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Amino acid utilization and isotope discrimination of amino nitrogen in nitrogen metabolism of rat liver in vivo.

Urea and plasma protein differ in natural 15N abundance up to 10%. The origin of this difference is the branched nitrogen metabolism in the liver. One main branch is the protein synthesis pathway, the other the urea synthesis pathway. By this branching 15N of precursor amino acids is depleted in urea while it is enriched in protein. With the 15N abundance of precursor amino acids, which may be taken from jejunum tissue, utilization of amino acids in liver metabolism can be calculated from isotope discrimination in either pathway. This was investigated by feeding different proteins to rats. When feeding high quality protein (whey protein) utilization of amino acids in liver metabolism at requirement intake was better than at zero protein intake (> 85% vs. 70%). From this we conclude that the pattern of amino acids available from the metabolic pool at zero protein intake is characterized by an imbalance. This endogenous imbalance can be complemented by exogenous dietary amino acids so that nitrogen excretion may even be smaller than the so-called "obligatory" losses of intakes not exceeding requirement. Thus, the quality of dietary protein is reflected not only by N balance. It also may be quantified by analysis of isotope discrimination in nitrogen metabolism of the liver. In addition, the quality of amino acid pattern available from the metabolic pool is indicated by this method.

Amino Acids↗

Activation of the glnA, glnK, and nac promoters as Escherichia coli undergoes the transition from nitrogen excess growth to nitrogen starvation.

The nitrogen-regulated genes and operons of the Ntr regulon of Escherichia coli are activated by the enhancer-binding transcriptional activator NRI approximately P (NtrC approximately P). Here, we examined the activation of the glnA, glnK, and nac promoters as cells undergo the transition from growth on ammonia to nitrogen starvation and examined the amplification of NRI during this transition. The results indicate that the concentration of NRI is increased as cells become starved for ammonia, concurrent with the activation of Ntr genes that have less- efficient enhancers than does glnA. A diauxic growth pattern was obtained when E. coli was grown on a low concentration of ammonia in combination with arginine as a nitrogen source, consistent with the hypothesis that Ntr genes other than glnA become activated only upon amplification of the NRI concentration.

Arginine↗

Expression of the DAL80 gene, whose product is homologous to the GATA factors and is a negative regulator of multiple nitrogen catabolic genes in Saccharomyces cerevisiae, is sensitive to nitrogen catabolite repression.

We have cloned the negative regulatory gene (DAL80) of the allantoin catabolic pathway, characterized its structure, and determined the physiological conditions that control DAL80 expression and its influence on the expression of nitrogen catabolic genes. Disruption of the DAL80 gene demonstrated that it regulates multiple nitrogen catabolic pathways. Inducer-independent expression was observed for the allantoin pathway genes DAL7 and DUR1,2, as well as the UGA1 gene required for gamma-aminobutyrate catabolism in the disruption mutant. DAL80 transcription was itself highly sensitive to nitrogen catabolite repression (NCR), and its promoter contained 12 sequences homologous to the NCR-sensitive UASNTR. The deduced DAL80 protein structure contains zinc finger and coiled-coil motifs. The DAL80 zinc finger motif possessed high homology to the transcriptional activator proteins required for expression of NCR-sensitive genes in fungi and the yeast GLN3 gene product required for functioning of the NCR-sensitive DAL UASNTR. It was also homologous to the three GATAA-binding proteins reported to be transcriptional activators in avian and mammalian tissues. The latter correlations raise the possibility that both positive and negative regulators of allantoin pathway transcription may bind to similar sequences.

Allantoin↗

[Effect of deficiency of dietary nitrogen on cellulose digestibility and nitrogen-fixing flora activity in the sibling vole Microtus rossiaemeridionalis].

The effect of food composition on nitrogen-fixing activity of the symbionts living in the digestive tract of the sibling vole Microtus rossiaemeridionalis was experimentally studied. In the control, the rodents were allowed to select any of three food mixes: protein-rich, protein-free with low cellulose content, and protein-free with high cellulose content. The voles consumed the food in a proportion corresponding to 16% of protein and about 6% of cellulose. Exclusion of the protein-rich food considerably increased cellulose consumption (up to 12%) and digestibility (from 35 to 64%). The changes in the consumed food composition were reflected in localization of nitrogenase activity in the digestive tract: the control level of nitrogen-fixing activity in the cecum was three times that in the proventriculus, while an inverse proportion was observed in the experimental groups. In all cases, the colon was the major locus of nitrogen fixation.

Animals↗

[Use of the combined Kjeldahl-wet digestion/Berthelot-reaction for determining nitrogen in biological materials. 2. Design and testing of a partially automated apparatus for routine determination of nitrogen].

The authors describe the design and the mode of operation of a partially automated analyzer system, based on the combination of the Kjeldahl wet digestion with the Berthelot reaction, for the determination of nitrogen in biological materials. The results from three years of operation are discussed from the view-points of variability of the materials to be tested, of accuracy and of precision. This analytical technique (with an accuracy of 98% and a precision of 4%) is suited for determining amino and amido nitrogen as well as non-aromatically bound ring nitrogen. The statistical evaluation of the analytical results revealed that the deviation of the measured values is mainly caused by the high-temperature digestion.

Amides↗

Nitrogen metabolism in the facultative methylotroph Arthrobacter P1 grown with various amines or ammonia as nitrogen sources.

The metabolism of trimethylamine (TMA) and dimethylamine (DMA) in Arthrobacter P1 involved the enzymes TMA monooxygenase and trimethylamine-N-oxide (TMA-NO) demethylase, and DMA monooxygenase, respectively. The methylamine and formaldehyde produced were further metabolized via a primary amine oxidase and the ribulose monophosphate (RuMP) cycle. The amine oxidase showed activity with various aliphatic primary amines and benzylamine. The organism was able to use methylamine, ethylamine and propylamine as carbon- and nitrogen sources for growth. Butylamine and benzylamine only functioned as nitrogen sources. Growth on glucose with ethylamine, propylamine, butylamine and benzylamine resulted in accumulation of the respective aldehydes. In case of ethylamine and propylamine this was due to repression by glucose of the synthesis of the aldehyde dehydrogenase(s) required for their further metabolism. Growth on glucose/methylamine did not result in repression of the RuMP cycle enzyme hexulose-6-phosphate synthase (HPS). High levels of this enzyme were present in the cells and as a result formaldehyde did not accumulate. Ammonia assimilation in Arthrobacter P1 involved NADP-dependent glutamate dehydrogenase (GDH), NAD-dependent alanine dehydrogenase (ADH) and glutamine synthetase (GS) as key enzymes. In batch cultures both GDH and GS displayed highest levels during growth on acetate with methylamine as the nitrogen source. A further increase in the levels of GS, but not GDH, was observed under ammonia-limited growth conditions in continuous cultures with acetate or glucose as carbon sources.

Amines↗

Variation of the amino acid scores and of the nitrogen-to-protein conversion factors in barley grain as a function of nitrogen content as compared with wheat and rye.

Barley grains (9 samples from 7 cultivars) with nitrogen contents (N) ranging from 1.45 to 4.01% of dry matter were analysed for their amino acid (AA) composition with high accuracy from six different hydrolysates per sample. AA levels in grain increased as linear functions of N with correlation coefficients close to unity. A comparison with literature data confirmed that the AA composition of any grain sample of normal barley can be predicted from its N for all phenotypes and genotypes. AAs in grain protein changed as hyperbolic functions of N which increased for Phe, Pro and Glx but more or less strongly decreased for the other AAs. By plotting AA scores against N, barley proteins were shown to be always richer than wheat and rye in Val and Phe + Tyr; sometimes richer than both other species for N less than 2 (Lys); 2.2 (Leu and Ile); 3.4 (Thr); sometimes intermediate to wheat and rye above the latter N values. They were also intermediate in sulphur AAs for N less than 1.9 and drastically poorer for N greater than 1.9. However, they were richer than both other species in Trp for N greater than 1.6. The hyperbolic variations of non-protein nitrogen and nitrogen-to-protein conversion factors were determined as a function of N and also compared with those of wheat and rye.

Amino Acids↗

The anaerobic fungus Piromyces sp. strain E2: nitrogen requirement and enzymes involved in primary nitrogen metabolism.

The anaerobic fungus Piromyces sp. strain E2 appeared restricted in nitrogen utilization. Growth was only supported by ammonium as source of nitrogen. Glutamine also resulted in growth, but this was due to release of ammonia rather than to uptake and utilization of the amino acid. The fungus was not able to grow on other amino acids, albumin, urea, allantoin, or nitrate. Assimilation of ammonium is very likely to be mediated by NADP-linked glutamate dehydrogenase (NADP-GDH) and glutamine synthetase (GS). One transaminating activity, glutamate-oxaloacetate transaminase (GOT), was demonstrated. Glutamate synthase (GOGAT), NAD-dependent glutamate dehydrogenase (NAD-GDH), and the transaminating activity glutamate-pyruvate transaminase (GPT) were not detected in cell-free extracts of Piromyces sp. strain E2. Specific enzyme activities of both NADP-GDH and GS increased four- to sixfold under nitrogen-limiting conditions.

Albumins↗

Nitrogen and energy requirements of the short-tailed fruit bat (Carollia perspicillata): fruit bats are not nitrogen constrained.

Nitrogen (N) and energy (E) requirements were measured in adult Carollia perspicillata which were fed on four experimental diets. Bats ate 1.3-1.8 times their body mass.day-1 and ingested 1339.5-1941.4 kJ.kg-0.75.day-1. Despite a rapid transit time, dry matter digestibility and metabolizable E coefficient were high (83.3% and 82.4%, respectively), but true N digestibility was low (67.0%). Mass change was not correlated with E intake, indicating that bats adjusted their metabolic rate to maintain constant mass. Bats were able to maintain constant mass with digestible E intake as low as 1168.7 kJ.kg-0.75.day-1 or 58.6 kJ.. Metabolic fecal N and endogenous urinary N losses were 0.87 mg N.g-1 dry matter intake and 172.5 mg N.kg-0.75.day-1, respectively, and bats required 442 mg N.kg-0.75.day-1 (total nitrogen) or 292.8 mg N.kg-0.75.day-1 (truly digestible nitrogen) for N balance. Based on E and N requirements and digestibilities, it was calculated that non-reproductive fruit bats were able to meet their N requirements without resorting to folivory and without over-ingesting energy. It was demonstrated that low metabolic fecal requirements allowed bats to survive on low-N diets.

Animals↗

Detrital control on the release of dissolved organic nitrogen (DON) and dissolved inorganic nitrogen (DIN) from the forest floor under chronic N deposition.

The role of detrital quantity and quality in forest floor N leaching was investigated in a litter manipulation experiment at a deciduous forest under chronic N deposition. Dissolved inorganic nitrogen (DIN) comprised the bulk of nitrogen leaching from the control except a short period following autumn litterfall. The dominance of DIN was strengthened by litter exclusion, whereas the addition of glucose or fresh litter led to a small increase in dissolved organic nitrogen (DON) and either a temporary or gradual reduction in NO(3)(-) release, respectively. Changes in soluble organic C and microbial C in the forest floor implied that increased availability of C sources might have enhanced microbial immobilization of DIN, either temporarily following glucose application or over the longer term following litter addition. The results suggest that detrital quantity and quality can play a crucial role in determining the balance between DIN and DON in N-enriched forest soils.

Adsorption↗

Effects of nitrogen deposition on nitrogen cycling in an Aleppo pine stand in Athens, Greece.

High levels of nitrogen deposition measured in throughfall fluxes in 2002, reaching 38 kg ha(-1) year(-1), affected the biogeochemical cycle of N in an Aleppo pine (Pinus halepensis) stand situated in Athens, the capital of Greece. The N fluxes in bulk deposition were far lower than those in throughfall indicating high levels of dry deposition in the city of Athens. The amounts of N in litterfall and those stored in the forest floor and mineral soil were not high, while the mean residence time of N in the forest floor, derived from fluxes equations, decreased considerably due to the high levels of N fluxes in throughfall deposition. Nitrogen concentration in needle tissues was high and this resulted in a high ratio of N/Zn. Zinc availability was low due to the alkaline nature of soil in the study area. Fast N cycling may affect the living status of Aleppo pine, which is an oligotrophic plant species capable of occupying poor soil sites. Nitrogen eutrophication may adversely affect forests grown on calcareous soils by developing nutrition imbalances between N and micronutrients, the solubility of which depends on soil pH values.

Environmental Monitoring↗

Apparatus and method for interfacing capillary electrophoresis and chemiluminescence nitrogen detection for the analysis of nitrogen-containing compounds.

We have developed an interface that allows the specific detection of nitrogen-containing compounds by using a chemiluminescence nitrogen detector. The feasibility of using this interface was demonstrated by separating and detecting two nitrogen-containing compounds, p-aminosalicylic acid and L-phenylalanine. Although baseline separation was achieved, the theoretical plates were lower when compared to UV detection (25000 vs. approximately 85000). A sensitivity of 75 ng (approximately 500 pmol) per injection was achieved with this system which is adequate for pharmaceutical and biotech applications.

Aminosalicylic Acid↗

Cyanobacteria in Uruguayan rice fields: diversity, nitrogen fixing ability and tolerance to herbicides and combined nitrogen.

It has been established that cyanobacteria play a vital role in the maintenance of flooded rice field fertility. To evaluate the potential use of nitrogen-fixing cyanobacteria as a natural biofertilizer for rice in Uruguay, the diversity, abundance and nitrogen fixing ability of these microorganisms were studied in the field and in the laboratory. The effect of urea fertilization on population density and diversity of heterocystous cyanobacteria was determined on a 3-year assay. The highest number of cyanobacteria, 1.6x10(4) CFU x m(-2), was found at the control 8 weeks after flooding. About 90% of the heterocystous cyanobacteria found in both treatments belong to the genera Nostoc and Anabaena. Maximal nitrogenase activity was reached after 12 weeks of flooding in both treatments, with an average of about 20 micromol C2H4 x m(-2) x h(-1). To improve the understanding of the environmental factors that can limit nitrogenase activity in rice fields, two of the most abundant cyanobacteria isolates were tested for tolerance to combined nitrogen and two herbicides. In both isolates 0.2 mM ammonium inhibited nitrogenase activity after 24 h of culture. The addition of field-recommended doses of quinclorac and propanil affected oxygen photoevolution but nitrogenase activity was only inhibited by propanil.

Acetylene↗

ESTs from the basidiomycete Schizophyllum commune grown on nitrogen-replete and nitrogen-limited media.

Lambda phage cDNA libraries were constructed using mRNAs from the basidiomycete Schizophyllum commune grown on media with high or low nitrogen concentrations. A total of 440 clones were sequenced, representing 373 distinct transcripts. Of these, 166 showed significant similarity to annotated genes in GenBank. Those that could be tentatively identified using BLAST searches were classified by function using the Gene Ontology (GO) database. Genes with products involved in cell-cycle processes were more frequent in the nitrogen-limited libraries, while genes with products involved in protein biosynthesis were more frequent in the nitrogen-replete library. Overall, clones showed much greater similarity to the one publicly available basidiomycete genome, Phanerochaete chrysosporium, than to any of the ascomycete genomes.

Bacteriophage lambda↗

Joint action of elevated ambient nitrite and nitrate on hemolymph nitrogenous compounds and nitrogen excretion of tiger shrimp Penaeus monodon.

Penaeus monodon (12.13+/-1.14 g) exposed individually to six different nitrite and nitrate regimes (0.002, 0.36 and 1.46 mM nitrite combined with 0.005 and 7.32 mM nitrate), at a salinity of 25 ppt, were examined for hemolymph nitrogenous compounds and whole shrimp's nitrogen excretions after 24 h. Nitrogen excretion increased directly with ambient nitrite and nitrate. Hemolymph nitrite, nitrate, urea and uric acid levels increased, while hemolymph ammonia, oxyhemocyanin and protein were inversely related to ambient nitrite. Exposure of P. monodon to elevated nitrite in the presence of 7.32 mM nitrate did not alter hemolymph nitrite, ammonia, uric acid, oxyhemocyanin and protein levels, but caused an increase in hemolymph nitrate and a decrease in hemolymph urea as compared to exposure to elevated nitrite only. Following exposure to elevated nitrite, nitrite was oxidized to nitrate and P. monodon showed uricogenesis and uricolysis. The shrimp also used strategies to avoid joint toxicities of nitrite and metabolic ammonia by removing ammonia or reducing ammonia production under the stress of elevated nitrite.

Ammonia↗

Nitrogen-regulated genes for the metabolism of cyanophycin, a bacterial nitrogen reserve polymer: expression and mutational analysis of two cyanophycin synthetase and cyanophycinase gene clusters in heterocyst-forming cyanobacterium Anabaena sp. PCC 7120.

Two gene clusters each encoding the cyanophycin-metabolism enzymes cyanophycin synthetase and cyanophycinase are found in the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. In cluster cph1, the genes cphB1 and cphA1 were expressed in media containing ammonium, nitrate, or N(2) as nitrogen sources, but expression was higher in the absence of combined nitrogen taking place both in vegetative cells and heterocysts. Both genes were cotranscribed from three putative promoters located upstream of cphB1, and, additionally, the cphA1 gene was expressed monocistronically from at least two promoters located in the intergenic cphB1-cphA1 region. Both constitutive promoters and promoters dependent on the global nitrogen control transcriptional regulator NtcA were identified. In cluster cph2, the cphB2 and cphA2 genes, which are found in opposite orientations, were expressed as monocistronic messages in media containing ammonium, nitrate, or N(2), but expression was higher in the absence of ammonium. Expression of the cph2 genes was lower than that of cph1 genes. Analysis of cph gene insertional mutants indicated that cluster cph1 genes contributed more than cluster cph2 genes to cyanophycin accumulation in the whole filament as well as in heterocysts. Diazotrophic growth was more severely impaired in cyanophycinase than in cyanophycin synthetase mutants, indicating that cyanophycin, although normally synthesized in the heterocysts, is not required for heterocyst function and that the inability to degrade this polymer is detrimental for the diazotrophic growth of the cyanobacterium.

Anabaena↗

Ammonium-nitrogen transformation and nitrogen retention in broiler manure supplemented with a soil amendment containing nitrifying bacteria.

The effect of a soil amendment on ammonium nitrogen transformation and nitrogen retention in broiler manure was evaluated. Prior to incubation, broiler manure was mixed with autoclaved soil or non-autoclaved soil in different ratios to make 1 kg mixtures; broiler manure:non-autoclaved soil=9:1, 5:5, and 1:9 or broiler manure:autoclaved soil=9:1, 5:5, and 1:9. The non-autoclaved soil treatment reduced either numerically or significantly NH(4)(+)-N concentration compared to the autoclaved soil treatment during the 8-wk incubation. Total-N concentration of the non-autoclaved soil treatments was lower than the autoclaved soil treatments from 4 to 8 wk. The lowest manure to non-autoclaved soil treatment (M:S=1:9) had considerably more nitrite and nitrate; however, the higher ratio manure to non-autoclaved soil treatments (M:S=9:1 and 5:5) had slightly higher total nitrite and nitrate levels compared to the same ratio of autoclaved soil treatments. The moisture level of the 9:1, 5:5, and 1:9 M:S treatments were approximately 70, 45, and 30%, respectively. The results indicated that nitrifying bacteria in the non-autoclaved soil reduced the ammonium nitrogen concentrations of poultry manure by converting NH(3) or NH(4)(+) to NO(2)(-) or NO(3)(-). However, the higher moisture levels in treatments with greater manure to soil ratios (M:S=9:1 and 5:5) created anaerobic conditions that allowed for denitrification and greater N losses.

Agriculture↗

[Studies on the determination of the part of bacterial nitrogen in relation to total nitrogen content in the duodenum of cows].

A method for estimation of content of bacterial nitrogen in duodenal intestine was described. The method of HUTTON et al. (1971) in modificated form for the own research was the best method. In two trials roughage (chopped dried forage) and ground corn were given to cows with duodenal bridge fistula in different order (90 min-intervall). Portion of bacterial nitrogen to total nitrogen was 72.5% (corn leads to roughage) and 64.7% (roughage leads to corn).

Animal Feed↗