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SLC26A9 is expressed in gastric surface epithelial cells, mediates Cl-/HCO3- exchange, and is inhibited by NH4+.

HCO3- secretion by gastric mucous cells is essential for protection against acidic injury and peptic ulcer. Herein we report the identification of an apical HCO3- transporter in gastric surface epithelial cells. Northern hybridization and RT-PCR demonstrate the expression of this transporter, also known as SLC26A9, in mouse and rat stomach and trachea (but not kidney). In situ hybridization in mouse stomach showed abundant expression of SLC26A9 in surface epithelial cells with apical localization on immunofluorescence labeling. Functional studies in HEK-293 cells demonstrated that SLC26A9 mediates Cl-/HCO3- exchange and is also capable of Cl--independent HCO3- extrusion. Unlike other anion exchangers or transport proteins reported to date, SLC26A9 activity is inhibited by ammonium (NH4+). The inhibitory effect of NH4+ on gastric HCO3- secretion was also indicated by reduced gastric juxtamucosal pH (pHjm) in rat stomach in vivo. This report is the first to describe the inhibition of HCO3- transport in vitro and the reduction of pHjm in stomach in vivo by NH4+. Given its critical localization on the apical membrane of surface epithelial cells, its ability to transport HCO3-, and its inhibition by NH4+, we propose that SLC26A9 mediates HCO3- secretion in surface epithelial cells and is essential for protection against acidic injury in the stomach. Disease states that are associated with increased ammonia (NH3)/NH4+ generation (e.g., Helicobacter pylori) may impair gastric HCO3- secretion and therefore predispose patients to peptic ulcer by inhibiting SLC26A9.

Animals↗

New process for simultaneous removal of nitrogen and sulphur under anaerobic conditions.

A granular activated carbon (GAC) anaerobic fluidised-bed reactor treating vinasse from an ethanol distillery of sugar beet molasses was operated for 90 days, the first 40 days of start-up followed by 50 days of operation at constant organic loading rate of 1.7g COD/Ld. The reactor showed good performance in terms of organic matter removal but an anomalous behaviour in terms of unusual high concentrations of molecular nitrogen in the biogas. The analysis of the different nitrogenous and sulphur compounds and the mass balances of these compounds in the liquid and gas phases clearly indicated an uncommon evolution of nitrogen and sulphur in the reactor. About 50% of the nitrogen entering the reactor as total Kjeldahl nitrogen (TKN) was removed from the liquid phase appearing as N2 in the gas phase. Simultaneously, only 20% of the S-SO4(2-) initially present in the influent appears as S-S2- in the effluent or S-H2S in the biogas, indicating that 80% of the sulphur is removed. This behaviour has not been reported previously in the literature. These observations may suggest a new anaerobic removal process of ammonia and sulphate according to an uncommon mechanism involving simultaneous anaerobic ammonium oxidation and sulphate reduction.

Anaerobiosis↗

Mutant strains (nit) of Salmonella typhimurium with a pleiotropic defect in nitrogen metabolism.

We have isolated mutant strains (nit) of Salmonella typhimurium that are defective in nitrogen metabolism. They have a reduced ability to use a variety of compounds including glutamate, proline, arginine, N-acetyl-glucosamine, alanine, and adenosine as sole nitrogen source. In addition, although they grow normally on high concentrations of ammonium chloride (greater than 1 mM) as nitrogen source, they grow substantially more slowly than wild type at low concentrations (less than 1 mM). We postulated that the inability of these strains to utilize low concentrations of ammonium chloride accounts for their poor growth on other nitrogen sources. The specific biochemical lesion in strains with a nit mutation is not known; however, mutant strains have no detectable alteration in the activities of glutamine synthetase, glutamate synthetase, or glutamate dehydrogenase, the enzymes known to be involved in assimilation of ammonia. A nit mutation is suppressed by second-site mutations in the structural gene for glutamine synthetase (glnA) that decrease glutamine synthetase activity.

Acetylglucosamine↗

Renal response to acid load after phenformin.

Normally the kidneys respond to an acid load by increased ammonia production. In six patients with adult-type diabetes this response was reduced by a mean 50% after a therapeutic dose of phenformin. The reduced ability to compensate for acid loads may be one factor leading to metabolic acidosis and lactoacidosis sometimes associated with phenformin therapy.

Adult↗

[Ethonium in the treatment of peptic ulcer patients].

Results are reported of clinical trials in 52 patients of ethonium belonging to quaternary ammonia compounds with consideration of the patients' age and concomitant disorders of the hepatobiliary system. It was established that ethonium possesses a marked antiradical and antioxidant effect in ulcer disease, furthering potentiation of the activity of peroxidedysmutase, protective enzymes of the glutathione system, improvement of bioenergetic processes in the gastric mucosa, enhancement of ulcer healing, prevention of ulcer recurrence.

Adult↗

Ammonia emission factors for N fertilizers applied to two contrasting grassland soils.

Ammonia volatilization from nitrogen (N) fertilizer applied throughout the year to two soil types was measured using a system of small wind tunnels. Losses from urea ranged from 12 to 46% of the applied N. Small losses, averaging <1%, were measured from ammonium nitrate (AN) and calcium nitrate applications. Factors influencing these losses are discussed. Using these results and those from other workers, emission factors for urea and AN applications to grassland in the UK were determined as 23.0 and 1.6% of the applied N, respectively. Emission factors for these fertilizers when applied to arable land were estimated as 11.8 and 0.8%, respectively. The emission factor for all other applied N (as straight and compound fertilizers) was assumed to be similar to that for AN. Calculations showed that fertilizer applications to agricultural land in the UK contributes 34 kt NH3-N per year, equivalent to 17% of the total annual NH3 emission.

Journal Article↗

Functional importance of Asp56 from the alpha-polypeptide of Phaseolus vulgaris glutamine synthetase. An essential residue for transferase but not for biosynthetic enzyme activity.

Replacement of Asp56 by site-directed mutagenesis of the alpha-gene from Phaseolus vulgaris glutamine synthetase heterologously expressed in Escherichia coli produces a complete loss of transferase enzyme activity, thus revealing essentiality of the residue for this particular enzyme activity. This happens independent of Asp56 being replaced by Ala or Glu, suggesting that the essentiality of this residue cannot be attributed to its negative electrical charge. However, a high level of glutamine synthetase biosynthetic specific activity (referred to glutamine synthetase protein, as determined immunologically), is present in D56A and D56E mutants, suggesting that Asp56 is an example of a residue that has a different role in the catalytic mechanism of both enzyme activities of this protein. Km for ATP, glutamate and Mg2+, as well as energy of activation, can be altered as a consequence of the performed mutations. However, the Km and catalytic efficiency for ammonium remains unaffected. Therefore, the catalytic role of Asp56 in the alpha-polypeptide of higher plant glutamine synthetase is quite different from the role proposed for its highly conserved homologue in bacteria (Asp50 in E. coli), which has been associated with binding and deprotonation of ammonium. On the other hand, we also show other results indicating that Asp56 is important in the spatial conformation of the active site and/or the protein, Asp56 being a crucial residue in the salting-out aggregation properties of the enzyme.

Binding Sites↗

Initial-phase optimization for bioremediation of munition compound-contaminated soils.

We examined the bioremediation of soils contaminated with the munition compounds 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine, and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetraazocine by a procedure that produced anaerobic conditions in the soils and promoted the biodegradation of nitroaromatic contaminants. This procedure consisted of flooding the soils with 50 mM phosphate buffer, adding starch as a supplemental carbon substrate, and incubating under static conditions. Aerobic heterotrophs, present naturally in the soil or added as an inoculum, quickly removed the oxygen from the static cultures, creating anaerobic conditions. Removal of parent TNT molecules from the soil cultures by the strictly anaerobic microflora occurred within 4 days. The reduced intermediates formed from TNT and hexahydro-1,3,5-trinitro-1,3,5-triazine were removed from the cultures within 24 days, completing the first stage of remediation. The procedure was effective over a range of incubation temperatures, 20 to 37 degrees C, and was improved when 25 mM ammonium was added to cultures buffered with 50 mM potassium phosphate. Ammonium phosphate buffer (50 mM), however, completely inhibited TNT reduction. The optimal pH for the first stage of remediation was between 6.5 and 7.0. When soils were incubated under aerobic conditions or under anaerobic conditions at alkaline pHs, the TNT biodegradation intermediates polymerized. Polymerization was not observed at neutral to slightly acidic pHs under anaerobic conditions. Completion of the first stage of remediation of munition compound-contaminated soils resulted in aqueous supernatants that contained no munition residues or aminoaromatic compounds.

Ammonia↗

Co-digestion of ruminal content and blood from slaughterhouse industries: influence of solid concentration and ammonium generation.

At the present time, organic solid wastes from industries and agricultural activities are considered to be promising substrates for biogas production via anaerobic digestion. Moreover solids stabilisation is required before reutilization or disposal. Slaughterhouses are among the most important industries in Uruguay and produce 150,000 tons of ruminal content (RC) and 30,000 tons of blood per year. In order to determine the influence of the solids and blood contents, the ammonia inhibition and the inoculum adaptation co-digestion batch tests were performed. A set of experiences with TS concentration of 2.5%, 5% and 7.5% and different ratios of RC/blood were carried out using an inoculum from an UASB reactor. In all experiences fast blood hydrolisation was observed. A higher methane production was detected in the experiences with higher TS content. However, the fraction of solids degradation was lower in these experiences. A plateau in the biogas production was found. The free ammonia level, which was above the reported inhibitory levels, could explain this behaviour. After the inhibition period the biogas production restarted probably due to the biomass acclimatisation to the ammonia. In order to determine the inoculum adaptation a new experiment was performed. The inoculum used was the sludge coming from the first set of experiences. Based upon batch tests a 3.5 m3 pilot reactor was designed and started up. Ammonia inhibition was avoided by the start-up strategy and in two weeks the biogas production was 3.5 m3/d. The VS stabilisation with a solid retention time of 20 days was of 43%. The pilot reactor working at steady state had a TS concentration of 3-4% with a ratio of RC/blood of 10:1 at the entrance.

Abattoirs↗

Effect of increasing degradable intake protein on intake and digestion of low-quality, tallgrass-prairie forage by beef cows.

Five ruminally and duodenally fistulated Angus x Hereford cows were used in a 5 x 5 Latin square to monitor intake, ruminal fermentation responses, and site and extent of digestion associated with providing increasing amounts of supplemental degradable intake protein (DIP). Cows had ad libitum access to low-quality, tallgrass-prairie forage (1.9% CP, 77% NDF) that was fed twice daily. The supplemental DIP (sodium caseinate; 90% CP) was infused intraruminally at 0630 and 1830 immediately before feeding forage. Levels of DIP were 0, 180, 360, 540, and 720 g/d. Each period consisted of 14 d of adaptation and 6 d of sampling. Forage OM intake increased quadratically (P < .01) with increasing supplemental DIP reaching a peak at the 540 g/d level. True ruminal OM and NDF digestion increased with the addition of 180 g/d supplemental DIP, but exhibited only moderate and somewhat variable responses when greater amounts of supplemental DIP were infused (cubic, P < or = .03). Microbial N flow and efficiency increased linearly (P < .01) with increasing supplemental DIP. However, a quadratic effect (P < .01) was observed for total duodenal N flow, which was maximized at 540 g/d supplemental DIP. A linear (P = .02) treatment effect was observed for ruminal fluid dilution rate. Total ruminal VFA and ammonia concentrations increased (P < .01) in response to DIP supplementation. In conclusion, increasing supplemental DIP generally improved forage utilization; intake of digestible OM was maximized when it contained approximately 11% DIP.

Animals↗

Settleability and kinetics of a nitrifying sludge in a sequencing batch reactor.

A physiological study of a nitrifying sludge was carried out in a sequencing batch reactor (SBR). Pseudo steady-state nitrification conditions were obtained with an ammonium removal efficiency of 99% +/- 1% and 98% +/- 2% conversion of NH4+-N to NO3 - -N. The rate of biomass production was negligible (1.3 +/- 0.1 mg microbial protein-N.L(-1).d(-1)). The sludge presented good settling properties with sludge volume index values lower than 20 mL.g(-1) and an exopolymeric protein/carbohydrate ratio of 0.53 +/- 0.34. Kinetic results indicated that the nitrifying behavior of the sludge changed with the number of cycles. After 22 cycles, a decrease in the specific rate of NO3- -N production coupled with an increase in the NO2- -N accumulation were observed. These results showed that the activity of the nitrite oxidizing bacteria decreased at a longer operation time. Ammonia oxidizing bacteria were found to exhibit the best stability. After 4 months of operation, the specific rates of NH4+-N consumption and NO3- -N production were 1.72 NH4+-N per microbial protein-N per hour (g.g(-1).h(-1)) and 0.54 NO3- -N per microbial protein-N per hour (g.g(-1).h(-1)), respectively.

Bacteria↗

Quantitative aqueous ammonium ion analysis by transmission infrared spectroscopy.

An investigation was undertaken to determine whether ammonium ion could be quantitated in aqueous solution by using commercially available infrared filter instruments such as the Multispec M1 analyzer. Ammonium salt solutions were scanned using an infrared spectrophotometer modified specifically to facilitate the study of aqueous systems and were shown to have 2 main absorption bands at 3.49 microns (2865 cm-1) and 6.89 microns (1451 cm-1). The 3.49 microns band did not correspond to any individual band noted in the literature and was concluded to be a composite of fundamental bands and Fermi resonances due to hydrogen bonding affecting the ion in solution. The response of the 6.89 microns band to NH4+ concentration was approximately twice that of the 3.49 microns band, and quantitation of the ammonium ion was assessed by the dual wavelength method as employed in filter-based commercial infrared analyzers. Good quantitation was obtained using 5.56 microns as a reference wavelength, and the slope of the standard curve of ammonium sulfate was not significantly affected by pH in the pH 3-8 region. A 6.86 microns sample filter and a 5.56 microns reference filter were installed in a Multispec M1 analyzer and a linear response was obtained for concentrations of up to 0.6% ammonium sulfate. Accurately weighed samples of ammonium sulfate were converted to ammonia by using the micro-Kjeldahl procedure and then distilled into standard acid, diluted to volume, and assessed by titration and transmission infrared analysis. The infrared method was more accurate than the titration method in replicating the initial weights.(ABSTRACT TRUNCATED AT 250 WORDS)

Ammonium Sulfate↗

The role of glutamine in regulation of ammonium transport in Azotobacter vinelandii.

Under N2-fixing conditions, Azotobacter vinelandii expresses a specific transport system for methylammonium (ammonium) [E. M. Barnes, Jr. and P. Zimniak (1981) J. Bacteriol. 146, 512-516]. This activity is decreased markedly by culture of cells in the presence of 10 mM ammonium or 2 mM methylammonium; in both cases, the Vmax values for methylammonium uptake were 25% of those of N2-fixing cells. Mixing experiments with assay medium indicate that transport activity is controlled by intracellular rather than extracellular metabolites. Glutamine synthetase activity of cells cultured with ammonium was 33% that of N2-fixing cultures, but activity was unaffected by incubation with methylammonium. Thus ammonium transport and ammonium fixation are regulated independently. When ammonium was removed from the medium, cells recovered over 90% of the initial transport activity after 1 h; this recovery was not affected by addition of chloramphenicol. The loss of uptake activity in cells incubated with ammonium or methylammonium correlated with over sixfold increases in intracellular levels of glutamine and gamma-glutamylmethylamide, respectively. Recovery of transport was accompanied by similar reductions in pools of these compounds. Over one-half of methylammonium transport activity could be blocked by direct addition of 10 mM glutamine or gamma-glutamylmethylamide to transport assays; these concentrations were similar to those observed in vivo. The glutamine analog, 6-diazo-5-oxo-L-norleucine, was the most potent inhibitor found (68% inhibition at 10 microM). These results indicate that the regulation of ammonium transport by ammonium and methylammonium is due to inhibition of the transporter by intracellular gamma-glutamyl amides rather than by repression of transporter synthesis.

Amino Acids↗

Modeling the partial nitrification in sequencing batch reactor for biomass adapted to high ammonia concentrations.

Partial nitrification has proven to be an economic way for treatment of industrial N-rich effluent, reducing oxygen and external COD requirements during nitrification/denitrification process. One of the key issues of this system is the intermediate nitrite accumulation stability. This work presents a control strategy and a modeling tool for maintaining nitrite build-up. Partial nitrification process has been carried out in a sequencing batch reactor at 30 degrees C, maintaining strong changing ammonia concentration in the reactor (sequencing feed). Stable nitrite accumulation has been obtained with the help of an on-line oxygen uptake rate (OUR)-based control system, with removal rate of 2 kg NH4 (+)-N x m(-3)/day and 90%-95% of conversion of ammonium into nitrite. A mathematical model, identified through the occurring biological reactions, is proposed to optimize the process (preventing nitrate production). Most of the kinetic parameters have been estimated from specific respirometric tests on biomass and validated on pilot-scale experiments of one-cycle duration. Comparison of dynamic data at different pH confirms that NH3 and NO2- should be considered as the true substrate of nitritation and nitratation, respectively. The proposed model represents major features: the inhibition of ammonia-oxidizing bacteria by its substrate (NH3) and product (HNO2), the inhibition of nitrite-oxidizing bacteria by free ammonia (NH3), the INFluence of pH. It appears that the model correctly describes the short-term dynamics of nitrogenous compounds in SBR, when both ammonia oxidizers and nitrite oxidizers are present and active in the reactor. The model proposed represents a useful tool for process design and optimization.

Adaptation, Physiological↗

The effect of intragastric ammonia production on titratable gastric acid output in Helicobacter pylori-infected patients with chronic gastritis.

The purpose of this study was to assess whether intragastric neutralization of HCl by ammonia in Helicobacter pylori-infected patients could meaningfully affect the titratable acid output as a measure of gastric acid secretion in a relation to the severity of infection. In 79 patients with different degrees of Helicobacter pylori infection and chronic gastritis, the basal acid output (BAO) and maximal acid output (MAO) after pentagastrin (6 microg/kg s.c.) was estimated. Cl- and NH4+ contents in these fractions were also assayed. H+/Cl- ratio in the MAO fraction was diminished in markedly infected patients (68.1 +/- 3.9%, vs 84.1 +/- 3.3% in noninfected patients; P < 0.005). Ammonium content was maximal in patients with marked infection (0.912 +/- 0.086 vs 0.149 +/- 0.034 mmol/hr in MAO [P < 0.001] and 0.475 +/- 0.063 vs 0.105 +/- 0.016 mmol/hr in BAO of noninfected patients [P < 0.001]), with intermediate values in mild and moderate infection. The NH4 +/(H+ + NH4+) ratio reached 27.01 +/- 7.34% in the BAO of moderately infected patients, vs 10.22 +/- 3.81% in noninfected patients (P = 0.05), and 7.25 +/- 1.06% in the MAO of markedly infected patients, vs 1.14 +/- 0.33% in noninfected patients (P < 0.001). The intragastric ammonia production affects the titratable acid output in Helicobacter pylori-infected patients dependent on the severity of infection. Therefore this factor should be taken into consideration in the evaluation of gastric secretory function in Helicobacter pylori-infected patients.

Adult↗

Pathophysiology of gastric acid secretion in patients with chronic renal failure: influence of Helicobacter pylori infection.

OBJECTIVES: The incidence of gastroduodenal diseases is high in patients with chronic renal failure (CRF). However, gastric acidity in CRF has been reported to range in level from low to high. Moreover, it remains unknown whether Helicobacter pylori infection influences gastric acidity in such patients. Thus, we aimed to clarify the pathophysiological perturbation in gastric acidity and to determine the influence of H. pylori infection in CRF. DESIGN: Case-control study. SETTING: A university hospital. SUBJECTS: Twenty-seven patients with CRF and 24 control patients, presenting with either gastrointestinal symptoms, positive faecal occult blood, or anaemia (haemoglobin <10 g dL(-1)). MEASURES: The patients underwent gastroduodenal endoscopy with simultaneous determination of H. pylori infection. Gastric ammonium concentration, serum pepsinogen I and II, and basal gastrin level were measured. Thereafter, gastric acid secretion was monitored by 24-h intragastric acidity measurement with calculation of pH-3 holding time (%) (hours showing pH>3/24 h). RESULTS: In the CRF group, pH-3 holding time of H. pylori (+) subgroup was significantly greater than that of H. pylori (-) subgroup (71.2 +/- 32.4% vs. 32.8 +/- 30.0%, mean +/- SD; P=0.03). Pepsinogen I/II ratio was inversely correlated with pH-3 holding time in the control and CRF groups. Gastric ammonium concentration in CRF/H. pylori (+) subgroup (14.1 +/- 9.2 mmol L(-1)) was significantly higher than in CRF/H. pylori (-) (2.5 +/- 2.7 mmol L(-1); P=0.002) and control/H. pylori (+) subgroups (6.1 +/- 4.2 mmol L(-1); P=0.01). Serum gastrin level was significantly higher in the CRF group than in the control group (297 +/-343 pg mL(-1) vs. 116 +/- 69 pg mL(-1); P=0.02) as a whole. However, there was no significant correlation between serum creatinine and gastrin levels in the CRF group. Gastrin level in CRF/H. pylori (+) subgroup was significantly higher than in CRF/H. pylori (-), control/H. pylori (+), and control/H. pylori (-) subgroups (423 +/-398 pg mL(-1) vs. 113 +/- 79, 124 +/- 78, and 96 +/-43 pg mL(-1), respectively; P=0.01-0.03). Significant positive correlations amongst pH-3 holding time, ammonium and gastrin concentrations were found in the CRF group, but not in the control group. CONCLUSIONS: CRF without H. pylori infection primarily shows a tendency for high gastric acidity, but without hypergastrinaemia. Persistent H. pylori infection in CRF leads to decreased acidity and, consequently, to fasting hypergastrinaemia via a feedback mechanism. The hypoacidity in CRF with H. pylori infection appears to result from neutralization of acid by ammonia as well as from gastric atrophy. Thus, H. pylori infection status critically determines perturbation in gastric acidity and fasting gastrin level in CRF.

Adult↗

Hyperammonemia decreases body fat content in rat.

We have developed an animal model of hyperammonemia consisting of feeding rats a diet containing 20% (w/w) ammonium acetate. Ingestion of this diet markedly affects carcass composition, with a 46% reduction in lipid content. The ammonium diet alters levels of several key compounds involved in lipid metabolism. Long-chain acylcarnitine is increased in liver by approx. 60% while free carnitine and acetylcarnitine are unaffected. The hepatic content of acetyl-CoA increases by approx. 50%. The level of ketone bodies in blood increases by 32% but remains unchanged in liver. Our data indicate that hyperammonemia alters lipid metabolism and results in a significant decrease in body lipid content.

Acetates↗

Simultaneous determination of serum cations, anions and uremic toxins by ion chromatography using an immobilized enzyme.

A procedure is described for the analysis of serum cations and uremic toxins by simultaneous conductivity (CD) and ultraviolet (UV) photometric detection and using phosphoric acid as the eluent. In addition, studies were made on the determination of urea in serum using immobilized urease, and on the simultaneous determination of cationic and anionic compounds. The ammonium ion produced from urea by immobilized urease was determined by CD. No significant differences in amounts were found between the present method and conventional colorimetric methods. There was no significant reduction in immobilized urease activity after two months of continuous use. Simultaneous analysis of cationic and anionic compounds in serum was performed by switching valves in order to change the eluents and to change the channel to the analytical column. No marked base-line variation was observed and satisfactory recovery of anions was achieved.

Ammonia↗