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Query: UNIPROT:P06889 (Mol)
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The intermediary role and relative importance of cAMP in follicle-stimulating hormone (FSH) hormonal action were reinvestigated at the level of the rat granulosa cell employing Rp-cAMPS, a novel antagonistic analog of cAMP. This approach may not only provide for direct documentation of cAMP dependence, but may also, by inference, highlight the potential relative importance of other putative intracellular second messenger systems. Initial cell-free validation studies indicated that Rp-cAMPS is capable of effectively competing with cAMP for binding to and activation of the regulatory subunit of the granulosa cell A-kinase holoenzyme. Subsequent whole-cell studies employed cultured rat granulosa cells, the cAMP-phosphodiesterase activity of which was suppressed with ZK62711. Basal progesterone accumulation was relatively low, remaining unaffected by treatment with a maximally effective dose of Rp-cAMPS by itself (10(-3) M). Whereas treatment with FSH (30 ng/ml) resulted in a substantial increase in progesterone accumulation, concurrent treatment with increasing concentrations (10(-6)-10(-3) M) or Rp-cAMPS brought about dose-dependent decrements in the FSH effect with a median effective dose of 1.8 +/- (SE) 0.4 x 10(-5) M and a maximal, but incomplete inhibitory effect of 70 +/- (SE) 6%. Higher concentrations of FSH (greater than or equal to 100 ng/ml) progressively diminished, but did not abolish the Rp-cAMPS blockade. Removal of Rp-cAMPS resulted in progressive resumption of FSH responsiveness suggesting reversibility of action. Significantly, Rp-cAMPS proved highly effective in blocking the action of its agonistic diastereomer Sp-cAMPS. However, Rp-cAMPS was unable to block the action of the lactogenic receptor agonist prolactin, the second messenger of which remains uncertain. Taken together, these findings provide additional direct support to the notion that cAMP may be an intracellular second messenger of FSH. However, to the extent that Rp-cAMPS is incapable of complete neutralization of FSH action, our findings further suggest that cAMP may play a central, albeit non-exclusive role in FSH-supported granulosa cell differentiation and that other putative second messenger systems may also be at play.
Mol Cell Endocrinol 1990 Jul 30
PMID:Blockade of granulosa cell differentiation by an antagonistic analog of adenosine 3',5'-cyclic monophosphate (cAMP): central but non-exclusive intermediary role of cAMP in follicle-stimulating hormone action. 217 13

The comparison of the Km and Vmax values for the primers was carried out. The primers were either completely complementary to the template or contained non-complementary bases at different positions with respect to the 3'-end. The addition of NaF, selectively inhibiting 3'----5'-exonuclease activity of the enzyme, was shown to result in the increase of Vmax values by 10% and 30% for complementary and partially complementary primers, respectively, Km values of the latters being unchanged. Km values for d[(pT)10pC] is about 146-fold greater than that for d[(pT)11]. Km values for d[(pT)7pC(pT)2] (20 microM) and d[[(pT)2pC]3pT] (20 microM); d[(pT)4pC(pT)5] (5.0 microM); d[(pC)(pT)7] (1.3 microM) and d[(pT)2pC(pT)7] (1.2 microM) are comparable with those for d[(pT)2] (22 microM), d[(pT)5] (4.1 microM) and d[(pT)7] (1.2 microM), respectively, but not with the decathymidylate d[(pT)10] (0.2 microM). We suggest that it is not the length of the primers but the number of bases in the fragment beginning with the first nucleotide from the 3'-end and ending in the non-complementary base, that determines the efficiency of interaction of the primers containing non-complementary bases with the enzyme. The addition of one link to d(pT)n (n less than or equal to 10) resulted in a 1.8-fold increase in the affinity. When 11 less than n less than 25 the affinity is decreased so that d(pT)22-23 have minimal affinity to the enzyme. The primers containing more than 50 units were found to have about the same affinity (calculated on base concentration) as d(pT)10-11.
Mol Biol (Mosk)
PMID:[The effect of bases non-complementary to the template on the efficacy of primer interaction with the Klenow fragment of DNA polymerase I from Escherichia coli]. 219 84

3'-Azido-2',3'-dideoxyuridine (AzdU, CS-87) is a potent inhibitor of human immunodeficiency virus replication in human peripheral blood mononuclear cells (PBMC) with limited toxicity for human bone marrow cells (BMC). In the present study, metabolism of AzdU was investigated in human PBMC and BMC after exposure of cells to 2 or 10 microM [3H]AzdU. 3'-Azido-2',3'-dideoxyuridine-5'-monophosphate (AzdU-MP) was the predominant metabolite, representing approximately 55 to 65% of intracellular radioactivity in both PBMC and BMC at all times. The AzdU-5'-diphosphate and -5'-triphosphate intracellular levels were 10- to 100-fold lower than the AzdU-MP levels and, of note, AzdU-5'-triphosphate was not detected in human BMC. Using anion exchange chromatography, a new peak of radioactivity, distinct from any known anabolites, was detected. This chromatographic peak was found to be resistant to alkaline phosphatase but was hydrolyzed by 5'-phosphodiesterase, yielding AzdU-MP. Incubation of [3H]AzdU and D-[1-14C]glucose in PBMC and BMC produced a double-labeled peak with the same retention time as the anabolite, suggesting formation of a hexose derivative of AzdU. A novel high performance liquid chromatography method was developed that allowed for the separation of nucleosides, nucleotides, and carbohydrate derivatives thereof. Using this highly specific method, the putative AzdU-hexose actually was separated into two chromatographic peaks. These novel metabolites were identified as 3'-azido-2',3'-dideoxyuridine-5'-O-diphosphoglucose and 3'-azido-2',3'-dideoxyuridine-5'-O-diphospho-N-acetylglucosamine. Following 48 hr of incubation with [3H] AzdU, as much as 20 and 30% of these AzdU metabolites accumulated in PBMC and BMC, respectively. When AzdU was removed from the cell cultures, intracellular AzdU diphosphohexose concentrations decayed in a monophasic manner, with an elimination half-life of 14.3 hr. By 48 hr, levels of 0.3 pmol/10(6) cells were still detected, reflecting a gradual anabolism of these metabolites. Elimination of AzdU-MP and AzdU-5'-diphosphate was characterized by a two-phase process, with a short initial half-life of 0.83 and 0.24 hr and a long terminal half-life of 14.10 and 8.24 hr, respectively. Similar diphosphohexoses of deoxyuridine (dUrd) were also detected in human PBMC and BMC after exposure to [3H]dUrd, suggesting that dUrd derivatives are metabolized in a similar manner. In summary, the discovery of novel metabolic pathways for dUrd analogs demonstrates that AzdU has unique metabolic features that may contribute to the low toxicity of this anti-HIV agent in human BMC and also affect its mechanism of action.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Pharmacol 1990 Dec
PMID:Cellular metabolism of 3'-azido-2',3'-dideoxyuridine with formation of 5'-O-diphosphohexose derivatives by previously unrecognized metabolic pathways for 2'-deoxyuridine analogs. 225 Jun 66

Three linear DNA plasmids were found in isolate RI-64 of anastomosis group 4 (AG-4) of Rhizoctonia solani. These plasmids, designated pRS64-1, -2, and -3, possessed the same size of 2.7 kb. Restriction mapping and Southern hybridization analysis of pRS64-1, -2, and -3 revealed the presence of homologous regions at both termini. The plasmid DNAs were resistant to both 3'-exonuclease and 5'-exonuclease even after treatment with proteinase K or alkali. The length of both terminal fragments that were generated by restriction endonuclease digestion was doubled under the denaturation condition, indicating that the linear plasmid DNAs have hairpin loops at both termini. Southern blotting analysis of total DNA showed the presence of two types of dimeric forms of pRS64 DNA. One is a head-to-head dimer and the other is a tail-to-tail dimer. The role of these unique DNA structures in replication of the plasmids is discussed.
Mol Gen Genet 1990 Jan
PMID:Linear plasmid DNAs of the plant pathogenic fungus Rhizoctonia solani with unique terminal structures. 232 20

Isolation and general properties of 3'-5' exonucleases I and II (EC 3.1.4.26), which are specific to single-stranded DNA, are described. Such enzymes, being components of replication complexes, could correct replication errors. Homogeneous exonucleases I and II consist of a single subunit with molecular mass of 50 and 40 kDa, respectively. These enzymes are located preferentially in the nuclear membrane and chromatin. They form complexes with nuclear DNA polymerases and some other proteins and are not observed practically in a free state. Molecular masses of the complexes amount from 70 to 1.500 kDa. The complexes dissociate as a result of solution hydrophobization and can be reconstituted after the decrease of hydrophobization. The heavy membrane complex form of 3'----5' exonuclease I manifests enzymatic activities of DNA polymerase alpha (EC 2.7.7.7), non-specific nucleoside triphosphatase (EC 3.1.3.2), nucleotidase (EC 3.1.3.31) and faint activity of endonuclease (EC 3.1.4.5). Complexes under study do not display activity of thymidine kinase (EC 2.7.1.21), marker protein of replitase, neither in G0 nor in S-period.
Mol Biol (Mosk)
PMID:[Homogeneous 3'----5'-exonucleases and their multienzyme complexes from the rat liver]. 234 19

Platelet aggregation and secretion are associated with a rise in intracellular calcium concentration ([Ca2+]i). Adenosine has been postulated as an endogenous inhibitor of platelet aggregation. The antiaggregatory effects of adenosine are related to activation of adenylate cyclase. We studied the effect of adenosine on the rise in [Ca2+]i and platelet aggregation produced by thrombin. Human platelets were obtained from dextrose/citrate-treated plasma. [Ca2+]i was determined by fluorescence-dye techniques (fura-2). Adenosine inhibited the slope of the first phase of aggregation and the rise in [Ca2+]i produced by thrombin, in a dose-dependent manner. The dose that produced 50% inhibition of both aggregation and the rise in [Ca2+]i was approximately 500 nM. The effects of adenosine on [Ca2+]i were shared by its stable analogs, 5'-N-ethylcarboxamidoadenosine being approximately 10-fold more potent than (-)N6-phenylisopropyladenosine, suggesting that these effects were mediated through adenosine A2 receptors. Furthermore, caffeine antagonized the inhibitory effects of adenosine on platelet aggregation and [Ca2+]i. The effects of adenosine on [Ca2+]i appear to be mediated through a rise in intracellular cAMP, because they were prevented by the adenylate cyclase inhibitor 2',5'-dideoxyadenosine (1 mM) and were potentiated by phosphodiesterase inhibition with papaverine (1 microM). Adenosine also inhibits the rise in [Ca2+]i produced by thrombin in a calcium-free medium, suggesting that adenosine inhibits both calcium influx and the release of calcium from intracellular stores.
Mol Pharmacol 1990 Jun
PMID:Adenosine inhibits the rise in intracellular calcium and platelet aggregation produced by thrombin: evidence that both effects are coupled to adenylate cyclase. 235 5

Iodide inhibits cyclic AMP accumulation in the thyroid by a process which is prevented by inhibition of iodide uptake and of thyroid peroxidase. By a similar process, it also exerts other independent effects such as the enhancement of iodinated protein release. Iodide inhibited the stimulation of adenylate cyclase by prostaglandin E1, cholera toxin and forskolin. The action of iodide was not relieved by phosphodiesterase inhibitors and was not additive with the effect of norepinephrine or adenosine. Iodide did not decrease the cellular level of ATP. The data are compatible with an inhibition of adenylate cyclase beyond the level of the receptor, presumably at the level of the catalytic unit or its interaction with the positive transducing unit NS. The effect of iodide required TSH for its expression but not for its installation. It was decreased under all conditions in which iodide organification was decreased: decreased iodide or increased methimazole concentration, absence of calcium in the medium, etc. However, the relation between iodide binding to proteins and effect was not linear. The effect was not relieved by washing in the absence of iodide and in the presence of perchlorate, but it was partly reversible in the presence of methimazole propylthiouracyl or thiourea. It was not relieved by cooling to 20 degrees C and cytochalasin b, which block stimulated thyroglobulin hydrolysis and iodothyronine release, nor by actinomycin D, cycloheximide, puromycin, mepacrine or indomethacin. The data suggest that iodide binds to a saturable cell component by a reaction which is reversible only in the presence of thiol-containing drugs.
Mol Cell Endocrinol 1985 May
PMID:Further characterization of the iodide inhibitory effect on the cyclic AMP system in dog thyroid slices. 240 38

The putative neuropeptide, molt-inhibiting hormone (MIH), regulates crustacean growth by periodically suppressing the secretion of ecdysteroid molting hormone from peripheral glands (Y-organs). A mediating role for cyclic AMP (cAMP) in MIH action was evaluated with isolated Y-organs of the crab, Cancer antennarius. MIH activity in eyestalk extracts inhibited ecdysteroid secretion but increased cAMP levels dose-dependently in 24-h incubations. The cAMP rise preceded the onset of ecdysteroid suppression. Dibutyryl cAMP, activators of adenylate cyclase (forskolin, choleragen), and an inhibitor of phosphodiesterase (IBMX), but not AMP or cGMP, mimicked the inhibitory action of MIH.
Mol Cell Endocrinol 1985 Sep
PMID:Cyclic AMP mediates the negative regulation of Y-organ ecdysteroid production. 241 11

The relationship between long-term electrical activity and protein phosphorylation was investigated in single, identifiable neurons in the abdominal ganglion of Aplysia californica by the intracellular injection of radiolabeled ATP followed by sodium dodecyl sulfate (SDS) gel electrophoresis. Natural and pharmacological treatments that alter the impulse activity of neurons L6 and R15 for prolonged periods did not appear to affect the phosphorylation of most of the 15 major phosphoproteins examined in these cells. Long-term excitation of L6 induced by the phosphodiesterase inhibitor IBMX correlated with phosphorylation of a 29,000-dalton protein. Long-term inhibition of L6 induced by afterdischarge of peptidergic bag-cell neurons appeared to cause dephosphorylation of a 29,000-dalton protein. Burst augmentation of R15 induced by bag-cell afterdischarge did not cause detectable changes in the phosphorylation of the major proteins we examined. These data are consistent with other studies of neural and nonneural tissues which have found a correlation between activity and the level of phosphorylation of a 29,000-dalton protein.
Cell Mol Neurobiol 1985 Dec
PMID:Activity-related changes in protein phosphorylation in an identified Aplysia neuron. 241 15

The H1-histamine receptor antagonist [3H]mepyramine bound with high affinity (Kd = 3-5 nM) to membranes derived from 1321N1 human astrocytoma cells. The H1-receptor antagonists triprolidine and diphenhydramine inhibited [3H]mepyramine binding with Kj values of 1-5 nM, whereas the Kj of the H2-histamine receptor antagonist cimetidine was greater than 100 microM. Histamine also inhibited [3H]mepyramine binding to 1321N1 cell membranes, and the histamine inhibition curve was shifted to the right and steepened in the presence of 1 microM guanosine 5'-O-(3-thiotriphosphate). Treatment of 1321N1 cells with pertussis toxin had no effect on the capacity of histamine to inhibit [3H]mepyramine binding either in the absence or presence of guanosine 5'-O-(3-thiotriphosphate). Therefore, agonist-occupied histamine receptors in these cells apparently interact with a guanine nucleotide regulatory protein that is not the inhibitory guanine nucleotide regulatory protein of adenylate cyclase. Although adenylate cyclase activity was not affected by histamine in a cell-free preparation, incubation of 1321N1 cells with histamine resulted in an attenuation of cyclic AMP accumulation. Analysis of cyclic AMP degradation in the presence of histamine indicated that the effects of histamine on cyclic AMP accumulation are mediated through activation of phosphodiesterase. This idea was supported by the fact that the phosphodiesterase inhibitor 1-isobutyl 3-methylxanthine blocked attenuation of cyclic AMP accumulation by histamine in a noncompetitive manner. Histamine also markedly increased phosphoinositide breakdown and 45Ca2+ efflux in 1321N1 cells. These histamine-induced effects apparently are mediated through H1-receptors, since triprolidine, but not cimetidine, potently inhibited histamine action. As for histamine interaction with its receptor, pertussis toxin had no effect on histamine-induced phosphoinositide breakdown, 45Ca2+ efflux, or attenuation of cyclic AMP accumulation. Taken together, these data indicate that 1321N1 human astrocytoma cells are a useful model system for the study of H1-histamine receptors and the biochemical responses mediated through these receptors.
Mol Pharmacol 1986 Feb
PMID:H1-histamine receptors on human astrocytoma cells. 241 44


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