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

Two recombinant retroviral vectors encoding the cDNA of the human adenosine deaminase (ADA; EC 3.5.4.4) gene and the bacterial neomycin resistance (Neo) gene have been used to transduce bone marrow cells obtained from four patients affected by the ADA-deficient variant of severe combined immunodeficiency. By utilizing the long-term marrow culture system, freshly isolated bone marrow cells were subjected to multiple infection cycles with cell-free supernatants containing high titers of viral vector and then maintained in long-term marrow culture in the absence of any overt selection pressure. By using this experimental protocol, about 30-40% of the hematopoietic progenitors were productively transduced with the viral vector, as judged by the appearance of G418-resistant colonies derived from granulocyte/macrophage and multipotent hematopoietic progenitor cells. The vector-encoded human ADA gene was expressed efficiently in both the myeloid and lymphoid progeny of the cultured bone marrow cells, reaching levels between 15% and 100% as compared to the levels of ADA in normal bone marrow cells. The efficiency of gene transfer and ADA production was proportional to the number of infection cycles. Furthermore, transduction of the ADA vectors into the bone marrow cells derived from an ADA-deficient patient restored the capacity of the cells to respond to phytohemagglutinin and interleukin 2.
Proc Natl Acad Sci U S A 1989 Sep
PMID:Retroviral vector-mediated high-efficiency expression of adenosine deaminase (ADA) in hematopoietic long-term cultures of ADA-deficient marrow cells. 254 45

In rat adipocytes, the breakdown of phosphoinositides labelled by a 3 h incubation with [3H]inositol resulted in the accumulation of labelled inositol mono-, bis- and trisphosphates in the presence of oxytocin, vasotocin or vasopressin. Oxytocin at a concentration of 1 nM markedly increased phosphoinositide breakdown. Incubation of adipocytes both during the 3 h labelling and the 10 min breakdown period in a low adenosine medium (presence of adenosine deaminase) or high adenosine medium (presence of 0.1 microM N6-(phenylisopropyl)adenosine) (PIA) did not affect basal or ligand-stimulated phosphoinositide breakdown. The addition of 1 microM PIA only during the measurement of phosphoinositide breakdown variably stimulated basal breakdown but significantly potentiated that due to oxytocin. Isoproterenol similarly had little effect on basal but inhibited oxytocin stimulation of phosphoinositide breakdown. Insulin did not affect basal or ligand-stimulated phosphoinositide breakdown in the low or high adenosine medium. However, in adipocytes incubated in the absence of added adenosine deaminase or PIA, insulin stimulated basal accumulation of inositol phosphates by about 20% and inhibited that due to oxytocin by about 20%. There was no significant effect of insulin on the stimulation by vasopressin or vasotocin of phosphoinositide breakdown. These results indicate that, in adipocytes, phosphoinositide breakdown stimulated by oxytocin is enhanced by adenosine, inhibited by isoproterenol and, under some conditions is inhibited by insulin.
Biochim Biophys Acta 1989 Sep 04
PMID:Regulation of oxytocin-induced phosphoinositide breakdown in adipocytes by adenosine, isoproterenol and insulin. 255 83

The effect of neuropeptide Y (NPY) on cell contractions of ventricular myocytes isolated from the adult rat heart was investigated. Maximum changes in cell length (dL) during stimulated (0.5 Hz) contractions were determined in presence of the phosphodiesterase inhibitor Ro 20-1724 (0.5 mM) and adenosine deaminase (5 U/ml). Under these basal conditions NPY (10(-6) M) reduced dL by 39% of control. Isoproterenol (10(-6) M) increased dL by 105% of control; the EC50 was 2 x 10(-9) M. NPY reduced the increase in dL achieved by isoproterenol in a dose dependent manner. The IC50 value was 1 x 10(-9) M and NPY (10(-6) M) produced complete inhibition. In the absence of the phosphodiesterase inhibitor the IC50 was 4 x 10(-9) M. The EC50 of isoproterenol and IC50 of NPY producing accumulation of cAMP in myocytes (Millar et al. 1988) exceeded the respective values of dL by one order of magnitude. Prior treatment of the myocytes with pertussis toxin abolished the potency of NPY to antagonize the increase in dL by isoproterenol while not interfering with the response to the beta-agonist. These results demonstrate a negative inotropic effect of NPY on the ventricular myocardial cell. Complete abolition of the effect of NPY by pertussis toxin indicate that this effect is mediated by a sarcolemmal receptor for NPY linked to adenylate cyclase via an inhibitory guanine nucleotide binding protein.
Naunyn Schmiedebergs Arch Pharmacol 1989 Sep
PMID:The negative inotropic effect of neuropeptide Y on the ventricular cardiomyocyte. 255 54

Adenosine deaminase was found to bind 6-hydroxy-1,6-dihydropurine ribonucleoside (II), formed by reversible addition of water to purine ribonucleoside (I) in a reaction analogous to formation of a tetrahedral intermediate in substrate deamination, with an apparent Ki value of 3 x 10(-13) M at 20 degrees C. 1,6-Dihydropurine ribonucleoside (IV), synthesized by photolysis of purine ribonucleoside in the presence of NaBH4, exhibited a Ki value of 5.4 x 10-6 M. After correction for differences between the relative free energies of solvation of II and IV, the 6-hydroxyl group of II was estimated to contribute more than 16 kcal to the free energy of binding, approaching the enthalapy of formation of a single hydrogen bond to charged group in the vapor phase. The relatively weak binding of IV and of substrate water suggests that entropic effects, arising from the cooperative action of binding determinants contained within these separate molecules, contribute more than 10 kcal/mol to the free energy of binding of II in which these binding determinants are contained within a single molecule. In free solution, the entropy of reversible hydration of I was evaluated by measuring the temperature dependence of equilibria of protonation of I and of pseudobase formation from I-methylpurinium ribonucleoside as -35 eu, comparable with the entropy of activation for the uncatalyzed hydrolysis of adenosine. In the active site of adenosine deaminase, this thermodynamic obstacle is evidently climbed spontaneously as a result of attractive interactions between the active site and the critical hydroxyl group at the 6-position.(ABSTRACT TRUNCATED AT 250 WORDS)
Biochemistry 1989 Sep 19
PMID:Contribution of a single hydroxyl group to transition-state discrimination by adenosine deaminase: evidence for an "entropy trap" mechanism. 255 14

As a model of somatic cell gene therapy, a normal adenosine deaminase (ADA) gene was introduced into a B-lymphoblastoid cell line (LCL) established from a patient with ADA deficiency by microcell-mediated chromosome transfer (MMCT). A LCL derived from his mother was used as a gene donor. Seven fusion experiments were performed and hybrid cells were pipetted into 123 wells. After selection in the presence of deoxyadenosine, cells grew in 12 wells at Week 9 after fusion. Among these wells, ADA activity of hybrids was low in 4 wells, 130-280% of that of the donor LCL in 7 wells and very high in one well. Hybrid cells in 4 wells with ADA-positive cells were investigated for the time-course of expression of ADA activity. In one well, ADA activity was expressed until Week 36, while, in 3 wells, ADA activity decreased or was lost between 21-36 weeks after fusion. These findings indicate the transfer of chromosome 20 containing a normal ADA gene into recipient cells and the deletion of this chromosome from some part of the hybrid cells. Karyotyping at Week 35 or 37 revealed 47 chromosomes in about 30% of the cells in 2 wells, which suggests that these hybrids were relatively stable in culture.
Nihon Ketsueki Gakkai Zasshi 1989 Sep
PMID:Transfer of the adenosine deaminase (ADA) gene of a B-lymphoblastoid cell line (LCL) to an ADA-deficient LCL by a microcell-mediated chromosome transfer technique. 258 53

Certain disorders of the immune system seem to be associated with skeletal defects. The association was first recorded by McKusick et al. (Bulletin of the Johns Hopkins Hospital 116:285-326, 1964). A number of relationships between lymphocytes and osteocytes can be proposed. These include a common environment for development, common metabolic needs and effects upon osteocytes by products (cytokines) elaborated from lymphocytes or monocytes during immune responses. Thus, bony defects of varying degrees of severity are seen in short-limb dwarfs, cartilage-hair hypoplasia, and adenosine deaminase (ADA) deficiency. Cytokine activation of osteoclasts accounts for the lytic lesions seen in malignancies and the excessive bone resorption which accompanies autoimmune disorders such as rheumatoid arthritis. Correction of primary immune deficiency is accomplished by bone marrow transplantation. If the bony abnormality is subtle (as in some cases of ADA deficiency) the skeletal problem is resolved; if the bone defect is major as in short-limb dwarfism, no improvement is seen.
Am J Med Genet 1989 Sep
PMID:Associations of the skeletal and immune systems. 268 81

In 15%-20% of children with severe combined immunodeficiency (SCID), the underlying defect is adenosine deaminase (ADA) deficiency. The overall goal of our research has been to identify the precise molecular defects in patients with ADA-deficient SCID. In this study, we focused on a patient whom we found to have normal sized ADA mRNA by Northern analysis and an intact ADA structural gene by Southern analysis. By cloning and sequencing this patient's ADA cDNA, we found a C-to-T point mutation in exon 11. This resulted in the amino acid substitution of a valine for an alanine at position 329 of the ADA protein. Sequence analysis revealed that this mutation created a new BalI restriction site. Using Southern analyses, we were able to directly screen individuals to determine the frequency of this mutation. By combining data on eight families followed at our institution with data on five other families reported in the literature, we established that five of 13 patients (seven of 22 alleles) with known or suspected point mutations have this defect. This mutation was found to be associated with three different ADA haplotypes. This argues against a founder effect and suggests that the mutation is very old. In summary, a conservative amino acid substitution is found in a high proportion of patients with ADA deficiency; this can easily be detected by Southern analysis.
Am J Hum Genet 1989 Sep
PMID:A high proportion of ADA point mutations associated with a specific alanine-to-valine substitution. 277 32

The mechanism of the depletion of ATP, recorded in the erythrocytes of adenosine deaminase-deficient children and of leukemia patients treated with deoxycoformycin, was investigated in normal human erythrocytes treated with this inhibitor of adenosine deaminase. Deoxyadenosine, which accumulates in both clinical conditions, provoked a dose-dependent accumulation of dATP, depletion of ATP, and increases in the production of inosine plus hypoxanthine. Concomitantly, there was an increase of AMP and IMP, but not of adenosine, indicating that catabolism proceeded by way of AMP deaminase. A series of nucleoside analogues (9-beta-D-arabinofuranosyladenine, N6-methyladenosine, 6-methylmercaptopurine ribonucleoside, tubercidin, ribavirin, and N-1-ribosyl-5-aminoimidazole-4-carboxamide riboside) also stimulated adenine nucleotide catabolism and increased AMP and IMP to various extents. The effects of deoxyadenosine and of the nucleoside analogues were prevented by 5'-iodotubercidin, an inhibitor of adenosine kinase. Strikingly, they were reversed if the inhibitor was added after the accumulation of nucleotide analogues and initiation of adenine nucleotide catabolism. Further analyses revealed linear relationships between the rate of phosphorylation of deoxyadenosine and nucleoside analogues and the increase in AMP and between the elevation of the latter above a threshold concentration of 10 microM and the rate of adenine nucleotide catabolism. Kinetic studies with purified erythrocytic AMP deaminase, at physiological concentrations of its effectors, showed that the enzyme is nearly inactive up to 10 microM AMP and increases in activity above this threshold. We conclude that the main mechanism whereby deoxyadenosine and nucleoside analogues stimulate catabolism of adenine nucleotides by way of AMP deaminase in erythrocytes is elevation of AMP, secondary to the phosphorylation of the nucleosides.
Cancer Res 1989 Sep 15
PMID:Mechanism of adenosine triphosphate catabolism induced by deoxyadenosine and by nucleoside analogues in adenosine deaminase-inhibited human erythrocytes. 278 93

Adenosine deaminase-deficient mutants of a mouse lymphoma cell line S49 have been isolated by a two-step selection process. In the first step, we derived mutant lines containing haploid levels of adenosine deaminase activity from wild-type cells. The selective medium contained tritiated deoxyadenosine, deoxycytidine, and deoxycoformycin. Wild-type cells were killed, presumably because of suicidal incorporation of tritiated deoxyadenosine via the adenosine deaminase pathway. The second step was to derive, from the partially deficient mutants, sublines that were virtually lacking adenosine deaminase, using tritiated deoxyadenosine and deoxycytidine. Four mutant clones were found to contain less than 5% of the enzyme activity of wild-type cells and virtually no immunoreactive adenosine deaminase protein. Northern blot analysis showed that the levels of adenosine deaminase mRNA were drastically reduced. Back-selection for adenosine deaminase-positive revertants can be accomplished by using a medium containing deoxyadenosine (as a sole source of purine), aminopterin, and thymidine or, alternatively, by using deoxyadenosine alone in a serum-free medium.
Somat Cell Mol Genet 1989 Sep
PMID:Isolation and characterization of S49 mouse lymphoma cell mutants deficient in adenosine deaminase. 278 37

The enzyme adenosine deaminase (adenosine aminohydrolase EC 3.5.4.4) from calf intestinal mucosa is commercially available at high purity grade yet, at the sensitivity at which fluorescence studies may be undertaken, a nonpeptidic fluorescence is detectable at lambda exmax = 350 nm and lambda emmax = 420 nm. A sevenfold decrease of this nonpeptidic fluorescence was obtained upon irradiation by the third harmonic (355 nm) of a Nd:YAG laser for 16 min, at 5 mJ/pulse, with a pulse width of 6 ns at a repetition rate of 10 Hz. The decline of fluorescence was accompanied by a negligible loss of enzymatic activity. Moreover, the integrity of the protein was ascertained by (i) its fluorescence (lambda exmax = 305 nm, lambda emmax = 335 nm) and lifetime distribution and (ii) its kinetics in the presence of the substrate adenosine and two inhibitors, all of which remained essentially unaltered. Laser photobleaching is a simple way to achieve a fluorescence grade adenosine deaminase.
Anal Biochem 1989 Sep
PMID:Laser photobleaching leads to a fluorescence grade adenosine deaminase. 281 1


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>