Effects of deoxycoformycin in mice. III. A murine model reproducing multi-system pathology of human adenosine deaminase deficiency.
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Adult AKR/J mice were treated with 10 micrograms/g or 100 micrograms/g 2'-deoxycoformycin, an adenosine deaminase inhibitor with chemotherapeutic potential. The thymus and adrenal glands were decreased in weight more than any other organ. Histologic and cytofluorographic analyses indicated preferential depletion of peanut-agglutinin-positive, cortical thymocytes, as well as acute, dose-dependent damage to the adrenal cortex and medulla. The effect of 2'-deoxycoformycin on the thymus was proven to be independent of the adrenal glands by use of adrenalectomized mice. Dose-dependent liver necrosis, hemolysis, and leukemoid reactivity were observed. These findings illustrate a differential sensitivity of thymocyte subpopulations and suggest, in addition, preferential sensitivity of certain nonlymphoid tissues to 2'-deoxycoformycin administered in vivo. (+info)
Adenosine deaminase inhibitors enhance cerebral anoxic hyperemia in the rat.
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Cerebral blood flow in the rat was monitored by a venous outflow technique with an extracorporeal circulation, which allows for the continuous recording of flow over periods of several hours. The adenosine deaminase inhibitors erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) (1.0-100 micrograms/kg) and deoxycoformycin (0.1-1 micrograms/kg) potentiated the reactive hyperemia elicited by a brief (24-s) anoxic challenge. Basal flow rate was unaltered by EHNA administration and slightly enhanced by deoxycoformycin. The results are consistent with the hypothesis that adenosine plays a significant role in cerebral vascular regulation and suggest that low doses of these deaminase inhibitors may be useful in the treatment of cerebral vascular insufficiency. (+info)
Effects of nucleoside transport inhibitors on the salvage and toxicity of adenosine and deoxyadenosine in L1210 and P388 mouse leukemia cells.
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Incubation of deoxycoformycin-treated L1210 leukemia cells with dipyridamole or nitrobenzylthioinosine, inhibitors of nucleoside transport, enhanced the long-term incorporation of 2'-deoxyadenosine and adenosine into the nucleotide pool and the toxicity of 2'-deoxyadenosine for the cells. In contrast, 2'-deoxyadenosine uptake in deoxycoformycin-treated P388 leukemia cells, which was about 10 times greater than that in L1210 cells, was inhibited by dipyridamole and nitrobenzylthioinosine, and 2'-deoxyadenosine toxicity was not significantly affected by the transport inhibitors. P388 cells also were about 6 times more resistant to 2'-deoxyadenosine than were L1210 cells, in spite of the greater uptake of the nucleoside. We found that purine nucleoside transport in L1210 and P388 cells exhibited similar kinetic properties and sensitivity to dipyridamole and nitrobenzylthioinosine (both influx and efflux) and that the stimulation of 2'-deoxyadenosine uptake by the inhibitors in L1210 cells is not mediated at the level of its transport into the cells but rather reflects an enhanced intracellular net accumulation of deoxyadenosine nucleotides. (+info)
Differential sensitivity of leukemic cells to growth inhibition by deoxyadenosine and deoxycoformycin.
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Both established cell lines and human leukemic cells in circulating blood which were incubated in vitro with 2'-deoxyadenosine (AdR) plus adenosine deaminase inhibitor, 2'-deoxycoformycin (dCF), showed different metabolic responses depending upon the histologic and immunologic types of leukemia. The leukemic T-cell lines in tissue culture were 200-fold more sensitive than B-cell lines to the toxic effect of deoxyadenosine. The increased sensitivity of T-cell lines to AdR plus dCF was associated with the accumulation of deoxyadenosine triphosphate (dATP) in the cells. In established cell lines, an inverse correlation was observed between ED 50 of AdR plus dCF and the relative increase of dATP levels in the cells after the incubation of the cells with AdR plus dCF. In circulating leukemic cells that had been incubated with AdR and dCF, dATP arose in all groups but the correlation was not found between the sensitivity of AdR and the relative dATP accumulation. The failure to find the correlation in patients's leukemic cells may be attributed to the heterogeneity of the response of the blasts to AdR and dCF. (+info)
Purification and characterization of adenosine deaminase from a genetically enriched mouse cell line.
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Mammalian adenosine deaminase has been shown by genetic and biochemical evidence to be essential for the development of the immune system. For the purpose of studying the function and structure of this enzyme, we have isolated by genetic selection a mouse cell line, B-1/50, in which adenosine deaminase levels were increased 4,300-fold over the parent cell line. The enzyme was purified from these cells in large quantity and high yield by a simple two-step purification scheme. The enzyme derived from the B-1/50 cells was indistinguishable from that of the parental cells as judged by several biochemical criteria. The Km (30 microM) and Ki (4 nM) values using adenosine as substrate and 2'-deoxycoformycin as inhibitor, respectively, were identical for the enzyme derived from the parental cells as well as the adenosine deaminase gene amplification mutants. The enzyme from both cell types exhibited multiple isoelectric focusing forms which co-purified using our purification protocol. Electrophoretic analysis using sodium dodecyl sulfate-polyacrylamide gels showed that adenosine deaminase migrated with an apparent molecular weight of 41,000 or 36,000 depending on whether the enzyme was reduced or oxidized, respectively. This shift was reversible, indicating that proteolysis was not responsible for the faster migrating form. Monospecific antibodies raised against purified adenosine deaminase cross-reacted with the enzyme derived from the parental cells and precipitated 37% of the total soluble protein in the B-1/50 cells. Continued genetic selection resulted in the isolation of cells in which adenosine deaminase was overproduced by 11,400-fold and accounted for over 75% of the soluble protein. (+info)
2'-Chloropentostatin: discovery, fermentation and biological activity.
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2'-Chloropentostatin (2-CP) is a new nucleoside antibiotic produced by Actinomadura sp. ATCC 39365. A selectively sensitive assay organism, Enterococcus faecalis PD 05045 (MIC 0.005 micrograms/ml) was instrumental in the discovery of this compound. 2-CP is a tight-binding inhibitor of adenosine deaminase (Ki = 1.1 X 10(-10) M). (+info)
Recovery of 2'-deoxycoformycin-inhibited adenosine deaminase of mouse erythrocytes and leukemia L1210 in vivo.
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The antibiotic 2'-deoxycoformycin, a potent inhibitor of adenosine deaminase, has potential as a chemotherapeutic agent. Injection of 2'-deoxycoformycin i.v. (0.2 mg/kg) to mice bearing ascites L1210 leukemia cells completely inhibits adenosine deaminase in both erythrocytes and L1210 cells. The recovery of the enzymic activity is markedly different in the two tissues. The recovery is very slow in erythrocytes (13% in 48 hr), whereas 80% recovery occurs during the same time interval in L1210 cells. This marked difference in the recovery of the enzyme in different tissues may play a role in the pharmacological and chemotherapeutic behavior of this drug. (+info)
Studies on inhibition of adenosine deaminase by isocoformycin in vitro and in vivo.
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Isocoformycin is a structural isomer of coformycin which has been demonstrated to be a potent inhibitor of adenosine deaminase. Isocoformycin showed a weaker inhibition of this enzyme than coformycin; the binding of coformycin to enzyme was irreversible, but isocoformycin inhibition was competitive with substrate. The Ki value of isocoformycin was 4.5 approximately 10 X 10(-8) M. Following intraperitoneal injection of isocoformycin in mice, the adenosine deaminase activity of homogenates of several organs was determined and the following ED50 values (50% inhibition doses) were observed: 29 mg/kg for thymus, 13 mg/kg for spleen, 80 mg/kg for liver and 20 mg/kg for kidney. The inhibition of adenosine deaminase in rabbit blood in vitro was also tested in comparison with coformycin. (+info)