Biphasic Behavior of Anammox Regulated by Nitrite and Nitrate in an Estuarine Sediment

The production of N₂ gas via anammox was investigated in sediment slurries at in situ NO₂⁻ concentrations in the presence and absence of NO₃⁻. With single enrichment above 10 μM ¹⁴NO₂⁻ or ¹⁴NO₃⁻ and ¹⁵NH₄⁺, anammox activity was always linear (P < 0.05), in agreement with previous findings. In contrast, anammox exhibited a range of activity below 10 μM NO₂⁻ or NO₃⁻, including an elevated response at lower concentrations. With 100 μM NO₃⁻, no significant transient accumulation of NO₂⁻ could be measured, and the starting concentration of NO₂⁻ could therefore be regulated. With dual enrichment (1 to 20 μM NO₂⁻ plus 100 μM NO₃⁻), there was a pronounced nonlinear response in anammox activity. Maximal activity occurred between 2 and 5 μM NO₂⁻, but the amplitude of this peak varied across the study (November 2003 to June 2004). Anammox accounted for as much as 82% of the NO₂⁻ added at 1 μM in November 2003 but only for 15% in May 2004 and for 26 and 5% of the NO₂⁻ added at 5 μM for these two months, respectively. Decreasing the concentration of NO₃⁻ but holding NO₂⁻ at 5 μM decreased the significance of anammox as a sink for NO₂⁻. The behavior of anammox was explored by use of a simple anammox-denitrification model, and the concept of a biphasic system for anammox in estuarine sediments is proposed. Overall, anammox is likely to be regulated by the availability of NO₃⁻ and NO₂⁻ and the relative size or activity of the anammox population.     

Oxygen sensitivity of anammox and coupled N-cycle processes in oxygen minimum zones

Nutrient measurements indicate that 30-50% of the total nitrogen (N) loss in the ocean occurs in oxygen minimum zones (OMZs). This pelagic N-removal takes place within only ~0.1% of the ocean volume, hence moderate variations in the extent of OMZs due to global warming may have a large impact on the global N-cycle. We examined the effect of oxygen (O(2)) on anammox, NH(3) oxidation and NO(3)(-) reduction in (15)N-labeling experiments with varying O(2) concentrations (0-25 μmol L(-1)) in the Namibian and Peruvian OMZs. Our results show that O(2) is a major controlling factor for anammox activity in OMZ waters. Based on our O(2) assays we estimate the upper limit for anammox to be ~20 μmol L(-1). In contrast, NH(3) oxidation to NO(2)(-) and NO(3)(-) reduction to NO(2)(-) as the main NH(4)(+) and NO(2)(-) sources for anammox were only moderately affected by changing O(2) concentrations. Intriguingly, aerobic NH(3) oxidation was active at non-detectable concentrations of O(2), while anaerobic NO(3)(-) reduction was fully active up to at least 25 μmol L(-1) O(2). Hence, aerobic and anaerobic N-cycle pathways in OMZs can co-occur over a larger range of O(2) concentrations than previously assumed. The zone where N-loss can occur is primarily controlled by the O(2)-sensitivity of anammox itself, and not by any effects of O(2) on the tightly coupled pathways of aerobic NH(3) oxidation and NO(3)(-) reduction. With anammox bacteria in the marine environment being active at O(2) levels ~20 times higher than those known to inhibit their cultured counterparts, the oceanic volume potentially acting as a N-sink increases tenfold. The predicted expansion of OMZs may enlarge this volume even further. Our study provides the first robust estimates of O(2) sensitivities for processes directly and indirectly connected with N-loss. These are essential to assess the effects of ocean de-oxygenation on oceanic N-cycling.     

Correlation between anammox activity and microscale distribution of nitrite in a subtropical mangrove sediment

The distribution of anaerobic ammonium oxidation (anammox) in nature has been addressed by only a few environmental studies, and our understanding of how anammox bacteria compete for substrates in natural environments is therefore limited. In this study, we measure the potential anammox rates in sediment from four locations in a subtropical tidal river system. Porewater profiles of NO(x)(-) (NO2- plus NO3-) and NO2- were measured with microscale biosensors, and the availability of NO2- was compared with the potential for anammox activity. The potential rate of anammox increased with increasing distance from the mouth of the river and correlated strongly with the production of nitrite in the sediment and with the average concentration or total pool of nitrite in the suboxic sediment layer. Nitrite accumulated both from nitrification and from NO(x)(-) reduction, though NO(x)(-) reduction was shown to have the greatest impact on the availability of nitrite in the suboxic sediment layer. This finding suggests that denitrification, though using NO2- as a substrate, also provides a substrate for the anammox process, which has been suggested in previous studies where microscale NO2- profiles were not measured.     

New anaerobic, ammonium-oxidizing community enriched from peat soil

Anaerobic ammonium-oxidizing (anammox) bacteria have been recognized as an important sink for fixed nitrogen and are detected in many natural environments. However, their presence in terrestrial ecosystems has long been overlooked, and their contribution to the nitrogen cycling in natural and agricultural soils is currently unknown. Here we describe the enrichment and characterization of anammox bacteria from a nitrogen-loaded peat soil. After 8 months of incubation with the natural surface water of the sampling site and increasing ammonium and nitrite concentrations, anammox cells constituted 40 to 50% of the enrichment culture. The two dominant anammox phylotypes were affiliated with "Candidatus Jettenia asiatica" and "Candidatus Brocadia fulgida." The enrichment culture converted NH(4)(+) and NO(2)(-) to N(2) with the previously reported stoichiometry (1:1.27) and had a maximum specific anaerobic ammonium oxidation rate of 0.94 mmol NH(4)(+)·g (dry weight)(-1)·h(-1) at pH 7.1 and 32°C. The diagnostic anammox-specific lipids were detected at a concentration of 650 ng·g (dry weight)(-1), and pentyl-[3]-ladderane was the most abundant ladderane lipid.     

Constitutive nitric oxide synthase from cerebellum is reversibly inhibited by nitric oxide formed from L-arginine.

The objective of this study was to determine whether constitutive nitric oxide (NO) synthase from rat cerebellum could be regulated by the two products of the reaction, NO and L-citrulline, utilizing L-arginine as substrate. NO synthase activity was determined by monitoring the formation of 3H-citrulline from 3H-L-arginine in the presence of added cofactors. The rate of citrulline formation in enzyme reaction mixtures was non-linear. Addition of superoxide dismutase (SOD; 100 units) inhibited NO synthase activity and made the rate of product formation more non-linear, whereas addition of oxyhemoglobin (HbO2; 30 microM) increased NO synthase activity, made the rate of product formation linear and also abolished the effect of SOD. Added NO (10 microM) inhibited NO synthase activity and this inhibition was potentiated by SOD and abolished by HbO2. Added L-citrulline (1 mM) did not alter NO synthase activity. The two NO donors, S-nitroso-N-acetylpenicillamine (200 microM) and N-methyl-N'-nitro-N-nitrosoguanidine (200 microM) mimicked the inhibitory effect of NO and inhibition of NO synthase activity by NO was reversible. These observations indicate clearly that NO formed during the NO synthase reaction or added to the enzyme reaction mixture causes a reversible inhibition of NO synthase activity. Thus, NO may function as a negative feedback modulator of its own synthesis.     

Kinetics, diffusional limitation and microscale distribution of chemistry and organisms in a CANON reactor

In the Completely Autotrophic Nitrogen removal Over Nitrite (CANON) process, aerobic and anaerobic ammonia oxidizing bacteria cooperate to remove ammonia in one oxygen-limited reactor. Kinetic studies, microsensor analysis, and fluorescence in situ hybridization on CANON biomass showed a partial differentiation of processes and organisms within and among aggregates. Under normal oxygen-limited conditions ( approximately 5 microM O2), aerobic ammonia oxidation (nitrification) was restricted to an outer shell (<100 microm) while anaerobic ammonia oxidation (anammox) was found in the central anoxic parts. Larger type aggregates (>500 microm) accounted for 68% of the anammox potential whereas 65% of the nitrification potential was found in the smaller aggregates (<500 microm). Analysis with O2 and NO2- microsensors showed that the thickness of the activity zones varied as a function of bulk O2 and NO2- concentrations and flow rate.     

Physiological and biochemical alterations in Anabaena 7120 under iron stress

Various physiological and biochemical process like growth, NO3- -uptake, nitrate reductase, glutamine synthetase and ATPases (Mg2+ and Ca2+ dependent) in the cyanobacterium Anabaena 7120 were observed under iron stress. Growth was found to be maximum in 50 microM Fe3+ added cells however, 20 microM Fe3+ (the Fe3+ concentration generally used for routine culturing of cyanobacterial cell in Chu 10 medium) incubation resulted in lower growth. Fe3+ starvation on the other hand showed very poor growth up to 4th day but once the growth started it reached at significant level on 7th day. Higher Fe3+ concentration reflected reduced growth with lethality at 500 microM Fe3+. Chlorophyll a fluorescence under Fe3+ stress reflected almost the similar results as in case of growth. However, the pigment was found to be more sensitive as compared to protein under Fe3+ stress. Similar results have been observed in case of NO3-uptake with only 80% reduction in nutrient uptake in 500 microM Fe3+ incubated cells. Nitrate reductase activity was lower in Fe3+ starved cells as compared to significant enzyme activity in 20 and 50 microM Fe3+ incubated cells. Similar to nitrate reductase, glutamine synthetase also showed maximum level in 50 microM Fe3+ added cells, however, higher Fe3+ concentration (300-500 microM ) resulted in reduced enzymatic activity. Glutamine synthetase activity was less sensitivity as compared to nitrate reductase activity under Fe3+ stress. ATPase (Mg2+ and Ca2+ dependent) always showed higher level with increasing Fe3+ concentration.     

Anaerobic ammonium oxidation (anammox) irreversibly inhibited by methanol

Methanol inhibition of anaerobic ammonium oxidation (anammox) activity was characterized. An enrichment culture entrapped in a polyethylene glycol gel carrier was designed for practical uses of wastewater treatment. Batch experiments demonstrated that anammox activity decreased with increases in methanol concentration, and relative activity reached to 29% of the maximum when 5 mM methanol was added. Also, batch experiments were conducted using anammox sludge without immobilization. Anammox activity was evaluated by quantifying ¹⁴N¹⁵N (²⁹N) emission by combined gas chromatography-quadrupole mass spectrometry, and the anammox activity was found to be almost as sensitive to methanol as in the earlier trials in which gel carriers were used. These results indicated that methanol inhibition was less severe than previous studies. When methanol was added in the influent of continuous feeding system, relative activity was decreased to 46% after 80 h. Although the addition was halted, afterwards the anammox activity was not resumed in another 19 days of cultivation, suggesting that methanol inhibition to anammox activity was irreversible. It is notable that methanol inhibition was not observed if anammox activity was quiescent when substrate for anammox was not supplied. These results suggest that methanol itself is not inhibitory and may not directly inhibit the anammox activity.     

In situ activity and spatial organization of anaerobic ammonium-oxidizing (anammox) bacteria in biofilms

We investigated autotrophic anaerobic ammonium-oxidizing (anammox) biofilms for their spatial organization, community composition, and in situ activities by using molecular biological techniques combined with microelectrodes. Results of phylogenetic analysis and fluorescence in situ hybridization (FISH) revealed that "Brocadia"-like anammox bacteria that hybridized with the Amx820 probe dominated, with 60 to 92% of total bacteria in the upper part (<1,000 microm) of the biofilm, where high anammox activity was mainly detected with microelectrodes. The relative abundance of anammox bacteria decreased along the flow direction of the reactor. FISH results also indicated that Nitrosomonas-, Nitrosospira-, and Nitrosococcus-like aerobic ammonia-oxidizing bacteria (AOB) and Nitrospira-like nitrite-oxidizing bacteria (NOB) coexisted with anammox bacteria and accounted for 13 to 21% of total bacteria in the biofilms. Microelectrode measurements at three points along the anammox reactor revealed that the NH(4)(+) and NO(2)(-) consumption rates decreased from 0.68 and 0.64 micromol cm(-2) h(-1) at P2 (the second port, 170 mm from the inlet port) to 0.30 and 0.35 micromol cm(-2) h(-1) at P3 (the third port, 205 mm from the inlet port), respectively. No anammox activity was detected at P4 (the fourth port, 240 mm from the inlet port), even though sufficient amounts of NH(4)(+) and NO(2)(-) and a high abundance of anammox bacteria were still present. This result could be explained by the inhibitory effect of organic compounds derived from biomass decay and/or produced by anammox and coexisting bacteria in the upper parts of the biofilm and in the upstream part of the reactor. The anammox activities in the biofilm determined by microelectrodes reflected the overall reactor performance. The several groups of aerobic AOB lineages, Nitrospira-like NOB, and Betaproteobacteria coexisting in the anammox biofilm might consume a trace amount of O(2) or organic compounds, which consequently established suitable microenvironments for anammox bacteria.     

In vitro assay and light regulation of nitrate reductase in red alga Gracilaria chilensis

Nitrate reductase (NR) is the first enzyme in the nitrogen assimilation pathway. The in vitro NR activity of Gracilaria chilensis was assayed under different conditions to reveal its stability and biochemical characteristics, and an optimized in vitro assay is described. Maximal NR activities were observed at pH 8.0 and 15 degrees C. The apparent Km value for NADH was 8 microM and for nitrate 680 microM. Crude extracts of G. chilensis stored at 4 degrees C showed a 50% decrease of NR activity after 24 h. The highest NR activity value (253.20+/-2.60 x 10(-3) U g(-1)) was obtained when 100% von Stosch medium (500 microM NO3-) was added before extraction of apical parts. Algae under light:dark cycles of 12:12h exhibited circadian fluctuation of NR activity and photosynthesis with more than 2 times higher levels in the light phase. No evidence of endogenous diel rhythm controlling NR activity or photosynthesis was observed. Light pulses lasting 10 or 60 min during the darkness increased the NR activity by 30% and 45%, respectively. The results indicate that NR and photosynthesis are regulated mainly by light and not by a biological clock.     

Nitric oxide donors regulate nitric oxide synthase in bovine pulmonary artery endothelium

This study examined the notion that exogenous generation of nitric oxide (NO) modulates NOS gene expression and activity. Bovine pulmonary artery endothelial cells (BPAEC) were treated with the NO donors, 1 mM SNAP (S-nitroso-N-acetylpenicillamine), 0.5 mM SNP (sodium nitroprusside) or 0.2 microM NONOate (spermine NONOate) in medium 199 containing 2% FBS. Controls included untreated cells and cells exposed to 1 mM NAP (N-acetyl-D-penicillamine). NOS activity was assessed using a fibroblast-reporter cell assay; intracellular Ca2+ concentrations were assessed by Fura-2 microfluorometry; and NO release was measured by chemiluminescence. Constitutive endothelial (e) and inducible (i) NOS gene and protein expression were examined by northern and western blot analysis, respectively. Two hours exposure to either SNAP or NONOate caused a significant elevation in NO release from the endothelial cells (SNAP = 51.4 +/- 5.9; NONOate = 23.8 +/- 4.2; control = 14.5 +/- 2.8 microM); but A23187 (3 microM)-stimulated NO release was attenuated when compared to controls. Treatment with either SNAP or NONOate for 2 h also resulted in a significant increase in NOS activity in endothelial homogenates (SNAP = 23.6 +/- 2.5; NONOate= 29.8 +/- 7.7; control = 14.5 +/- 2.5fmol cGMP/microg per 10(6) cells). Exposure to SNAP and SNP, but not NONOate, for 1 h caused an increase in intracellular calcium. Between 4 and 8 h, SNAP and NONOate caused a 2- to 3-fold increase in eNOS, but not iNOS, gene (P < 0.05) and protein expression. NAP had little effect on either eNOS gene expression, activity or NO production. Our data indicate that exogenous generation of NO leads to a biphasic response in BPAEC, an early increase in intracellular Ca2+, and increases in NOS activity and NO release followed by increased expression of the eNOS gene, but not the iNOS gene. We conclude that eNOS gene expression and activity are regulated by a positive-feedback regulatory action of exogenous NO.     

Tillage and Nitrogen Application Effects on Nitrous and Nitric Oxide Emissions from Irrigated Corn Fields

A 2-year study was conducted to investigate the potential of no-till cropping systems to reduce N₂O and NO emissions under different N application rates in an irrigated corn field in northeastern Colorado. Flux measurements were begun in the spring of 2003, using vented (N₂O) and dynamic (NO) chambers, one to three times per week, year round, within plots that were cropped continuously to corn (Zea mays L.) under conventional-till (CT) and no-till (NT). Plots were fertilized at planting in late April with rates of 0, 134 and 224 kg N ha⁻¹ and corn was harvested in late October or early November each year. N₂O and NO fluxes increased linearly with N application rate in both years. Compared with CT, NT did not significantly affect the emission of N₂O but resulted in much lower emission of NO. In 2003 and 2004 corn growing seasons, the increase in N₂O-N emitted per kg ha⁻¹ of fertilizer N added was 14.5 and 4.1 g ha⁻¹ for CT, and 11.2 and 5.5 g ha⁻¹ for NT, respectively. However, the increase in NO-N emitted per kg ha⁻¹ of fertilizer N added was only 3.6 and 7.4 g ha⁻¹ for CT and 1.6 and 2.0 g ha⁻¹ for NT in 2003 and 2004, respectively. In the fallow season (November 2003 to April 2004), much greater N₂O (2.0–3.1 times) and NO (13.1–16.8 times) were emitted from CT than from NT although previous N application did not show obvious carry-over effect on both gas emissions. Results from this study reveal that NT has potential to reduce NO emission without an obvious change in N₂O emission under continuous irrigated corn cropping compared to CT.     

Effects of specific inhibitors on anammox and denitrification in marine sediments

The effects of three metabolic inhibitors (acetylene, methanol, and allylthiourea [ATU]) on the pathways of N2 production were investigated by using short anoxic incubations of marine sediment with a 15N isotope technique. Acetylene inhibited ammonium oxidation through the anammox pathway as the oxidation rate decreased exponentially with increasing acetylene concentration; the rate decay constant was 0.10+/-0.02 microM-1, and there was 95% inhibition at approximately 30 microM. Nitrous oxide reduction, the final step of denitrification, was not sensitive to acetylene concentrations below 10 microM. However, nitrous oxide reduction was inhibited by higher concentrations, and the sensitivity was approximately one-half the sensitivity of anammox (decay constant, 0.049+/-0.004 microM-1; 95% inhibition at approximately 70 microM). Methanol specifically inhibited anammox with a decay constant of 0.79+/-0.12 mM-1, and thus 3 to 4 mM methanol was required for nearly complete inhibition. This level of methanol stimulated denitrification by approximately 50%. ATU did not have marked effects on the rates of anammox and denitrification. The profile of inhibitor effects on anammox agreed with the results of studies of the process in wastewater bioreactors, which confirmed the similarity between the anammox bacteria in bioreactors and natural environments. Acetylene and methanol can be used to separate anammox and denitrification, but the effects of these compounds on nitrification limits their use in studies of these processes in systems where nitrification is an important source of nitrate. The observed differential effects of acetylene and methanol on anammox and denitrification support our current understanding of the two main pathways of N2 production in marine sediments and the use of 15N isotope methods for their quantification.     

Effects of reactive oxygen and nitrogen species on cyclooxygenase-1 and -2 activities

The effects of reactive oxygen species (superoxide anion radical--O(2)*-, hydrogen peroxide--H(2)O(2) and hydroxyl radical--*OH; the reaction products of xanthine plus xanthine oxidase system) and reactive nitrogen species [nitric oxide--NO*; from 1-hydroxyl-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene--NOC7 and peroxynitrite--ONOO(-)] on the activities of purified cyclooxygenase (COX)-1 and -2 were studied. Xanthine plus xanthine oxidase suppressed the COX-1 and -2 activities in a xanthine oxidase concentration-dependent fashion. This effect was reversed by addition of catalase to the reactive oxygen species-generating system but not by superoxide dismutase or mannitol, indicating that H(2)O(2) is the responsible metabolite. NOC7 activated the COX-1 activity but inhibited the COX-2 activity at concentrations ranging from 1 to 50 microM. Experiments utilizing a NO* antidote, carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide revealed that the observed effects of NOC7 are caused by NO*.ONOO(-), a product of NO* and O(2)*-, both activated and inhibited the COX-1 and -2 activities, depending on ONOO(-) concentration. At a low concentration of ONOO(-) (5 microM) there was enhancement of the COX-1 and -2 activities, but with higher concentrations there was suppression of these two enzyme activities (COX-1, at 200 microM; COX-2, >50 microM). These results suggest that H(2)O(2), NO* and ONOO(-) can have different modulatory effects on the COX-1 and -2 activities.     

PVA-alginate immobilized cells for anaerobic ammonium oxidation (anammox) process

The feasibility of an anaerobic ammonium oxidation (anammox) process combined with a cell-immobilization technique for autotrophic nitrogen removal was investigated. Anammox biomass was cultivated from local activated sludge and achieved significant anammox activity in 6 months. The development of a mature anammox biomass was confirmed by fluorescence in situ hybridization (FISH) analysis and off-line activity measurements. The abundance fraction of the anammox bacteria determined by FISH analysis was estimated by software. The anaerobic ammonia oxidizers occupied almost half of the total cells. Additionally, the anammox biomass was granulated as spherical gel beads of 3-4 mm in diameter by using a cell-immobilization technique. The nitrogen removal activity was proved to be successfully retained in the beads, with about 80% of nitrogenous compounds (NH(4) (+), NO(2) (- )and total nitrogen) removed after 48 h. These results offer a promising technique for the preservation of anammox microorganisms, the protection of them against the unfavorable surroundings, and the prevention of biomass washout towards the implementation of sustainable nitrogen elimination biotechnology. This is the first report on the immobilization of anammox biomass as gel beads.     

Accelerating effect of hydroxylamine and hydrazine on nitrogen removal rate in moving bed biofilm reactor

In biological nitrogen removal, application of the autotrophic anammox process is gaining ground worldwide. Although this field has been widely researched in last years, some aspects as the accelerating effect of putative intermediates (mainly N?H? and NH?OH) need more specific investigation. In the current study, experiments in a moving bed biofilm reactor (MBBR) and batch tests were performed to evaluate the optimum concentrations of anammox process intermediates that accelerate the autotrophic nitrogen removal and mitigate a decrease in the anammox bacteria activity using anammox (anaerobic ammonium oxidation) biomass enriched on ring-shaped biofilm carriers. Anammox biomass was previously grown on blank biofilm carriers for 450 days at moderate temperature 26.0 (±0.5) °C by using sludge reject water as seeding material. FISH analysis revealed that anammox microorganisms were located in clusters in the biofilm. With addition of 1.27 and 1.31 mg N L?1 of each NH?OH and N?H?, respectively, into the MBBR total nitrogen (TN) removal efficiency was rapidly restored after inhibitions by NO??. Various combinations of N?H?, NH?OH, NH??, and NO?? were used as batch substrates. The highest total nitrogen (TN) removal rate with the optimum N?H? concentration (4.38 mg N L?1) present in these batches was 5.43 mg N g?1 TSS h?1, whereas equimolar concentrations of N?H? and NH?OH added together showed lower TN removal rates. Intermediates could be applied in practice to contribute to the recovery of inhibition-damaged wastewater treatment facilities using anammox technology.     

Nitric oxide inhibits Ca(2+) mobilization through cADP-ribose signaling in coronary arterial smooth muscle cells

The present study was designed to determine whether the cADP-ribose-mediated Ca(2+) signaling is involved in the inhibitory effect of nitric oxide (NO) on intracellular Ca(2+) mobilization. With the use of fluorescent microscopic spectrometry, cADP-ribose-induced Ca(2+) release from sarcoplasmic reticulum (SR) of bovine coronary arterial smooth muscle cells (CASMCs) was determined. In the alpha-toxin-permeabilized primary cultures of CASMCs, cADP-ribose (5 microM) produced a rapid Ca(2+) release, which was completely blocked by pretreatment of cells with the cADP-ribose antagonist 8-bromo-cADP-ribose (8-Br-cADPR). In intact fura 2-loaded CASMCs, 80 mM KCl was added to depolarize the cells and increase intracellular Ca(2+) concentration ([Ca(2+)](i)). Sodium nitroprusside (SNP), an NO donor, produced a concentration-dependent inhibition of the KCl-induced increase in [Ca(2+)](i), but it had no effect on the U-46619-induced increase in [Ca(2+)](i). In the presence of 8-Br-cADPR (100 microM) and ryanodine (10 microM), the inhibitory effect of SNP was markedly attenuated. HPLC analyses showed that CASMCs expressed the ADP-ribosyl cyclase activity, and SNP (1-100 microM) significantly reduced the ADP-ribosyl cyclase activity in a concentration-dependent manner. The effect of SNP was completely blocked by addition of 10 microM oxygenated hemoglobin. We conclude that ADP-ribosyl cyclase is present in CASMCs, and NO may decrease [Ca(2+)](i) by inhibition of cADP-ribose-induced Ca(2+) mobilization.     

Anammox bacteria disguised as denitrifiers: nitrate reduction to dinitrogen gas via nitrite and ammonium

Anaerobic ammonium-oxidizing (anammox) bacteria oxidize ammonium with nitrite and produce N(2). They reside in many natural ecosystems and contribute significantly to the cycling of marine nitrogen. Anammox bacteria generally live under ammonium limitation, and it was assumed that in nature anammox bacteria depend on other biochemical processes for ammonium. In this study we investigated the possibility of dissimilatory nitrate reduction to ammonium by anammox bacteria. Physically purified Kuenenia stuttgartiensis cells reduced (15)NO(3) (-) to (15)NH(4) (+) via (15)NO(2) (-) as the intermediate. This was followed by the anaerobic oxidation of the produced ammonium and nitrite. The overall end-product of this metabolism of anammox bacteria was (15)N(15)N dinitrogen gas. The nitrate reduction to nitrite proceeds at a rate of 0.3 +/- 0.02 fmol cell(-1) day(-1) (10% of the 'normal' anammox rate). A calcium-dependent cytochrome c protein with a high (305 mumol min(-1) mg protein(-1)) rate of nitrite reduction to ammonium was partially purified. We present evidence that dissimilatory nitrate reduction to ammonium occurs in Benguela upwelling system at the same site where anammox bacteria were previously detected. This indicates that anammox bacteria could be mediating dissimilatory nitrate reduction to ammonium in natural ecosystems.     

Maturational modulation of endothelium-dependent vasodilatation in ovine cerebral arteries

To address the hypothesis that maturation enhances endothelial vasodilator function in cerebral arteries, relaxant responses to ADP and A-23187 were determined in ovine carotid and cerebral arteries harvested from 25 newborn lambs (3-7 days) and 23 adult sheep. Maturation significantly increased pD(2) values for A-23187 (newborn range: 4.9 +/- 0.3 to 5.4 +/- 0.3; adult range: 6.0 +/- 0.2 to 7.1 +/- 0.2) and the maximal vasodilator response to A-23187 by 10-18%. In contrast, maturation decreased maximum responses to ADP by 5-25% with no change in pD(2). The magnitudes of endothelium-dependent relaxation were not affected by 10 microM indomethacin but were virtually abolished by 100 microM N(G)-nitro-L-arginine methyl ester/L-nitro arginine, indicating that nitric oxide (NO) is the primary endothelium-dependent vasodilator in these arteries. Maturation also modestly decreased endothelial NO synthase (eNOS) abundance in both carotid (32%) and cerebral (26%) arteries. Together, these findings reinforce the view that receptor coupling to endothelial activation is tightly regulated and may offset underlying changes in maximal endothelial vasodilator capacity. This capacity, in turn, appears to increase with postnatal age despite major growth and expansion of endothelial cell size and vascular wall volume. In ovine cerebral arteries, endothelial vasodilator capacity appears completely dependent on eNOS activity but not on cyclooxygenase activity. In turn, eNOS activity appears to be postnatally regulated by mechanisms independent of changes in eNOS abundance alone.     

Phosphatidylinositol-specific phospholipase C activity of chromaffin granule-binding proteins

Using [U-14C]phosphatidylinositol as substrate, Ca2+-dependent phospholipase C activity was detected in a group of bovine adrenal medullary proteins that bind to chromaffin granule membranes in the presence of Ca2+ ("chromobindins," Creutz, C. E., Dowling, L. G., Sando, J. J., Villar-Palasi, C., Whipple, J. H., and Zaks, W. J. (1983) J. Biol. Chem. 258, 14664-14674). The activity was maximal at neutral pH and represented an 80- to 240-fold enrichment of adrenal medullary cytosol phospholipase C activity measured at pH 7.3. The stimulation of activity by Ca2+ was complex; no activity was present in the absence of Ca2+, 25% activation occurred at 1 microM Ca2+, and full activation at 5 mM Ca2+. The enzyme bound to chromaffin granule membranes in the presence of 2 mM Ca2+ but was released at 40 microM Ca2+, suggesting that intrinsic enzyme activity may be regulated by [Ca2+] at 1 microM, but additional activation at higher concentrations of Ca2+ is seen in vitro as a result of Ca2+-dependent binding of the active enzyme to substrate-containing membranes. This enzyme may generate diacylglycerol and phosphorylated inositol to act as intracellular messengers in the vicinity of the chromaffin granule membrane during the process of exocytosis.