The effect of bromodeoxyuridine and dibutyryl cyclic AMP on the induction of placental alkaline phosphate in human choriocarcinoma cells.
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ABSTRACT:
SUBMITTER: Hamilton TA
PROVIDER: S-EPMC1163207 | biostudies-other | 1981 Jul
REPOSITORIES: biostudies-other
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Project description:BeWo choriocarcinoma cells synthesize two alkaline phosphatase isoenzymes: germ-cell alkaline phosphatase and tissue-unspecific alkaline phosphatase. We have made use of the differential heat-stabilities of these two isoenzymes to study the induction of germ-cell alkaline phosphatase by sodium butyrate and cyclic AMP (cAMP). Sodium butyrate causes a large induction of germ-cell alkaline phosphatase activity (approx. 35-fold after 96 h) after an initial lag period of 12-24 h. We showed that butyrate increases germ-cell alkaline phosphatase mRNA. Dibutyryl cAMP also induces germ cell alkaline phosphatase (approx. 2.5-fold after 96 h). When optimal concentrations of butyrate and dibutyryl cAMP were added simultaneously to cells, they caused a synergistic induction of activity. This suggested that these compounds use separate mechanisms to induce germ-cell alkaline phosphatase activity and that it is the cAMP moiety of dibutyryl cAMP that induces enzyme activity. This was confirmed by the use of two additional cAMP analogues, 8-(4-chlorophenylthio) cAMP and 8-bromo cAMP, and of two compounds, 3-methyl-1-isobutylxanthine and cholera toxin, which raise the endogenous concentration of cAMP. All four compounds caused a 2-fold increase in enzyme activity. Treatment of cells with 8-(4-chlorophenylthio) cAMP, 8-bromo cAMP and cholera toxin increased germ-cell alkaline phosphatase mRNA between 2- and 7-fold. These data suggest that this alkaline phosphatase isoenzyme is regulated at the level of its mRNA by cAMP, in a manner distinct from that of butyrate.
| S-EPMC1137655 | biostudies-other
Project description:This series examines differential gene expression during appressorium formation induced by exogeous cyclic AMP in the rice blast fungus Magnaporthe grisea. RNA was extracted from spores germinated on hydrophilic (non-inductive) side of GelBond as well as from spores elaborating appressia on hydrophilic surfaces in the presence of cAMP after 9 hrs incubation. RNA from two biological replications of each treatment was pooled in equal amounts and labeled with both cy3 and cy5 dyes using the Agilent Technologies Low Input Linear Amplification Kit. Hybridizations were performed on Agilent Technologies whole genome oligo rice blast arrays (product G4137B) using manufacturer prescribed protocols and reagents in an reference design in which each treatment was paired with every other. This series contains a total of 4 hybridizations; each treatment was used in 4 hybridizations (2 with Cy3 and 2 Cy5). Spot fluorescence was normalized using Lowess within and between microarray slides in Bioconductor. Keywords: Cell development
2008-01-15 | GSE10173 | GEO
Project description:This series examines differential gene expression during appressorium formation induced by exogeous cyclic AMP in the rice blast fungus Magnaporthe grisea. RNA was extracted from spores germinated on hydrophilic (non-inductive) side of GelBond as well as from spores elaborating appressia on hydrophilic surfaces in the presence of cAMP after 9 hrs incubation. RNA from two biological replications of each treatment was pooled in equal amounts and labeled with both cy3 and cy5 dyes using the Agilent Technologies Low Input Linear Amplification Kit. Hybridizations were performed on Agilent Technologies whole genome oligo rice blast arrays (product G4137B) using manufacturer prescribed protocols and reagents in an reference design in which each treatment was paired with every other. This series contains a total of 4 hybridizations; each treatment was used in 4 hybridizations (2 with Cy3 and 2 Cy5). Spot fluorescence was normalized using Lowess within and between microarray slides in Bioconductor. Keywords: Cell development Two-condition experiment, cyclic AMP treated vs. non treated spores. 4 biological replicates with dye-swaps
2010-05-26 | E-GEOD-10173 | biostudies-arrayexpress
Project description:The effects of verapamil, a calcium antagonist, on lipolysis in isolated rat adipocytes were studied. Verapamil (100 microM) potentiated lipolysis due to dibutyryl cyclic AMP (Bt2cAMP) at submaximal concentrations, with or without extracellular Ca2+. Lipolysis due to 0.5 mM-Bt2cAMP was potentiated by verapamil in a dose-dependent manner up to 200 microM, whereas at concentrations higher than 100 microM the stimulatory effect of verapamil was progressively diminished with or without extracellular Ca2+. Verapamil showed only an inhibitory effect on lipolysis due to adrenaline (0.1-10 microM) or 3-isobutyl-1-methylxanthine (IBMX; 25-200 microM). The stimulatory effect of verapamil on lipolysis due to Bt2cAMP was not blocked by alpha-adrenergic antagonists. These results suggest (i) that verapamil has a biphasic effect on lipolysis due to Bt2cAMP and only an inhibitory effect on that due to adrenaline or IBMX, and (ii) that extracellular Ca2+ or alpha-adrenergic receptors are not involved in the action of verapamil.
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Project description:Biochemical and immunochemical studies were undertaken to quantify the effects of cyclic AMP on cyclic AMP-dependent protein kinase subunit levels in nuclei of H4IIE hepatoma cells. Dibutyryl cyclic AMP (10 microM) caused a significant biphasic (10 and 120 min after stimulation) increase in total nuclear protein kinase activity. The increase observed 10 min after dibutyryl cyclic AMP stimulation was primarily due to an approx. 3-fold increase of catalytic (C) subunit activity, whereas the change observed 120 min after stimulation consisted of an increase in both C subunit and cyclic AMP-independent protein kinase activities. Analysis of nuclear protein extracts by photoaffinity labelling with 8-azido cyclic [32P]AMP identified only the type II regulatory subunit (RII), but not the type I regulatory subunit (RI). Analysis of nuclear RII variants by two-dimensional gel electrophoresis demonstrated that dibutyryl cyclic AMP caused the appearance of two RII variant forms which were not present in the nuclei of unstimulated cells. Using affinity-purified polyclonal antibodies and immunoblotting procedures, we identified an approx. 2-fold increase in the RII and C subunits in nuclear extracts of dibutyryl cyclic AMP-treated hepatoma cells. Finally, the RI, RII and C subunits were quantified by an e.l.i.s.a. which indicated that dibutyryl cyclic AMP increased nuclear RII and C subunits levels biphasically, reaching peak values 10 and 120 min after the initial stimulation. Nuclear RI subunit levels were not affected. These results provide qualitative as well as quantitative evidence for a modulation by cyclic AMP of the nuclear RII and C subunit levels in rat H4IIE hepatoma cells, and indicate a relatively rapid but temporarily limited dibutyryl cyclic AMP-induced translocation of the RII and C subunits to nuclear sites.
| S-EPMC1138730 | biostudies-other
Project description:Synthetic lipopeptides activate superoxide-anion (O2-) formation in human neutrophils in a pertussis-toxin (PTX)-sensitive manner, suggesting the involvement of G-proteins of the Gi family in the signal-transduction pathway. We compared G-protein activation by lipopeptides and the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLP) in dibutyryl-cyclic-AMP-differentiated HL-60 cells. The lipopeptide (2S)-2-palmitoylamino-6-palmitoyloxymethyl-7-palmitoyloxy heptanoyl-SK4 (Pam3AhhSK4) and fMLP activated high-affinity GTPase, i.e. the enzymic activity of G-protein alpha-subunits, in HL-60 membranes in a time- and protein-dependent manner, but they had no effect on Mg(2+)-ATPase and Na+/K(+)-ATPase. Pam3AhhSK4 and fMLP increased Vmax. of GTP hydrolysis. Pam3AhhSK4 activated GTP hydrolysis with half-maximal and maximal effects at about 2 microM and 10 microM respectively. Other lipopeptides activated GTP hydrolysis as well. Lipopeptides were less effective than fMLP to activate GTPase. In membranes from PTX-treated cells, the stimulatory effects of lipopeptides and fMLP on GTPase were abolished. In N-ethylmaleimide-treated membranes, the relative stimulatory effect of Pam3AhhSK4 on GTP hydrolysis was enhanced, whereas that of fMLP was diminished. fMLP and Pam3AhhSK4 activated GTPase in an over-additive manner in N-ethylmaleimide-treated membranes. Unlike fMLP, Pam3AhhSK4 did not enhance incorporation of GTP azidoanilide into, and cholera-toxin-catalysed ADP-ribosylation of Gi-protein alpha-subunits in, HL-60 membranes and did not induce rises in cytosolic Ca2+ concentration. Pam3AhhSK4 and fMLP stimulated phosphatidic acid formation in a PTX-sensitive manner. Pam3AhhSK4 itself did not activate O2- formation, but potentiated the stimulatory effects of fMLP. Our data suggest that (i) lipopeptides activate the GTPase of Gi-proteins, (ii) lipopeptides and fMLP activate Gi-proteins differently, (iii) lipopeptides stimulate phospholipase D via Gi-proteins, and (iv) phosphatidic acid formation is not sufficient for activation of O2- formation.
| S-EPMC1137680 | biostudies-other
Project description:Hepatic phosphoenolpyruvate carboxykinase (PEPCK) is significantly increased in the hyperthyroid starved rat, and moderately decreased in the hypothyroid starved rat. As tri-iodothyronine by itself has only a small and sustained effect on the induction of this enzyme, as was previously shown in the isolated perfused organ, the effect of hypo- and hyper-thyroidism on the increase in cytosolic PEPCK provoked by dibutyryl cyclic AMP (Bt2cAMP) was investigated in vivo and in the isolated perfused liver. Compared with euthyroid fed controls, in hypothyroid fed rats Bt2cAMP provoked in 2 h only a small increase in translatable mRNA coding for PEPCK. In contrast, in hyperthyroid animals PEPCK mRNA as measured by translation in vitro was already increased in the fed state, and further enhanced by Bt2cAMP injection to values as in euthyroid controls. Under all thyroid states a close correlation between PEPCK mRNA activity and PEPCK synthesis was observed. In the isolated perfused liver from the hyperthyroid fed rat, the increase in PEPCK provoked by Bt2cAMP or Bt2cAMP + isobutylmethylxanthine was considerably enhanced compared with those obtained in livers of hypothyroid rats. Also, adrenaline provoked a stimulated induction of PEPCK in hyperthyroid rats compared with hypothyroid rats. To summarize, our data indicate that the primary action of thyroid hormones on the synthesis of hepatic cytosolic PEPCK is to accelerate the cyclic AMP- or adrenaline-induction of the enzyme, acting primarily at a pretranslational level.
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Project description:Escherichia coli cyclic-AMP receptor protein (CRP) represents one of the paradigms of bacterial gene regulation. Yet despite decades of intensive study, new information continues to emerge that prompts reassessment of this classic regulatory system. Moreover, in recent years CRPs from several other bacterial species have been characterized, allowing the general applicability of the CRP paradigm to be tested. Here the properties of the E. coli, Mycobacterium tuberculosis and Pseudomonas putida CRPs are considered in the context of the ecological niches occupied by these bacteria. It appears that the cyclic-AMP-CRP regulatory system has been adapted to respond to distinct external and internal inputs across a broad sensitivity range that is, at least in part, determined by bacterial lifestyles.
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Project description:Vibrio cholerae is a facultative human pathogen. The ability of V. cholerae to form biofilms is crucial for its survival in aquatic habitats between epidemics and is advantageous for host-to-host transmission during epidemics. Formation of mature biofilms requires the production of extracellular matrix components, including Vibrio polysaccharide (VPS) and matrix proteins. Biofilm formation is positively controlled by the transcriptional regulators VpsR and VpsT and is negatively regulated by the quorum-sensing transcriptional regulator HapR, as well as the cyclic AMP (cAMP)-cAMP receptor protein (CRP) regulatory complex. Transcriptome analysis of cyaA (encoding adenylate cyclase) and crp (encoding cAMP receptor protein) deletion mutants revealed that cAMP-CRP negatively regulates transcription of both VPS biosynthesis genes and genes encoding biofilm matrix proteins. Further mutational and expression analysis revealed that cAMP-CRP negatively regulates transcription of vps genes indirectly through its action on vpsR transcription. However, negative regulation of the genes encoding biofilm matrix proteins by cAMP-CRP can also occur independent of VpsR. Transcriptome analysis also revealed that cAMP-CRP regulates the expression of a set of genes encoding diguanylate cyclases (DGCs) and phosphodiesterases. Mutational and phenotypic analysis of the differentially regulated DGCs revealed that a DGC, CdgA, is responsible for the increase in biofilm formation in the Deltacrp mutant, showing the connection between of cyclic di-GMP and cAMP signaling in V. cholerae.
| S-EPMC2566190 | biostudies-literature
Project description:Our previous studies [Chao (1992) Biochem. J. 282, 203-207; C.C.-K. Chao, unpublished work] has suggested a correlation between the levels of constitutive UV-damaged-DNA-recognitionproteins (UVDRP) and cellular DNA repair in different cell types. In the present study, UVDRP were induced in F9 and NIH3T3 cells by 0.1 microM retinoic acid (RA) and 1 mM dibutyryl cyclic AMP (dbcAMP), which is sufficient to induce differentiation in murine F9 stem cells. The induction of UVDRP in F9 and NIH3T3 cells was optimized after 6 and 2 days incubation with RA/dbcAMP respectively. Since NIH3T3 cells were not induced to differentiate by RA/dbcAMP, the upregulation of the UVDRP in mammalian cells would thus seem not to be mediated directly by differentiation. Using a plasmid re-activation assay to estimate DNA repair, we did not find a correlation between DNA repair and UVDRP in RA/dbcAMP-treated cells. The results suggest that UVDRP may have a function other than, or in addition to, its role in DNA repair.
| S-EPMC1216927 | biostudies-other