The effects of amino acids on albumin synthesis by the isolated perfused rat liver

Albumin synthesis was measured in the isolated perfused rat liver by using the livers of both well-fed and starved rats. Starvation markedly decreased albumin synthesis. The livers from starved rats were unable to increase synthesis rates after the addition to the perfusates of single amino acids or the addition of both glucagon and tryptophan. Arginine, asparagine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan and valine, added together to ten times their normal peripheral blood concentrations, restored synthesis rates to normal. The plasma aminogram (i.e. the relative concentrations, of amino acids) was altered by depriving rats of protein for 48h. The use of blood from the deprived rats as perfusate, instead of normal blood, decreased albumin synthesis rates significantly by livers obtained from well-fed rats. The addition of single amino acids, including the non-metabolizable amino acid, alpha-aminoisobutyric acid, to the above mixture increased albumin synthesis rates to normal values. It is concluded that amino acids play an important role in the control of albumin synthesis and that more than one mechanism is probably involved.     

THE EFFECTS OF PHENYLALANINE ON AMINO ACID METABOLISM AND PROTEIN SYNTHESIS IN BRAIN CELLS IN VITRO1

Incubation of brain cell suspensions with 14 mM-phenylalanine resulted in rapid alterations of amino acid metabolism and protein synthesis. Both thc rate of uptake and the final intracellular concentration of several radioactively-labelled amino acids were decreased by high concentrations oi phenylalanine. By prelabelling cells with radioactive amino acids, phenylalanine was also shown to effect a rapid loss of the labelled amino acids from brain cells. Amino acid analysis after the incubation of the cells with phenylalanine indicated that several amino acids were decreased in their intracellular concentrations with effects similar to those measured with radioisotopic experiments (large neutral > small and large basic > small neutral > acidic amino acids). Although amino acid uptake and efflux were altered by the presence of 14 mwphenylalanine, little or no alteration was detected in the resulting specific activity of the intracellular amino acids. High levels of phenylalanine did not significantly altcr cellular catabolism of either alanine, lysine, leucine or isoleucine. As determined by the isolation of labcllcd aminoacyl-tRNA from cells incubated with and without phenylalanine, there was little or no alteration in the level of this precursor for radioactive alanine and lysine. There was, however, a detectable decrease in thc labelling of aminoacyl-tRNA for leucine and isoleucine. Only aftcr correcting for the changes of the specific activity of the precursors and thcir availability to translational events, could the effects of phenylalanine on protein synthesis be established. An inhibition of the incorporation into protein for each amino acid was approximately 20%.     

Nutrient media for plant parasitic nematodes. V. Amino acid nutrition of Aphelenchoides rutgersi

Nutrition of axenically cultured A. Rutgersi was investigated by deletion and addition of various levels of essential amino acids. Reproduction was lacking when isoleucine, leucine, methionine, phenylalanine, threonine, histidine, tryptophan, and lysine were individually deleted from M-10. Normal reproduction was observed over a range of concentrations but declined to nothing at still higher concentrations of tryptophan and histidine. On the basis of these tests and analyses of both the nematode and host tissue, M-12 was devised and tested. The amino acid group of M-12 contained 7 fewer amino acids and concentrations of another 15 amino acids were adjusted; but no significant differences in reproduction occurred.     

The effects of chronic hyperphenylalaninaemia on mouse brain protein synthesis can be prevented by other amino acids.

A prolonged elevation in the concentrations of circulating phenylalanine was maintained in newborn mice by daily injections of phenylalanine and a phenylalanine hydroxylase inhibitor, alpha-methylphenylalanine. The result of this chronic hyperphenylalaninaemia was an accumulation of vacant or inactive monoribosomes that persisted for 18 h of each day. An elongation assay in vitro with brain postmitochondrial supernatants demonstrated that, in addition, there was an equally prolonged decrease in the rates of polypeptide-chain elongation by the remaining brain polyribosomes. Analyses of the free amino acid composition in the brains of hyperphenylalaninaemic mice showed a loss of several amino acids from the brain, particularly the large, neutral amino acids, which are co- or counter-transported across plasma membranes with phenylalanine. When a mixture of these amino acids (leucine, isoleucine, valine, threonine, tryptophan, tyrosine, methionine) was injected into hyperphenylalaninaemic mice, there was an immediate cessation of monoribosome accumulation in the brain and there was no inhibition of the rates of polypeptide-chain elongation. Although the concentrations of the large, neutral amino acids in the brain were partially preserved by treatment of hyperphenylalaninaemic mice with the amino acid mixture, the elevated concentrations of phenylalanine remained unaltered. The amino acid mixture had no detectable effect on brain protein synthesis in the absence of the hyperphenylalaninaemic condition.     

The effect of amino acid composition of serum-free medium on DNA synthesis in primary hepatocyte cultures in the presence of epidermal growth factor

Summary The presence of optimal nutritional elements in cell culture medium is very important in studies of cultured cells. For this reason, several researchers have experimented with adding or increasing the concentration of one or more amino acids to the medium they were using to determine “essential” amino acids and optimal concentrations. We studied how leaving out one amino acid at a time from Dulbecco’s modified Eagle’s medium would affect epidermal growth factor-induced DNA synthesis in primary hepatocytes of the rat. Our “modified” DMEM contained only eight amino acids: arginine, cysteine, isoleucine, leucine, lysine, phenylalanine, tryptophan, and valine. Proline was found to be an essential amino acid in normal DMEM but not in the modified DMEM, and some other amino acids reduced DNA synthesis in this medium. This study showed that perhaps no single amino acid such as proline can be called “essential,” but rather an optimal balance of amino acids is required for each major function of each cell type cultured.     

Protein content and amino acids composition of bee-pollens from major floral sources in Al-Ahsa,eastern Saudi Arabia

Protein content and amino acids composition of bee-pollens from major pollen floral sources in Al-Ahsa, Saudi Arabia were determined to investigate the nutritive value of pollen protein relative to requirements of honeybees and adult humans. The major pollen sources were alfalfa, date palm, rape, summer squash, and sunflower. Bee-pollens from alfalfa and date palm showed high content of crude protein and amino acid concentrations. Bee-pollen from sunflower had low content of those components. Eighteen amino acids were found in bee-pollens from the five major floral sources. The highest concentrations of individual amino acids valine, leucine, isoleucine, phenylalanine and proline were obtained from alfalfa bee-pollen; lysine, arginine, cysteine, tryptophan and tyrosine from date palm; methionine, histidine, glycine and alanine from summer squash; threonine, serine and glutamic acid from sunflower; and aspartic acid from rape bee-pollen. The amino acid composition obtained from sunflower bee-pollen showed the lowest concentrations of the essential amino acids: isoleucine, leucine, methionine, phenylalanine and valine. Apart from methionine, arginine and isoleucine, the essential amino acids of bee-pollen from alfalfa, date palm, summer squash and rape exceeded the honeybees’ requirements. Methionine was the limiting amino acid in bee-pollens from the five selected sources. Concentrations of essential amino acids in the tested bee-pollens were variable and significantly correlated to their botanical origin of pollen. Bee-pollens from alfalfa, date palm and summer squash was found to be rich source of protein and amino acids for bees and for humans.     

Rapid repletion of brain serotonin in malnourished corn-fed rats following L-tryptophan injection.

The concentration of tryptophan in serum, and the levels of tryptophan, serotonin (5-HT), and 5-hydroxyindole-acetic acid (5-HIAA) in brain are substantially reduced in rats that consume for 6 weeks a diet in which corn is the only source of protein. Single injections of L-tryptophan (25, 50, or 100 mg/kg) cause dose-related increases in brain tryptophan, 5-HT, and 5-HIAA in corn-fed animals. At each dose, brain tryptophan content rises to a proportionately greater extent in corn-fed rats than in well-nourished controls, even though serum tryptophan concentrations attain higher levels in controls. This difference may reflect the greatly reduced serum concentrations in corn-fed rats of other large neutral amino acids that compete with tryptophan for uptake into the brain (tyrosine, phenylalanine, leucine, isoleucine, and valine). However, the substantial decrease in serum albumin levels also diminishes the binding of tryptophan to serum albumin; thus it is not yet possible to state which of these changes is responsible for the much greater increments in brain tryptophan observed in corn-fed rats after tryptophan injection. The fact that tryptophan administration rapidly restores brain 5-hydroxyindole levels in corn-fed animals suggests that the reductions in 5-HT and 5-HIAA levels associated with this type of malnutrition may be largely caused by inadequate availability of substrate.     

INCREASE IN LARGE NEUTRAL AMINO ACID TRANSPORT INTO BRAIN BY INSULIN

The administration of oral glucose to fasted rats produced a decline of all large neutral amino acid levels in serum, including that of the free fraction of tryptophan. In addition to this well known effect, it also decreased the brain concentrations of leucine, isoleucine and valine, while increasing those of tryptophan, tyrosine and phenylalanine. The total concentration of large neutral amino acids in serum was decreased by 44%, while it was slightly increased in brain. Analogous results were obtained in 4 rats injected with exogenous insulin. Moreover, the administration of either glucagon or isoproterenol to rats force-fed with glucose produced a decline in total serum tryptophan concentration proportional to that of the rise in FFA, while it increased free serum tryptophan and brain tryptophan levels. It can be concluded that insulin stimulates the transport of large neutral amino acids from blood to brain and that the level of free serum tryptophan also controls the entry of tryptophan into the brain under the influence of insulin.     

THE INFLUENCE OF HIGH PHENYLALANINE AND TYROSINE ON THE CONCENTRATIONS OF ESSENTIAL AMINO ACIDS IN BRAIN

—High circulating levels of phenylalanine caused depletions of threonine, valine, methionine, isoleucine, leucine, histidine, tryptophan, and tyrosine in immature and adult rat brains. The branched-chain amino acids were most affected. Their reductions ranged between 38–64 per cent of control values when phenylalanine was administered either parenterally or in the diet. The pattern of cerebral amino acid depletions found in phenylalanine-injected infant rats was similar to that of the adults. Phenylalanine loading caused depletions in serum amino acid levels in adult rats, but in infant rats the serum levels were either unchanged or somewhat elevated. Tyrosine, when administered to adult rats either parenterally or via the diet, caused cerebral depletions in essential amino acids, but the depletions were not as striking as with phenylalanine. In both the infant and adult rat, brain-blood ratios of most of the essential amino acids were significantly reduced by phenylalanine loading.     

Development of improved defined media for Clostridium botulinum serotypes A, B, and E.

The minimal nutritional growth requirements were determined for strains Okra B and Iwanai E, which are representatives of groups I and II, respectively, of Clostridium botulinum. These type B and E strains differed considerably in their nutrient requirements. The organic growth factors required in high concentrations by the Okra B strain (group I) were arginine and phenylalanine. Low concentrations (less than or equal to 0.1 g/liter) of eight amino acids (methionine, leucine, valine, isoleucine, glycine, histidine, tryptophan, and tyrosine) and of five vitamins (pyridoxamine, p-aminobenzoic acid, biotin, nicotinic acid, and thiamine) were also essential for biosynthesis. The 10 required amino acids could be replaced by intact protein of known composition by virtue of the bacterium's ability to synthesize proteases. Glucose or other carbohydrates were not essential for Okra B, although they did stimulate growth. Quantitatively, the most essential nutrients for Okra B were arginine and phenylalanine. In contrast, the nonproteolytic strain, Iwanai E (group II), did not require either arginine or phenylalanine. It required glucose or another carbohydrate energy source for growth and did not utilize arginine or intact protein as a substitute source of energy. Iwanai E utilized ammonia as a nitrogen source, although growth was stimulated significantly by organic nitrogenous nutrients, especially glutamate and asparagine. Iwanai E also required biosynthesis levels of seven amino acids (histidine, isoleucine, leucine, tryptophan, tyrosine, valine, and serine), adenine, and six vitamins (biotin, thiamine, pyridoxamine, folic acid, choline, and nicotinamide). Calcium pantothenate also stimulated growth. On the basis of the nutritional requirements, chemically defined minimal media have been constructed for C. botulinum serotypes A, B, E, and F (proteolytic).(ABSTRACT TRUNCATED AT 250 WORDS)     

Translation kinetics in cultured mouse hepatoma cells. Regulation of albumin synthesis by amino acids

The synthesis of albumin in the liver has been shown to correlate with the availability of essential amino acids in the diet. We have investigated this phenomenon in the highly differentiated mouse hepatoma cell line, Hepa. Cells were grown for three days in complete medium with daily changes. The cells were then incubated for 22 h in media containing varying concentrations of individual essential amino acids. The deficient media were then changed; 1.5 h later the cells were labeled for 0.5 h with [3H]leucine. Albumin was immunoprecipitated and total protein was acid-precipitated from postribosomal supernatants of detergents-solubilized cells. With the exception of isoleucine, the relative rates of albumin synthesis decreased as a function of amino acid concentration from 4.3% in complete medium to 2.5% in totally deficient media. This specific reduction in albumin synthesis was confirmed by analysis of labeled Hepa proteins displayed on sodium dodecyl sulfate/polyacrylamide gels. Essential amino acid limitation reduced total protein synthesis by 50%. This is the result of a decrease in the translation efficiency of total mRNA from 5 to 3 polypeptides/message min-1 and is consistent with a reduction in the initiation rate. In contrast, the 70% decrease in albumin synthesis was a result of a reduced number of functional albumin messages/cell. The translation efficiency of these albumin messages remained unchanged at 1.     

Correlation between brain tryptophan and plasma neutral amino acid levels following food consumption in rats

Brain tryptophan increases significantly within two hr of the time that rats begin to consume a diet containing carbohydrate and fat, but fails to rise if the diet also contains 18–24% protein. The effects of particular diets on brain tryptophan are not well correlated with plasma tryptophan concentrations alone, but do correlate well with the ratio of plasma tryptophan to individual neutral amino acids (leucine, isoleucine, valine, tyrosine, phenylalanine) or to their sums. (These amino acids compete with tryptophan for uptake into the brain.) Carbohydrate ingestion raises brain tryptophan by elevating plasma tryptophan and depressing the plasma levels of the competing neutral amino acids; protein consumption prevents an increase in brain tryptophan by raising the plasma concentrations of the competing amino acids more than of tryptophan.     

Regulation of Chlamydia psittaci (strain guinea pig inclusion conjunctivitis) growth in McCoy cells by amino acid antagonism

Chlamydiae have amino acid requirements for growth in tissue culture as defined by those amino acids whose individual omission from the growth medium prevents chlamydial multiplication. We have tested the hypothesis that this inhibition of growth arises as a result of antagonism between particular amino acids such that inhibition occurs when the concentration of one amino acid is reduced in the presence of the antagonist amino acid at high concentration. Using the Chlamydia psittaci strain guinea pig inclusion conjunctivitis (GPIC), in the presence of cycloheximide, the requirement for valine was abrogated by the simultaneous omission of isoleucine, that for phenylalanine by simultaneous omission of tryptophan and that for leucine by simultaneous omission of isoleucine plus valine. The antagonism shown between leucine and isoleucine plus valine appears to be unique among bacteria. In the absence of cycloheximide, GPIC had an additional need for tryptophan, tyrosine and isoleucine; these amino acid requirements were shown for both infected McCoy, HeLa and BHK cells. The results are consistent with a mechanism for regulation of parasite growth which depends on the balance of amino acid concentrations in the extracellular environment.     

Albumin synthesis and catabolism following partial hepatectomy in the rat. The effects of amino acids and adrenocortical steroids on albumin synthesis after partial hepatectomy.

Plasma albumin levels were measured in partially hepatectomized, sham operated and control rats. The levels fell in both the partially hepatectomized and sham operated groups; while the latter group returned to normal within a few days, the low plasma albumin in the partially hepatectomized animals was sustained. Albumin synthesis rates in the isolated perfused rat liver were measured in the three groups of animals at varying intervals after partial hepatectomy. There was a significant depression of albumin synthesis rate in terms of both liver and whole animal weights when compared to the sham operated and control animals. This depression was almost completely reversed by the addition of arginine, asparagine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan and valine added together to 10 times their normal plasma concentrations. The addition of hydrocortisone had no effect on the albumin synthesis rate after partial hepatectomy. Studies in vivo in the three groups of animals (partially hepatectomized, sham operated and control animals) revealed a fall in the albumin catabolic rate after partial hepatectomy coinciding with the fall in the albumin synthesis rate. An hypothesis whereby the amino acids may have their stimulatory effect is proposed.     

Substrate-dependent regulation of intracellular amino acid concentrations in cultured bovine aortic endothelial cells

Amino acid deprivation induces adaptive changes in amino acid transport and the intracellular amino acid pool in cultured cells. In this study intracellular amino acid levels were determined in cultured bovine aortic endothelial cells (EC) deprived of L-arginine or total amino acids for 1, 3, 6 and 24 h. Amino acid concentrations were analyzed by reverse phase HPLC after precolumn derivatisation. Under normal culture conditions levels of L-arginine L-citrulline, total essential and non-essential amino acids were 840 +/- 90 microM, 150 +/- 40 microM, 11.4 +/- 0.9 mM and 53.3 +/- 3.4 mM (n = 9), respectively. In EC deprived of L-arginine or all amino acids for 24 h L-arginine and L-citrulline levels were 200 microM and 50 microM, and 670 microM and 100 microM Deprivation of L-arginine or total amino acids induced rapid (1 h) decreases (30 - 50%) in the levels of other cationic (lysine, ornithine) and essential branched-chain (valine, isoleucine, leucine) and aromatic (phenylalanine, tryptophan) amino acids. L-glutamine was reduced markedly in EC deprived of total amino acids for 1 h - 6 h but actually increased 3-fold in EC deprived of L-arginine for 6 h or 24 h. Arginine deprivation resulted in a rapid decrease in the total intracellular amino acid pool, however concentrations were restored after 24 h. Increased amino acid transport and/or reduced protein synthesis may account for the restoration of amino acid levels in EC deprived of L-arginine. The sustained reduction in the free amino acid pool of EC deprived of all amino acids may reflect utilization of intracellular amino acids for protein synthesis.     

Development of improved defined media for Clostridium botulinum serotypes A, B, and E

The minimal nutritional growth requirements were determined for strains Okra B and Iwanai E, which are representatives of groups I and II, respectively, of Clostridium botulinum. These type B and E strains differed considerably in their nutrient requirements. The organic growth factors required in high concentrations by the Okra B strain (group I) were arginine and phenylalanine. Low concentrations (less than or equal to 0.1 g/liter) of eight amino acids (methionine, leucine, valine, isoleucine, glycine, histidine, tryptophan, and tyrosine) and of five vitamins (pyridoxamine, p-aminobenzoic acid, biotin, nicotinic acid, and thiamine) were also essential for biosynthesis. The 10 required amino acids could be replaced by intact protein of known composition by virtue of the bacterium's ability to synthesize proteases. Glucose or other carbohydrates were not essential for Okra B, although they did stimulate growth. Quantitatively, the most essential nutrients for Okra B were arginine and phenylalanine. In contrast, the nonproteolytic strain, Iwanai E (group II), did not require either arginine or phenylalanine. It required glucose or another carbohydrate energy source for growth and did not utilize arginine or intact protein as a substitute source of energy. Iwanai E utilized ammonia as a nitrogen source, although growth was stimulated significantly by organic nitrogenous nutrients, especially glutamate and asparagine. Iwanai E also required biosynthesis levels of seven amino acids (histidine, isoleucine, leucine, tryptophan, tyrosine, valine, and serine), adenine, and six vitamins (biotin, thiamine, pyridoxamine, folic acid, choline, and nicotinamide). Calcium pantothenate also stimulated growth. On the basis of the nutritional requirements, chemically defined minimal media have been constructed for C. botulinum serotypes A, B, E, and F (proteolytic).(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential amino acid requirements for sporulation in Bacillus subtilis

The amino acid requirements for sporulation were studied by use of auxotrophic mutants of Bacillus subtilis 168. Cells were grown to T(0) in medium containing the test amino acid and were then transferred to a minimal medium lacking that amino acid. Omission of leucine caused no reduction in sporulation. Omission of methionine, lysine, and phenylalanine appeared to cause reduced levels of sporulation, and sporulation was completely inhibited when isoleucine, tryptophan, and threonine were omitted. The amino acids in this third class showed a sequence of requirements, with tryptophan required earlier than isoleucine, which in turn was required earlier in the sporulation process than threonine. Isoleucine omission did not affect the early sporulation functions of extracellular protease formation or septum formation, but prevented the increased levels of protein synthesis and oxygen consumption that normally accompany early sporulation stages. Isoleucine did not appear to be metabolized to other compounds in significant amounts during sporulation. The role of isoleucine in the sporulation process remains unclear.     

Variation in the free amino acid content of skeletal muscle of Ophicephalus punctatus (Bloch)

The skeletal muscle of Ophicephalus punctatus contains nine essential free amino acids, arginine, histidine, isoleucine, leucine, methionine, phenylalanine, threonine, valine and lysine, and eight non-essential amino acids, alanine, aspartic acid, cystine, glutamic acid, glycine, tyrosine, proline and serine. Histidine and lysine dominated the free amino acids pool. Seasonal variation was detected in the levels of histidine, arginine, leucine, phenylalanine, glycine, cystine and serine with highest values occurring in April and again in November. Changes were also detected in the concentrations of certain amino acids as the fish grew in size. Levels of free amino acids did not significantly differ between sexes. Factors effecting variation are discussed.     

β-Adrenergic Effects on Plasma and Brain Large Neutral Amino Acids Are Unaltered by Chronic Administration of Antidepressants

Effects of isoproterenol (3 mg kg-1, i.p. for 60 min) and salbutamol (3, 10 mg kg-1, i.p. for 60 min) on large neutral amino acid concentrations in rat plasma and brain were assessed. Phenylalanine, leucine, isoleucine, and valine were measured by gas chromatography with electron-capture detection; tyrosine and tryptophan were measured by HPLC with electrochemical detection. These drugs induced increases in brain tryptophan, tyrosine, phenylalanine, and valine and decreases in plasma tryptophan, tyrosine, leucine, isoleucine, and valine. Effects of salbutamol (3 mg kg-1, i.p. for 60 min) were assessed following chronic administration of phenelzine sulfate and desipramine.HCl (each drug 10 mg kg-1 per day, s.c. via Alzet 2ML4 osmotic minipumps for 28 days). There were no effects of these antidepressants on basal levels of large neutral amino acids in brain and plasma. In both brain and plasma, salbutamol-induced changes in large neutral amino acids were unaffected by these antidepressants. The results indicate that beta-adrenoceptor-regulated availability of plasma and brain large neutral amino acids is unaffected by chronic administration of tricyclic or monoamine oxidase inhibitor antidepressants.     

Alanine and glutamine synthesis and release from skeletal muscle. II. The precursor role of amino acids in alanine and glutamine synthesis.

The synthesis and release of alanine and glutamine have been studied in the intact rat epitrochlaris skeletal muscle preparation. Aspartate, cysteine, leucine, valine, methionine, isoleucine, serine, theronine, and glycine increased significantly the formation and release of alanine from muscle. Cysteine, leucine, valine, methionine, isoleucine, tyrosine, lysine, and phenylalanine increased the rate of glutamine synthesis. Only ornithine, arginine, and tryptophan were without effect on the synthesis of either alanine or glutamine. Half-maximal stimulation of alanine and glutamine formation by added amino acids was observed with concentrations ranging between 0.5 and 1.0 mM. Increases in alanine and glutamine formation were not accompanied by changes in pyruvate production or glucose uptake. The progressive decline in alanine and glutamine synthesis noted on prolonged incubation was prevented by the addition of amino acids to the incubation medium. Stimulation of alanine synthesis by added amino acids was unaffected by inhibition of glycolysis with iodoacetate. Inhibition of alanine aminotransferase with aminooxyacetate significantly decreased alanine formation. Pyruvate and ammonium chloride did not increase further the rate of either alanine or glutamine formation above that produced by added amino acids. These data indicate that most amino acids are precursors for alanine and glutamine synthesis in skeletal muscle. A general mechanism is presented for the de novo formation of alanine from amino acids in skeletal muscle, and the importance of proteolysis for the supply of amino acid precursors for alanine and glutamine synthesis is discussed.