Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

We have studied the expression of mRNA encoding adenosine deaminase (ADA; EC 3.5.4.4), purine nucleoside phosphorylase (PNP; EC 2.4.2.1), and terminal deoxynucleotidyltransferase (TdT; EC 2.7.7.31) in different leukemic cell lines of B- and T-cell lineage. Incubation of leukemic cells in the presence of the phorbol esters, 12-O-tetradecanoyl-phorbol-13-acetate or phorbol 12,13-dibutyrate, resulted in reduction of ADA and TdT mRNA levels, while PNP mRNA levels increased under the same treatment. The effect of TPA on the activity of these enzymes correlated well with its effects on their mRNA levels. TPA caused a 40% decrease in ADA and a 60% decrease in TdT enzyme activity, after 6 h of treatment. In contrast, PNP activity increased up to 200% after 12 h of incubation with the phorbol ester. The changes induced by the phorbol esters in the levels of mRNA of ADA, PNP, and TdT, and their enzyme activities in human leukemic cell lines mimic the changes in the activities of these enzymes in developing T-lymphocytes during differentiation in vivo, suggesting a role for protein kinase C in the regulation of ADA, PNP, and TdT gene expression during lymphoid cell differentiation.
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PMID:Phorbol esters induce changes in adenosine deaminase, purine nucleoside phosphorylase, and terminal deoxynucleotidyl transferase messenger RNA levels in human leukemic cell lines. 211 May 2

A novel fluorescent competitive inhibitor of adenosine deaminase (EC 3.5.4.4) (ADA), 1-N6-etheno-[erythro-9-(2-hydroxy-3-nonyl)] adenine (epsilon-EHNA), is protonated at the active site of the enzyme. In epsilon-EHNA [K1 = (4.06 +/- 1.00) 10(-6) M] part of the competitive inhibition of EHNA is combined with spectroscopic properties of etheno-adenines. Computer subtraction of the fluorescence excitation spectrum of ADA from that of its equimolar complex with epsilon-EHNA yielded the corrected excitation spectrum of epsilon-EHNA at the active site of the enzyme. This spectrum mimics that of epsilon-EHNA at pH 5.5 in buffer solution and is suggested to indicate a shift in protonation equilibrium at the active site.
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PMID:The protonated form of 1-N6-etheno-[erythro-9-(2-hydroxy-3-nonyl)] adenine is identified at the active site of adenosine deaminase. 215 76

We have previously characterized mutant adenosine deaminase (ADA; adenosine aminohydrolase, EC 3.5.4.4) enzymes in seven children with partial ADA deficiency. Six children shared common origins, suggesting a common progenitor. However, we found evidence for multiple phenotypically different mutant enzymes. We hypothesized that many of the mutations would be at CpG dinucleotides, hot spots at which spontaneous deamination of 5-methylcytosine results in C to T or G to A transitions. Digestion of DNA from these children with Msp I and Taq I, enzymes recognizing CpG dinucleotides, identified three different mutations, each correlating with expression of a different mutant enzyme. Sequencing of cDNA clones and genomic DNA amplified by polymerase chain reaction confirmed the presence of C to T or G to A transitions at CpG dinucleotides (C226 to T, G446 to A, and C821 to T, resulting in Arg76 to Trp, Arg149 to Gln, and Pro274 to Leu). A "null" mutation, also found in two ADA-deficient severe combined immunodeficient children, was serendipitously detected as gain of a site for Msp I. Simultaneous loss of a site for Bal I defined the precise base substitution (T320 to C, Leu107 to Pro), confirmed by sequence analysis. To determine the true frequency of hot spot mutation in these children, consecutively ascertained through a newborn screening program, we sequenced cDNA from the remaining alleles. Two others were hot spot mutations (C631 to T and G643 to A, resulting in Arg211 to Cys and Ala215 to Thr), each again resulting in expression of a phenotypically different mutant enzyme. Only one additional mutation (previously identified by us) is not in a hot spot. These seven mutations account for all 14 chromosomes in these children. There is thus a very high frequency of hot spot mutations in partial ADA deficiency. Most of these children carry two different mutant alleles. We were able to correlate genotype and phenotype and to dissect the activity of individual mutant alleles.
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PMID:Hot spot mutations in adenosine deaminase deficiency. 216 47

Four 2-amino-6-halo- and four 6-halo-2',3'-dideoxypurine ribofuranosides (ddPs) were synthesized and tested for in vitro activity to suppress the infectivity, cytopathic effect, Gag protein expression, and DNA synthesis of human immunodeficiency virus (HIV). The comparative order of in vitro anti-HIV activity of the eight 6-halo-ddPs was as follows: 2-amino-6-fluoro, 2-amino-6-chloro, 6-fluoro greater than 2-amino-6-bromo greater than 2-amino-6-iodo, 6-chloro greater than 6-bromo greater than 6-iodo. 2-Amino-6-fluoro-, 2-amino-6-chloro-, and 6-fluoro-ddPs showed a potent activity against HIV comparable to that of 2',3'-dideoxyinosine (ddI) or 2',3'-dideoxyguanosine (ddG) and completely blocked the infectivity of HIV without affecting the growth of target cells. The lipophilicity order was as follows: 2-amino-6-iodo greater than 2-amino-6-bromo greater than 2-amino-6-chloro greater than 2-amino-6-fluoro much greater than ddG greater than ddI. All eight 6-halo-ddPs were substrates for adenosine deaminase (ADA; adenosine aminohydrolase, EC 3.5.4.4). The relative rates of hydrolysis by ADA were as follows: ddA, 2-amino-6-fluoro much greater than 2-amino-6-chloro, 2-amino-6-bromo greater than 2-amino-6-iodo. Taken together, these compounds may represent an additional class of lipophilic prodrugs for ddI and ddG and may also provide a strategy for endowing therapeutic purine nucleosides with desirable lipophilicity.
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PMID:Lipophilic halogenated congeners of 2',3'-dideoxypurine nucleosides active against human immunodeficiency virus in vitro. 225 Dec 84

Recombinant retroviruses encoding human adenosine deaminase (ADA; adenosine aminohydrolase, EC 3.5.4.4) have been used to infect murine hematopoietic stem cells. In bone marrow transplant recipients reconstituted with the genetically modified cells, human ADA was detected in peripheral blood mononuclear cells of the recipients for at least 6 months after transplantation. In animals analyzed in detail 4 months after transplantation, human ADA and proviral sequences were detected in all hematopoietic lineages; in several cases, human ADA activity exceeded the endogenous activity. These studies demonstrate the feasibility of introducing a functional human ADA gene into hematopoietic stem cells and obtaining expression in multiple hematopoietic lineages long after transplantation. This approach should be helpful in designing effective gene therapies for severe combined immunodeficiency syndromes in humans.
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PMID:Expression of human adenosine deaminase in mice reconstituted with retrovirus-transduced hematopoietic stem cells. 229 99

A high titer retroviral vector was used to transfer a human adenosine deaminase (h-ADA) cDNA into murine bone marrow cells in vitro. The h-ADA cDNA was linked to the retroviral promoter, and the vector also contained a neomycin phosphotransferase gene as a selectable marker. Infected marrow was transplanted into syngeneic W/Wv recipients, and h-ADA expression was monitored for 5.5 months. Several weeks after transplantation, h-ADA was detected in the erythrocytes of all nine recipients, eight of which expressed levels equal to the endogenous enzyme. This level of expression persisted in two of six surviving mice, while expression in three others stabilized at lower, but readily detectable, levels. Only one mouse had no detectable h-ADA after 5.5 months. Vector DNA sequences with common integration sites were found in hematopoietic and lymphoid tissues of the mice at 5.5 months, providing evidence that hematopoietic stem cells had been infected. Furthermore, all mice transplanted with marrow that had been selected in G418 before infusion had multiple vector copies per genome. While this category included the two highest h-ADA expressors, it also included the negative mouse. Thus, multiple copies of the vector were not sufficient to guarantee long-term h-ADA expression. Mice were monitored for "helper virus" infections with an assay designed to detect a wide range of replication-competent retroviruses, including those endogenous to the mouse genome. No helper virus was detected in the two highest h-ADA expressors, ruling out helper-assisted vector spread as a cause of the high h-ADA expression. These results help provide a foundation for the development of somatic gene therapy techniques to be used in the treatment of human disease.
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PMID:Expression of human adenosine deaminase in mice after transplantation of genetically-modified bone marrow. 232 23

We developed a simple, rapid, and automated method for simultaneous measurement of adenosine deaminase (ADA, EC 3.5.4.4) isoenzymes in human serum, based on their apparent difference in Ki values for erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) as inhibitor. Serum ADA was partially purified by CM-Sephadex, gel-filtration, and affinity chromatography into two types of isoenzymes, designated ADA1 (300 kDa) and ADA2 (120 kDa). Because ADA2 has a higher Km for adenosine and higher Ki values for EHNA than does ADA1, the activity of ADA1 is almost completely inhibited by EHNA at 0.1 mM (analytical recovery 4.1%), whereas ADA2 is practically unaffected (analytical recovery 94.8%) by that concentration of EHNA. We measured the activities of ADA2 and total ADA in the presence and absence of 0.1 mM EHNA. ADA1 activities were calculated by subtracting the activity of ADA2 from that of total ADA. The mean within-assay CV was 5.7% for ADA1 and 2.7% for ADA2. The interassay CV was 2.8% for ADA1 and 3.1% for ADA2. Results of the present method correlated well (r = 0.9026 for ADA1, 0.9438 for ADA2) with those of the ion-exchange chromatography method. The upper limits of the reference intervals, as calculated from data for 320 healthy donors, are 7.2 U/liter for ADA1, and 14.6 U/liter for ADA2. This method is suitable for analysis of large numbers of samples in clinical laboratories for routine monitoring of the activities of ADA isoenzymes in serum.
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PMID:Automated enzymatic measurement of adenosine deaminase isoenzyme activities in serum. 238 28

The metabolism of adenosine and its effects on phosphoribosylpyrophosphate, PP-ribose-P, dependent nucleotide synthesis were studied using erythrocytes from patients with adenosine deaminase and hypoxanthine phosphoribosyltransferase deficiency as models. The phosphorylation of adenosine was progressively inhibited by concentrations of adenosine greater than 1 mumol L-1 for control and ADA deficient erythrocytes. There was essentially no initial rate of phosphorylation at 30 mumol L-1 adenosine. Adenosine, 1 mumol L-1, also caused a 60% reduction in PP-ribose-P concentration in ADA deficient erythrocytes. For HPRT deficient erythrocytes in which ADA activity was blocked by coformycin, 10 mumol L-1 inosine stimulated PP-ribose-P dependent nucleotide synthesis from adenine, whereas, 10 mumol L-1 adenosine inhibited nucleotide synthesis. These observations suggest that adenosine phosphorylation and PP-ribose-P dependent nucleotide synthesis are inhibited under conditions in which adenosine accumulates, such as in hereditary or pharmacologically induced ADA deficiency.
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PMID:Substrate inhibition of adenosine phosphorylation in adenosine deaminase deficiency and adenosine-mediated inhibition of PP-ribose-P dependent nucleotide synthesis in hypoxanthine phosphoribosyltransferase deficient erythrocytes. 245 96

The mechanism by which the expression of adenosine deaminase (ADA, EC 3.5.4.4) is regulated in murine tissues was analyzed. Elevated levels of the specific activity of this enzyme appeared to correlate directly with steady state levels of ADA mRNA. Transcriptional studies using nuclear run-on experiments revealed a pronounced asymmetric distribution of nascent transcripts across the ADA genome. The ability to detect nascent transcripts downstream of the promoter proximal region varied among tissues and correlated with tissue levels of ADA mRNA and enzymatic activity. These results indicate that processes affecting the elongation of nascent transcripts into completed primary transcripts play a role in tissue-specific expression of ADA.
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PMID:Adenosine deaminase gene expression. Tissue-dependent regulation of transcriptional elongation. 247 47

This study describes a type of retroviral vector called double-copy (DC) vector that was designed to improve the expression of transduced genes. The unique feature of DC vectors is that the transduced gene is inserted within the U3 region of the 3' long terminal repeat (LTR). Consequently, in the infected cell the gene is duplicated and transferred to the 5' LTR. The important result is that in its new position the gene is placed outside the retroviral transcriptional unit, eliminating or at least reducing the negative effects of the retroviral transcriptional unit. The utility of the DC vector design was tested by using a 2.1-kilobase-pair (kbp)-long adenosine deaminase (ADA; EC 3.5.4.4) minigene that was inserted into the 3' LTR of the N2 retroviral vector, generating a 2.7-kbp-long chimeric LTR. DNA blot analysis was used to show that the chimeric LTR was faithfully duplicated in cells infected with the corresponding virus, generating two copies of the ADA minigene, one copy in each LTR. Insertion of the ADA minigene into the 3' LTR of the N2 vector led to a 10- to 20-fold increase in ADA transcripts and human ADA isozyme synthesized in NIH 3T3 cells as compared to cells harboring the same vector in which the ADA minigene was inserted between the two LTRs. A similar increase in ADA expression was observed in two human lymphoid cell lines tested, HUT 78 and Raji. These results are consistent with previous observations that upstream promoters exert an inhibitory effect on promoters placed downstream and bear out the predictions used in the design of DC vectors. The use of DC vectors may contribute to the solution of the problems encountered in expressing retrovirally transduced genes in cultured cells and, in particular, when introduced into the live animal.
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PMID:Improved gene expression upon transfer of the adenosine deaminase minigene outside the transcriptional unit of a retroviral vector. 254 34


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