Serodiagnostic efficiency of phospholipid associated protein of Mycobacterium tuberculosis H37Rv.

The phospholipid-associated protein (55-67 kDa) fraction of Mycobacterium tuberculosis H37Rv was purified as the DE-V protein fraction. This DE-V fraction was used for diagnosis of tuberculosis by enzyme-linked immunosorbent assay (ELISA), detecting IgG antibody in sera collected from different categories of tuberculosis patients, i.e. with acid fast bacilli (AFB) culture-positive pulmonary tuberculosis, with AFB culture-negative, but radiologically suspected, pulmonary tuberculosis, extrapulmonary tuberculosis, and control groups of patients suffering from diseases other than tuberculosis (asthma and/or rhinitis, lepromatous leprosy) as well as from healthy volunteers. Encouraging operational ELISA validity could be achieved with 93% sensitivity, 100% specificity, 97% efficiency, 100% positive predictivity and 95% negative predictability even among the extrapulmonary and suspected pulmonary tuberculosis patients. The above assay was insensitive but with 100% specificity among control group of patients suffering from diseases other than tuberculosis. The DE-V protein fraction was associated with phosphatidyl inositol and phosphatidyl inositol mannosides. The dissociation of phospholipid-protein complex decreased ELISA specificity. ELISA reactivity of the DE-V fraction appeared to be thermostable; thus, it may have serodiagnostic utility in developing countries.

Calmodulin-like activity in mycobacteria.

Calmodulin-like activity has been reported for the first time in mycobacterial species, namely Mycobacterium tuberculosis BCG and M. smegmatis ATCC 14468. The activity was mainly located in the soluble fraction of the mycobacterial cells, Radioimmunoassay revealed maximum levels of calmodulin in young growing cells (early logarithmic phase of growth). Calmodulin-dependent phosphodiesterase activation assay revealed low activity (22%) of partially purified calmodulin either due to insufficient amount of calmodulin to activate phosphodiesterase or due to the presence of some factors interfering with the assay. Calmodulin antagonists, viz. trifluoperazine and phenothiazine, significantly inhibited the 32Pi incorporation into mycobacterial phospholipids. Similar inhibition was observed when EGTA (which removes calcium) was added to the medium. Significant inhibition of 32Pi incorporation in the presence of calmodulin antagonists suggested the involvement of calmodulin in mycobacterial phospholipid metabolism.

Serodiagnosis of tuberculosis using purified mycobacterial proteins.

With a view to detect specific M. tuberculosis infection, mycobacterial proteins were purified initially by ammonium sulphate precipitation followed by ion-exchange chromatography. Out of the three fractions, namely P1, P2 and P3 precipitated with increasing concentrations (0-25%, 26-65% and 66-100% respectively) of ammonium sulphate, P2 fraction was found to be more immunoreactive. P2 fraction proteins were further fractionated into five fractions by salt gradient using DEAE-cellulose DE-52 ion-exchange chromatography. Immunoreactivity against tuberculous patients sera of all the fractions was assessed using ELISA test. The last fraction (DE-V) eluted with high salt concentration was found to have a more specific immunoreactive set of proteins within the range of 55 kD to 67 kD molecular weight. Multiple non-specific proteins were distributed in all the other fractions. ELISA test using P2 fraction proteins against tuberculous patients sera showed significantly higher (p less than 0.01) titre even in the absence of any other bacteriological evidence. DE-V fraction of P2 proteins was found to have a significantly high specificity for detecting M. tuberculosis infection in clinically confirmed and suspected tuberculous patients indicating its application in the diagnosis of the disease.

Calmodulin-like protein and the phospholipids of Mycobacterium smegmatis.

When Mycobacterium smegmatis TMC1546 was grown at different concentrations of glucose supplemented to a synthetic medium already containing 2% v/v glycerol, the following changes were observed. Amount of calmodulin-like protein (CAMLP), total and individual phospholipids (PLs) namely phosphatidylethanolamine, cardiolipin, phosphatidylglycerol and phosphatidylinositol mannosides and total lipids and growth increased up to 5% w/v but decreased at higher concentrations of glucose (7.5% w/v and above). Cyclic AMP content of the whole cells decreased continuously with increase in glucose concentration in the medium. Incorporation of 32Pi into total phospholipids was inhibited by two calmodulin antagonists trifluoperazine and phenothiazine (50% at 40 microM) and the calcium-specific chelator ethylene glycol bis (beta-aminoethyl ether) N,N,N',N'-tetraacetate (EGTA) 35% at 2 mM. Total lipids, CAMLP and growth of this organism are also modulated in a similar way in response to the glucose concentration in the growth medium. Taking these observations together it is suggested that CAMLP has some effect on the metabolism of PLs.

A Ca2+-sensitive cyclic-3',5'-nucleotide phosphodiesterase from sheep lung modulated by a tightly bound endogenous calmodulin.

Highly purified sheep lung cyclic-3',5'-nucleotide phosphodiesterase was sensitive to Ca2+/EGTA but insensitive to exogenous calmodulin. The Ca2+-sensitivity was inhibited by trifluoperazine. Heat-treated enzyme could activate a calmodulin-deficient phosphodiesterase, suggesting the presence of endogenous calmodulin in sheep lung cyclic-3',5'-nucleotide phosphodiesterase, possibly associated with the enzyme in a Ca2+-independent manner.

Temperature and thiol-induced desensitization of a Ca2+-sensitive cyclic-3',5'-nucleotide phosphodiesterase from sheep lung.

Ca2+-sensitivity of sheep lung cyclic-3',5'-nucleotide phosphodiesterase is provided by endogenous tightly bound calmodulin. The calcium sensitivity of a highly purified enzyme was desensitized by increasing the assay temperature. It could also be desensitized to Ca2+-activation by thiols such as dithiothreitol. The thiol-induced desensitization could be partially reversed by dialysis and almost completely reversed by dilution. The results presented in this paper indicate that thiols are possibly involved in the interaction of calmodulin with cyclic-3',5'-nucleotide phosphodiesterase. This is the first report on temperature and thiol-induced desensitization of Ca2+-sensitivity of a cyclic-3',5'-nucleotide phosphodiesterase.

Purification and properties of aspartate transcarbamylase from Mycobacterium smegmatis.

Aspartate transcarbamylase (carbamoyl-phosphate: L-aspartate carbamoyltransferase, EC 2.1.3.2) has been purified from Mycobacterium smegmatis TMC 1546 using streptomycin sulphate precipitation, ammonium sulphate precipitation, DE-52 chromatography, second ammonium sulphate precipitation, Sephadex G-200 gel filtration, and aspartate-linked CNBr-activated Sepharose 4B affinity chromatography in successive order. The enzyme was purified 231.6-fold, and the preparation was found to be homogeneous on column chromatography and polyacrylamide gel electrophoresis. The purified enzyme had a molecular weight of 246,000 and was composed of two asymmetrical subunits. The kinetic and regulatory properties of aspartate transcarbamylase from M. smegmatis were also studied. The enzyme was found to be an allosteric in nature with carbamyl phosphate showing positive cooperativity and UMP exhibiting a negative cooperativity. CTP was found to be the most potent inhibitor among nucleotides. Phosphate acted as a non-competitive product inhibitor with respect to aspartate. Succinate and maleate exerted a competitive inhibition when aspartate was the variable substrate.

Role of various carbon and nitrogen sources in the regulation of enzymes of pyrimidine biosynthesis in Mycobacterium smegmatis TMC 1546.

Pyrimidine biosynthesis and its regulation in the presence of different carbon and nitrogen sources in the growth medium of Mycobacterium smegmatis were studied. M. smegmatis TMC 1546 cells grown in shake culture were found to have marginally higher pyrimidine enzyme biosynthesis activities than cells grown in static culture. The activity was highest at the mid-log phase of growth during both surface and shake cultures, suggesting that the cells were metabolically most active at this stage of growth. Replacement of glycerol by glucose and fructose slightly increased the activities of carbamyl phosphate synthetase and aspartate transcarbamylase. The enzyme activities involved in pyrimidine biosynthesis decreased when citrate was replaced by succinate, fumarate, pyruvate or acetate in the growth medium. The activities of the enzymes in pyrimidine biosynthesis were found to decrease when asparagine as a nitrogen source in the medium was replaced by glutamate, glutamine or ammonium chloride. The presence of ornithine in place of asparagine in the growth medium increased these enzyme activities, while the presence of arginine instead of asparagine in the growth medium decreased these enzyme activities, though the differences in activity were small. The activity of aspartate transcarbamylase in vitro was inhibited by arginine. In the case of cells grown in the presence of ornithine, the activity of aspartate transcarbamylase was induced by ornithine, but inhibited by arginine.

NADP-specific isocitrate dehydrogenase of Mycobacterium phlei ATCC 354: purification and characterization.

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.

Drug metabolism in experimental tuberculosis: I. Changes in hepatic and pulmonary monooxygenase activities due to infection.

Pulmonary and hepatic drug metabolizing enzyme activities of tuberculous guinea pigs were examined in detail. Experimental tuberculosis resulted in enlargement of liver and lung accompanied by decreased microsomal cytosolic protein. The tuberculosis infection resulted in decreased hepatic contents of cytochrome P-450 and cytochrome b5 NADPH-cytochrome C reductase in lung and liver. A parallel decrease in the microsomal mixed function oxidases (MFO) was observed in liver and lung of tuberculous guinea pigs. The hepatic and pulmonary activities of UDP-glucuronyl transferase were elevated in the infected animals. Glutathione S-transferase activity exhibited an increase in liver and decrease in the lung of tuberculous guinea pigs. Some of the changes observed in monooxygenase in tuberculosis were caused by reduced food consumption. In general, tuberculosis infection can be viewed to lower drug metabolizing capacity of the animal, probably due to the damage and disturbed membrane integrity.

Ornithine transcarbamylase from Mycobacterium smegmatis ATCC 14468: purification, properties, and reaction mechanism.

Ornithine transcarbamylase (EC 2.1.3.3) has been purified 980-fold from Mycobacterium smegmatis and has a molecular weight of 116,000. Initial velocity determinations indicated that the reaction proceeds via a sequential kinetic mechanism. The limiting Michaelis constants for carbamyl phosphate (KmA) and ornithine (KmB) and the dissociation constant for carbamyl phosphate (Kia) were found to be 0.20, 0.25, and 0.07 mM, respectively. Ornithine at higher concentrations acted as an uncompetitive inhibitor when carbamyl phosphate was the variable substrate. Phosphate was a competitive inhibitor with carbamyl phosphate as variable substrate and showed noncompetitive or mixed type inhibition when ornithine was the variable substrate. Norvaline acted as a competitive inhibitor with ornithine as variable substrate and as an uncompetitive inhibitor when carbamyl phosphate was the variable substrate. Such inhibitory patterns are characteristic of reactions that proceed via sequential ordered mechanisms. Although the enzyme activity was strongly inhibited by arginine, several arginine analogs had no effect on the enzyme activity. The results suggest that, even though the enzyme from M. smegmatis is unique in the sense that it is feedback inhibited by arginine, the reaction mechanism is similar to the ornithine transcarbamylase isolated from other microorganisms.

Changes in liver polyamines due to aflatoxin B1.

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.

Changes in the enzyme activities involved in nitrogen assimilation in Mycobacterium smegmatis under various growth conditions.

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.

3-MHPG as a non-predictor of antidepressant response to imipramine and electroconvulsive therapy.

The urinary excretion of 3-methoxy, 4-hydroxyphenyl-glycol, (3-MHPG) was measured in 20 unipolar depressed patients before treatment with imipramine and electroconvulsive therapy (ECT) to investigate the relationship between pretreatment urinary excretion of 3-MHPG and clinical response. There was no difference in 3-MHPG excretion for depressed patients and controls. There was no significant difference between the mean percentage reduction of Hamilton Depression Rating Scale Scores in "low" and "high" excretors of 3-MHPG in the imipramine and ECT group of patients after four weeks of treatment.

Metabolic potential of the adipose tissue of rats during hyper- and hypovitaminosis A.

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.

Nitrogen metabolism in Aspergillus parasiticus NRRL 3240 and A. flavus NRRL 3537 in relation to aflatoxin production.

The relationship between nitrogen assimilation, metabolism and aflatoxin formation has been investigated in a toxigenic and a non-toxigenic strain of Aspergillus parasiticus. Ammonia from the medium is mainly assimilated via NADP-requiring glutamate dehydrogenase. During growth NAD-requiring glutamate dehydrogenase followed an inverse pattern of activity with respect to NADP glutamate dehydrogenase. Alpha-ketoglutarate, the product of NAD glutamate dehydrogenase, stimulated acetate incorporation into aflatoxins. Glutamine synthetase, ornithine transcarbamylase, both utilizing glutamate as substrate were assayed under different growth conditions. An important regulatory role for glutamine synthetase is suggested. The metabolic route of asparagine utilization was also investigated. Both the known pathways, glutamate oxaloacetate transaminase and glutamate pyruvate transaminase are operative simultaneously.

Regulation of aflatoxin biosynthesis: effect of adenine nucleotides, cyclic AMP and N6-O2' -dibutyryl cyclic AMP on the incorporation of (1-14C)-acetate into aflatoxins by Aspergillus parasiticus NRRL-3240.

Adenosine triphosphate (ATP) was found to inhibit the incorporation of labelled acetate into aflatoxins while adenosine diphosphate (ADP) and adenosine monophosphate (AMP) stimulated the process. Exogenous cyclic AMP and its derivative, N6-O2' -dibutyryl cyclic AMP produced efficient (1-14C)-acetate incorporation into aflatoxins at all the concentrations tried (0.1, 0.5 and 1 mM). The stimulation of incorporation of labelled acetate into aflatoxins was significant at 0.1 and 1 mM concentration of the cyclic nucleotide but was found to be maximum at 0.5 mM concentration.

Benzo(a)pyrene hydroxylase activity in human bronchial mucus.

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.