Adenosine receptor blockade augments interstitial fluid levels of excitatory amino acids during cerebral ischemia.
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The excitotoxic hypothesis suggests that cerebral ischemic damage results in part from the accumulation of the excitatory and potentially toxic neurotransmitters glutamate and aspartate. Adenosine, which also increases during cerebral ischemia, is proposed to inhibit neurotransmitter release. The purpose of this study was to determine if adenosine receptor blockade exacerbates the accumulation of glutamate and aspartate during cerebral ischemia. Microdialysis probes, implanted bilaterally in the caudate nucleus of halothane-anesthetized rats, were used to (1) assess changes in interstitial fluid (ISF) glutamate, aspartate, adenosine, and adenosine metabolites; (2) measure local cerebral blood flow (H2 clearance); and (3) deliver 8-(p-sulfophenyl)theophylline (SPT), an adenosine receptor antagonist, locally to the brain. The probe on one side of the brain was perfused with artificial cerebrospinal fluid (CSF) containing 10(-3) M SPT, while the probe on the opposite side received only artificial CSF. Animals were exposed to 20 min of ischemia (carotid occlusion+arterial blood pressure = 50 mm Hg) followed by 60 min of reperfusion. Dialysate glutamate and aspartate increased during and after cerebral ischemia, but were increased to a greater extent in the presence of adenosine receptor blockade. Likewise, the increase in dialysate adenosine and adenosine metabolites was enhanced on the side of locally administered SPT. These data suggest that endogenous adenosine attenuates the accumulation of glutamate and aspartate during cerebral ischemia. (+info)
Regulation of hypoxanthine transport in Neurospora crassa.
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Hypoxanthine uptake and hypoxanthine phosphoribosyltransferase activity (EC 2.4.2.8) were determined in germinated conidia from the adenine auxotrophic strains ad-1 and ad-8 and the double mutant strain ad-1 ad-8. The mutant strain ad-1 appears to lack aminoimidazolecarboximide ribonucleotide formyltransferase (EC 2.1.2.3) or inosine 5'monophosphate cyclohydrolase (EC 3.5.1.10) activities, or both, whereas the ad-8 strain lacks adenylosuccinate synthase activity (EC 6.3.4.4). Normal (or wild-type) hypoxanthine transport capacity was found to the ad-1 conidia, whereas the ad-8 strains failed to take up any hypoxanthine. The double mutant strains showed intermediate transport capacities. Similar results were obtained for hypoxanthine phosphoribosyl-transferase activity assayed in germinated conidia. The ad-1 strain showed greatest activity, the ad-8 strain showed the least activity, and the double mutant strain showed intermediate activity levels. Ion-exchange chromatography of the growth media revealed that in the presence of NH+/4, the ad-8 strain excreted hypoxanthine or inosine, the ad-1 strain did not excrete any purines, and the ad-1 ad-8 double mutant strain excreted uric acid. In the absence of NH+/4, none of the strains excreted any detectable purine compounds. (+info)
Assay method for monitoring the inhibitory effects of antimetabolites on the activity of inosinate dehydrogenase in intact human CEM lymphocytes.
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A rapid and convenient method has been developed to monitor the inhibition of inosinate (IMP) dehydrogenase by antimetabolites in intact human CEM lymphocytes. This method is based on the determination of 3H release from [2,8-3H]hypoxanthine ([2,8-3H]Hx) or [2,8-3H]inosine ([2,8-3H]Ino). The validity of this procedure was assessed by evaluating IMP dehydrogenase inhibition in intact CEM cells by the well-known IMP dehydrogenase inhibitors ribavirin, mycophenolic acid and tiazofurin. As reference materials, several compounds that are targeted at other enzymes in de novo purine nucleotide anabolism (i.e. hadacidine, acivicin) or catabolism (i.e. 8-aminoguanosine, allopurinol) were evaluated. There was a strong correlation between the inhibitory effects of the IMP dehydrogenase inhibitors (ribavirin, mycophenolic acid, tiazofurin) on 3H release from [2,8-3H]Hx and [2,8-3H]Ino in intact CEM cells and their ability to decrease intracellular GTP pool levels. The other compounds (hadacidine, acivicin, 8-aminoguanosine, allopurinol) had no marked effect on 3H release from [2,8-3H]Hx. Using this method, we demonstrated that the novel ribavirin analogue, 5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide, is a potent inhibitor of IMP dehydrogenase in intact cells. (+info)
Hexamethylene bisacetamide stimulates herpes simplex virus immediate early gene expression in the absence of trans-induction by Vmw65.
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Hexamethylene bisacetamide (HMBA) and DMSO are known to induce differentiation of cultured erythroleukaemic cells and to enhance the reactivation of latent herpes simplex virus (HSV) after explantation of ganglia. We report that the presence of these compounds in cell culture medium overcomes the replication defect of in1814, an HSV-1 mutant with an insertion mutation that inactivates the virion trans-inducing factor, Vmw65 (VP16). The effect of HMBA was not cell type-specific and was attained even by a short exposure (1.5 to 5 h) to the agent early after infection. The presence of HMBA resulted in an increase in immediate early (IE) RNA accumulation after infection of cells in the presence of cycloheximide, such that RNA levels in in1814-infected cells approached the values observed in wild-type HSV-1-infected cells in the absence of HMBA. Transport of viral DNA to the cell nucleus was not affected by HMBA. The results suggest that HMBA- and DMSO-mediated enhancement of reactivation from latency is due to an increase in IE RNA production. In addition, these studies demonstrate a primary effect of HMBA on gene regulation which may be a paradigm for initial events during erythroleukaemic cell differentiation. (+info)
Structural characterization and corepressor binding of the Escherichia coli purine repressor.
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The Escherichia coli purine repressor, PurR, binds to a 16-bp operator sequence and coregulates the genes for de novo synthesis of purine and pyrimidine nucleotides, formation of a one-carbon unit for biosynthesis, and deamination of cytosine. We have characterized the purified repressor. Chemical cross-linking indicates that PurR is dimeric. Each subunit has an N-terminal domain of 52 amino acids for DNA binding and a C-terminal 289-residue domain for corepressor binding. Each domain was isolated after cleavage by trypsin. Sites for dimer formation are present within the corepressor binding domain. The corepressors hypoxanthine and guanine bind cooperatively to distinct sites in each subunit. Competition experiments indicate that binding of one purine abolishes cooperativity and decreases the affinity and the binding of the second corepressor. Binding of each corepressor results in a conformation change in the corepressor binding domain that was detected by intrinsic fluorescence of three tryptophan residues. These experiments characterize PurR as a complex allosteric regulatory protein. (+info)
Molybdenum cofactor (chlorate-resistant) mutants of Klebsiella pneumoniae M5al can use hypoxanthine as the sole nitrogen source.
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Selection for chlorate resistance yields mol (formerly chl) mutants with defects in molybdenum cofactor synthesis. Complementation and genetic mapping analyses indicated that the Klebsiella pneumoniae mol genes are functionally homologous to those of Escherichia coli and occupy analogous genetic map positions. Hypoxanthine utilization in other organisms requires molybdenum cofactor as a component of xanthine dehydrogenase, and thus most chlorate-resistant mutants cannot use hypoxanthine as a sole source of nitrogen. Surprisingly, the K. pneumoniae mol mutants and the mol+ parent grew equally well with hypoxanthine as the sole nitrogen source, suggesting that K. pneumoniae has a molybdenum cofactor-independent pathway for hypoxanthine utilization. (+info)
THE GENUS VEILLONELLA . II. NUTRITIONAL STUDIES.
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Rogosa, M. (National Institute of Dental Research, U.S. Public Health Service, Bethesda, Md.), and Ferial S. Bishop. The genus Veillonella. II. Nutritional studies. J. Bacteriol. 87:574-580. 1964.-A medium is described for the study of the vitamin, hypoxanthine, putrescine, or cadaverine requirements of 86 Veillonella isolates from man, rabbit, rat, and hamster. No organism required riboflavine or folic acid for growth. Niacin and calcium pantothenate were often stimulatory, but in nearly all cases were dispensable. Biotin and p-aminobenzoic acid were frequently stimulatory and sometimes indispensable for continued growth. V. parvula (antigenic group VI) required pyridoxal and thiamine and did not require putrescine or cadaverine. V. alcalescens (antigenic group IV) required pyridoxal, generally required thiamine, and also required putrescine or cadaverine. Of the isolates, 25 from the rat and 3 from the hamster (antigenic group II) generally behaved like V. parvula, except that a putrescine or cadaverine requirement was often observed. Spermine, spermidine, and agmatine could not replace putrescine or cadaverine. Although succinate is metabolized by resting cells, the organisms could not grow with succinate as an energy source. (+info)
Substrate specificity of the purine-2'-deoxyribonucleosidase of Crithidia luciliae.
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The purine-2'-deoxyribonucleosidase of Crithidia luciliae catalyses an efficient deoxyribosyl transfer between a variety of purine bases, benzimidazole and 5,6-dimethylbenzimidazole. Since the deoxyriboside of a deoxyribosyl acceptor is necessarily also a substrate, the trans-N-deoxyribosylase activity of the enzyme allows a study of its specificity to be extended to a large number of purines and purine analogues. Amongst 27 different deoxyribosyl acceptors, only hypoxanthine gave rise to isomeric products. The introduction of methyl groups at appropriate positions in either purine or benzimidazole lowered the Michaelis constant, KB, for deoxyribosyl acceptors: by about 10-fold for 6-methylpurine (KB 351 +/- 87 microM) compared with purine (KB 3.91 +/- 0.8 mM) and by about 10(3)-fold for 5,6-dimethylbenzimidazole (KB 7.0 +/- 0.79 microM) compared with benzimidazole (Km,app. 7.8 +/- 2.4 mM). The maximal rates of deoxyribosyl transfer to different acceptors, on the other hand, varied by only 4.5-fold, and can be ascribed to decreases in the rate of release of the newly formed purine deoxyriboside from the enzyme. (+info)