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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Although many of the new cardiotonic agents are known to increase cAMP and to inhibit with variable potency a low Km cAMP phosphodiesterase, there is still debate as to the mechanism(s) by which these agents act. In a rat adipocyte membrane model we demonstrate that only approximately 50% of the effect of the new cardiotonic agent sulmazole on cAMP accumulation can be attributed to phosphodiesterase inhibition and that the remaining production of cAMP involves stimulation of adenylate cyclase activity. Two distinct pathways for stimulation of adenylate cyclase are herein reported. Sulmazole, UD-CG 212 CL, enoximone, piroximone, amrinone, and milrinone are all shown to be competitive antagonists of inhibitory A1 adenosine receptors, with EC50 values of 11-909 microM. Elimination of the effects of endogenous adenosine with adenosine deaminase reveals a third distinct mechanism for activation of adenylate cyclase. This mechanism appears to involve Gi, the inhibitory guanine nucleotide-regulatory protein, in that sulmazole attenuates the capacity of GTP to inhibit adenylate cyclase activity, and covalent modification of Gi by pertussis toxin treatment abolishes the capacity of sulmazole to mediate stimulation. Thus, functional blockade of Gi activity is the likely mode of action. Restoration of sulmazole's stimulatory effect on adenylate cyclase activity in pertussis toxin-treated membranes can be accomplished by reconstituting purified preparations of either Gi or mixtures of Gi/Go into treated adipocyte membranes. Of note, this stimulatory effect is completely reversed by inhibitory receptor agonists. Thus, the new cardiotonic agent sulmazole mediates increases in cAMP accumulation by mechanisms other than phosphodiesterase inhibition, including A1 adenosine receptor antagonism and inhibition of Gi function.
Mol Pharmacol 1988 Apr
PMID:The new cardiotonic agent sulmazole is an A1 adenosine receptor antagonist and functionally blocks the inhibitory regulator, Gi. 312 27

Several sugar-modified 2,6-diaminopurine and guanine 2',3'-dideoxyribosides were synthesized and evaluated in vitro for their ability to inhibit the cytopathic effect and replication of human immunodeficiency virus (HIV), the causative agent of acquired immunodeficiency syndrome (AIDS). 3'-Azido-2,6-diaminopurine-2',3'-dideoxyriboside (AzddDAPR), 3'-fluoro-2,6-diaminopurine-2',3'-dideoxyriboside (FddDAPR), and 3'-fluoro-2',3'-dideoxyguanosine emerged as potent and selective anti-HIV agents in MT4 cells (50% effective antiviral dose: 0.3-4.5 microM). Their selectivity indexes, based on the ratio of the 50% cytotoxic dose to the 50% antiviral effective dose, were 157, 80, and 96, respectively, as compared to 106 for 2,6-diaminopurine-2',3'-dideoxyriboside (ddDAPR) and 132 for 2',3'-dideoxyadenosine (ddAdo), two other potent anti-HIV agents. The 9-beta-D-arabinoside and 9-beta-D-2'-deoxyxyloside derivatives of 2,6-diaminopurine were devoid of any antiretrovirus activity. Both AzddDAPR and FddDAPR, like the parent compounds ddDAPR and ddAdo, proved susceptible to deamination by beef intestine adenosine deaminase (Km, 11, 148, 29, and 73 microM, respectively). 2'-Deoxycoformycin, a potent inhibitor of adenosine deaminase, decreased the antiretrovirus and cytostatic activity of ddDAPR and FddDAPR to a greater extent than that of AzddDAPR. This suggests that ddDAPR and FddDAPR are primarily active as their guanine analogues, whereas AzddDAPR may be potentially active as a 2,6-diaminopurine derivative as well.
Mol Pharmacol 1988 Mar
PMID:Potent and selective activity of 3'-azido-2,6-diaminopurine-2',3'-dideoxyriboside, 3'-fluoro-2,6-diaminopurine-2',3'-dideoxyriboside, and 3'-fluoro-2',3'-dideoxyguanosine against human immunodeficiency virus. 325 4

In a previous study of three independent families of mutants selected for overproduction of adenylate deaminase (AMPD), we were not able to isolate a cDNA probe for the gene and so could not demonstrate its amplification directly. In addition to overproduction of AMPD, four proteins of unknown function, designated W, X, Y1, and Y2, accumulated, and by using the corresponding cDNA probes, we demonstrated amplification of all four genes. In independent mutant clones, sometimes all and sometimes only a subset of these genes were amplified. Assuming that all five genes are linked, the pattern of their coamplification suggested a genetic map in which AMPD lies between W and Y1. We show here that a two-step chromosome walk joins the W and Y1 genes, that the AMPD gene is the only expressed sequence between them, and that its amplification is indeed responsible for overproduction of the AMPD protein. In the course of this work, we cloned and studied two novel joints which mark rearrangements on either side of the AMPD gene. Each joint was generated independently in a single first-step mutant at single or low copy number. Remarkably, each joint was amplified preferentially in every second- and third-step mutant derived from the first-step line in which it was originally present, suggesting that the two independent rearrangements each generated amplification-prone structures.
Mol Cell Biol 1988 Jan
PMID:Preferential amplification of rearranged sequences near amplified adenylate deaminase genes. 333 58

We have obtained single crystals of a cloned mammalian adenosine deaminase (Mr = 41,000), a key enzyme in purine degradation and in normal development of the immune system, that are suitable for high-resolution structural analysis. The crystals belong to the space group C2 with unit cell parameters a = 101.68 A (1 A = 0.1 nm), b = 94.38 A, c = 85.51 A, and beta = 96.54 degrees. The asymmetric unit contains two enzyme molecules.
J Mol Biol 1988 Apr 05
PMID:Preliminary X-ray analysis of crystals of murine adenosine deaminase. 339 52

Simplified Moloney murine leukemia virus-based recombinant retrovirus vectors have been constructed which transduce human adenosine deaminase (ADA) cDNA. ADA transcription is under the control of the constitutive promoter for the human X chromosome phosphoglycerate kinase (pgk) gene. In these simplified vectors, dominant selectable markers are not included and selection is dependent on overproduction of functional ADA enzyme. Primary murine hematopoietic cells were infected with helper-free recombinant ADA virus generated from Psi-2 packaging cells. Protein analysis revealed that human ADA enzyme was expressed in progenitor-derived hematopoietic colonies in vitro and CFU-S-derived spleen colonies in vivo. Enzyme expression was dependent on transcription from the pgk promoter. ADA expression in primary murine hematopoietic cells directed by the internal promoter was not adversely affected by the presence of the Moloney virus long terminal repeat enhancer sequence. Use of these vectors allows systematic evaluation of the effects of specific sequences in recombinant retrovirus vectors on expression in primary murine hematopoietic cells in vivo.
Mol Cell Biol 1987 Oct
PMID:Retrovirus-mediated gene transfer of human adenosine deaminase: expression of functional enzyme in murine hematopoietic stem cells in vivo. 368 89

Neplanocin A and aristeromycin are carbocyclic adenosine analogs that differ only in that neplanocin A contains a double bond in the carbocyclic ring, whereas this ring in aristeromycin is saturated. We have compared the metabolism and some of the metabolic effects of neplanocin A and synthetic (+/-)-aristeromycin (C-Ado) in murine leukemia L1210 cells in culture. C-Ado, as shown earlier, was not only converted to its own phosphates but also was metabolized to phosphates of carbocyclic guanosine. Both rapidly proliferating and slowly proliferating or resting cells phosphorylated C-Ado, but C-Ado was not converted to phosphates of carbocyclic guanosine in detectable amounts in cells whose growth had reached a plateau. When the metabolism of neplanocin and C-Ado was examined in the same experiment, both analogs were converted to the triphosphate analogs of ATP; no conversion of neplanocin A to the corresponding carbocyclic analogs of guanine nucleotides was detected, whereas C-Ado was converted to the carbocyclic analog of GTP in amounts that approximated the GTP pool. This difference in metabolism was associated with a marked difference in effects of the two analogs on the utilization of hypoxanthine and guanine which was inhibited by C-Ado but not by neplanocin. The failure of neplanocin A to be converted to analogs of guanine nucleotides apparently is the result of poor capacity of its monophosphate to serve as a substrate for AMP deaminase; the Vmax for deamination of neplanocin-5'-monophosphate by this enzyme was only 5% of that for C-Ado monophosphate. In contrast, neplanocin A was a better substrate than C-Ado for adenosine deaminase.
Mol Pharmacol 1986 Apr
PMID:Differences in the metabolism and metabolic effects of the carbocyclic adenosine analogs, neplanocin A and aristeromycin. 370 57

Four genes encoding proteins designated as W, X, Y1, and Y2 were found previously to be amplified at different levels in a Chinese hamster fibroblast mutant line selected for overproduction of adenylate deaminase. To gain information on the molecular mechanisms responsible, we studied the levels of amplification and the structures of these four genes in several lineages of mutant cells with comparable activities of adenylate deaminase, the selected enzyme. Only the W gene was amplified in all the lines. In one line, the X, Y1, and Y2 genes were coamplified, while in others either the Y1 gene or the pair X and Y2 were coamplified. The results were consistent with linkage of all the genes--in a particular order--in an amplifiable sequence with variable endpoints. Novel joints with a nonrandom distribution were observed. We frequently detected rearranged copies of the W gene, but very few novel joints were present in the other three genes in the six highly amplified lines examined. Some of the novel joints in gene W were highly amplified; they were generated by reamplification of a rearrangement that appeared at an early selection step. In some lines, reamplification was accompanied by deletion or mass correction of preexisting units. We discuss mechanisms which might account for these observations.
Mol Cell Biol 1986 May
PMID:Segregation and rearrangement of coamplified genes in different lineages of mutant cells that overproduce adenylate deaminase. 378 79

Two transcription factors, COUP and S300-II, were isolated and partially purified from HeLa cell nuclear extracts. Both factors are required for the efficient transcription in vitro of the ovalbumin gene but not the simian virus 40 early genes. COUP factor binds to the chicken ovalbumin upstream promoter (COUP) sequence which lies between -70 to -90 base pairs upstream from the cap site. A series of competition experiments with a band-shifting assay was carried out to determine the relative affinity of COUP box transcription factor for various promoters. We found that a promoter DNA fragment isolated from the ovalbumin gene competes better than those isolated from the ovomucoid, Y, and alpha-genes. In contrast, the the simian virus 40 early genes, the beta-globin gene, and the adenosine deaminase gene promoters do not compete well in this assay. The molecular weight of the COUP factor was estimated by S-300 column chromatography, glycerol gradient centrifugation to be 90,000. However, two bands were observed in sodium dodecyl sulfate gel electrophoresis of cross-linked COUP factor to a 32P-labeled oligonucleotide containing the COUP sequence. The protein moieties of the major and minor bands were estimated to be 85,000 to 90,000 and 40,000 to 45,000, respectively. The S300-II factor with an apparent molecular weight of 45,000 in an S-300 column is required for function in an in vitro reconstituted transcription system. In contrast to the COUP factor, the S300-II factor does not have apparent specificity for binding to the ovalbumin gene promoter. The S300-II factor may function by interacting with RNA polymerase or other DNA-binding transcription factors.
Mol Cell Biol 1986 Dec
PMID:Identification of two factors required for transcription of the ovalbumin gene. 379 2

Human adenosine deaminase (ADA) is an important purine catabolic enzyme which irreversibly deaminates adenosine and deoxyadenosine. Severe genetic deficiency of ADA leads to an immunological deficiency state in which T-lymphoid cells are selectively destroyed by the accumulation of toxic levels of deoxyadenosine and deoxy-ATP. In preparation for transfer of ADA sequences into a variety of cell types, we explored expression of ADA cDNAs transfected into cultured cells within a simian virus 40-based expression vector. After transfection into monkey kidney (COS) cells, ADA cDNA encompassing the entire coding region of the protein generated human ADA activity. An unexpected finding, however, was the identification of a cDNA clone that failed to produce either human enzyme activity or immunoreactive ADA protein. As this pattern is typical of many naturally occurring mutant ADA alleles, we characterized the molecular defect in this clone. DNA sequence analysis revealed a single nucleotide substitution in amino acid position 50 (glycine-valine). Northern blotting with a unique 17-mer oligonucleotide demonstrated the absence of the mutant sequence in the mRNA from which the cDNA library giving rise to the mutant cDNA was constructed. Therefore, the substitution in the variant cDNA was created during cloning. These data define one critical region of the human ADA protein molecule and suggest a convenient strategy for characterization of the phenotypes associated with naturally occurring mutant alleles.
Mol Cell Biol 1985 Apr
PMID:Transient expression of human adenosine deaminase cDNAs: identification of a nonfunctional clone resulting from a single amino acid substitution. 383 97

(-)-N6-(R-4-Hydroxyphenylisopropyl)adenosine (HPIA) was iodinated with NaI and trace 125I. Mono- and diiodinated reaction products and the starting material were separated by high pressure liquid chromatography and the structures of the reaction products were verified by NMR. (-)-N6-(R-Phenylisopropyl)adenosine (PIA), IHPIA, and I2HPIA decreased rat atrial contractility with ED50 values of 24, 28, and 33 nM, respectively. The contractile effects of these compounds were competitively blocked by theophylline (KI = 7.9 microM), but were not affected by adenosine deaminase. IHPIA also inhibited (-)isoproterenol-stimulated cyclic AMP accumulation in adipocytes with an ED50 (10 nM) and to an extent (83%) nearly identical to PIA. [125I]HPIA prepared using carrier-free 125I bound to adenosine receptors on membranes from rat cerebral cortex, adipocyte ghosts, and heart ventricles. Binding was inhibited stereospecifically by PIA and by other adenosine analogues and alkylxanthines. The KD of [125I]HPIA determined kinetically using brain membranes at 21 degrees was 0.94 nM (K1 = 2.55 X 10(7) M-1 min-1; K-1 = 0.024 min-1) in good agreement with the equilibrium determination of 1.94 nM. The density of adenosine receptors in brain membranes was found to be 871 fmol/mg of protein. When normalized to protein, the density of receptors in heart membranes and adipocyte ghosts, respectively, was found to be 39- and 2.3-fold less than in brain membranes. We conclude that [125I]HPIA can be rapidly synthesized and purified, binds to adenosine R-sites and is an agonist radioligand resistant to adenosine deaminase. Computer modeling of the equilibrium binding resulting from the use of mixed stereoisomers of a radioligand indicates that the combined use of (-)[125I]HPIA and (+)[125I]HPIA would result in the generation of nonlinear Scatchard plots.
Mol Pharmacol 1984 Nov
PMID:Purification and characterization of (-)[125I]hydroxyphenylisopropyladenosine, an adenosine R-site agonist radioligand and theoretical analysis of mixed stereoisomer radioligand binding. 609 94


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