Amino acid uptake systems in Bacteroides ruminicola

Uptake of amino acids by Bacteroides ruminicola was observed in cells grown in a complete defined medium, containing ammonia as the nitrogen source. A high rate of uptake occurred only in fresh medium, as an inhibitory substance, possibly acetate, apparently accumulated during growth. All amino acids except proline were taken up and incorporated into cold trichloroacetic acid precipitable material. Different patterns of incorporation and different responses to 2,4-dinitrophenol and potassium ferricyanide indicated multiple uptake systems were involved. Kinetic inhibition patterns suggested six distinct systems were present for amino acid uptake, with specificities related to the chemical structures of the amino acids. Thus, the failure of free amino acids to act as sole nitrogen sources for growth of B. ruminicola is not due to the absence of transport systems for these compounds.     

Hind leg muscle amino acid balances in cold-exposed rats

The changes in hind leg tissue (muscle and skin) amono acid pool size and arteriovenous balance were measured in rats subjected to 0–90 min of cold exposure (4°C). Tissue free amino acid pools presented a different composition pattern from protein amino acids. Muscle rapidly reacted to cold exposure by releasing small amounts of some amino acids (alanine, aspartate), with only small changes in pool size during the first 30 min. Amino acid oxidation was very limited during the whole period of cold exposure, since at all times tested there was either nil ammonia efflux or net absorption of ammonia and glutamine; i.e. the muscle was in positive nitrogen balance throughout the period studied. Thus most of the amino acid nitrogen taken up from the blood and not found in the free amino pools must have been incorporated into protein, since it was not oxidized, as shown by the glutamine and ammonia blance. The data on amino acid incorporation into proteins indicate that hind leg protein turnover is rapidly and widely modulated from a low initial setting upon cold exposure to a higher protein synthesis rate immediately afterwards, suggesting that protein turnover may have an important role in short-term events in cold-exposed muscle, in addition to its influence in long-term adaptation.     

Utilization of amino acids and dipeptides by Lactobacillus plantarum from orange in nutritionally stressed conditions

Aims:  To investigate amino acid and dipeptide utilization by Lactobacillus plantarum N4 isolated from orange peel, in a nutritionally depleted medium based on MRS (Mann, Rogosa, Sharpe).
Methods and Results:  In MRS with 0·1 g l⁻¹ of meat extract and without peptone and yeast extract, growth increased when essential and stimulatory amino acids and nonessential amino acid were added to the medium. Replacement of the essential amino acid, leucine, and the nonessential amino acid, glycine, by leucyl-leucine (Leu-Leu) and/or glycyl-glycine (Gly-Gly) significantly enhanced growth. Essential amino acids were mainly consumed and the dipeptides were almost completely used at the end of growth. Leucine and glycine accumulated internally from the peptides were higher than from the free amino acids. Glucose utilization increased in the media containing dipeptides compared with the medium containing free amino acids.
Conclusions:  In a N-depleted medium, Leu-Leu and/or Gly-Gly were more effective than the respective amino acids in supporting growth of the micro-organism. The more efficient internal accumulation of glycine and especially leucine from dipeptides confirmed the ability of the strain to assimilate mainly complex nitrogen molecules rather than simple ones.
Significance and Impact of the Study:  The ability of Lact. plantarum N4 to efficiently use dipeptides could contribute to spoilage development in the natural medium of the organism, orange juice.     

Amino acid pools in cultured muscle cells

Compartmentalization of cellular amino acid pools occurs in cultures of cardiac and skeletal muscle cells, but the factors involved in this are not clear. We have further defined this problem by analyzing the intracellular free leucine and the transfer-RNA-(tRNA)-bound leucine pool in cultures of skeletal and cardiac muscle incubated with ³H-leucine in the presence and absence of serum and amino acids. Withdrawal of nitrogen substrates caused substantial changes in leucine pool relationships–in particular, a change in the degree to which intracellular free leucine and tRNA-leucine were derived from the culture medium. In separate experiments, the validity of our tRNA measurements was confirmed by measurements of the specific activity of newly synthesized ferritin after iron induction. We discuss the implications of these findings with regard-to factors involved in the control of amino acid flux through the cell, as well as with regard to design of experiments using isotopic amino acids to measure rates of amino acid utilization.     

Glucose-induced Release of Amino Acids from Saccharomyces carlsbergensis by Action on the Cytoplasmic Membrane

When washed yeast cells grown under appropriate conditions were suspended in glucose solution there was a sudden release of α-amino nitrogen into the medium. This released material was of low molecular weight, and its composition was closely similar to that of the intracellular free amino acid pool. During the leakage of amino acids, the yeast did not efficiently absorb labeled amino acids added to the test medium, despite the rapid uptake and metabolism of glucose. Uptake of a labeled amino acid and reabsorption of the released α-amino nitrogen occurred almost simultaneously. When these yeast cells were exposed to glucose in the presence of calcium ions, leakage was strongly inhibited. Butanol under the same conditions increased glucose-induced leakage of cell contents. The adenosine triphosphatase activity of intact yeast cells exposed to glucose was greater than that of cells exposed to water. Yeast cells treated with glucose prior to equilibration with sorbose exhibited less ability to retain the sorbose when washed at 0 C than did cells pretreated with water. It was concluded that glucose-induced leakage of amino acids was the result of two factors acting together. These were (i) a change in membrane permeability associated with glucose uptake, and (ii) a temporary shortage of energy for amino acid uptake or retention.     

Transient responses of hybridoma cells in continuous culture to step changes in amino acid and vitamin concentrations

The effects of step-change increase in the concentrations of amino acids and vitamins on the metabolism, growth, and antibody productivity of a murine hybridoma cell line grown in continuous culture on serum-free medium are presented. Additions of the amino acids cysteine with methionine, tryptophan, and isoleucine with valine and vitamin B(12) (as cyanocobalamin) resulted in significant increases in viable cell concentrations. Additions of aspartate with asparagine, and threonine with vitamin B(1) (as thiamine hydrochloride) resulted in significant increases in final antibody concentrations. Substantial decrease in the fraction of amino acid nitrogen excreted as ammonia occurred upon supplementation with three times the normal concentrations of branched chain amino acids. Decreases in the fraction of amino acid nitrogen converted to ammonia were paralleled by increases in the fraction converted to alanine. (c) 1994 John Wiley & Sons, Inc.     

Free amino Acid contents of stem and phylloxera gall tissue cultures of grape

Free amino acid constituents were determined of grape stem and Phylloxera leaf gall callus in tissue culture. Fast, medium and slow growing single cell clones of, respectively, stem and gall origins were grown on a mineral salt-sucrose medium supplemented with coconut milk and α-naphthaleneacetic acid. Stem and gall clones showed qualitative similarities and quantitative variations in the amino acids and nitrogenous constituents. Nineteen amino acids, glucosamine, ethanolamine, sarcosine, methionine sulfoxides and ammonia were identified. Two free polypeptides accounted for over 30% of the amino compounds in the stem and gall callus tissues which were not found in the intact plant parts. Stem clones of different growth rates grown on agar showed generally an excess of amino acid constituents over gall tissues of similar growth rates, except for the free polypeptides. Fast growing stem clones grown on agar medium contained lower amounts of certain amino acids than the fast growing gall clones, but when grown in liquid medium they contained higher amounts of these acids than the gall clones. The total and nonsoluble nitrogen of stem clones were higher than in the gall clones. Tissue cultures differed from the original plant parts with respect to their free polypeptides and high amino acid contents.     

Utilization of Ammonia Nitrogen by Intestinal Bacteria Isolated from Pigs

In a medium containing ammonia, proteose peptone, and cysteine as nitrogen sources, 17 of 24 Bacteroidaceae strains, 3 of Selenomonas strains, 1 of 7 curved rods, 3 of 7 Spirochaetaceae strains, 8 of 20 Eubacterium strains, 8 of 13 Peptococcaceae strains, 3 of 4 Clostridium strains, 19 of 20 Enterobacteriaceae strains, and 1 of 8 Streptococcus strains utilized ammonia nitrogen preferentially to proteose peptone nitrogen. To determine the ability of intestinal microbes to synthesize amino acids from ammonia, ammonia utilization by Bacteroides ruminicola strain 9 was studied in defined media containing ammonia and other nitrogen sources. In another medium containing ammonia, proteose peptone, and cysteine as nitrogen sources, ammonia was preferentially utilized even when the proteose peptone nitrogen content was eight times greater than that of ammonia nitrogen. In a medium containing ammonia, an amino acid, and cysteine, the lowest uptake of ammonia nitrogen was observed when the medium contained aspartic acid, glutamic acid, threonine, or alanine; but ammonia was utilized more effectively than any of the amino acids. Incorporation of ¹⁵N from [¹⁵N]ammonia into bacterial amino acids was studied. ¹⁵N was incorporated into every amino acid of B. ruminicola strain 9, and the highest uptake was observed in aspartic acid and alanine.     

The importance of amino acids as carbon sources for Micromonospora echinospora (ATCC 15837)

AIMS: This study set out to investigate the effect of amino acids on the uptake of glucose by Micromonospora eichinospora (ATCC 15837). METHODS AND RESULTS: The specific rate of glucose uptake was found to be reduced when organic nitrogen components were present in the medium. Radioactive uptake studies revealed that the Km for glucose in this organism was 53 mm, indicating a low affinity for uptake compared with other actinomycete sugar transport systems. Individual amino acids negatively influenced the rate of glucose transport, suggesting a relationship between amino acid metabolism and glucose uptake in this organism. The sugar transport system was found to be an active process being inhibited by ionophores and KCN. CONCLUSIONS: The data suggest a direct link between amino acid metabolism and glucose uptake at the level of sugar transport. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that the uptake of glucose, a major carbon source for many antibiotic fermentations, is significantly reduced in the presence of amino acids. This fact should inform the medium design and feeding regimes of fermentations involving similar actinomycetes.     

Monitoring hybridoma metabolism in continuous suspension culture at the intracellular level

A model mouse hybridoma cell line was grown in continuous culture experiments in a serum-free low-protein lipid-free medium. The steady-state responses of cell numbers, extra- and intracellular metabolite concentrations, substrate and (by) product consumption/production rates, and yield coefficients were investigated as a function of step changes in the glutamine concentration of the feed medium. In addition to the commonly performed analysis of metabolites in culture supernatants, we prepared perchloric acid extracts of cells and determined the amount and the composition of intracellular amino acids and organic acids. Significant differences were found with respect to intracellular metabolite pools for cells growing at nearly identical specific growth rates. To our knowledge this is the first time that data on the intracellular concentrations (pools) of amino acids and Krebs cycle intermediates are reported in the literature that were obtained under carefully defined culture conditions such as those attained in continuous culture experiments.     

Stimulation of amino acid accumulation in neuroblastoma and astrocytoma cells byl-histidine

Uptake of amino acids by cultured neuroblastoma and astrocytoma cells was studied in the presence and absence ofl-histidine. Intracellularly accumulated histidine was assumed to induce accumulation of radioactively labeled amino acids from medium by means of exchange transport. Neuroblastoma cells accumulated more histidine than astrocytoma cells and were more sensitive to the enhancement of the uptake of other large neutral amino acids by histidine. Histidine also increased glutamic acid uptake in astrocytoma cells, but reduced it in neuroblastoma cells. The greatest differences between the cell lines in amino acid uptake without histidine were found with acidic amino acids (astrocytoma cells accumulated them more than neuroblastoma cells) and with taurine (the reverse was found). The uptake and exchange mechanisms for some neutral and acidic amino acids may thus be dissimilar in the plasma membranes of cultured cells of neuronal and glial origin.     

Formation and distribution of free amino acids in Cunninghamella elegans mycelium during growth on media with glucose and dodecane]

The growth, assimilation of organic nitrogen compounds, and production and distribution of free amino acids were studied during the growth of Cunninghamella elegans (--) 1204 on the defined medium 12 containing glucose and dodecane and without an additional carbon source (control). The culture utilized all leucine, glycine, and asparagine after three days of the growth, irrespective of the source of carbon in the medium. Glutamic acid was assimilated only in the presence of glucose. Some 15 free amino acids and 2 amides were detected in the cells, and also some correlations in the composition of the pool in the presence of different sources of carbon in the medium. The substitution of dodecane for glucose decelerates metabolism of free intracellular amino acids and stimulates their accumulation in the cells, especially if there is a deficiency of carbon sources: the overall content of amino acids and amides on the medium with glucose is from 2.2 to 4.1 mg/g; with dodecane, from 9.8 to 16.5 mg/g; in the control, from 16.4 to 25.8 mg/g.     

Metabolic flux rearrangement in the amino acid metabolism reduces ammonia stress in the α1-antitrypsin producing human AGE1.HN cell line

This study focused on metabolic changes in the neuronal human cell line AGE1.HN upon increased ammonia stress. Batch cultivations of α(1)-antitrypsin (A1AT) producing AGE1.HN cells were carried out in media with initial ammonia concentrations ranging from 0mM to 5mM. Growth, A1AT production, metabolite dynamics and finally metabolic fluxes calculated by metabolite balancing were compared. Growth and A1AT production decreased with increasing ammonia concentration. The maximum A1AT concentration decreased from 0.63g/l to 0.51g/l. Central energy metabolism remained relatively unaffected exhibiting only slightly increased glycolytic flux at high initial ammonia concentration in the medium. However, the amino acid metabolism was significantly changed. Fluxes through transaminases involved in amino acid degradation were reduced concurrently with a reduced uptake of amino acids. On the other hand fluxes through transaminases working in the direction of amino acid synthesis, i.e., alanine and phosphoserine, were increased leading to increased storage of excess nitrogen in extracellular alanine and serine. Glutamate dehydrogenase flux was reversed increasingly fixing free ammonia with increasing ammonia concentration. Urea production additionally observed was associated with arginine uptake by the cells and did not increase at high ammonia stress. It was therefore not used as nitrogen sink to remove excess ammonia. The results indicate that the AGE1.HN cell line can adapt to ammonia concentrations usually present during the cultivation process to a large extent by changing metabolism but with slightly reduced A1AT production and growth.     

Effects of dissolved oxygen levels and the role of extra- and intracellular amino acid concentrations upon the metabolism of mammalian cell lines during batch and continuous cultures

The effects of dissolved oxygen and the concentration of essential amino acids upon the metabolism of two mammalian cell lines (rCHO producing human active (t-PA) and a mouse-mouse hybridoma) were investigated in batch, chemostat, and perfusion cultures. Intracellular amino acid concentrations were measured for both cell lines during repeated batch cultures and the K_S-values for the essential amino acids were calculated using Monod equations via computer simulation. The K_S-values were in the range of 10 mmol L⁻¹ and the pool of most intracellular amino acids remained constant at about 10–100 fold higher in concentration than in the medium. No significant differences were observed between the hybridoma and CHO cell. The specific nutrient uptake rates corresponded with the cell specific growth rate and the effects of reduced dissolved oxygen concentrations only became evident when the DO dropped below 5% of air saturation (critical concentration below 1%). Nevertheless, a correlation between nutrient concentration and specific oxygen uptake was detected. This revised version was published online in June 2006 with corrections to the Cover Date.     

Mechanisms of transport of p-borono-phenylalanine through the cell membrane in vitro

The mechanisms of transport of p-(dihydroxyboryl)-phenylalanine (BPA) through the cell membrane were investigated in vitro, evaluating especially the systems responsible for the transport of neutral amino acids as potential carriers for BPA. Rat 9L gliosarcoma cells and Chinese hamster V79 cells were exposed to BPA under controlled conditions and in a defined medium that was free of amino acids. The time course of (10)B (delivered by BPA) uptake and efflux was measured under different conditions. To analyze the intracellular boron content, direct-current plasma atomic emission spectroscopy (DCP-AES) was used after separating the cells from extracellular boron in the cell medium using an oil filtration technique. The dependence of factors such as cell type, temperature, composition and concentration of amino acids and specific substrates for amino acid transport systems in the culture medium or in intracellular compartments on BPA uptake and efflux were studied. The results of this study support the hypothesis that BPA is transported by the L system and that transport can be further stimulated by amino acids preaccumulated in the cell by either the L or A amino acid transport system. Copyright [bj54] by Radiation Research Society     

Regulation of amino acid transport in growing cells of Streptomyces hydrogenans

(1) The active uptake of different amino acids by growing cells of Streptomyces hydrogenans was shown to be correlated with the physiological age of the cells. During the lag phase of growth the transport capacity increased and attained its highest level when the growth rate was maximum. During further growth the transport capacity declined progressively. The lowest transport activity was observed when the culture shifted into the stationary growth phase. (2) Such modulation of transport capacity was independent on the presence or absence of amino acids in the growth medium of the cells. (3) The size and the composition of the pool of free intracellular amino acids was also undergoing substantial variations during the growth cycle of the culture. In the lag phase, the levels of all amino acids decreased markedly and attained their lowest values at the end of this phase. During further growth the pool size was slowly replenished. (4) Removal of the pool resulted in a considerable gain of transport capacity. Therefore, it was concluded that active amino acid transport in growing Streptomyces hydrogenans is under feedback control by intracellular amino acids. (5) Quantitatively, the modulation of the pool size could not fully account for the variation of the transport capacity. Since a pool-independent stimulation of transport was found to be correlated with the increase of the growth rate of the cells, the possibility is discussed that the stimulation of transport is either due to increased levels of distinct RNA species, which might provide positive feedback signals for transport, or by increased rates of de novo synthesis of transport limiting proteins.List of Abbreviations AIB 2-aminoisobutyric acid - CM complete medium - MM mineral medium     

Landscape patterns of free amino acids in arctic tundra soils

Concentrations of free amino acids were measured in soils from four major ecosystem types in arctic Alaska. Total free amino acid concentrations were several-fold higher than ammonium (the major form of inorganic nitrogen) in water extracts of soils. The dominant free amino acids in these soils were glycine, aspartic acid, glutamic acid, serine, and arginine. Concentrations of total amino acids ranged 5-fold across communities, being highest in tussock tundra and lowest in wet meadows. Incubation experiments indicate that the turnover of amino acids is rapid, which suggests high rates of gross nitrogen mineralization in these soils. The high concentrations and dynamic nature of soil free amino acids suggest that this nitrogen pool is a significant component of nitrogen cycling in these tundra ecosystems.     

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.