets/ml) with 15μUnits insulin/ml produced 1.41nmol NO. The PRP from the IS and HS victims produced 0.38nmol NO and 0.08nmol NO respectively. Pretreatment of PRP from IS or HS subjects with 15μM aspirin followed by 15μUnits of insulin/ml resensitized the platelets to the inhibitory effect of insulin. Mice hepatocytes treated with 0.14μM DCN2 abolished the glucose induced insulin synthesis by NO that can be reversed by using 15μM aspirin. It can be concluded that presence of DCN2 in stroke causes a condition similar to type I diabetes and nullified the effect of insulin in the inhibition of platelet aggregation in both IS and HS. The effect was reversed by 15μM aspirin.

Exposure to nitric oxide protects against oxidative damage but increases the labile iron pool in sorghum embryonic axes.

Sodium nitroprusside (SNP) and diethylenetriamine NONOate (DETA NONOate), were used as the source of exogenous NO to study the effect of NO upon germination of sorghum (Sorghum bicolor (L.) Moench) seeds through its possible interaction with iron. Modulation of cellular Fe status could be an important factor for the establishment of oxidative stress and the regulation of plant physiology. Fresh and dry weights of the embryonic axes were significantly increased in the presence of 0.1 mM SNP, as compared to control. Spin trapping EPR was used to assess the NO content in axes from control seeds after 24 h of imbibition (2.4±0.2 nmol NO g−1 FW) and seeds exposed to 0.01, 0.1, and 1 mM SNP (3.1±0.3, 4.6±0.2, and 6.0±0.9 nmol NO g−1 FW, respectively) and 1 mM DETA NONOate (6.2±0.6 nmol NO g−1 FW). Incubation of seeds with 1 mM SNP protected against oxidative damage to lipids and maintained membrane integrity. The content of the deferoxamine–Fe (III) complex significantly increased in homogenates of axes excised from seeds incubated in the presence of 1 mM SNP or 1 mM DETA NONOate as compared to the control (19±2 nmol Fe g−1 FW, 15.2±0.5 nmol Fe g−1 FW, and 8±1 nmol Fe g−1 FW, respectively), whereas total Fe content in the axes was not affected by the NO donor exposure. Data presented here provide experimental evidence to support the hypothesis that increased availability of NO drives not only protective effects to biomacromolecules, but to increasing the Fe availability for promoting cellular development as well.

Urinary excretion of nitrite and nitrate in experimental glomerulonephritis reflects systemic immune activation and not glomerular synthesis.

In immune-induced glomerulonephritis (gn), glomeruli (gl) synthesize nitric oxide (NO), and urinary nitrite (NO2-) excretion is increased. In mammals on a low nitrate (NO3-) diet, urinary NO3- is a measure of endogenous NO3- synthesis. Excretion is increased after administration of macrophage activators, reflecting induction of NO production. To determine whether increased urinary NO2- gn is due to glomerular synthesis we studied urinary NO2-/NO3- in accelerated nephrotoxic nephritis induced by preimmunization with rabbit immunoglobulin G (IgG), followed by rabbit anti-rat nephrotoxic globulin, and in control rats similarly preimmunized with rabbit IgG, but followed by normal rabbit serum. Both urinary NO2- and NO3- were increased by i.p. preimmunization with rabbit IgG (peak 463 +/- 171 nmol NO2-/60.3 +/- 9.4 nmol NO3-/24 h, P < 0.001 for both NO2- and NO3- compared with preimmunization levels). Repeat immunization with i.v. rabbit anti-rat nephrotoxic globulin (nephritic rats) or normal rabbit globulin (control rats) again increased urinary NO2- and NO3-. There was no statistically significant difference in urinary NO2- and NO3- levels between nephritic rats where globulin had nephrotoxic activity and the control rats injected with normal rabbit globulin, despite increased NO2- synthesis in ex vivo nephritic glomeruli after nephrotoxic globulin (7.9 +/- 1.9 nmol/2000 gl/48 h; controls 3.2 +/- 1.0 nmol/2000 gl/48 h). Thus neither urinary NO2- nor NO3- levels reflect local activation of the NO pathway in glomeruli. As reported for other stimulants, we show here that systemic stimulation with foreign antigen increased NO synthesis.

Seasonal variations in nitrate reductase activity and internal N pools in intertidal brown algae are correlated with ambient nitrate concentrations.

Nitrogen metabolism was examined in the intertidal seaweeds Fucus vesiculosus, Fucus serratus, Fucus spiralis and Laminaria digitata in a temperate Irish sea lough. Internal NO(3) (-) storage, total N content and nitrate reductase activity (NRA) were most affected by ambient NO(3) (-), with highest values in winter, when ambient NO(3) (-) was maximum, and declined with NO(3) (-) during summer. In all species, NRA was six times higher in winter than in summer, and was markedly higher in Fucus species (e.g. 256 +/- 33 nmol NO(3) (-) min(-1) g(-1) in F. vesiculosus versus 55 +/- 17 nmol NO(3) (-) min(-1) g(-1) in L. digitata). Temperature and light were less important factors for N metabolism, but influenced in situ photosynthesis and respiration rates. NO(3) (-) assimilating capacity (calculated from NRA) exceeded N demand (calculated from net photosynthesis rates and C : N ratios) by a factor of 0.7-50.0, yet seaweeds stored significant NO(3) (-) (up to 40-86 micromol g(-1)). C : N ratio also increased with height in the intertidal zone (lowest in L. digitata and highest in F. spiralis), indicating that tidal emersion also significantly constrained N metabolism. These results suggest that, in contrast to the tight relationship between N and C metabolism in many microalgae, N and C metabolism could be uncoupled in marine macroalgae, which might be an important adaptation to the intertidal environment.

Production of NO and N 2O in the presence and absence of C 2H 2 by soil slurries and batch cultures of denitrifying bacteria

We evaluated the potential of the C 2H 2-catalyzed NO oxidation reaction to influence N 2O production during denitrification. We measured the total amount of free NO and N 2O produced by slurries of sandy loam soil and by batch cultures of denitrifying bacteria under both anaerobic and low O 2 conditions. The maximum amount of free NO released by anaerobic Nos + (able to reduce N 2O to N 2) and Nos − (unable to reduce N 2O to N 2) batch cultures of Cytophaga johnsonae strains catalyzing denitrification of nitrate was 17–79 nmol NO per bottle. In all cases the maximum headspace concentrations of NO-N measured in anaerobic cultures in the absence of C 2H 2 was less than 0.21% of those of N 2O-N measured in the presence of 10 kPa C 2H 2. Peak NO production was delayed when between 2.0 and 4.5% O 2 was present. Less NO accumulated in cultures in the presence of both O 2 and C 2H 2, and the maximum amount of NO-N measured in the absence of C 2H 2 was less than 0.13% of the total amount of N 2O-N measured in the presence of C 2H 2. For an agricultural sandy loam soil, the maximum concentrations of free NO released from slurries were 598–897 ng NO-N g −1 of dry soil in the absence of C 2H 2 and 118–260 ng NO-N g −1 of dry soil in the presence of 10 kPa C 2H 2. The maximum concentration of NO-N released in the absence of C 2H 2 was between 0.32 and 8.1% of the maximum concentration of N 2O-N accumulated in the presence of 10 kPa C 2H 2. We conclude that scavenging of NO by the C 2H 2-catalyzed NO oxidation reaction in the presence of trace amounts of O 2 does not cause a serious underestimate of long-term measurements of active denitrification in anaerobic soils containing adequate carbon and nitrate sources.

Biodegradation of monohalogenated alkanes by soil NH(3)-oxidizing bacteria.

Although cooxidative biodegradation of monohalogenated hydrocarbons has been well studied in the model NH(3)-oxidizing bacterium, Nitrosomonas europaea, virtually no information exists about cooxidation of these compounds by native populations of NH(3)-oxidizing bacteria. To address this subject, nitrifying activity was stimulated to 125-400 nmol NO(3)(-) produced g(-1) soil h(-1) by first incubating a Ca(OH)(2)-amended, silt loam soil (pH 7.0+/-0.2) at field capacity (270 g H(2)O kg(-1) soil) with 10 micro mol NH(4)(+) g(-1) soil for 14 days, followed by another 10 days of incubation in a shaken slurry (2:1 water:soil, v/w) with periodic pH adjustment and maintenance of 10 mM NH(4)(+). These slurries actively degraded both methyl bromide (MeBr) and ethyl chloride (EtCl) at maximum rates of 20-30 nmol ml(-1) h(-1) that could be sustained for approximately 12 h. Although the MeBr degradation rates were linear for the first 10-12 h of incubation, they could not be sustained regardless of NH(4)(+) level and declined to zero over 20 h of incubation. The transformation capacity of the slurry enrichments (~1 micro mol MeBr ml(-1) soil slurry) was similar to the value measured previously in cell suspensions of N. europaea with similar NH(3)-oxidizing activity. Several MeBr-degrading characteristics of the nitrifying enrichments were found to be similar to those documented in the literature for MeBr-degrading methanotrophs and facultatively methylotrophic bacteria.

Nitrite Concentration Effects on NO3-N Uptake and Reduction, Growth, and Fruit Yield in Strawberry

Strawberries (Fragaria xananassa Duch. 'Osogrande') were grown hydroponically with three NO3-N concentrations (3.75, 7.5, or 15.0 mM) to determine effects of varying concentration on NO3-N uptake and reduction rates, and to relate these processes to growth and fruit yield. Plants were grown for 32 weeks, and NO3-N uptake and nitrate reductase (NR) activities in roots and shoots were measured during vegetative and reproductive growth. In general, NO3-N uptake rates increased as NO3-N concentration in the hydroponics system increased. Tissue NO3- concentration also increased as external NO3-N concentration increased, reflecting the differences in uptake rates. There was no effect of external NO3-N concentration on NR activities in leaves or roots during either stage of development. Leaf NR activity averaged approximately 360 nmol NO2 formed/g fresh weight (FW)/h over both developmental stages, while NR activity in roots was much lower, averaging approximately 115 nmol NO2 formed/g FW/h. Vegetative organ FW, dry weight (DW), and total fruit yield were unaffected by NO3-N concentration. These data suggest that the inability of strawberry to increase growth and fruit yield in response to increasing NO3-N concentrations is not due to limitations in NO3-N uptake rates, but rather to limitations in NO3- reduction and/or assimilation in both roots and leaves.

Inhibition of inducible nitric oxide synthase gene expression and nitric oxide synthesis in vascular smooth muscle cells by granulocyte-colony stimulating factor in vitro

Clinical and experimental evidence suggests that granulocyte-colony stimulating factor (G-CSF) acts as an anti-inflammatory modulator with beneficial effects in severe inflammatory diseases, e.g., sepsis and septic shock. Excessive production of nitric oxide (NO) is regarded as a potent mediator of the vascular changes leading to systemic hypotension that occurs during sepsis. Therefore, the aim of the present study was to investigate the influence of G-CSF on inducible nitric oxide synthase (iNOS) gene expression and NO synthesis in vascular smooth muscle cells (VSMC). Qualitative and quantitative analyses of iNOS cDNA revealed that G-CSF significantly reduced interferon-γ/lipopolysaccharide (IFN-γ/LPS) dependent iNOS gene expression (P<0.05) following 6, 18, 24, and 48 h incubation periods. In addition, the co-application of G-CSF resulted in a decreased IFN-γ/LPS mediated iNOS protein generation as detected by immunoblotting methods after 24 and 48 h. Measurement of the stable NO metabolites showed a significant reduction of nitrite/nitrate concentrations following co-incubation of VSMC with G-CSF+IFN-γ/LPS (242.57±10.73 nmol NO2−/NO3−/mg cell protein, n=8) as compared to IFN-γ/LPS treatment (306.20±19.26 nmol NO2−/NO3−/mg cell protein, n=8, P<0.05) following a 24-h incubation protocol. This inhibitory effect of G-CSF was still present after a 48 h incubation period (G-CSF+IFN-γ/LPS: 319.56±6.26 nmol NO2−/NO3−/mg cell protein; IFN-γ/LPS: 489.20±27.15 nmol NO2−/NO3−/mg cell protein (P<0.05), n=8, respectively). The present findings suggest that inhibition of iNOS gene expression and NO generation in VSMC might be one of the protective anti-inflammatory effects of G-CSF during sepsis.

Efficient formation of nitric oxide from selective oxidation of N-aryl N'-hydroxyguanidines by inducible nitric oxide synthase.

Inducible nitric oxide synthase (NOS II) efficiently catalyzes the oxidation of N-(4-chlorophenyl)N'-hydroxyguanidine 1 by NADPH and O2, with concomitant formation of the corresponding urea and NO. The characteristics of this reaction are very similar to those of the NOS-dependent oxidation of endogenous Nomega-hydroxy-L-arginine (NOHA), i.e., (i) the formation of products resulting from an oxidation of the substrate C=N(OH) bond, the corresponding urea and NO, in a 1:1 molar ratio, (ii) the absolute requirement of the tetrahydrobiopterin (BH4) cofactor for NO formation, and (iii) the strong inhibitory effects of L-arginine (L-arg) and classical inhibitors of NOSs. N-Hydroxyguanidine 1 is not as good a substrate for NOS II as is NOHA (Km = 500 microM versus 15 microM for NOHA). However, it leads to relatively high rates of NO formation which are only 4-fold lower than those obtained with NOHA (Vm = 390 +/- 50 nmol NO min-1 mg protein-1, corresponding roughly to 100 turnovers min-1). Preliminary results indicate that some other N-aryl N'-hydroxyguanidines exhibit a similar behavior. These results show for the first time that simple exogenous compounds may act as NO donors after oxidative activation by NOSs. They also suggest a possible implication of NOSs in the oxidative metabolism of certain classes of xenobiotics.

Nitrate Reductase Activities in Rice Genotypes in Irrigated Lowlands

Nitrate reductase (NR) is a key enzyme involved in nitrate assimilation in crops. In irrigated lowlands, specific activities of NADH‐ and NADPH‐NR in rice (Oryza saliva L.) have not been investigated. The objectives of this study were to evaluate the nitrate reductase activity (NRA) patterns of rice genotypes with different NRA levels under irrigated lowland conditions, and to determine the effects of NR deficiency on nitrate use, grain yield, biomass, and other agronomic characteristics. Seven cultivars and seven NR mutants were grown under irrigated lowland conditions; bioassayed in vitro for NRA at different growth stages; and evaluated for grain yield, biomass, and other agronomic characters. NADH‐NRA was present at minimal levels in all cultivars before transplanting. At 15 d after transplanting (DAT), NADH‐NRA in all cultivars reached optimum levels and gradually declined until maturity. A similar pattern was observed in most of the NR mutants but with lower levels of activity. NADPHNRA in cultivars and mutants gradually increased from 15 to 45 DAT and then fluctuated between 5 to 10 nmol NO−2 min−1 g−1 fresh weight until maturity. Nitrate availability in the soil may significantly influence NRA and nitrate accumulation in rice in irrigated lowlands. Effects of low or deficient levels of NR on traits evaluated varied among mutants. A decrease of 27 to 73% in grain yield was observed in mutants relative to original cultivars. This study demonstrates that rice assimilates nitrate in anaerobic and flooded conditions, which may have implications for genetic improvement and nitrate or nitrogen management strategies in rice.

Production of Nitric Oxide by Mitochondria

The production of NO. by mitochondria was investigated by electron paramagnetic resonance using the spin-trapping technique, and by the oxidation of oxymyoglobin. Percoll-purified rat liver mitochondria exhibited a negligible contamination with other subcellular fractions (1-4%) and high degree of functionality (respiratory control ratio = 5-6). Toluene-permeabilized mitochondria, mitochondrial homogenates, and a crude preparation of nitric oxide synthase (NOS) incubated with the spin trap N-methyl-D-glucamine-dithiocarbamate-FeII produced a signal ascribed to the NO. spin adduct (g = 2.04; aN = 12.5 G). The intensity of the signal increased with time, protein concentration, and L-Arg, and decreased with the addition of the NOS inhibitor NG-monomethyl-L-arginine. Intact mitochondria, mitochondrial homogenates, and submitochondrial particles produced NO. (followed by the oxidation of oxymyoglobin) at rates of 1.4, 4.9, and 7.1 nmol NO. x (min.mg protein)-1, respectively, with a Km for L-Arg of 5-7 microM. Comparison of the rates of NO. production obtained with homogenates and submitochondrial particles indicated that most of the enzymatic activity was localized in the mitochondrial inner membrane. This study demonstrates that mitochondria are a source of NO., the production of which may effect energy metabolism, O2 consumption, and O2 free radical formation.

Characterization of Human Liver Inducible Nitric Oxide Synthase Expressed inEscherichia coli

We have cloned the human liver inducible isoform of nitric oxide synthase (NOS) into anEscherichia coliexpression vector and have expressed and purified the enzyme. The protein has been expressed with and without a polyhistidine tail. In both cases, expression of functional protein requires coexpression with calmodulin and inclusion of tetrahydrobiopterin (H4B) in the purification buffers. Unlike the constitutive isoforms of NOS, this isoform is unstable in the absence ofl-arginine (l-Arg) and H4B toward loss of the heme group and the formation of a low-spin species spectroscopically distinct from that of the cofactor-bound protein. The enzyme purified in the presence of bothl-Arg and H4B is highly active, with aVmaxof ∼800 nmol NO min−1mg−1and aKmforl-Arg of 22 μm. The cytochrome c reductase activity is 38,000 nmol·min−1mg−1. Similar values are obtained for the enzyme with and without the polyhistidine tail. Ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetic acid does not inhibit the activity of the protein, nor is the activity of the enzyme increased by the addition of exogenous calmodulin and/or Ca2+. These findings contrast with an earlier report, based on experiments with extracts of COS-1 cells expressing the recombinant enzyme, that the enzyme responds to changes in the Ca2+concentration. The human hepatic isoform is similar in its properties to the inducible NOS isoform purified from macrophages.

Nitrification rates, ammonium and nitrate distribution in upper layers of the water column and in sediments of the Indian sector of the Southern Ocean

During the ANTARES 1/F-JGOFS cruise (April–May 1993) concentrations of inorganic nitrogen, nitrification rates (ammonium and nitrite oxidation) and 14C carbonate incorporation by nitrifying bacteria were measured at 0, 50 and 100 m in the water column, and at the water-sediment interface between the polar front (52°S) and the subantarctic and subtropical fronts (42°S) in the Indian Ocean Sector of the Antarctic Ocean. The 0–100 m water layer showed a global increase of NH 4 + (from 0.4 to 0.6 μM) and a global decrease of NO 2 − + NO 3 − (from 25 to 5 μM) along the south-north transect. Anomalies were detected for both ammonium and nitrite + nitrate concentrations in the SubTropical Front (STF) and SubAntarctic Front (SAF). Pelagic nitrifying activities (N oxidation) did not demonstrate any latitudinal gradient in the water column. In terms of integrated rates between 0 and 100 m, the nitrification increased by a factor 1.8–2 in the STF-SAF area. The N oxidation processes mostly depended on the substrate availability. For both ammonium and nitrite oxidizers the C fixation was well correlated ( r = 0.68, p = 0.001 and r = 0.98, p = 0.0001, respectively) with N oxidation, in the range of 15 N (ammonium oxidizers) and 20 N (nitrite oxidizers) oxidized for 1 C incorporated. For nitrite oxidizers, C incorporation was principally influenced by temperature ( Q 10 = 1,73). From the calculation of nitrogen fluxes, the nitrifiers would be able to compete with the primary producers for the regenerated ammonium, while the flux of nitrate produced by nitrification could sustain 10 to 100% of the primary producer requirements for this nitrogen source. In the sediment pore waters, the inorganic nitrogen compounds displayed two different latitudinal distributions. The ammonium concentrations were lower (around 4 μM) south of the 48°S than in the northern part (values reaching 10 μM in the first centimetre). The nitrite + nitrate concentrations were close to 40–45 μM at each end of the transect, and increased to 50–55 μM near 45°S. The benthic nitrification, measured from 0 to 5 cm depth in the sediment cores, showed high rates in the upper 3 cm. The rates of ammonium oxidation demonstrated a good correlation ( r = 0.64, p = 0.0001) with the ammonium content of the interstitial water. The nitrite oxidation rates were well correlated ( r = 0.92, p = 0.0001) with the ammonium oxidation rates. The integrated rates (0–5 cm) of ammonium oxidation were of about 35 nmol NO 2 − produced cm −2 day −1 in the south, and increased by a factor 1.3–1.9 after 44°S. Both ammonium and nitrite oxidation rates showed a maximum near 44°S, corresponding to the SAF-STF frontal zone.

Nitrate deposition in northern hardwood forests and the nitrogen metabolism of Acer saccharum marsh.

It is generally assumed that plant assimilation constitutes the major sink for anthropogenic Nitrate NO 3- deposited in temperate forests because plant growth is usually limited by nitrogen (N) availability. Nevertheless, plants are known to vary widely in their capacity for NO 3- uptake and assimilation, and few studies have directly measured these parameters for overstory trees. Using a combination of field and greenhouse experiments, we studied the N nutrition of Acer saccharum Marsh. in four northern hardwood forests receiving experimental NO 3- additions equivalent to 30 kg N ha-1 year-1. We measured leaf and fine-root nitrate reductase activity (NRA) of overstory trees using an in vivo assay and used 15N to determine the kinetic parameters of NO 3- uptake by excised fine roots. In two greenhouse experiments, we measured leaf and root NRA in A. saccharum seedlings fertilized with 0-3.5 g NO 3- -N m-2 and determined the kinetic parameters of NO 3- and NH 4+ uptake in excised roots of seedlings. In both overstory trees and seedlings, rates of leaf and fine root NRA were substantially lower than previously reported rates for most woody plants and showed no response to NO 3- fertilization (range = non-detectable to 33 nmol NO 2- g-1 h-1). Maximal rates of NO 3- uptake in overstory trees also were low, ranging from 0.2 to 1.0 μmol g-1 h-1. In seedlings, the mean V max for NO 3- uptake in fine roots (1 μmol g-1 h-1) was approximately 30 times lower than the V max for NH 4+ uptake (33 μmol g-1 h-1). Our results suggest that A. saccharum satisfies its N demand through rapid NH 4+ uptake and may have a limited capacity to serve as a direct sink for atmospheric additions of NO 3- .

Resistance to glomerular injury in the diabetic biobreeding rat.

This study was designed to determine whether the diabetic BioBreeding rat develops significant renal injury following long-term moderate to severe hyperglycaemia. Diabetic and control rats were followed from the onset of diabetes (2-4 months) to 18 months of age. Frank proteinuria and/or albuminuria were always absent. Glomerular filtration rate, measured by inulin clearance (ml min-1 (100 g body weight)-1), was significantly higher in diabetic rats than in controls at 10, 12 and 18 months of age. Advanced glycosylation end-product cross-links assessed by percentage solubility of tail tendon collagen were moderately increased in diabetic compared with control animals. Urinary excretion of advanced glycosylation end-products in unfractionated urine and in urine fractionated for low molecular mass peptides (< 10 kDa) was 11-fold greater in the diabetic rats than in the control group. Urinary excretion of nitric oxide metabolites (nmol NO2- and NO3- (24 h)-1) were significantly (P < 0.05) greater in diabetic rats than in controls after 8 months of age. Mild histopathology resembling human diabetic nephropathy, including increased mesangial volume and glomerular basement membrane thickness, was detected at 18 months of age. The findings of hyperfiltration and mild glomerular morphological changes in diabetic BioBreeding rats are similar to the abnormalities seen in stage 2 human diabetic nephropathy. We hypothesize that two factors which may contribute to the resistance or tolerance to renal injury in the BioBreeding diabetic rat are increased nitric oxide production and the decreased accumulation of advanced glycosylation end-products.

Simultaneous generation of nitric oxide and superoxide by inflammatory cells in rats

It has recently been shown that peroxynitrite anion is a powerful oxidant that can initiate lipid peroxidation. As this oxidant is the product of the reaction between nitric oxide and superoxide, we have studied whether cells isolated from an inflammatory exudate can release both radicals simultaneously under physiological conditions. The carrageenin-induced granuloma model in rats was used. Cells from the inflammatory pouch were stimulated with opsonized zymosan in the absence or in the presence of exogenous l-arginine. Nitric oxide production without exogenous l-arginine was detectable after 15 min (0.29 nmol NO 2 −) and increased with time (1.65 nmol NO 2 − at 4 h). When nitrite released from cells was expressed as a rate a burst was shown in the first few minutes. Between 0 and 15 min, cells produced NO 2 − at the following rates: 20 pmol NO 2 −/1 × 10 6 cells/min without exogenous l-arginine and 83 pmol NO 2 −/1 × 10 6 cells/min with exogenous l-arginine. Production was further stimulated with opsonized zymosan (92 pmol NO 2 −/1 × 10 6 cells/min), and inhibited by L-NMMA and L-NIO. The production of superoxide increased for up to 2 h and then stabilized. A significant increase in nitrite was observed in the presence of SOD, whereas L-NIO increased superoxide generation. These results suggest that peroxynitrite anion may be formed by inflammatory cells.

Inhibition of methane oxidation by ammonium in the surface layer of a littoral sediment

The diffusive flux of CH4 from the sediment into the water was measured in intact sediment cores taken from the littoral of Lake Constance. CH4 fluxes were measured under oxic and anoxic conditions. Emissions under anoxic conditions were used to estimate the potential diffusive flux. The results showed that about 79% of the CH4 diffusing upwards was oxidized at the sediment surface. Kinetic measurements of CH4 oxidation in diluted surface sediment gave Vmax = 1.2 μmol CH4 mlsed−1h−1. This was high enough to explain the oxidation rates calculated from the flux measurements. NH4+ was added to the water overlaying the sediment cores to measure its influence on CH4 oxidation. This treatment resulted in increased concentrations of pore water NH4+ which were measured with a NH4+ microelectrode. Pore water NH4+ concentrations of 20 mM. Measurements of potential NH4+ oxidation rates were below the detection limit of 0.5 nmol NO2− produced mlsed−1 h−1. Thus, the NH4+ oxidizing activity was about 3 magnitudes lower than the CH4 oxidizing activity, and it is unlikely that NH4+ oxidizers are important for CH4 oxidation in this littoral sediment.