Production of branched-chain volatile fatty acids by certain anaerobic bacteria

Net production of isobutyric acid, isovaleric acid, and 2-methylbutyric acid by cultures of Bacteroides ruminicola and Megasphaera elsdenii on media that contained Trypticase or casein hydrolysate continued (up to 5 days) after growth had ceased. Only trace quantities of these acids were produced in a medium that contained a mixture of amino acids that did not include the branched-chain amino acids. M. elsdenii produced increased quantities of the branched-chain fatty acids in a medium that contained Trypticase when glucose was reduced or eliminated from the culture medium. However, B. ruminicola produced increased quantities of branched-chain fatty acids and of phenylacetic acid from Trypticase when glucose was supplied at 3 mg/ml rather than at 1 mg/ml. Single strains of Streptococcus bovis, Selenomonas ruminantium, Bacteroides amylophilus, and Butyrivibrio fibrisolvens did not produce branched-chain fatty acids.     

Production of branched-chain volatile fatty acids by certain anaerobic bacteria.

Net production of isobutyric acid, isovaleric acid, and 2-methylbutyric acid by cultures of Bacteroides ruminicola and Megasphaera elsdenii on media that contained Trypticase or casein hydrolysate continued (up to 5 days) after growth had ceased. Only trace quantities of these acids were produced in a medium that contained a mixture of amino acids that did not include the branched-chain amino acids. M. elsdenii produced increased quantities of the branched-chain fatty acids in a medium that contained Trypticase when glucose was reduced or eliminated from the culture medium. However, B. ruminicola produced increased quantities of branched-chain fatty acids and of phenylacetic acid from Trypticase when glucose was supplied at 3 mg/ml rather than at 1 mg/ml. Single strains of Streptococcus bovis, Selenomonas ruminantium, Bacteroides amylophilus, and Butyrivibrio fibrisolvens did not produce branched-chain fatty acids.     

Nutritional Interdependence Among Rumen Bacteria, Bacteroides amylophilus, Megasphaera elsdenii, and Ruminococcus albus

Nutritional interdependence among three representatives of rumen bacteria, Bacteroides amylophilus, Megasphaera elsdenii, and Ruminococcus albus, was studied with a basal medium consisting of minerals, vitamins, cysteine hydrochloride, and NH₄⁺. B. amylophilus grew well in the basal medium supplemented with starch and produced branched-chain amino acids after growth ceased. When cocultured with B. amylophilus in the basal medium supplemented with starch and glucose, amino acid-dependent M. elsdenii produced an appreciable amount of branched-chain fatty acids, which are essential growth factors for cellulolytic R. albus. A small addition of starch (0.1 to 0.3%) to the basal medium containing glucose and cellobiose brought about successive growth of the three species in the order of B. amylophilus, M. elsdenii, and R. albus, and successive growth was substantiated by the formation of branched-chain amino acids and fatty acids in the culture. Supplementation with 0.5% starch, however, failed to support the growth of R. albus. On the basis of these results, the effects of supplementary starch or branched-chain fatty acids on cellulose digestion in the rumen was discussed.     

Incorporation of C14 from amino acids and peptides into protein by Clostridium perfringens type D

Hauschild, Andreas H. W. (University of Toronto, Toronto, Ontario, Canada). Incorporation of C(14) from amino acids and peptides into protein by Clostridium perfringens type D. J. Bacteriol. 90:1569-1574. 1965.-Uptake of C(14) from C(14)-labeled amino acids and peptides by Clostridium perfringens was measured in culture media containing acid or papain hydrolysates of C(14)-labeled Chlorella protein. Between 2 and 4 hr of growth, the rate of C(14) uptake from peptides was higher than from free amino acids. Peptides extracted from cells with hot ethyl alcohol contained six to nine times more C(14) after 4 hr of growth with C(14)-labeled peptides than with C(14)-labeled amino acids. Incorporation of C(14)-labeled glycine, serine, threonine, alanine, and proline into both cellular and exocellular protein was two to five times higher when these were supplied as components of dialyzable peptides rather than as free amino acids.     

Studies on the uptake of (14C) amino acids derived from both dietary (14C) protein and dietary (14C) amino acids by rainbow trout, Salmo gairdneri Richardson

Previous studies have shown that rainbow trout fed on diets containing whole protein have superior growth rates compared to fish fed on diets of similar amino acid composition but containing a high proportion of free amino acids. The influence of several nutritional factors on the uptake of radioactivity from food pellets containing either [U-^I4C] protein or [U-¹⁴C] amino acids into the systemic blood of trout has been investigated. The time taken for radioactivity in the free amino acid fraction of blood to reach a peak after a meal containing [U-¹⁴C] protein had been given was much shorter, and the level of radioactivity in the blood higher, in trout with almost empty stomachs than in fish with almost full stomachs; uptake of radioactivity into blood amino acids was also more rapid and reached much higher concentrations when pellets containing [U-¹⁴C] amino acids were fed than when [U-¹⁴C] protein was fed. Incorporation of radioactivity into blood protein continued for a much longer period and reached higher levels when a pellet containing [U-¹⁴C] protein was fed than when a pellet containing [U-¹⁴C] amino acids was fed. Previous dietary history (low or high protein intake) did not appear to affect the rate of absorption of amino acids from either protein or free amino acid pellets. The uptake rates from pellets containing free amino acids could be slowed by mixing the dietary amino acids with albumin. The distribution, postabsorption, of radioactivity in the different fractions of blood and liver suggested that incorporation of carbon residues into glycogen and lipid from an amino acid diet was greater than from a protein diet. The converse was true of incorporation of radioactivity into tissue protein.     

Amylolytic activity of selected species of ruminal bacteria.

A variety of species of ruminal bacteria were screened for the ability to grow in starch-containing medium and produce amylase. Of those tested, the highest levels of amylase were produced by Streptococcus bovis JB1 and Ruminobacter amylophilus H18. Other strains that grew well on starch and produced amylase included Butyrivibrio fibrisolvens A38 and 49 and Bacteroides ruminicola 23 and B14. Varying the carbohydrate source provided for growth resulted in changes in the growth rate and level of amylase produced by these strains. All strains grew rapidly in starch-containing medium, and the rates of growth were generally more rapid than those observed for maltose-grown cultures. For S. bovis JB1, B. ruminicola 23 and B14, and B. fibrisolvens 49 and A38, amylase was produced when growth was on maltose or starch, but this activity was greatly reduced in glucose-grown cultures. The distribution of amylolytic activity between cellular and extracellular fractions was sometimes affected by the carbohydrate provided for growth. If S. bovis JB1 and B. fibrisolvens 49 were grown on starch, amylase was largely associated with cell pellets; however, if grown on maltose these strains produced activities that were almost entirely present in the extracellular fluid fractions. Although not as dramatic, a similar shift in the location of amylase activities was noted for the two B. ruminicola strains when grown on the same substrates. Growth on maltose or starch had little influence on either the predominantly cell-associated activity of B. fibrisolvens A38 or the activity of R. amylophilus H18, which was equally divided between cell pellet and extracellular fluid fractions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amylolytic activity of selected species of ruminal bacteria

A variety of species of ruminal bacteria were screened for the ability to grow in starch-containing medium and produce amylase. Of those tested, the highest levels of amylase were produced by Streptococcus bovis JB1 and Ruminobacter amylophilus H18. Other strains that grew well on starch and produced amylase included Butyrivibrio fibrisolvens A38 and 49 and Bacteroides ruminicola 23 and B14. Varying the carbohydrate source provided for growth resulted in changes in the growth rate and level of amylase produced by these strains. All strains grew rapidly in starch-containing medium, and the rates of growth were generally more rapid than those observed for maltose-grown cultures. For S. bovis JB1, B. ruminicola 23 and B14, and B. fibrisolvens 49 and A38, amylase was produced when growth was on maltose or starch, but this activity was greatly reduced in glucose-grown cultures. The distribution of amylolytic activity between cellular and extracellular fractions was sometimes affected by the carbohydrate provided for growth. If S. bovis JB1 and B. fibrisolvens 49 were grown on starch, amylase was largely associated with cell pellets; however, if grown on maltose these strains produced activities that were almost entirely present in the extracellular fluid fractions. Although not as dramatic, a similar shift in the location of amylase activities was noted for the two B. ruminicola strains when grown on the same substrates. Growth on maltose or starch had little influence on either the predominantly cell-associated activity of B. fibrisolvens A38 or the activity of R. amylophilus H18, which was equally divided between cell pellet and extracellular fluid fractions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amino acid requirement for the growth-hormone stimulation of incorporation of precursors into protein and nucleic acids of liver slices

1. Incorporation of [(14)C]leucine into protein in rat liver slices, incubated in vitro, increased as the concentration of unlabelled amino acids in the incubation medium was raised. A plateau of incorporation was reached when the amino acid concentration was 6 times that present in rat plasma. Labelling of RNA by [(3)H]orotic acid was not stimulated by increased amino acid concentration in the incubation medium. 2. When amino acids were absent from the medium, or present at the normal plasma concentrations, no effect of added growth hormone on labelling of protein or RNA by precursor was observed. 3. When amino acids were present in the medium at 6 times the normal plasma concentrations addition of growth hormone stimulated incorporation of the appropriate labelled precursor into protein of liver slices from normal rats by 31%, and into RNA by 22%. A significant effect was seen at a hormone concentration as low as 10ng/ml. 4. Under the same conditions addition of growth hormone also stimulated protein labelling in liver slices from hypophysectomized rats. Tissue from hypophysectomized rats previously treated with growth hormone did not respond to growth hormone in vitro. 5. No effect of the hormone on the rate or extent of uptake of radioactive precursors into acid-soluble pools was found. 6. Cycloheximide completely abolished the hormone-induced increment in labelling of both RNA and protein. 7. It was concluded that, in the presence of an abundant amino acid supply, growth hormone can stimulate the synthesis of protein in rat liver slices by a mechanism that is more sensitive to cycloheximide than is the basal protein synthesis. The stimulation of RNA labelling observed in the presence of growth hormone may be a secondary consequence of the hormonal effect on protein synthesis. 8. The mechanism of action of growth hormone on liver protein synthesis in vitro was concluded to be similar to its mechanism of action in vivo.     

Synergistic Effects of Metabolically Related Amino Acids on the Growth of a Multicellular Plant: II. Studies of 14C-Amino Acid Incorporation 1,2

The synergistic inhibition of the growth of Marchantia polymorpha gemmalings by lysine and threonine and its prevention by methionine has been investigated utilizing (14)C-labeled amino acids. Experiments involving the uptake of (14)C-lysine or (14)C-threonine in the presence or absence of methionine indicated that the synergistic growth effects were not a result of altered amino acid uptake. These data, as well as direct chemical analysis, indicated that growth inhibition was correlated with an inhibition of protein synthesis. Experiments utilizing (14)C-aspartic acid revealed that the presence of lysine and threonine resulted in increased (14)CO(2) production and an accumulation of soluble (14)C-aspartic acid and labeled ninhydrin-positive compounds. These metabolic alterations were prevented when methionine was also included in the growth media. A model depicting a sequence of events which involve the interaction of regulatory mechanisms is suggested to account for the effects of specific amino acids on plant growth.     

Protein synthesis and amino acid pool during yeast-mycelial transition induced by N-acetyl-D-glucosamine in Candida albicans

Protein synthesis at different stages of yeast-mycelial transition induced by N-acetyl-D-glucosamine in Candida albicans was evaluated by following incorporation of radioactive amino acids into the acid-insoluble cellular material. In passing from the early germ-tube formation (60-90 min) to the mature hyphal cell (240-270 min) there was a marked decrease in the capacity for protein synthesis. Apparently, this decrease was not due to a decreased amino acid uptake into the soluble cellular pool or to exhaustion of carbon/energy source in the inducing medium with consequent arrest of growth. Protein synthesis, however, did not decay when amino acids at high concentration were added to the medium fostering the yeast-mycelial transition and this effect was potentiated by glucose. Analysis of the intracellular amino acid pool showed that both germ-tubes and hyphal cells were relatively depleted of several amino acids as compared to the yeast-form cells, whereas in the hyphae there was a higher concentration of glutamic acid/glutamine, the latter being the predominant component. These modulations in amino acid pool composition were not seen when yeasts were converted to hyphae in an amino acid-rich induction medium. This study emphasizes that yeast-form cells of C. albicans may efficiently convert to the mycelial form even under a progressively lowered rate of protein synthesis, and suggests that initiation of hyphal morphogenesis in the presence of N-acetyl-D-glucosamine is somehow separated from cellular growth.     

Action of insulin and growth hormone on protein synthesis in muscle from non-hypophysectomized rabbits

The separate effects of insulin and growth hormone on the uptake and incorporation of five amino acids into diaphragm muscle from non-hypophysectomized rabbits has been examined. Both growth hormone and insulin, when present in the medium separately, stimulated the incorporation into protein of the amino acids, leucine, arginine, valine, lysine and histidine. Insulin also stimulated amino acid uptake, but growth hormone did not. When insulin and growth hormone were present in the incubation medium together, the uptake and incorporation of valine, the only amino acid studied under these conditions, tended to be greater than the sum of the separate effects of the two hormones.     

Nitrogen limitation and amino-acid metabolism of Chlorella symbiotic with green hydra

Chlorella algae symbiotic in the digestive cells of Hydra viridissima Pallas (green hydra) were found to contain less amino-N and smaller pools of free amino acids than their cultured counterparts, indicating that growth in symbiosis was nitrogen-limiting. This difference was reflected in uptake of amino acids and subsequent incorporation into protein; symbiotic algae incorporated a greater proportion of sequestered radioactivity, supplied as ¹⁴C-labelled alanine, glycine or arginine, than algae from nitrogen-sufficient culture, presumably because smaller internal pools diluted sequestered amino acids to a lesser extent. Further experiments with symbiotic algae showed that metabolism of the neutral amino acid alanine differed from that of the basic amino acid arginine. Alanine but not arginine continued to be incorporated into protein after uptake ceased, and while internal pools of alanine were exchangeable with alanine in the medium, those of arginine were not exchangeable with external arginine. Thin-layer chromatography of ethanol-soluble extracts of algae incubated with [¹⁴C]alanine or [¹⁴C]arginine showed that both were precursors of other amino acids. The significance of nitrogen-limiting growth of symbiotic algae is discussed in terms of host-cell regulation of algal cell growth and division.     

Nitrogen nutrition in the cyanobacterium Nostoc ANTH, a symbiotic isolate from Anthoceros: uptake and assimilation of inorganic-n and amino acids

Amino acid uptake and utilization of various nitrogen sources (amino acids, nitrite, nitrate and ammonia) were studied in Nostoc ANTH and i ts mu tant (Het(-)Nif(-)) isolate defective in heterocyst formation and N2-fixation. Both parent and its mutant grew at the expense of glutamine, asparagine and arginine as a source of fixed-nitrogen. Growth was better in glutamine-and asparagine-media as compared to that in arginine media. Glutamine and asparagine repressed heterocyst formation, N2-fixation and nitrate reduction in Nostoc ANTH, but arginine did so only partially. The poor growth in arginine-medium was not due to poor uptake rates, since the uptake rates were not significantly different from those for glutamine or asparagine. The glutamine synthetase activity remained unaffected during cultivation in media containing any one of the three amino acids tested. The uptake of amino acids was substrate-inducible, energy-dependent and required de novo protein synthesis. Nitrate and ammonium repressed ammonium uptake, but did not repress uptake of amino acids. In N2-medium (BG-11(0)), the uptake of ammonium and amino acids in the mutant was significantly higher than its parent strain. This was apparently due to nitrogen limitation since the mutant was unable to fix N2 and the growth medium lacked combined-N.     

Positional cues for the starch/lipid balance in maize kernels and resource partitioning to the embryo

This study tests the hypotheses that in vivo oxygen levels inside developing maize grains locally affect assimilate partitioning and ATP distribution within the kernel. These questions were addressed through combined topographical analysis (O2- and ATP-mapping), metabolite profiling, and isotope flux analysis. Internal and external oxygen levels were also experimentally altered. Under ambient conditions, mean O2 concentration immediately inside starchy endosperm dropped to only 1.4% of atmospheric saturation (approximately 3.8 microm), but was 10-fold higher in the oil-storing embryo. Increasing the O2 supply to intact kernels stimulated their O2 demand, shifted ATP localization within the kernel, and elevated their ATP/ADP ratio. Enhanced O2 availability also increased steady-state levels of glycolytic intermediates and those of the citric acid cycle, as well as some related pools of free amino acids. Subsequent analyses indicated that starch formation within endosperm, but not lipid biosynthesis within embryo, was adapted to the endogenous low oxygen. Increasing the O2 supply did not change ADP-glucose levels, activity of ADP-glucose pyrophosphorylase, 13C-labeling of ADP-glucose, or flux of 14C-sucrose into starch. In contrast, enhanced O2 availability increased 14C-label uptake into the embryo, 13C-labeling of acetyl-coenzyme A, and finally 14C-incorporation into lipids. Lipid accumulation in embryo appeared highest in regions with higher ATP. Consistent with labeling data, a decrease in O2 supply most strongly affected the embryo, whereas rising O2 levels expanded ATP-rich zones toward the starch-storing endosperm and the scutellar part of embryo. The latter might be responsible for higher 14C-label uptake into the embryo and flux toward lipid. Collectively, data indicate that the in vivo oxygen distribution in maize kernels markedly affects ATP gradients, metabolite levels, and favors assimilate partitioning toward starch within the O2-depleted endosperm. Clear advantages are thus evident for peripheral localization of the protein and lipid storing structures in maize kernels.     

Extraction of protein and amino acids from deoiled rice bran by subcritical water hydrolysis

This study investigated the production of value-added protein and amino acids from deoiled rice bran by hydrolysis in subcritical water (SW) in the temperature range between 100 and 220 degrees C for 0-30 min. The results suggested that SW could effectively be used to hydrolyze deoiled rice bran to produce useful protein and amino acids. The amount of protein and amino acids produced are higher than those obtained by conventional alkali hydrolysis. The yields generally increased with increased temperature and hydrolysis time. However, thermal degradation of the product was observed when hydrolysis was carried out at higher temperature for extended period of time. The highest yield of protein and amino acids were 219 +/- 26 and 8.0 +/- 1.6 mg/g of dry bran, and were obtained at 200 degrees C at hydrolysis time of 30 min. Moreover, the product obtained at 200 degrees C after 30 min of hydrolysis exhibited high antioxidant activity and was shown to be suitable for use as culture medium for yeast growth.     

Peptides containing 2-aminopimelic acid. Synthesis and study of in vitro effects on bacterial cells

Taking advantage of the peptide transport strategy, we have designed and synthesized several new peptides containing 2-aminopimelic acid (Apm), an inhibitor of the diaminopimelate pathway in bacteria: L-Lys-ambo-Apm, ambo-Apm-L-Lys, L-Lys-L-Ala-ambo-Apm, ambo-Apm-L-Ala-L-Lys, L-Ala(Cl)-ambo-Apm and ambo-Apm-L-Ala(Cl). In the two latter cases, Apm was associated with antibacterial amino acid beta-chloro-L-alanine [L-Ala(Cl)], an inhibitor of alanine racemase and transaminase B. The peptides displayed weak or no antibacterial activities; nevertheless, those containing L-Ala(Cl) had low MIC values in the presence of amino acids restoring protein synthesis. When tested on exponential phase Escherichia coli cells grown in minimal medium, the peptides were without effect or bacteriostatic, but important bacteriolytic effects could be observed, especially for the L-Ala(Cl)-containing peptides, when the growth medium was supplemented with specific amino acids. It was demonstrated that the weak or nil effect of the L-lysine-containing peptides was due to a poor uptake.     

The simultaneous biosynthesis and uptake of amino acids by Lactococcus lactis studied by (13)C-labeling experiments

Uniformly (13)C labeled glucose was fed to a lactic acid bacterium growing on a defined medium supplemented with all proteinogenic amino acids except glutamate. Aspartate stemming from the protein pool and from the extracellular medium was enriched with (13)C disclosing a substantial de novo biosynthesis of this amino acid simultaneous to its uptake from the growth medium and a rapid exchange flux of aspartate over the cellular membrane. Phenylalanine, alanine, and threonine were also synthesized de novo in spite of their presence in the growth medium.     

Ectopic expression of an amino acid transporter (VfAAP1) in seeds of Vicia narbonensis and pea increases storage proteins

Storage protein synthesis is dependent on available nitrogen in the seed, which may be controlled by amino acid import via specific transporters. To analyze their rate-limiting role for seed protein synthesis, a Vicia faba amino acid permease, VfAAP1, has been ectopically expressed in pea (Pisum sativum) and Vicia narbonensis seeds under the control of the legumin B4 promoter. In mature seeds, starch is unchanged but total nitrogen is 10% to 25% higher, which affects mainly globulin, vicilin, and legumin, rather than albumin synthesis. Transgenic seeds in vitro take up more [14C]-glutamine, indicating increased sink strength for amino acids. In addition, more [14C] is partitioned into proteins. Levels of total free amino acids in growing seeds are unchanged but with a shift toward higher relative abundance of asparagine, aspartate, glutamine, and glutamate. Hexoses are decreased, whereas metabolites of glycolysis and the tricarboxylic acid cycle are unchanged or slightly lower. Phosphoenolpyruvate carboxylase activity and the phosphoenolpyruvate carboxylase-to-pyruvate kinase ratios are higher in seeds of one and three lines, indicating increased anaplerotic fluxes. Increases of individual seed size by 20% to 30% and of vegetative biomass indicate growth responses probably due to improved nitrogen status. However, seed yield per plant was not altered. Root application of [15N] ammonia results in significantly higher label in transgenic seeds, as well as in stems and pods, and indicates stimulation of nitrogen root uptake. In summary, VfAAP1 expression increases seed sink strength for nitrogen, improves plant nitrogen status, and leads to higher seed protein. We conclude that seed protein synthesis is nitrogen limited and that seed uptake activity for nitrogen is rate limiting for storage protein synthesis.     

Functional characterization and cloning of amino acid transporter B(0,+) (ATB(0,+)) in primary cultured rat pneumocytes

Cationic amino acid transport in primary cultured rat pneumocytes exhibiting characteristics of alveolar epithelial type I-like cells are described. Asymmetry and activator ion dependency of (3)H-L-arginine uptake were characterized from the apical or basolateral fluid of pneumocytes grown on permeable support. Substrate specificity of transport was evaluated as a function of (3)H-L-arginine uptake inhibition in the presence of other amino acids. Transepithelial transport studies estimated (3)H-L-arginine flux in the apical-to-basolateral and basolateral-to-apical directions. Full length cDNA of rat amino acid transporter B(0,+) (rATB(0,+)) was cloned and its relative expression level studied. Results indicate that uptake of (3)H-L-arginine from apical fluid is dependent on Na(+) and Cl(-). Zwitterionic and cationic amino acids (excluding L-proline and anionic amino acids) inhibited uptake of (3)H-L-arginine from apical, but not basolateral incubation fluid. Apical-to-basolateral transepithelial flux of (3)H-L-arginine was 20x higher than basolateral-to-apical transport. Kinetic studies of (3)H-L-arginine uptake from apical fluid revealed maximal velocity (V(max)) and Michaelis-Menten constants (K(t)) of 33.32 +/- 2.12 pmol/mg protein/15 min and 0.50 +/- 0.11 mM, respectively, in a cooperative process having a coupling ratio of 1.18 +/- 0.16 with Na(+) and 1.11 +/- 0.13 with Cl(-). Expression of rATB(0,+) mRNA was identified by RT-PCR and Northern analysis. Corresponding cloned 3.2 kb rATB(0,+) cDNA sequence exhibits pronounced homology in deduced amino acid sequence to mouse (95% identity and 97% similarity) and human (89% identity and 95% similarity) ATB(0,+) homologues. We conclude that rat pneumocytes express ATB(0,+), which may partly contribute towards recovering cationic and neutral amino acids from alveolar luminal fluid.     

Amino Acid Transport in Pseudomonas aeruginosa

Properties of the transport systems for amino acids in Pseudomonas aeruginosa were investigated. Exogenous (14)C-labeled amino acids were shown to equilibrate with the internal native amino acid pool prior to incorporation into protein. When added at low external concentrations, the majority of the amino acids examined entered the protein of the cell unaltered. The rates of amino acid transport, established at low concentrations with 18 commonly occurring amino acids, varied as much as 40-fold. The transport process became saturated at high external amino acid concentrations, was temperature-sensitive, and was inhibited by sodium azide and iodoacetamide. Intracellular to extracellular amino acid ratios of 100- to 300-fold were maintained during exponential growth of the population in a glucose minimal medium. When the medium became depleted of glucose, neither extracellular nor intracellular amino acids could be detected.