Mode of utilization of amino acids as growth substrates by Azospirillum brasilense

The study was undertaken to analyze the rate of uptake and utilization of various amino acids by Azospirillum brasilense Sp81 (RG) in a basal mineral salts solution under non-nitrogen fixing condition. These amino acids including other nitrogenous compounds were tested for both N- and C-sources. The kinetic constants (Km and Vmax) of uptake of some amino acids (e.g. lysine, arginine, proline, glutamine and glutamic acid) were exploited using a Hanes-Woolf plot, and discussed in the context of nitrogen starvation or both carbon and nitrogen starvation. To summarize all the kinetic data for these amino acids strongly suggested that the mode of these amino acids utilization in this bacterium followed the same general pattern, although the quantitative differences were there. A single amino acid was able to satisfy the nitrogen needs of this bacterium in basal mineral salts solution, and this possibility could be considered for the cost-effective growth medium for this bacterium in the biotechnological industry.     

Studies on the biosynthesis of biotin. Production of biotin and biotin-like compounds by a pseudomonad

1. Filtrates from cultures of a strain of Pseudomonas aeruginosa, grown in a basal glucose-ammonium chloride-vitamins-salts medium, possessed biotin activity as detected by microbiological assays. Exponential-phase culture filtrates contained biotin and desthiobiotin in the approximate ratio 1:3, with smaller amounts of biotin sulphoxide and three unidentified compounds with biotin activity. 2. The addition of malonate, adipate or pimelate to the basal medium stimulated the production of compounds with biotin activity; this effect was enhanced when these compounds were included in the medium as the major carbon source. Succinate, glutarate, suberate, fumarate or oxaloacetate did not stimulate the production of compounds with biotin activity. The ratio of biotin to desthiobiotin in filtrates from cultures grown in medium containing malonate as the carbon source was about 1:1. Experiments in which mixtures of malonate and pimelate were included in the medium as the carbon sources showed that these acids probably make a similar contribution in biotin biosynthesis. 3. A number of heterocyclic compounds, including several containing the ureido group (-NH-CO-NH-), were included in the basal medium but none of them stimulated the production of compounds with biotin activity to any marked degree. 4. Several amino acids, particularly cysteine (or cystine) and lysine, when added individually as supplements to the basal medium, stimulated the production of compounds with biotin activity. Filtrates from cultures grown in medium supplemented with cysteine contained approximately equal proportions of biotin and desthiobiotin. A much greater stimulation in the production of compounds with biotin activity was obtained when certain amino acids were included in the medium as the major source of nitrogen or carbon and nitrogen; ornithine, citrulline and argininosuccinate had the most marked effect. The ratio of biotin to desthiobiotin in filtrates from these cultures was usually greater than in filtrates from cultures grown in basal medium. 5-Aminovalerate also caused some stimulation when used as the nitrogen source, but urea was inactive. The effect of binary mixtures of certain amino acids was also examined. 5. The results are discussed in relation to the possible role of the stimulatory compounds during biotin biosynthesis.     

Rapid Microbiological Assay of Amino Acids

The rapid microbiological method for determination of amino acids was established. It is composed of 3 steps of culture; inoculum culture, intermediate culture, and assay culture. The inoculum culture is the same as that of ordinary method using Leuc. mesenteroides P–60. For the intermediate culture, which is carried out between the inoculum and assay cultures, the basal medium supplemented with appropriate amount of the amino acid to be determined is employed. The large amount of cells at logarithm phase grown in the intermediate culture are dispersed and used as inoculum for the assay culture. By this technique the assay can be performed by 2.5 to 3.5 hr of assay culture after 2 to 3 hr-intermediate culture.

The technique can be applied to the determination of amino acids in the mixture and the results agree with those obtained by ordinary method.     

Uptake and excretion of amino acids and utilization of glucose by Schistosoma japonicum eggs

The utilization of amino acids and glucose in the external nutrients and the excretion of nitrogenous compounds by Schistosoma japonicum eggs were investigated with the eggs cultured in a chemically defined medium (MEMSE-J). Of the 15 amino acids in MEMSE-J, arginine and glutamine markedly decreased in concentration during cultivation of S. japonicum eggs. The nitrogenous excretory products of developing eggs were demonstrated to be at least four amino acids (alanine, proline, glutamic acid and ornithine), urea and ammonia. Glucose was consumed at an estimated rate of 32 ng/living egg/day during the period of egg growth and differentiation. When 14C-labelled glucose was included in the culture medium, the radioactivity was incorporated into three amino acids (alanine, proline and glutamic acid), which were excreted by S. Japonicum eggs. The results were discussed with reference to the possible role in stimulating fibrosis in the granuloma of schistosomiasis.     

Nutritional requirements and media development for Lactococcus lactis IL1403

Lactic acid bacteria are extensively used in food technology and for the production of various compounds, but they are fastidious in nutrient requirements. In order to elucidate the role of each component precisely, defined multicomponent media are required. This study focuses on determining nutrient auxotrophies and minimizing media components (amino acids, vitamins, metal ions, buffers and additional compounds) for the cultivation of Lactococcus lactis subsp. lactis IL1403, using microtitre plates and test tubes. It was shown that glutamine and asparagine were the most important media components for achieving higher biomass yields while the branched-chain amino acids were necessary to increase specific growth rate. The amino acid and glucose ratio was reduced to achieve minimal residual concentration of amino acids in the medium after the growth of cells, whereas the specific growth rate and biomass yield of cells were not considerably affected. As the percentage of each consumed amino acid compared to initial amount is larger than measurement error, these optimized media are important for achieving more precise data about amino acid utilization and metabolism.     

Effect of glucose on pyruvate utilization by Rhodotorula glutinis

Summary The utilization of glucose and pyruvate by the yeast Rhodotorula glutinis in a medium containing both carbon sources has been studied. Glucose is readily consumed whereas the uptake of pyruvate is completely blocked by the presence of the sugar.The content of pyruvate kinase and phosphoenolpyruvate carboxykinase in R. glutinis cells growing on glucose plus pyruvate are drastically affected with time by the disappearance of the sugar from the culture medium. After complete exhaustion of glucose, the level of pyruvate kinase drops sharply down to a minimum whereas that of phosphoenolpyruvate carboxykinase rises abruptly up to a maximum.Feeding experiments with labelled compounds show that glucose affects the utilization of the amino acids alanine and aspartate, and conversely that the amino acids influence the utilization of the sugar. Glucose breakdown and its incorporation into polysaccharides is controlled by the amino acids and gluconeogenesis from the amino acids is controlled by the sugar.     

Determination of bioavailability of some long-chain N-substituted derivatives of L-methionine and L-lysine

The bioavailability of N-acyl-L-methionine derivatives has been determined using microbiological assay with Tetrahymena pyriformis. It was found that palmitoyl- and stearoyl-L-methionine, stearoyl-L-methionine ethyl ester, and stearoyl-L-methionine sodium salt were partially utilized (14-38%) for growth of the microorganism. These compounds partially inhibited utilization of free methionine added to the media. The shorter derivatives, acetyl-, hexanoyl-, lauroyl-, and myristoyl-L-methionine completely inhibited the growth of T. pyriformis. This effect was not reversed when DL-methionine was added to the media. N6-fatty acyl L-lysine derivatives gave low availability values (3-18%) in microbiological assessment with T. pyriformis. N2-Acetyl-and N6-acetyl-lysine did not inhibit the utilization of the added parent amino acid. Nutritional evaluation of L-methionine derivatives by the rat growth method using net protein ratio (NPR) as the performance index indicated complete availability of stearoyl-L-methionine, stearoyl-L-methionine sodium salt, and partial availability of stearoyl-L-methionine ethyl ester (52%), stearoyl-L-methionylglycine (32%), and lauroyl-L-methionine (75%).     

Optimization of an Escherichia coli formate dehydrogenase assay for selenium compounds.

A microbiological assay to detect different chemical compounds of selenium for potential future use in the study of the distribution of these chemical forms in foods is being developed. This assay is based on the detection, by infrared analysis, of CO2 in a culture of Escherichia coli when the bacteria are grown in the presence of various selenium compounds. The CO2 production is the result of selenium-dependent formate dehydrogenase activity, which catalyzes oxidation of formic acid produced during glucose metabolism. Smooth response curves were generated over several orders of magnitude for selenocystine, selenite, and selenomethionine. The assay detects selenium concentrations (above background) as low as 1.5 nM for selenocystine and selenite and 4 nM for selenomethionine in minimal medium. Detection of selenomethionine was enhanced (to a sensitivity of 1.5 nM) by the addition of methionine to minimal medium and was enhanced even further (to a sensitivity of 0.8 nM) by the addition of a defined mixture of amino acids. Selenomethionine could be assayed in the presence of an amino acid concentration which is proportional to the amino acid/elemental selenium ratio found in a wheat gluten reference material (NIST SRM 8418). This implies that the assay can detect selenium compounds in a variety of foods at low concentrations, avoiding the background CO2 production caused by high concentrations of non-selenium-containing amino acids. The observation that methionine enhanced selenomethionine availability for formate dehydrogenase synthesis supports studies in animals demonstrating that methionine controls selenomethionine incorporation into selenoenzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amino acid utilization by L-M strain mouse cells in a chemically defined medium

Summary The amino acid requirements of strain L-M mouse cells grown in a chemically defined medium (2×Eagle) containing only the 13 essential amino acids (EAA) were investigated. Medium and acid hydrolysate samples were analyzed for amino acid content by the method of ion exchange chromatography. The extent of utilization of the EAA differed;e.g. after 120 hr of cell growth without medium change, glutamine was exhausted from the medium; methionine, leucine, isoleucine, cystine, arginine, and valine were depleted 60 to 80%; other EAA were used to lesser extents. Although the EAA were used in excess of their requirements for protein synthesis, a correlation could generally be made between utilization and protein amino acid composition. Glutamine appeared to be, a growth-limiting factor. Use of U-¹⁴C-labeled glutamine indicated that over one-half of the metabolized glutamine was converted to carbon dioxide, 17% to cell material, and 15% was extracted from the amino acid pools. Nonessential amino acids (NEAA), viz. alanine, aspartic acid, glutamic acid, glycine, proline, and serine, were released into the medium during growth, and some were reutilized. Exogenous provision of these did not improve cell growth. In contrast to the other NEAA, only serine showed net utilization when provided exogenously. When glutamic acid largely replaced the glutamine in the medium, it exerted a sparing effect on the glutamine requirement for protein synthesis. Suggestions are given for the improvement of Eagle medium for cell growth. Supported by Research Grants CA 03720 and CA 11802 from the National Institutes of Health. Predoctoral, fellow supported, by Grant F01-GM-42156-02 from the National Institutes of Health.     

Viability of some metabolic processes in the isolated toad brain adapted to two osmotic environments.

The viability of the isolated toad brain in an aerated Ringer-like medium has been evaluated by the following criteria: 1) amino acid content before and after incubation; 2) accumulation of amino acids in the incubation medium; 3) a comparison of glucose utilization and [U-14C]glucose metabolism with that occurring in vivo; 4) tissue swelling; and 5) tissue lactate contents. On the basis of these criteria, the isolated toad brain, from toads adapted to a fresh-water or a salt-water environment, retains considerable metabolic integrity for at least 2 hr of incubation at 25 degrees C. Specifically, there was no swelling of the tissue, no apparent accumulation of lactate in the tissue, glucose appeared to be utilized at a rate not too different from that calculated for the toad brain in vivo, and the distribution of label from [U-14C]glucose had an overall pattern which resembled that observed in vivo. The tissue levels of amino acids were generally stable in vitro; however, there was a marked decline in the content of aspartate. The accumulation of amino acids in the medium varied considerably from one amino acid to another. Thus, there was very little net efflux of aspartate, GABA, and glutamate from the tissue but considerable net efflux of glutamine. This efflux of amino acids was greater from brains of hyperosmotically adapted toads than from the brains of toads adapted to fresh water by amounts proportional to their initial tissue contents.     

Nutrient medium for the isolation and cultivation of pneumococcus

A culture medium for the isolation and cultivation of pneumococci, produced in a solid or liquid form and based on raw material unsuitable for use as foodstuff (human placenta), has been developed. The amino acid composition of the medium has been studied. The medium has been found to contain 19 amino acids, to be free from ballast serum proteins and blood, and to ensure the good growth of pneumococci isolated from pathological material, the formation of the normal capsule, as well as active biological properties. The medium has proved to create elective and selective conditions enhancing the effectiveness of investigations and simplifying the isolation of pneumococci in the microbiological examination of patients.     

刺五加和短梗五加叶片初生代谢的发育特异性调控研究

利用GC-MS技术对刺五加（Acanthopanax senticosus（Rupr. et Maxim.） Harms）和短梗五加（Acanthopanax sessiliflorus（Rupr. et Maxim.） Seem）叶片初级代谢产物进行了代谢组学分析,运用PCA和OPLS-DA方法分析后,将刺五加和短梗五加叶片划分为3个发育时期：生长期（Growth period）、旺盛期（Exuberant period）、凋落期（Autumn period）。刺五加叶片3个时期筛选出糖类、氨基酸、有机酸、脂肪酸、多元醇等共53个差异化合物,短梗五加叶片3个时期筛选出糖类、氨基酸、有机酸、脂肪酸、多元醇等共51个差异化合物。进一步分析表明,刺五加和短梗五加叶片在旺盛期和凋落期之间差异化合物最多,其中刺五加主要是糖类、有机酸;短梗五加则除了上述两类化合物外,还包括脂肪酸、多胺类化合物。两者作为同属植物,在差异化合物的组成类型方面具有较高的相似性。结果初步揭示了2种同属药用植物叶片不同时期初级代谢物的积累模式,为2种植物叶片利用提供理论基础。     

The effect of nitrogen and carbon sources on proteinase production by Pseudomonas fluorescens

Some factors influencing the production of an extracellular proteinase by Pseudomonas fluorescens NCDO 2085 were studied. Proteinase production was optimal at 20C and pH 69 in static culture when calcium was included in the medium. Proteinase was not detectable in basal medium but could be induced by organic nitrogen compounds. The proteinase was produced in the exponential phase of growth on protein substrates but not until early stationary phase during growth on amino acids. The organism did not utilize lactose, the most abundant carbohydrate in milk. Citrate was readily utilized as an energy source but had a strong repressive effect on proteinase production. A medium containing sodium caseinate and pyruvate supported good growth and enzyme production. All the amino acids utilized as a sole carbon source, with the exception of serine, could induce proteinase production. Asparagine was the most effective amino acid inducer. Particular combinations of amino acids could induce or repress proteinase production. The regulation of proteinase production by Ps. fluorescens NCDO 2085 appears to be based on a balance between induction by low concentrations of low molecular weight degradation products and sensitivity to end product catabolite repression. The results suggest that the function of the proteinase is to ensure a supply of carbon rather than amino acids for protein synthesis.     

A chemically defined fermentation medium for the growth of Micromonospora purpurea

A chemically defined medium for Micromonospora purpurea has been devised, consisting of glucose, a nitrogen source, calcium carbonate, magnesium sulfate, dibasic potassium phosphate, and the required trace quantities of iron, copper, zinc, and manganese. Using washed cell inocula, dry mycelial weights of more than 16 mg./ml. were obtained in 7-day shaken-flask fermentations. Nutrient requirements for M. purpurea are discussed and growth data presented. Sucrose, maltose, starches and dextrins could be substituted for glucose, and resulted in good growth of the organism. A number of amino acids and inorganic nitrogen-containing compounds were capable of utilization as sole nitrogen sources. Weekly serial transfers of the culture in defined medium have shown no diminution in mycelial weights over a four-month period.     

The effect of nitrogen and carbon sources on proteinase production by Pseudomonas fluorescens

Some factors influencing the production of an extracellular proteinase by Pseudomonas fluorescens NCDO 2085 were studied. Proteinase production was optimal at 20 degrees C and pH 6.9 in static culture when calcium was included in the medium. Proteinase was not detectable in basal medium but could be induced by organic nitrogen compounds. The proteinase was produced in the exponential phase of growth on protein substrates but not until early stationary phase during growth on amino acids. The organism did not utilize lactose, the most abundant carbohydrate in milk. Citrate was readily utilized as an energy source but had a strong repressive effect on proteinase production. A medium containing sodium caseinate and pyruvate supported good growth and enzyme production. All the amino acids utilized as a sole carbon source, with the exception of serine, could induce proteinase production. Asparagine was the most effective amino acid inducer. Particular combinations of amino acids could induce or repress proteinase production. The regulation of proteinase production by Ps. fluorescens NCDO 2085 appears to be based on a balance between induction by low concentrations of low molecular weight degradation products and sensitivity to end product catabolite repression. The results suggest that the function of the proteinase is to ensure a supply of carbon rather than amino acids for protein synthesis.     

Effect of yeast extract and succinic acid on growth and sporulation of alternaria species

Summary Alternaria solani andA. nyctanthi, these pathogens causing leafspot disease were able to metabolize a variety of nitrogen compounds when grown on different culture media. The amount of growth varied with the nitrogen source. Peptone produced the best zonation when added in definite proportion to the yeast extract medium. Ammonium compounds were found to be moderately effective for growth but poor for sporulation. The effect of adding succinic acid in media containing ammonium sources and the role of pH in the utilization of nitrite nitrogen was investigated.The fungus gave more vegetative growth on a mixture of aminoacids than in culture media in which the same amino acids were supplied singly to study the effect produced on growth and sporulation.     

Phosphate is required for inhibition by glucose of development of hamster 8-cell embryos in vitro

The influence of sodium dihydrogen phosphate (Pi) and glucose on the development of hamster 8-cell embryos mediated by pyruvate (P) or amino acids (A) or lactate (L) was investigated using modified Tyrode's medium, TLP-PVA. When pyruvate was tested as the only energy substrate in medium TP-PVA for embryo development, blastocyst formation ranged from 81.3 to 90.9% whether or not the medium contained 0.35 mM Pi or 5 mM glucose; but, when these two compounds were present together, blastocyst formation fell to 51.8%. Similarly, in TA-PVA medium containing four amino acids: Phe, Ile, Met, and Gln), embryo development to blastocyst ranged from 74.1% to 90.4% whether or not the medium contained 0.35 mM Pi or 5 mM glucose; but, when these compounds were present together, blastocyst formation fell to 16.0%. In TL-PVA medium, 10 mM sodium lactate supported embryo development (84.4% blastocysts); the addition of 0.35 mM Pi decreased blastocyst development to 65.6%. However, addition of glucose to Pi-free TL-PVA medium did not decrease blastocyst formation (81.3%); when the medium contained 0.35 mM Pi, glucose curtailed blastocyst development to 7.5%. When glucose and Pi interactions were studied at different concentrations, glucose up to 1 mM was not inhibitory in Pi-free TL-PVA medium (74.3% blastocysts), but 0.25 mM glucose in the presence of 0.35 mM Pi markedly inhibited embryo development (7.7% blastocysts). Phosphate at a relatively high concentration (1 mM) was inhibitory (37.9% blastocysts), even in the absence of glucose.(ABSTRACT TRUNCATED AT 250 WORDS)     

Organic sulphur sources for the growth of the dermatophyte microsporum gypseum

The suitability of 30 organic compounds (of them 19 sulphur-containing amino acids) at a concentration of 1mm as sulphur sources for the growth of the dermatophyteMicrosporum gypseum was investigated. The dry mass of the mycelium after an 11-d growth served as a measure of utilizability. Of sulphur amino acids cystine, cysteine, reduced and oxidized glutathione, cysteic and cysteinesulphinic acids, S-sulphocysteine, lanthionine, taurine and serine sulphate were the best sources. Methionine and methionine-sulphone were utilized slightly less effectively. Other compounds were medium to poor sources and only S-carboxymethylcysteine was not utilized at all. All organic compounds that are not of amino acid type were poor sulphur sources or were utilized at all. Sodium dodecyl sulphate inhibited germination and growth completely.     

Amino acid utilization by L-M strain mouse cells in a chemically defined medium

Summary The amino acid requirements of strain L-M mouse cells grown in a chemically defined medium (2×Eagle) containing only the 13 essential amino acids (EAA) were investigated. Medium and acid hydrolysate samples were analyzed for amino acid content by the method of ion exchange chromatography. The extent of utilization of the EAA differed;e.g. after 120 hr of cell growth without medium change, glutamine was exhausted from the medium; methionine, leucine, isoleucine, cystine, arginine, and valine were depleted 60 to 80%; other EAA were used to lesser extents. Although the EAA were used in excess of their requirements for protein synthesis, a correlation could generally be made between utilization and protein amino acid composition. Glutamine appeared to be a growth-limiting factor. Use of U-¹⁴C-labeled glutamine indicated that over one-half of the metabolized glutamine was converted to carbon dioxide, 17% to cell material, and 15% was extracted from the amino acid pools. Nonessential amino acids (NEAA), viz. alanine, aspartic acid, glutamic acid, glycine, proline, and serine, were released into the medium during growth, and some were reutilized. Exogenous provision of these did not improve cell growth. In contrast to the other NEAA, only serine showed net utilization when provided exogenously. When glutamic acid largely replaced the glutamine in the medium, it exerted a sparing effect on the glutamine requirement for protein synthesis. Suggestions are given for the improvement of Eagle medium for cell growth. Supported by Research Grants CA 03720 and CA 11802 from the National Institutes of Health. Predoctoral fellow supported by Grant F01-GM-42156-02 from the National Institutes of Health. Present address: Department of Community Medicine. Basic Science Building, University of California, San Diego, La Jolla, Calif. 92037.     

A new method for the measurement of protein turnover.

A new technique for the determination of rate constants of protein degradation is described. By using the method, half-lives of total soluble protein of Lemna minor during growth on full culture medium and distilled water were measured. The method involves incubating Lemna on a growth medium containing 3H2O. After a short exposure (20 min) to 3H-labelled culture medium, 3H was found in soluble amino acids, especially aspartate, glutamate, glutamine and alanine. After transfer to a 3H-free medium for 30 min, 80% of the 3H originally present in soluble amino acids was lost. These results suggest that 3H enters and leaves amino acids at the alpha-carbon atom, a conclusion supported by the observed labelling of glutamates. The exchange of H and 3H on the alpha-carbon atom is catalysed by transaminases and the speed of this exchange ensures that when the 3H2O is removed, the 3H in free amino acids is rapidly lost, thereby eliminating problems connected with metabolic pools and recycling. After an exposure of 20 min to 3H-labelled medium all protein amino acids, except for arginine, were found to be radioactive. The loss of radioactivity from protein amino acids was used to measure protein degradation.