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NADP-dependent isocitrate dehydrogenase (EC 1.1.1.42) from Mycobacterium phlei ATCC 354 was purified to homogeneity by ammonium sulphate fractionation, followed by DEAE cellulose and Sephadex G-200 chromatography. The pH optimum of the enzyme was 8.5. The Km values for isocitrate and NADP were 74 and 53 microM, respectively. Mn2+ was essential for enzyme activity. The enzyme lost all activity on incubation at 70 degrees C for 15 min; isocitrate and NADP protected against this thermal inactivation. p-Chloromercuribenzoate inhibited the enzyme; pre-incubation of enzyme with isocitrate + Mn2+ prevented this inhibition. The purified enzyme showed concerted inhibition by glyoxylate + oxaloacetate and was inhibited by oxalomalate.

Uptake of radioactive tryptophan by Mycobacterium tuberculosis H(37)Rv grown in vitro and in vivo was investigated. K(m) values indicated low affinity, and sodium azide inhibited uptake. Rifampin at the minimal inhibitory concentration had no effect, whereas ethambutol inhibited uptake only in the bacilli grown under in vitro conditions. The significance of these results is discussed.

A comparative biochemical study of an aflatoxigenic strain Aspergillus parasiticus NRRL 3240 and a nonaflatoxigenic strain A. flavus NRRL 3237 was carried out in order to have a better idea of regulation of aflatoxin biosynthesis. The results obtained revealed continuous primary metabolic activity (protein synthesis) in the nonaflatoxigenic strain while the aflatoxigenic stain showed inhibition of protein and nucleic acid synthesis. The aflatoxigenic strain showed higher levels of oxygen uptake, RNA, NAD, FMN and activities of glycolytic enzymes. Furthermore, it had lower of lipids and reduced activity of glucose-6-phosphate dehydrogenase, which is a source for NADPH. The differences observed have been discussed in relation to aflatoxin biosynthesis and its regulation.

Phospholipids of Mycobacterium phlei ATCC 354 and Mycobacterium smegmatis CDC 46 consist of cardiolipin, phosphatidyl ethanolamine, tri-acylated dimannophosphoinositide, tetra-acylated dimannophosphoinositide and tetra-acylated pentamannosphosphoinositide. A comparative study of lipid patterns of M. phlei ATCC 354 and of M. smegmatis CDC 46 in relation to age of culture revealed higher total lipid level and increased activity of malate-vitamin K reductase, a phospholipid requiring enzyme, during the early logarithmic growth phase of the former. No appreciable change occurred in the latter. The high total lipid content coincides with an increase in phospholipid, brought about apparently by the increase in malate-vitamin K reductase. Changes in cardiolipin and phosphatidyl ethanolamine appeared to be unique to M. phlei ATCC 354. However, in both bacterial species, a decrease in glyceride and a progressive increase in tuberculostearic acid with a concomitant decrease in oleic acid, occurred with ageing.

Adenosine 3',5'-monophosphate (cAMP) is present in slow growing as well as fast growing mycobacteria. Apparently there does not seem to be any direct relationship between either intra- or extracellular cAMP content with the growth rate of bacilli. As compared to that of E. coli grown on a similar energy source, cAMP content is much higher in mycobacteria. cAMP content inside the cells remains unaltered throughout the growth period and this may be due to lack of complete utilization of the major energy source, glycerol. Glucose when added to the cells, suspended in phosphate buffer, caused a remarkable decrease in intracellular cAMP content, a phenomenon well established in other bacteria.

Changes in liver polyamines of rats and mice of both sexes injected with aflatoxin B1 (AFB1) were determined. AFB1 significantly enhanced liver polyamines of both susceptible and resistant animals, viz. rats and mice, respectively. Sex appears to have little influence on AFB1-mediated stimulation of liver polyamine levels. AFB1 significantly reduced liver polyamine in growing rats reflecting the inhibitory effect of this carcinogen on induced polyamine synthesis.

The effect of different combinations of aflatoxin B1, alpha-amanitin and actinomycin-D on the incorporation of orotic acid-6-14C into RNA was investigated using rat liver slices. The results support the view that the mode of action of aflatoxin B1 and alpha-amanitin are similar in some respects, and that aflatoxin B1 and actinomycin-D act according to different mechanisms. Evidence was also obtained about the formation of an active metabolite of aflatoxin B1 in this system.

Effect of feeding lysine and threonine deficient and supplemented rice diets to rats for fifteen days has been studied on the lipids of hepatic smooth (SER) and rough endoplasmic reticulum. The deficient diet reduced protein and phospholipids of SER where as it increased triglycerides contents of both SER and RER compared to rats fed the supplemented diet.

The interaction of aflatoxin B1 with a number of biologically important substances has been investigated by spectrophotometric and fluorimetric techniques. The ultra-violet absorption of tryptophan, adenosine, RNA and DNA were altered by the addition of aflatoxin B1. Some amino-acids, purine and pyrimidine derivatives, bovine-serum albumin and DNA quenched the fluorescence of aflatoxin B1. Fluorescence polarization data suggested that the interaction of aflatoxin B1 with bovine-serum albumin was stronger than that with DNA.

Effect of excess feeding and depletion of vitamin A on the ability of adipose tissue to maintain plasma free fatty acid levels has been studied in rats. Both in hypervitaminosis A (fed 9 mg of retinol for 2 consecutive days) and in vitamin A deficiency (kept on a vitamin A-deficient diet for 6 weeks) the rats showed elevated levels of plasma free fatty acids. Hypervitaminosis A caused a decrease in the fatty acid release from adipose tissue on in vitro incubation, probably due to lowered levels of cyclic AMP. On the other hand, adipose tissue from vitamin A-deficient animals showed an increased lipolytic rate as compared to that of the controls. No change in the lipogenic ability was indicated in either of the conditions as indicated by the activities of enzymes involved in this process. We conclude that the fatty acid homeostasis can be greatly influenced by the vitamin A status of the animals.

Aspergillus flavus NRRL 3251 grown on modified yeast extract-sucrose medium produced 1 mg of aflatoxin M(1) per 100 ml of medium.

The adenine nucleotide pools were studied in toxigenic (NRRL 3240) and non-toxigenic (NRRL 3537) strains of Aspergillus flavus group in relation to aflatoxin biosynthesis. The levels of ADP and AMP were comparatively low in the non-toxigenic strain throughout the growth period. In the toxigenic strain the levels of ADP and AMP increased significantly during the stationary phase. The ATP content of the toxigencic strain had higher ATP levels during the stationary phase compared to the toxigenic strain. The inorganic phosphate levels of the non-aflatoxigenic strain were higher when compared with the aflatoxigenic strain. High levels of inorganic phosphate have been reported to be inhibitory for secondary biosynthesis including aflatoxins. The energy charge values of the non-toxigenic strain, compared to the toxigenic strain, were lower during exponential phase but higher during stationary phase. The observed changes have been correlated with aflatoxin biosynthesis.

A Mg+2-(Ca+2)-activated adenosine triphosphatase activity has been demonstrated in saprophytic, human pathogenic and non-pathogenic, and atypical, species of mycobacteria. (Na+ + K+)-activated adenosine triphosphatase was absent in the species investigated. The effect of age of culture on enzyme activity was revealed in an increase up to mid logarithmic phase of growth, and a decline thereafter. Differences in oxygen tensin during growth did not alter enzyme activity. Isonicotinic acid hydrazide and streptomycin inhibited the enzyme activity.

The adenylic acid systems of Aspergillus parasiticus were studied in zinc-replete and zinc-deficient media. The adenosine 5'-triphosphate levels of the fungus were high during exponential phase and low during stationary phase in zinc-replete cultures. On the other hand, the levels of adenosine 5'-diphosphate and adenosine 5'-monophosphate were low during exponential phase of growth and high during stationary phase. The adenosine 5'-triphosphate levels during exponential phase may indicate higher primary metabolic activity of the fungus. On the other hand, high adenosine 5'-monophosphate levels during stationary phase may inhibit lipid formation and may enhance aflatoxin levels. The inorganic phosphorus content was low in a zinc-replete medium throughout the growth period, thereby favoring aflatoxin biosynthesis. The energy charge during the exponential phase was high but low during the stationary phase. In general the energy charge values were lower because of high adenosine 5'-monophosphate content.

Uptake of aflatoxin [14C]-B1 by the liver and kidney slices of different animal species clearly indicates that the transport of the toxin to different organs of the respective species is not the major cause for the reported variation in the species susceptibility to aflatoxin.

Pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from Mycobacterium smegmatis has been purified to homogeneity through a seven-step procedure with a yield of 16% and specific activity of 220 units/mg protein. The purified enzyme had a molecular weight of 230,700 and was composed of four subunits with identical molecular weights of 57,540. Analysis of amino acid composition revealed a low content of aromatic amino acids. The enzyme exhibited sigmoidal kinetics of varying concentrations of phosphoenolpyruvate, the degree of cooperativity and S0.5v value for phosphoenolpyruvate being strongly dependent on the pH of the reaction mixture. Among the nucleoside diphosphates acting as substrate for pyruvate kinase, ADP was the best phosphate acceptor, as judged by its lowest Km value. The enzyme showed an absolute requirement for divalent cations (either Mg2+ or Mn2+), but monovalent cations were not necessary for activity. Other divalent cations inhibited the Mg2+-activated enzyme to varying degrees (Ni2+ greater than Zn2+ greater than Cu2+ greater than Ca2+ greater than Ba2+). The differences in the kinetic responses of the enzyme to Mg2+ and Mn2+ are discussed.

In resting mycelia of Aspergillus parasiticus NRRL 3240, all the glycolytic intermediates with the exception of glucose and phosphoenolpyruvate stimulated the de novo synthesis of aflatoxin from exogenous acetate. Intracellular levels of glycolytic intermediates were measured in the toxigenic strain, A. parasiticus NRRL 3240, and the nontoxigenic strain, A. flavus NRRL 3537. In 48-h cultures, the concentrations of the intermediates beyond the phosphofructokinase reaction were higher in toxigenic mycelial extracts than in nontoxigenic cultures. There was a marked reduction in the specific activity of phosphofructokinase with increasing age in the toxigenic strain. The specific activity of this enzyme was two- to four-fold lower in zinc-deficient mycelia of A. parasiticus. In resting cultures, the uptake rate of labeled glucose was considerably lower in zinc-deficient mycelia than in zinc-replete or nontoxigenic cultures. On the basis of the results obtained, it can be said that intense formation of aflatoxin is associated with a sharp fall in the level of phosphoenolpyruvic and pyruvic acids. This was accompanied by a significantly higher pyruvate kinase activity.

The phospholipids and glycerides composition of spheroplasts of Mycobacterium smegmatis ATCC 14468 was examined. The percent total phospholipids in total lipids as well as cardiolipin were found to be higher in spheroplasts as compared to their parent forms. Increase in cardiolipin and free fatty acids content and decrease in triglycerides levels were observed during spheroplasts formation. The results suggest that increase in cardiolipin content in spheroplasts is an adaptational change concomitant with the loss of cell walls.

The effect of dieldrin on the bound and free activity of cathepsin, acid ribonuclease, acid phosphatase and aryl sulphatase has been investigated. Administration of dieldrin increased the activity of free cathepsin, acid ribonuclease, and acid phosphatase in rat liver whereas it did not affect the activity of aryl sulphatase. This indicates that dieldrin labilizes the lysosomes.

Glutamate dehydrogenase (aminating) and glutamine synthetase activities were assayed in Mycobacterium smegmatis following growth on various carbon and nitrogen sources. The activities (expressed as nmoles product formed/min/mg crude extract protein) of these two enzymes were higher in crude extracts from glucose-grown cells than in glycerol- or fructose-grown cells. In the presence of succinate, pyruvate, fumarate or acetate in the growth medium, both these enzyme activities were lower than those in citrate-grown cells. The glutamate dehydrogenase (GDH) activity was the same in asparagine and glutamine-grown cells. Ammonium chloride, alanine or glutamic acid, when used as nitrogen source, resulted in low GDH activity as compared to asparagine-grown cells. Glutamine synthetase activity was considerably lower (2-4 fold) when the cells were grown on alanine, glutamine, glutamic acid or ammonium chloride as the nitrogen source than those in asparagine-grown cells. Glutamate and ammonium chloride, when present in the growth medium, repressed both glutamate dehydrogenase and glutamine synthetase, though the degree of repression was small. The results suggest that only a weak transcriptional control operates for these enzyme activities in M. smegmatis.

Addition of Tween 80 to the growth medium brings about qualitative and quantitative changes in the lipids of Mycobacterium phlei ATCC 354. The results suggest that Tween 80 itself may be a variant in the system.

The effect of aflatoxin B1 on lipids of liver, kidney, adipose and plasma of rats was studied. A single dose administration (6 mg/kg body weight) increased liver and kidney weights and their total lipids within 24 h. Increase in liver lipids was confined mainly to phospholipid and cholesterol, whereas triglycerides showed a significant decrease. Adipose tissue triglycerides were, however, increased. Plasma showed decreases in triglycerides, free fatty acids and cholesterol. Incorporation studies with palmitate-1-14C revealed increased incorporation in adipose tissue lipids and decreased incorporation in liver and plasma lipids, thereby indicating an increased synthesis of lipids in adipose. Their mobilization to plasma was, however, inhibited, hence the low levels of triglyceride in liver. But the adrenals showed hypo-activity upon aflatoxin B1 administration.

Tolnaftate [2-napthyl-N-methyl-N-(m-tolyl)thionocarbamate], an antifungal drug, is widely used to control superficial fungal infections in humans and other animals. In this study the effect of tolnaftate on aflatoxin biosynthesis by Aspergillus parasiticus NRRL 3240 was investigated. Tolnaftate changed the morphology of A. parasiticus to yeastlike forms and inhibited aflatoxin formation. The formation of aflatoxin G was blocked considerably, indicating a metabolic block in the conversion of aflatoxin B to aflatoxin G. The incorporation of [1-14C]acetate into aflatoxin was significantly inhibited at a concentration of 1 mM tolnaftate. The presence of zinc in the resuspension buffer resulted in reversal of the tolnaftate-induced inhibition of aflatoxin G1 biosynthesis.

The growth and synthesis of macromolecules in spheroplasts of Mycobacterium smegmatis ATCC 14468 were found to be in a balanced state. Spheroplasts were not lysed during preparation and subsequent incubation, and were not contaminated with whole cells. Limited growth of spheroplasts was not due to depletion of medium constituents. High molar concentrations of osmotic stabilizer did not inhibit balanced growth. These studies establish that spheroplasts are metabolically active and can be used as a tool in cell wall synthesis and in genetic studies.

Sputum collected from patients with respiratory diseases were examined for presence of benzo(a)pyrene hydroxylase (BPH) activity. The human bronchial mucus used in these studies had significant capability to metabolize benzo(a)pyrene. Clarification of the sputum by agents such as N-acetylcysteine or pancreatin in presence of antibiotics was found to be essential for the detection of BPH activity. In vitro incubation of the clarified human bronchial mucus with benzoflavone caused inhibition, while 7,8-dimethyl-benzanthracene induced BPH enzyme activity.