Ammonia assimilation in Bacillus polymyxa. 15N NMR and enzymatic studies

Pathways of ammonia assimilation into glutamic acid and alanine in Bacillus polymyxa were investigated by 15N NMR spectroscopy in combination with measurements of the specific activities of glutamate dehydrogenase, glutamine synthetase, glutamate synthetase, alanine dehydrogenase, and glutamic-alanine transaminase. Ammonia was found to be assimilated into glutamic acid predominantly by NADPH-dependent glutamate dehydrogenase with a Km of 2.9 mM for NH4+ not only in ammonia-grown cells but also in nitrate-grown and nitrogen-fixing cells in which the intracellular NH4+ concentrations were 11.2, 1.04, and 1.5 mM, respectively. In ammonia-grown cells, the specific activity of alanine dehydrogenase was higher than that of glutamic-alanine transaminase, but the glutamate dehydrogenase/glutamic-alanine transaminase pathway was found to be the major pathway of 15NH4+ assimilation into [15N]alanine. The in vitro specific activities of glutamate dehydrogenase and glutamine synthetase, which represent the rates of synthesis of glutamic acid and glutamine, respectively, in the presence of enzyme-saturating concentrations of substrates and coenzymes are compared with the in vivo rates of biosynthesis of [15N]glutamic acid and [alpha,gamma-15N]glutamine observed by NMR, and implications of the results for factors limiting the rates of their biosynthesis in ammonia- and nitrate-grown cells are discussed

Glutamate biosynthesis in Bacillus azotofixans. 15N NMR and enzymatic studies

Pathways of ammonia assimilation into glutamic acid in Bacillus azotofixans, a recently characterized nitrogen-fixing species of Bacillus, were investigated through observation by NMR spectroscopy of in vivo incorporation of 15N into glutamine and glutamic acid in the absence and presence of inhibitors of ammonia-assimilating enzymes, in combination with measurements of the specific activities of glutamate dehydrogenase, glutamine synthetase, glutamate synthase, and alanine dehydrogenase. In ammonia-grown cells, both the glutamine synthetase/glutamate synthase and the glutamate dehydrogenase pathways contribute to the assimilation of ammonia into glutamic acid. In nitrate-grown and nitrogen-fixing cells, the glutamine synthetase/glutamate synthase pathway was found to be predominant. NADPH-dependent glutamate dehydrogenase activity was detectable at low levels only in ammonia-grown and glutamate-grown cells. Thus, B. azotofixans differs from Bacillus polymyxa and Bacillus macerans, but resembles other N2-fixing prokaryotes studied previously, as to the pathway of ammonia assimilation during ammonia limitation. Implications of the results for an emerging pattern of ammonia assimilation by alternative pathways among nitrogen-fixing prokaryotes are discussed, as well as the utility of 15N NMR for measuring in vivo glutamate synthase activity in the cell

Identifying Sources and Sinks in the Presence of Multiple Agents with Gaussian Process Vector Calculus

In systems of multiple agents, identifying the cause of observed agent dynamics is challenging. Often, these agents operate in diverse, non-stationary environments, where models rely on hand-crafted environment-specific features to infer influential regions in the system's surroundings. To overcome the limitations of these inflexible models, we present GP-LAPLACE, a technique for locating sources and sinks from trajectories in time-varying fields. Using Gaussian processes, we jointly infer a spatio-temporal vector field, as well as canonical vector calculus operations on that field. Notably, we do this from only agent trajectories without requiring knowledge of the environment, and also obtain a metric for denoting the significance of inferred causal features in the environment by exploiting our probabilistic method. To evaluate our approach, we apply it to both synthetic and real-world GPS data, demonstrating the applicability of our technique in the presence of multiple agents, as well as its superiority over existing methods.Comment: KDD '18 Proceedings of the 24th ACM SIGKDD International Conference on Knowledge Discovery & Data Mining, Pages 1254-1262, 9 pages, 5 figures, conference submission, University of Oxford. arXiv admin note: text overlap with arXiv:1709.0235

Effect of the nitrogen source on glutamine and alanine biosynthesis in Neurospora crassa. An in vivo 15N nuclear magnetic resonance study

The influences of different nitrogen sources on the relative rates of biosynthesis of glutamine and alanine have been studied by 15N nuclear magnetic resonance spectroscopy of intact Neurospora crassa mycelia suspensions. The rate of glutamine synthesis was fastest after growth in media deficient in free ammonium ion, whereas it was slowest following growth in media containing both glutamic acid and glutamine. The reverse trend was observed for the biosynthesis of alanine. A competition between the two biosynthetic pathways for the same substrate, glutamic acid, was found to limit the rate of alanine synthesis when glutamine synthesis was rapid. The observed in vivo rates of these reactions are compared to the reported specific activities of the enzymes catalyzing the reactions, and implications of these results for nitrogen regulation of these pathways under various physiological conditions are discussed

Conformational Preferences of 3-(Dimethylazinoyl)propanoic Acid as a Function of pH and Solvent; Intermolecular versus Intramolecular Hydrogen Bonding

The conformational equilibrium of 3-(dimethylazinoyl)propanoic acid (DMAPA, azinoyl = N^+(O^−) has a weak pH-dependence in D_2O, with a slight preference for trans in alkaline solutions. The acid ionization constants of the protonated amine oxide and carboxylic functional groups as determined by NMR spectroscopy were 7.9 × 10^(−4) and 6.3 × 10^(−6), respectively. The corresponding value of K_1/K_2 of 1.3 × 10^2 is not deemed large enough to provide experimental NMR evidence for a significant degree of intramolecular hydrogen bonding in D_2O. Conformational preferences of DMAPA are mostly close to statistical (gauche/trans = 2/1) in other protic solvents, e.g., alcohols. However, the un-ionized form of DMAPA appears to be strongly intramolecularly hydrogen-bonded and gauche in aprotic solvents

Assessing a commercially available sports drink on exogenous carbohydrate oxidation, fluid delivery and sustained exercise performance

Background: Whilst exogenous carbohydrate oxidation (CHOEXO) is influenced by mono- and disaccharide combinations, debate exists whether such beverages enhance fluid delivery and exercise performance. Therefore, this study aimed to ascertain CHOEXO, fluid delivery and performance times of a commercially available maltodextrin/ fructose beverage in comparison to an isocaloric maltodextrin beverage and placebo. Methods: Fourteen club level cyclists (age: 31.79 ± 10.02 years; height: 1.79 ± 0.06 m; weight: 73.69 ± 9.24 kg; VO2max: 60.38 ± 9.36 mL · kg·-1 min−1) performed three trials involving 2.5 hours continuous exercise at 50% maximum power output (Wmax: 176.71 ± 25.92 W) followed by a 60 km cycling performance test. Throughout each trial, athletes were randomly assigned, in a double-blind manner, either: (1) 1.1 g · min−1 maltodextrin + 0.6 g · min−1 fructose (MD + F), (2) 1.7 g · min−1 of maltodextrin (MD) or (3) flavoured water (P). In addition, the test beverage at 60 minutes contained 5.0 g of deuterium oxide (2H2O) to assess quantification of fluid delivery. Expired air samples were analysed for CHOEXO according to the 13C/12C ratio method using gas chromatography continuous flow isotope ratio mass spectrometry. Results: Peak CHOEXO was significantly greater in the final 30 minutes of submaximal exercise with MD + F and MD compared to P (1.45 ± 0.09 g · min−1, 1.07 ± 0.03 g · min−1and 0.00 ± 0.01 g · min−1 respectively, P < 0.0001), and significantly greater for MD + F compared to MD (P = 0.005). The overall appearance of 2H2O in plasma was significantly greater in both P and MD + F compared to MD (100.27 ± 3.57 ppm, 92.57 ± 2.94 ppm and 78.18 ± 4.07 ppm respectively, P < 0.003). There was no significant difference in fluid delivery between P and MD + F (P = 0.078). Performance times significantly improved with MD + F compared with both MD (by 7 min 22 s ± 1 min 56 s, or 7.2%) and P (by 6 min 35 s ± 2 min 33 s, or 6.5%, P < 0.05) over 60 km. Conclusions: A commercially available maltodextrin-fructose beverage improves CHOEXO and fluid delivery, which may benefit individuals during sustained moderate intensity exercise. The greater CHOEXO observed when consuming a maltodextrin-fructose beverage may support improved performance times