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Query: UNIPROT:P06889 (Mol)
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Our studies on the in vitro differentiation of a pleomorphic Trypanosoma brucei strain TREU667 indicate that the parasite differentiates directly from long-slender into procyclic form when incubated in Cunningham's medium at 26 degrees C. The intermediary and the short-stumpy bloodstream forms harvested from infected mice can also differentiate directly into procylic form in vitro with time courses similar to that for the long-slender form. Thus, none of the three bloodstream forms appear to be significantly better preadapted for differentiation. Tricarboxylic acid (TCA) cycle intermediates cis-aconitate and L-citrate can shorten the initial lag phase of the differentiation, but an essential trigger appears to be the temperature shift from 37 degrees C to 26 degrees C, when other TCA cycle intermediates such as L-proline, L-malate, alpha-ketoglutarate, fumarate and succinate are present in Cunningham's medium. The ornithine decarboxylase (ODC) activity in T. brucei showed a gradual increase and the ODC mRNA level remained constant during the differentiation. DL-alpha-Difluoromethylornithine (DFMO), putrescine, dibutyryl cAMP and theophylline all exerted no discernible effect on the in vitro process, which suggests that neither cAMP increase nor polyamine depletion could be counted among the triggers of T. brucei differentiation. A monomorphic T. brucei strain EATRO110 was tested in the same medium at 26 degrees C but was unable to differentiate.
Mol Biochem Parasitol 1991 Feb
PMID:The in vitro differentiation of pleomorphic Trypanosoma brucei from bloodstream into procyclic form requires neither intermediary nor short-stumpy stage. 205 26

Yeast mutants assigned to the pet complementation group G104 were found to lack alpha-ketoglutarate dehydrogenase activity as a result of mutations in the dihydrolipoyl transsuccinylase (KE2) component of the complex. The nuclear gene KGD2, coding for yeast KE2, was cloned by transformation of E250/U6, a G104 mutant, with a yeast genomic library. Analysis of the KGD2 sequence revealed an open reading frame encoding a protein with a molecular weight of 52,375 and 42% identities to the KE2 component of Escherichia coli alpha-ketoglutarate dehydrogenase complex. Disruption of the chromosomal copy of KGD2 in a respiratory-competent haploid yeast strain elicited a growth phenotype similar to that of G104 mutants and abolished the ability to mitochondria to catalyze the reduction of NAD+ by alpha-ketoglutarate. The expression of KGD2 was transcriptionally regulated by glucose. Northern (RNA) analysis of poly(A)+ RNA indicated the existence of two KGD2 transcripts differing in length by 150 nucleotides. The concentrations of both RNAs were at least 10 times lower in glucose (repressed)- than in galactose (derepressed)-grown cells. Different 5'-flanking regions of KGD2 were fused to the lacZ gene of E. coli in episomal plasmids, and the resultant constructs were tested for expression of beta-galactosidase in wild-type yeast cells and in hap2 and hap3 mutants. Results of the lacZ fusion assays indicated that transcription of KGD2 is activated by the HAP2 and HAP3 proteins. The regulated expression of KGD2 was found to depend on sequences that map to a region 244 to 484 nucleotides upstream of the structural gene. This region contains two short sequence elements that differ by one nucleotide from the consensus core (5'-TN[A/G]TTGGT-3') that has been proposed to be essential for binding of the HAP activation complex. These data together with earlier reports on the regulation of the KGD1 and LPD1 genes for the alpha-ketoglutarate and dihydrolipoyl dehydrogenases indicate that all three enzyme components of the complex are catabolite repressed and subject to positive regulation by the HAP2 and HAP3 proteins.
Mol Cell Biol 1990 Aug
PMID:Structure and regulation of KGD2, the structural gene for yeast dihydrolipoyl transsuccinylase. 211 21

The filarial parasite Litomosoides carinii was able to survive for longer than 15 h in basic filarial medium (BFM) containing either glutamine or alanine as a sole substrate. The filariids were more motile in BFM containing glucose, but even higher motility was recorded in media containing both glucose and glutamine. Incubations under aerobic conditions showed that radiolabelled glutamine was metabolised primarily to CO2. In addition, small amounts of lactate and acetate were excreted in almost equimolar quantities. Incubations where both glutamine and glucose were present demonstrated that the glutamine carbon utilised by the parasite could be completely recovered in the above three end products. The glutamine nitrogen could be recovered in the additional excretory products, alanine and ammonia. The glutamine-dependent viability of L. carinii was affected by known inhibitors of the mitochondrial respiratory chain. Glucose utilisation, and the production of CO2 from this substrate, were greatly stimulated by the presence of glutamine in the external medium. Various carbon balance studies, in conjunction with enzymatic analyses, suggest that in L. carinii, glutamine provides an input of carbon into the tricarboxylic acid (TCA) cycle, probably at the level of alpha-ketoglutarate. This increased availability of Krebs cycle intermediates will stimulate the rate of pyruvate oxidation via acetyl-CoA and the TCA cycle, and thus increase the rate of carbon flux through glycolysis. The energetic advantage associated with the utilisation of the glucose/glutamine substrate couple may explain the worm's enhanced motor activity compared to incubations with glucose as the sole energy source. Alanine was found to be degraded by the filariid to equivalent amounts of lactate, acetate and CO2, indicating a relatively low energetic efficiency. There was no detectable uptake of glutamate. A variety of other amino acids tested were neither metabolised nor able to maintain worm viability in vitro.
Mol Biochem Parasitol 1990 Jun
PMID:The role of amino acids in the energy generating pathways of Litomosoides carinii. 211 54

In the presence of Mg2+, pure glutamate dehydrogenase is more reactive with NADPH than with NADH and is markedly activated by elevations in the ADP/ATP ratio or the addition of leucine. Because these are properties of glutamate dehydrogenase in mitochondria but not properties of the pure enzyme studied in the absence of Mg2+, Mg2+ could be a ligand that confers upon glutamate dehydrogenase the regulatory properties of this enzyme found in situ. In the absence of the allosteric activators ADP, leucine, or succinyl-CoA, Mg2+ is an inhibitor and increases product inhibition by alpha-ketoglutarate in the forward reaction and substrate inhibition by alpha-ketoglutarate in the reverse reaction. However, the allosteric activators convert Mg2+ from an inhibitor into an activator of the forward reaction. In the reverse reaction, ADP also converts Mg2+ from an inhibitor into an activator and leucine eliminates inhibition by Mg2+. Because Mg2+ is an inhibitor in the absence of activator that also increases inhibition by alpha-ketoglutarate, whereas in the presence of activator Mg2+ has no effect or is itself an activator, Mg2+ magnifies the effect of the activator, and magnification increases with increases in the concentration of alpha-ketoglutarate. Leucine and its analog 2-aminobicyclo (2.2.1) heptane 2-carboxylic acid (BCH) have almost identical effects on both human and bovine glutamate dehydrogenase in both the presence and absence of Mg2+. However, advantages of BCH over leucine as a potential pharmacological activator of glutamate dehydrogenase are that BCH is not metabolized and, unlike leucine, BCH does not inhibit ornithine transcarbamylase. Isoleucine and valine alone have little effect on human glutamate dehydrogenase, but isoleucine slightly inhibits the enzyme in the presence of leucine.
Mol Pharmacol 1990 Jun
PMID:Regulation of glutamate dehydrogenase by Mg2+ and magnification of leucine activation by Mg2+. 235 6

Insertion of the fusion-generating phage Mud1 (Ap, lacZ) yielded two similar isolates, DC511 and DC512, which were unable to grow aerobically on acetate or alpha-ketoglutarate but which could use succinate, malate, fumarate, glycerol, and various sugars. These mutants were unable to grow anaerobically on most sugars unless provided with methionine, lysine, and delta-aminolevulinic acid, all of which require succinyl-CoA for their synthesis. The insertions of both mutants mapped at 17 min, in the suc operon. Enzyme assays indicated a lack of succinyl-CoA synthetase; however, full activity of the alpha-ketoglutarate dehydrogenase was retained. Beta-galactosidase expression by strains containing these gene fusions was reduced under anaerobic conditions. In aerobically grown cultures, both fusions were induced about fivefold in the presence of acetate. This type of regulation would be expected of a Krebs cycle enzyme.
Mol Gen Genet 1989 Jan
PMID:Anaerobic growth defects resulting from gene fusions affecting succinyl-CoA synthetase in Escherichia coli K12. 249 97

In the heart and other mammalian tissues, there are three exclusively intramitochondrial dehydrogenases that occupy key regulatory sites in oxidative metabolism which can be activated by increases in Ca2+ in the approximate range 0.05-5 microM; they are the pyruvate, NAD+-isocitrate and 2-oxoglutarate dehydrogenases. Activation of these enzymes can be demonstrated within intact mitochondria, incubated under expected physiological conditions, when the extramitochondrial concentration of Ca+ is raised within the expected physiological range. Recent studies with fura-2-loaded mitochondria have established that matrix Ca2+ is indeed in the 0.02-2 microM range as the enzymes are activated. There is now good evidence that in the rat heart, increases in cytoplasmic [Ca2+] caused by various inotropic agents result in increases in intramitochondrial Ca2+ and activation of these dehydrogenases. It is argued therefore that matrix Ca2+ may thus be a key regulator of oxidative phosphorylation under such circumstances. The major advantage of such a mechanism of dehydrogenase-based control of this process would be to the energy homeostasis of the cell by allowing stimulated ATP production without the need to decrease the ATP/ADP ratio. Therefore it is also proposed that the major function of the mitochondrial Ca2+-transport system is to regulate matrix Ca2+, and that the ability of mitochondria to buffer the extramitochondrial concentration of Ca2+ may thus only be reserved for pathophysiological conditions of abnormal sarcolemmal Ca2+ influx as perhaps may occur in ischaemia-reperfusion.
Mol Cell Biochem 1989 Sep 07
PMID:The role of Ca2+ ions in the regulation of intramitochondrial metabolism and energy production in rat heart. 268 6

Quantitative changes in the urinary excretion patterns of low molecular weight compounds were followed for up to 30 days after dosing of adult Sprague-Dawley rats with single intraperitoneal injections of CdCl2 (6-24 mumol/kg), using high resolution 1H NMR multicomponent urinalysis. There was a marked reduction in the rate of urinary excretion of citrate, 2-oxoglutarate, and succinate within 4.5 hr of the administration of 24 mumol/kg Cd2+. This continued for up to 4 days after dosing in male rats and was consistent with a renal tubular acidosis, caused by inhibition of carbonic anhydrase. Histological examination of the kidneys showed no evidence of structural abnormalities at any Cd2+ dose level. Creatinine excretion was not affected by Cd2+ treatment at any dose level but hippurate excretion was significantly reduced. Severe testicular damage was noted within 24 hr of Cd2+ treatment at doses of greater than 9 mumol/kg and the degree of damage appeared to be correlated with the presence of large amounts of creatine (up to 20 mM) in the urine. Analysis of homogenates of healthy testicular material indicated the presence of high concentrations of free creatine. Cadmium-induced creatinuria appears to result from direct release of creatine from the necrotic cells of the seminiferous tubules and, hence, the measurement of creatine excretion rates may provide a useful noninvasive indicator of testicular necrosis. Because NMR is nonselective in terms of metabolite detection, this work has shed new light on the changes in urinary composition arising from Cd toxicity. As such, the technique is potentially very valuable in the search for new metabolic markers of toxicity and organ dysfunction.
Mol Pharmacol 1989 Sep
PMID:Quantitative high resolution 1H NMR urinalysis studies on the biochemical effects of cadmium in the rat. 277 24

Rats were injected intraperitoneally with HgCl2 at doses of 2.5, 5, 7.5, and 10 mumol of Hg/kg. Urine was collected over a 24-hr period. At this time, plasma samples were taken and kidney damage was assessed by histological examination. Urinary gamma-glutamyltransferase levels were significantly elevated at Hg2+ doses of 7.5 and 10 mumol/kg, consistent with the detection of acute tubular necrosis by light microscopy. Resonances for a large number of low molecular weight metabolites were assigned in high resolution 1H NMR spectra of rat urine. Spectra from small volumes of urine (about 0.5 ml) were obtained in less than 5 min with no pretreatment. Significant Hg2+ dose-related decreases in the excretion of creatinine and citrate and increases of glucose, glycine, alanine, alpha-ketoglutarate, succinate, and acetate were detected. Elevated levels of lactate and creatinine in plasma of rats receiving the two highest doses were found by 1H NMR. There was a good correspondence between the histopathology, enzyme excretion, and 1H NMR urinary metabolite fingerprints in the assessment of Hg2+-induced renal damage. 1H NMR provided a sensitive measure of mercury-induced nephrotoxic lesions, and information on the molecular basis of mercury cytotoxicity was derived from the abnormal patterns of metabolite excretion. These suggested that primary metabolic effects of mercury were upon mitochondrial metabolism, in particular inhibition of certain citric acid cycle enzymes leading to decreased utilization of alpha-ketoglutarate and succinate by the renal tubular cells. The decrease in urinary citrate associated with Hg2+ dosing was attributed to intracellular, tubular acidosis with concomitant enhanced citrate reabsorption. The acidosis was assumed to arise from a combination of the inhibition of tubular carbonic anhydrase and a mild metabolic lactic acidosis due to increased activity of anaerobic pathways in the kidney. The possible extension of the 1H NMR techniques to the investigation of the nephrotoxic potential of other compounds and drugs is discussed.
Mol Pharmacol 1985 Jun
PMID:Proton NMR spectra of urine as indicators of renal damage. Mercury-induced nephrotoxicity in rats. 286 May 59

Specific messenger RNA for glutamate dehydrogenase was partially purified from a calf liver polysomal poly(A)-mRNA fraction by sucrose density gradient centrifugation. Enzyme activity in the translational incubation mixture was detected by measuring NADH oxidation in the presence of alpha-ketoglutarate and ammonia as a decrease in absorbency 340-442 nm in a dual wavelength Aminco DW-2 spectrophotometer.
Mol Biol Rep 1986
PMID:Identification of messenger RNA for glutamate dehydrogenase using a spectrophotometric probe. 287 75

The amino acid pool sizes of Trichomonas vaginalis are reported. Alanine, glutamic acid, proline and leucine account for 72% of the measured amino acids. Growth of T. vaginalis was unaffected by gostatin, an irreversible inhibitor of aspartate aminotransferase, when the enzyme activity within the cell had been completely inhibited and a specific elevation of the aspartate pool had occurred. In media lacking aspartate and glutamate, the amino acid substrates of the aspartate aminotransferase reaction, gostatin caused a larger increase in the aspartate pool. During incubation of cells with or without gostatin, aspartate and glutamate were produced in the medium, presumably by proteolysis of medium proteins. Hence any requirement for the aspartate aminotransferase reaction might have been bypassed. Glutamate-gamma-hydroxamate and aminooxyacetate inhibited growth of T. vaginalis but caused large changes in the pool-sizes of aspartate, glutamate, pyruvate plus oxaloacetate and 2-oxoglutarate, suggesting a more general interference with amino acid metabolism.
Mol Biochem Parasitol 1986 Oct
PMID:Modulation of amino acid and 2-oxo acid pools in Trichomonas vaginalis by aspartate aminotransferase inhibitors. 287 95


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