Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.5.4.4 (adenosine deaminase)
 5,136 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In cellular systems provided with activatory (Ra-site) receptors for adenosine, such as rat cerebral microvessels and rat liver plasma membranes, the adenosine-receptor antagonist 8-phenyltheophylline (10 microM) significantly decreased adenylate cyclase activity if ATP was the substrate and only if GTP was present. With dATP as substrate, adenylate cyclase activities in both preparations remained unaffected by 8-phenyltheophylline. In rat cerebral-cortical membranes, with inhibitory (Ri-site) receptors for adenosine, 8-phenyltheophylline significantly enhanced adenylate cyclase activity only in the presence of GTP and if ATP was the substrate. In rat cardiac ventricular membranes, which are devoid of any adenylate cyclase-coupled adenosine receptor, the methylxanthine had no GTP-dependent effect, irrespective of the substrate used. All assay systems contained sufficiently high amounts of adenosine deaminase (2.5 units/ml), since no endogenous adenosine, formed from ATP, was found chromatographically. In order to demonstrate a direct influence of phosphorylated adenosine derivatives on adenylate cyclase activity, we investigated AMP in a dATP assay system. AMP was verified chromatographically to remain reasonably stable under the adenylate cyclase assay conditions. In the microvessels, AMP increased enzyme activity in the range 0.03-1.0 mM, an effect competitively antagonized by 8-phenyltheophylline. In the cortical membranes, 0.1 mM-AMP inhibited adenylate cyclase, which was partially reversed by the methylxanthine. The presence of GTP was again necessary for all observations. In the ventricular membranes, AMP had no effect. Since the efficacy of adenosine-receptor agonists and, probably, that of other hormones on adenylate cyclase activity can be more efficiently measured with dATP as the enzyme substrate, this nucleotide seems preferable for adenylate cyclase measurements in systems susceptible to modulation by adenosine.
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PMID:Phosphorylated adenosine derivatives as low-affinity adenosine-receptor agonists. Methodological implications for the adenylate cyclase assay. 633 7

From human CCRF-CEM T-cells growing in continuous culture, we have selected, isolated, and characterized a clonal cell line, APHID-D2, with altered ribonucleotide reductase activity. In comparative growth rate experiments, the APHID-D2 cell line is less sensitive than the parental cell line to growth inhibition by deoxyadenosine in the presence of 10 microM erythro-9-(2-hydroxy-3-nonyl)adenine, an inhibitor of adenosine deaminase. The APHID-D2 cell line has elevated levels of all four dNTPs. The resistance of the APHID-D2 cell line to growth inhibition by deoxyadenosine and the abnormal dNTP levels can be explained by the fact that the APHID-D2 ribonucleotide reductase, unlike the parental ribonucleotide reductase, is not normally sensitive to inhibition by dATP. These results suggest that the allosteric site of ribonucleotide reductase which binds both dATP and ATP is altered in the APHID-D2 line. The isolation of a mutant clone of human T-cells which contains a ribonucleotide reductase that has lost its normal sensitivity to dATP and which is resistant to deoxyadenosine-mediated growth inhibition suggests that a primary pathogenic target of accumulated dATP in lymphocytes from patients with adenosine deaminase deficiency may be the cellular ribonucleotide reductase.
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PMID:Characterization of a cultured human T-cell line with genetically altered ribonucleotide reductase activity. Model for immunodeficiency. 633 93

Deoxyadenosine toxicity toward lymphocytes may produce immune dysfunction in patients with adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) deficiency. The relationship between endogenous deoxynucleoside synthesis in adenosine deaminase-deficient cells and sensitivity to adenosine and deoxyadenosine toxicity is unclear. The human histiocytic lymphoma cell line (DHL-9) naturally lacks adenosine deaminase, and has minimal levels of thymidine kinase. Dividing DHL-9 cells excrete deoxyadenosine and thymidine into the extracellular space. The present experiments have analyzed nucleoside synthesis and excretion in a mutagenized clone of DHL-9 cells, selected for increased resistance to deoxyadenosine toxicity. The deoxyadenosine-resistant cells excreted both deoxyadenosine and thymidine at a 6-7-fold higher rate than wild-type lymphoma cells. The deoxyadenosine overproduction was accompanied by a reduced ability to form dATP from exogenous deoxyadenosine, and a 2.5-fold increase in ribonucleotide reductase activity. The pace of adenosine excretion, the growth rate, and the levels of multiple other enzymes involved in deoxyadenosine and adenosine metabolism were equivalent in the two cell types. These results suggest that the excretion of deoxyadenosine and thymidine, but not adenosine, is exquisitely sensitive to alterations in the rate of endogenous deoxynucleotide synthesis. Apparently, small changes in deoxynucleotide synthesis can significantly influence cellular sensitivity to deoxyadenosine toxicity.
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PMID:Deoxynucleoside overproduction in deoxyadenosine-resistant, adenosine deaminase-deficient human histiocytic lymphoma cells. 637 66

We have studied the effects of various immunosuppressive drugs on the growth of human-derived T (MOLT-4) and B (MGL-8) lymphoblasts. In addition, we have examined whether the lymphotoxic effect of any of these drugs could be attributed to inhibition of either adenosine deaminase (ADA) or purine nucleoside phosphorylase (PNP). Results indicated that 1-beta-D-arabinofuranosylcytosine (Ara-C), methotrexate and chlorambucil were four to seven times more toxic for T than for B cells, while azathioprine, 6-thioguanine, 6-mercaptopurine, and 5-fluorouracil were highly toxic for both T and B cells. Cyclophosphamide and oxisuran were lymphotoxic only at concentrations exceeding 300 microM. Deoxyadenosine (50 microM), deoxyguanosine (10 microM) and deoxycoformycin (10 microM) failed to enhance T cell toxicity when individually combined with each drug. None of the drugs tested inhibited T or B lymphoblast ADA or PNP activity. With the exception of Ara-C, neither dATP nor dGTP accumulated in T lymphoblasts incubated in the presence of any of the drugs. We conclude that the cell culture system used in this investigation is useful for identifying lymphotoxic and T cell-specific immunosuppressive agents. However, none of the drugs studied appeared to function as an inhibitor of, or a competitive substrate for, either ADA or PNP.
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PMID:Effect of immunosuppressive agents on human T and B lymphoblasts. 640 81

Low ATP/ADP ratios have been reported consistently for nucleotide levels of mononuclear cells separated from peripheral blood by conventional techniques. We have established that these low values (mean 2.3:1) were not due to cell damage or poor viability, but resulted from heavy platelet contamination, which is unavoidable when heparinized blood is used. The results reflect the low ATP/ADP ratios (mean 1.6:1) characteristic of platelets. Platelet-free extracts from defibrinated blood had very high ATP/ADP ratios (mean 17.4:1). The initial finding of detectable amounts of deoxy-ATP and deoxy-GTP in mononuclear cells from children with two distinct inherited immunodeficiency disorders [adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) deficiency respectively] many have been due to contamination by nucleated erythrocytes as well as platelets in non-defibrinated preparations. Defibrination before nucleotide extraction of mononuclear cells from a patient with T-cell leukaemic/lymphoma treated with the ADA inhibitor deoxycoformycin enabled the demonstration of grossly raised deoxy-ATP levels relative to deoxy-ADP levels (ratio 16.1:1), associated with severe ATP depletion. This reciprocal relationship between ATP and dATP was found by us previously in the erythrocytes in inherited ADA deficiency. These findings underline the importance of extracts uncontaminated by platelets, or nucleated erythrocytes, in the evaluation of lymphocyte nucleotide levels in inherited or acquired immunodeficiency syndromes.
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PMID:Importance of platelet-free preparations for evaluating lymphocyte nucleotide levels in inherited or acquired immunodeficiency syndromes. 641 55

The mechanism responsible for the lymphocytotoxicity associated with congenital adenosine deaminase (ADA) deficiency has been ascribed to an accumulation of dATP. Elevated levels of dATP can then lead to inhibition of DNA synthesis by inhibiting ribonucleotide reductase and causing a depletion of the other deoxynucleotide triphosphates (dNTP). This hypothesis was derived principally from studies with murine and human lymphoblastoid cell lines (LCL) and apparently confirmed in a limited number of investigations with lectin-stimulated lymphocytes. Our biochemical studies of lectin-stimulated mouse and human lymphocytes were not consistent with the dATP model and suggested that AdR exerted effects on lymphocyte activation that preceded the initiation of DNA synthesis. In the current studies, we focused on the effects of AdR on the early events in T lymphocyte activation, because we found they were the most sensitive to AdR toxicity. AdR blocked neither the production of T cell growth factor (TCGF) by lectin-stimulated lymphocytes nor the expression of TCGF receptors as detected by the anti-Tac monoclonal antibody that recognizes the human TCGF receptor. AdR did, however, block the early TCGF-dependent events leading to the entry into the cell cycle. By using the metachromatic fluorescence stain acridine orange, we found that AdR blocked the increased synthesis of RNA that characterizes the entry into the G1 phase of the cell cycle from the G0, resting state. Because these early effects were caused by the lowest doses of AdR, and because they preceded the synthesis of DNA by 15 to 20 hr, it suggested that these effects may be principally responsible for the in vivo toxicity associated with ADA deficiency. Furthermore, none of the other proposed biochemical mechanisms, e.g., inhibition of methylation, diminution of ATP levels, or incorporation of AdR into polyadenylated RNA, appeared adequate to explain AdR toxicity during T lymphocyte activation.
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PMID:Deoxyadenosine (AdR) inhibition of newly activated lymphocytes: blockade at the G0-G1 interface. 642 32

The metabolism of 8-14C-labelled 2'-deoxyadenosine (dAR) and 2'-deoxyguanosine (dGR) has been investigated using lymphocytes in long-term culture transformed by Epstein-Barr (EB) virus (B-cells) from eight patients with different inherited purine enzyme defects. The use of such lines enabled accurate mapping of the route of metabolism by acting as a 'trap' for the radiolabel at specific points. With either substrate (25 microM) most of the label was recovered in the medium. Using dAR, less than 30% of the radiolabel was incorporated into cellular nucleotides. For dGR, values were less than 18%. Studies with dAR alone confirmed the principal route of metabolism was to hypoxanthine, with further metabolism (by lines with intact salvage pathways) to ATP and GTP in the ratio of approximately 4:1. Lack of accumulation of deoxyinosine in the purine nucleoside phosphorylase (PNP) deficient line, or hypoxanthine in the hypoxanthine guanine phosphoribosyltransferase (HGPRT) deficient line, using dAR together with the adenosine deaminase (ADA) inhibitor 2'-deoxycoformycin (dCF) at 10 microM, confirmed the effectiveness of ADA inhibition. Nevertheless, some ATP was still formed by all lines in the presence of dCF by a route as yet unknown. Only the ADA deficient lines formed dATP with dAR alone. However, some dATP was formed by all lines in the presence of dCF. A partially HGPRT deficient line formed extremely high dATP levels, well in excess of those formed by the T-cell line CEM. Studies with dGR revealed some interesting differences, a large proportion of the substrate being metabolized predominantly to xanthine by most enzyme deficient lines. In the PNP deficient line most of the substrate remained unmetabolized, but some dGTP was formed. No other enzyme deficient line formed any dGTP--with or without the PNP inhibitor 8-aminoguanosine (8-NH2GR)--with one exception. Again this was the partially HGPRT deficient line, which with the inhibitor again formed more dGTP than the T-cell line. Within the cells most of the substrate was metabolized to GTP, except in the PNP, and totally HGPRT deficient lines. Levels of GTP formed were not altered by the inhibitor, reflecting the lack of effective PNP inhibition by 8-NH2GR. Some counts were also found in ATP and IMP, confirming the existence of this route in mammalian cells of lymphoid origin. The results also support previous studies by us using cell lines with intact purine pathways, which demonstrated that, contrary to current beliefs, some B-cell lines are capable of accumulating high levels of deoxynucleotides.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Metabolism of deoxynucleosides by lymphocytes in long-term culture deficient in different purine enzymes. 642 79

Enzyme inhibitors used to simulate the inherited immunodeficiency diseases, adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) deficiency, have been assessed in cultured human lymphocytes. Only 2'-deoxycoformycin (dCF) completely inhibited ADA in T and B cells at concentrations in excess of 5 microM. Erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) and 8-amino guanosine (8-NH2GR) did not inhibit ADA or PNP completely at any concentration. Detailed metabolic experiments comparing viability and deoxynucleotide accumulation showed that B cell lines of malignant origin also accumulated high levels of dATP from 2'-deoxyadenosine (dAR), and dGTP from 2'-deoxyguanosine (dGR) as effectively as T cells--even without inhibitors, however, dAR reduced cell viability only when ADA was inhibited by dCF, whilst dGR was equally toxic with or without inhibitor, even to a line which accumulated no dGTP. These experiments indicate that cultured lymphocytes, using either EHNA or 8-NH2GR as enzyme inhibitor, are not valid models of the toxicity to the immune system in inherited ADA or PNP deficiency. They demonstrate that the ability to accumulate high levels of dATP or dGTP is not exclusive to T cells and that the in vitro toxicity of dAR or dGR could relate to the use of excess substrate and/or accumulation in different nucleotide, not deoxynucleotide pools.
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PMID:B cells as well as T cells form deoxynucleotides from either deoxyadenosine or deoxyguanosine. 642 86

2'-Deoxycoformycin, a tight-binding adenosine deaminase inhibitor, was administered to 11 adult patients with refractory lymphoproliferative diseases. Total doses ranged from 1.0 to 13.5 mg/kg. Inhibition of lymphoblast adenosine deaminase was obtained in all cases and tumor cytoreduction was noted in eight of ten cases, but no clinically meaningful remissions were obtained. Major toxicities occurred in five patients and included pulmonary edema, renal failure, central nervous system toxicity, hypotension, and death. Toxicity prevented retreatment in several cases in which marked cytoreduction occurred. Deoxyadenosine triphosphate accumulated to a variable extent in the red blood cells of all patients, and a reciprocal decrease in erythrocyte adenosine triphosphate was noted in all cases but one. All patients who suffered major organ toxicity had red blood cell deoxyadenosine triphosphate/adenosine triphosphate ratios greater than 1.3. These data suggest that the degree of replacement of adenosine triphosphate by deoxyadenosine triphosphate in erythrocytes reflects the biochemical milieu which may result in systemic toxicity following treatment with 2'-deoxycoformycin.
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PMID:Alterations in erythrocyte adenine nucleotide pools resulting from 2'-deoxycoformycin therapy. 660 Jun 52

The toxicity of the deoxyribonucleosides, 2'-deoxyadenosine, 2'-deoxyguanosine, and thymidine, for human T lymphoblasts is mediated by the accumulation of the corresponding deoxyribonucleoside triphosphate (dATP, dGTP, or dTTP, respectively). We have examined whether leukemic cells of non-T-cell origin are capable of accumulating deoxyribonucleotides in culture and whether this capability correlates with the activities of purine metabolizing enzymes in these cells. We have found that non-T, non-B acute lymphoblastic leukemia cells with low ecto-5'-nucleotidase and high adenosine deaminase activities increase their dATP pools by greater than tenfold when exposed to deoxyadenosine and an inhibitor of adenosine deaminase in culture. Cells from 2 of 9 patients with chronic lymphocytic leukemia and 4 of 11 patients with acute nonlymphoblastic leukemia achieved similar elevations in dATP, but there was no relationship between dATP accumulation and adenosine deaminase, purine nucleoside phosphorylase, or ecto-5'-nucleotidase activities. Treatment of four individuals with acute lymphoblastic leukemia with the adenosine deaminase inhibitor, 2'-deoxycoformycin, resulted in elevations in plasma deoxyadenosine concentrations and in increments in lymphoblast dATP levels that were similar to those measured in lymphoblasts cultured with deoxyadenosine and deoxycoformycin prior to treatment. In vitro incubations of leukemic cells with deoxyribonucleosides may provide a rational basis for the use of these compounds as chemotherapeutic agents.
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PMID:Deoxyribonucleoside triphosphate accumulation by leukemic cells. 660 41


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