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Insulin exposure stimulates an increase in glycerol-3-phosphate dehydrogenase (G3PDH) activity in isolated human lymphocytes that correlates to an increase in G3PDH mRNA and requires new protein synthesis. Synthetic diacylglycerol or phorbol ester can mimic the effect of insulin on G3PDH activity, suggesting that protein kinase C may be involved in regulation of G3PDH levels. In addition, lithium chloride, an inositol phosphate phosphatase inhibitor, and calcium uptake inhibitors can abolish insulin stimulation of G3PDH activity. For obese subjects in whom insulin resistance in vitro can be demonstrated, the extent of insulin stimulation of G3PDH activity is decreased compared to normal weight individuals, and treatment by a very low calorie diet restores insulin stimulation of G3PDH activity. Thus, insulin stimulation of G3PDH activity is dependent upon the metabolic state of the subject from whom the cells are obtained.

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Sulfite has been identified as an essential metabolite by means of growth studies using a chemically-defined, protein-free medium for culture of human peripheral lymphocytes. Sulfite reduced the amount of cysteine required for optimum growth by at least four-fold. In some subjects, sulfite stimulated growth even in the presence of optimal amounts of cysteine indicating that lymphocytes of some individuals are unable to convert cysteine to sulfite in adequate amounts.

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Quantitative growth responses of lymphocytes directly isolated from individual subjects in a newly developed chemically-defined, protein-free medium are used to demonstrate that supplements of both L-asparagine and a purine source, but neither alone, significantly reduce the quantitative requirement for L-glutamine for growth. This system is useful for exploring individual differences in quantitative glutamine requirements and adequacy of asparagine and purine biosynthesis.

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At concentrations greater than approximately 0.5 microM, dethiobiotin can cause the bioassay for biotin, which employs Lactobacillus plantarum, to over value the actual biotin level. This can be as much as 30-fold at 10 microM DL-dethiobiotin and 5 pM biotin. Dethiobiotin does this by exerting a sparing effect on the biotin response by the assay organism. We demonstrate one way to determine the actual biotin concentration in the presence of interfering levels of dethiobiotin.

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A chemically defined, protein-free medium (designated CFBI 1000, where CFBI = Clayton Foundation Biochemical Institute) that supports human peripheral lymphocyte proliferation has been developed. This medium allows exploration of individual metabolic differences by varying the medium composition as well as providing a base to explore further the mechanisms of lymphocyte activation in a system initially free of added macromolecular species other than mitogen. The peripheral blood lymphocyte is an ideal system for metabolic studies because it is easily obtained, is a primary resting cell that can be activated to proliferate, and presumably reflects both the genetic makeup and biochemical environmental history of the individual at the time the cells were formed. Examination of the role of various factors in lymphocyte activation and subsequent events may be simplified by the utilization of a medium that is protein-free and chemically defined. The CFBI 1000 medium supports the growth response of human peripheral lymphocytes to mitogen as measured by [3H]thymidine incorporation to an extent comparable to other media used widely in assessment of lymphocyte proliferation.

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In this study cells were grown in 34S-sulfate or L-[sulfane-34S]thiocystine, and the effects of unlabeled methionine and cystine on incorporation of sulfur into methionine, cystine and thiamin were determined. Unlabeled methionine effectively suppresses the incorporation of 34S into methionine but not into cysteine or thiamin. In contrast, cystine blocks incorporation of 34S only to approximately the relative ratio of 32S to 34S indicating, that cysteine is closely related to the origin of the sulfur in thiamin, and therefore the sulfane sulfur of thiocystine is also an effective source of the thiamin sulfur.

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Two steps in the biosynthesis of biotin in Escherichia coli, incorporation of the nitrogen atom of methionine into 7-keto-8-aminopelargonic acid and of the sulfur atom into dethiobiotin, were examined. Sulfur and nitrogen metabolism were monitored by gas chromatography-mass spectrometry of volatile derivatives of internal (protein-bound) amino acids and excreted biotin. We were able to show that internal cysteine and excreted biotin were labeled to the same extent with 34S from either of two exogenous sulfur sources, 34SO4(2)-or L-[sulfane-34S]thiocystine. Internal methionine was eliminated from consideration, while cysteine, or possibly a closely related intermediate, was implicated as providing the sulfur atom for biotin biosynthesis. Also, in experiments designed to follow the metabolism of the nitrogen atom of methionine, it was found that biotin excreted into the culture medium by this organism grown with 95 atom % [15N]methionine contained greater than 70 atom % excess 15N in one of the nitrogens over that obtained from cultures grown with methionine of natural abundance 15N. These results provide evidence for the direct transfer of the methionine nitrogen as the role of S-adenosylmethionine in the conversion of 7-keto-8-aminopelargonic acid to 7,8-diaminopelargonic acid.

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The comparative ability of Escherichia coli K-12 hpb lambda-, a biotin overproducing strain, to incorporate 35S from isotopically labeled L-methionine, L-cystine, and the sulfane sulfur of thiocystine was determined. Comparison of the specific activity of sulfur in biotin produced by the organism with that of the 35S-labeled amino acids demonstrates that the sulfur of cystine is transferred to biotin with an efficiency of at least 75%, that the sulfur of methionine does not contribute to biotin significantly, and that the sulfane sulfur of thiocystine contributes approximately one-third of the sulfur to newly synthesized biotin and is essentially equivalent in utilization to that of the other two sulfur atoms in the molecule.

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A large number of iron transport agents, siderophores, which stimulated the growth of Arthrobacter flavescens JG-9, were isolated during a study of the antitumor activity associated with the metabolic products of the fungus Epicoccum purpurascens. The production of the siderophores was significantly enhanced in a variety of media by culture of the fungus in the near absence of ferric iron. A novel method of purification involving a carboxylic ion-exchange resin separated the siderophores into four subgroups. The first subgroup, which contained the majority of the activity, was subsequently resolved in a similar manner with the carboxylic resin into seven individual siderophores. Of these, two were characterized as ferricrocin and coprogen whereas the others appeared to represent new compounds. One of the latter was given the name triornicin and exhibited slight antitumor activity in mice injected with Ehrlich ascites tumor cells.

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Spectroscopic analysis of the tumor inhibitory factor triornicin produced by epicoccum purpurascens indicated that it was of similar structure to the known siderophore desferricoprogen, which is also produced by the fungus. The 1H and 13C NMR spectra indicated the replacement of an (E)-5-hydroxy-3-methyl-2-pentenoyl moiety of the desferricoprogen structure with an acetyl function. Cleavage of triornicin with basic methanol produced two fragments. The first was identified as a natural siderophore, dimerumic acid, which was also produced by basic cleavage of desferricoprogen. The second compound was identified as N alpha, N delta-diacetyl-N delta-hydroxyornithine. The structure of these fragments serves to define the structure of triornicin as a new siderophore.

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The transport of phenylalanine by the general aromatic transport system in spheroplasts of Escherichia coli 9723 has been found to be stimulated by exogenous tRNA. Neither periodate-treated tRNA nor phenylalanine-charged tRNA stimulated, and the latter inhibited, phenylalanine uptake. Among preparations of specific tRNAs, tRNAPhe and tRNATyr were effective in stimulating the uptake of phenylalanine and tyrosine, respectively, and tRNAGlu and tRNAVal gave no detectable stimulation of phenylalanine or tyrosine transport. The preparation of tRNATyr was 10 times as active as unfractionated tRNA and gave as much as 167% stimulation of tyrosine transport. Correspondingly, the preparation of tRNAPhe was at least 3.5 times as active as the unfractionated tRNA and 2.5 times as active as the preparation of tRNATyr in stimulation of phenylalanine transport. Preliminary results in fractionation of the active component of tRNA for stimulating phenylalanine uptake show that the major activity resides in minor isoacceptor(s) tRNAPhe rather than the major component tRNAPhe, and the slight activity of preparations of tRNATyr is probably due to a contamination of the active tRNAPhe. Other preliminary results indicate that this type of stimulation occurs with uptake of other amino acids and their tRNA.

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A method by which three acetohydroxy acid synthetase activities are separated from extracts of Escherichia coli 9723 has been developed. Isoleucine specifically represses synthesis of one of the enzymes, which is not sensitive to valine inhibition, and isoleucine also simultaneously enhances the production of a second activity, which is valine inhibitable. The valine-inhibitable activity is repressed by leucine and valine, a combination of which is more effective than either alone. The third acetohydroxy acid synthetase, which is more active at pH 6 than at 8, is not controlled by the branched-chain amino acids. In a mutant of E. coli 9723 selected for the ability of valine to inhibit growth, the isoleucine-repressible acetohydroxy acid synthetase activity was no longer present, but isoleucine addition still resulted in enhanced production of the valine-inhibitable activity.

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Ethanol administered to weanling rats in various concentrations as the sole drinking fluid in conjunction with a semipurified dry diet was consumed at a level of not more than 20% of total energy. Suppression of fluid intake and concomitant reduction of food intake resulted in decreased growth. Analysis of body composition indicated that the difference in weight in rats given 20% solutions of ethanol as the sole drinking fluid and in rats pair-fed diets without ethanol was primarily due to dehydration. With total liquid formulations having good resistance to separation, there were no significant differences in weight gain between rats given 20% or 30% of total energy as ethanol and rats pair-fed diets without ethanol. Ethanol fed at 40% of total energy caused inhibition of growth which could not be ascribed solely to decreased consumption of food and water. These data indicate that the method of administering ethanol to weanling rats affects not only the levels at which ethanol is toxic but also the amount of ethanol voluntarily consumed by the rats. The use of pair-feeding techniques and of homogenous liquid formulations is essential for studies of the metabolic effects of ethanol.

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