Utilization of exogenous purine compounds in Bacillus cereus. Translocation of the ribose moiety of inosine. (1/498)

Intact cells of Bacillus cereus catalyze the breakdown of exogenous AMP to hypoxanthine and ribose 1-phosphate through the successive action of 5'-nucleotidase, adenosine deaminase, and inosine phosphorylase. Inosine hydrolase was not detectable, even in crude extracts. Inosine phosphorylase causes a "translocation" of the ribose moiety (as ribose 1-phosphate) inside the cell, while hypoxanthine remains external. Even though the equilibrium of the phosphorolytic reaction favors nucleoside synthesis, exogenous inosine (as well as adenosine and AMP) is almost quantitatively transformed into external hypoxanthine, since ribose 1-phosphate is readily metabolized inside the cell. Most likely, the translocated ribose 1-phosphate enters the sugar phosphate shunt, via its prior conversion into ribose 5-phosphate, thus supplying the energy required for the subsequent uptake of hypoxanthine in B. cereus.  (+info)

Tissue distribution and characteristics of xanthine oxidase and allopurinol oxidizing enzyme. (2/498)

Tissue distribution and levels of allopurinol oxidizing enzyme and xanthine oxidase with hypoxanthine as a substrate were compared with supernatant fractions from various tissues of mice and from liver of mice, rats, guinea pigs and rabbits. The allopurinol oxidizing enzyme activities in liver were quite different among the species and the sex difference of the enzyme activity only in mouse liver. In mice, the highest activity of allopurinol oxidizing enzyme was found in the liver with a trace value in lung, but the enzyme activity was not detected in brain, small intestine and kidney, while the highest activity of xanthine oxidase was detected in small intestine, lung, liver and kidney in that sequence. The allopurinol oxidizing enzyme activity in mouse liver supernatant fraction did not change after storage at -20 degrees C or dialysis against 0.1 M Tris-HCl containing 1.15% KCl, but the activity markedly decreased after dialysis against 0.1 M Tris-HCl. On the contrary, the xanthine oxidase was activated 2 to 3 times the usual activity after storage at -20 degrees C or dialysis of the enzyme preparation. These results indicated that allopurinol was hydroxylated to oxipurinol mainly by the enzyme which is not identical to xanthine oxidase in vivo. A possible role of aldehyde oxidase involved in the allopurinol oxidation in liver supernatant fraction was dicussed.  (+info)

Oxypurinol administration fails to prevent free radical-mediated lipid peroxidation during loaded breathing. (3/498)

The purpose of the present study was to determine whether it is possible to alter the development of fatigue and ablate free radical-mediated lipid peroxidation of the diaphragm during loaded breathing by administering oxypurinol, a xanthine oxidase inhibitor. We studied 1) room-air-breathing decerebrate, unanesthetized rats given either saline or oxypurinol (50 mg/kg) and loaded with a large inspiratory resistance until airway pressure had fallen by 50% and 2) unloaded saline- and oxypurinol-treated room-air-breathing control animals. Additional sets of studies were performed with animals breathing 100% oxygen. Animals were killed at the conclusion of loading, and diaphragmatic samples were obtained for determination of thiobarbituric acid-reactive substances and assessment of in vitro force generation. We found that loading of saline-treated animals resulted in significant diaphragmatic fatigue and thiobarbituric acid-reactive substances formation (P < 0.01). Oxypurinol administration, however, failed to increase load trial time, reduce fatigue development, or prevent lipid peroxidation in either room-air-breathing or oxygen-breathing animals. These data suggest that xanthine oxidase-dependent pathways do not generate physiologically significant levels of free radicals during the type of inspiratory resistive loading examined in this study.  (+info)

The mechanism of action of methotrexate in cultured L5178Y leukemia cells. (4/498)

This study investigates the relationships between the methotrexate (MTX)-induced purineless state and thymineless state and between the thymineless state and the kill of L5178Y cells. As an index of the thymineless state, we measured the effect of MTX on conversion of deoxyuridylate to thymidylate. This was measured as the rate of incorporation of tritiated deoxyuridine into DNA, but it was corrected for changes in incorporation of tritiated thymidine. Thus we derived the "calculated tritiated deoxyuridine rate." During the MTX treatment, the calculated tritiated deoxyuridine rate decreased rapidly at first and then more slowly. The slow 2nd-phase block was not reversed by hypoxanthine. As the 2nd-phase block deepened, the lymphoblasts continued to die (loss of cloning ability) but recovered the ability to incorporate tritiated thymidine into DNA. After 7 hr of MTX treatment, the kinetics of the 2nd-phase block in calculated tritiated deoxyuridine rate correlated closely with the kinetics of cell kill. Thus, MTX may inhibit dihydrofolate reductase enzyme, rapidly deplete S-phase L5178Y of reduced folates, and thus produce a purineless and thymineless state. As treatment continues, MTX intensifies the thymineless state, possibly by direct inhibition of thymidylate synthetase enzyme, and the cells die predominantly a thymineless death. The purineless state initially contributes to cell kill but later does not, possibly because it partially reverses spontaneously.  (+info)

Isolation of mammalian cell mutants deficient in glucose-6-phosphate dehydrogenase activity: linkage to hypoxanthine phosphoribosyl transferase. (5/498)

Mutants of Chinese hamster ovary cells deficient in glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP 1-oxidoreducatse, EC 1.1.1.49) activity were isolated after mutagenesis with ethyl methane sulfonate. The mutants were induced at frequencies of about 10-4 and do not differ in growth properties from wild-type cells. They were isolated by means of a sib selection technique coupled with a histochemical stain of colonies for enzyme activity. The lack of enzyme activity is not due to a dissociable inhibitor, and is recessive in hybrid cells. Multiple mutants that lack hypoxanthine phosphoribosyltransferase activity (IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) and adenine phosphoribosyltransferase activity (AMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.7) were isolated by further mutagenesis. By following segregation of wild-type phenotypes from heterozygous multiply marked hybrid cells, it was shown that the genes responsible for glucose-6-phosphate dehydrogenase activity and hypoxanthine phosphoribosyltransferase activity are linked in Chinese hamster cells, in agreement with the location of both on the X chromosome in humans. No linkage to adenosine phosphoribosyltransferase was found. The isolation of mutant cells carrying linked markers should prove useful for studying chromosomal events such as segregation, breakage, recombination, and X-chromosome reactivation.  (+info)

Purine metabolism in murine virus-induced erythroleukemic cells during differentiation in vitro. (6/498)

Purine metabolism was studied in murine virus-induced erythroleukemia cells stimulated to differentiate in vitro in the presence of dimethylsulfoxide. The activities of the enzymes that catalyze the synthesis of the first intermediate of the de novo purine pathway, phosphoribosyl-1-amine, were decreased while the enzymes that catalyze the conversion of purine bases to purine ribonucleotides remained unchanged at the time the cells acquired the specialized function of hemoglobin synthesis. In addition, cytidine deaminase (cytidine aminohydrolase, EC 3.5.4.5) activity increased with erythropoietic maturation, as it does during murine erythropoiesis in vivo. Stimulation of cellular proliferation of stationary erythroleukemic cells resulted in a marked increase in the activities of purine biosynthetic enzymes. These data provide a convincing example of repression and derepression of the PRA synthesizing enzymes in mammalian cells in vitro, and further evidence that the regulatory mechanisms operative in the normal development of erythrocytes can be activated by exposure of erythroleukemic cells to dimethylsulfoxide.  (+info)

Consequences of methotrexate inhibition of purine biosynthesis in L5178Y cells. (7/498)

Addition of 1 muM methotrexate to cultures of L5178Y cells results in an initial inhibition of thymidine, uridine, and leucine incorporation into acid-insoluble material followed, after about 10 hr, by a partial recovery in the extent of incorporation of these precursors. Acid-soluble adenosine triphosphate and guanosine triphosphate concentrations are greatly reduced initially, but guanosine triphosphate concentrations appear to recover partially by 10 hr. Acid-soluble uridine triphosphate and cytidine triphosphate concentrations initially increase after methotrexate treatment but then, with time, they too decline. Hypoxanthine and guanine are more effective than is adenine in overcoming the methotrexate-induced inhibition of thymidine incorporation. These results suggest that, in the presence of methotrexate, guanine nucleotides become limiting for nucleic acid synthesis before adenine nucleotides do. The block of purine de novo synthesis in L5178Y cells by methotrexate is almost complete and is not reversed with time. This suggests that the additional purine nucleotides that are available for nucleic acid synthesis 8 to 10 hr after addition of methotrexate are being derived from nucleic acid breakdown. Consistent with this is the observed reduction in the number of polyribosomes and hence, presumably in messenger RNA levels.  (+info)

Growth of human diploid fibroblasts in the absence of glucose utilization. (8/498)

Normal human diploid fibroblasts were able to undergo one to two cell divisions without glucose utilization in Eagle's minimum essential medium plus 10% dialyzed fetal calf serum if the medium was supplemented with hypoxanthine, thymidine, and uridine (supplemented medium termed HTU-MEM). Under these conditions, the added purine and pyrimidines were required for nucleic acid synthesis, as shown by the inability of Lesch-Nyhan fibroblasts to grow in HTU-MEM. Normal human diploid fibroblasts continued to produce lactate in HTU-MEM, but at a greatly reduced rate. Since cells grew in HTU-MEM without glucose utilization, the probable energy and carbon source was glutamine, which is present in relatively high concentration. Furthermore, the rate of glutamine utilization per cell division was 2-fold greater in HTU-MEM than in medium with 5.5 mM glucose. These results suggest that glutamine can be a major energy source for cells grown in vitro.  (+info)