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:3.1.4.1 (phosphodiesterase)
18,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The regulation of the expression of the human corticotropin-releasing-hormone gene (hCRH) was studied in a mouse anterior pituitary cell line (AtT20) after transiently transfection with a chimeric gene containing the hCRH gene promoter fused to the bacterial chloramphenicol acetyltransferase (CAT) gene. Expression of the chimeric hCRH-CAT gene in AtT20 cells was enhanced by the cAMP analog (8-bromo-cAMP) about 5-fold but not by phorbol 12-myristate-13-acetate. The cAMP phosphodiesterase inhibitor isobutylmethylxanthine also strongly stimulated 15-fold the expression of the chimeric hCRH-CAT gene. Coincubation of cAMP analog and isobutylmethylxanthine resulted in a moderate 2-fold synergistic enhancement of CAT activity. Sequence comparison of the hCRH gene revealed a core sequence for a cAMP responsive element 5'-TGACGTCA-3' at -221 relative to the cap site. This regulatory element also confers cAMP inducibility on a heterologous promoter when placed upstream of the thymidine kinase promoter from herpes simplex virus. Finally, treatment with 0.5 microM dexamethasone reduced CAT activity about 2.0-fold in cAMP-stimulated cells. This result suggests that cAMP and glucocorticoids coordinately control hCRH gene expression.
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PMID:Glucocorticoid repression of 3',5'-cyclic-adenosine monophosphate-dependent human corticotropin-releasing-hormone gene promoter activity in a transfected mouse anterior pituitary cell line. 169 84

During placental development cytotrophoblast stem cells fuse to form the syncytiotrophoblast, a multinucleate cytoplasm with a brush border in contact with the maternal blood. Biochemical differentiation including the expression of placental-specific proteins and hormones accompanies this maturation. However, the biochemical mechanisms responsible for these events are unknown. We have defined a system in which single cytotrophoblast-like cells of the human choriocarcinoma (BeWo) cell line undergo fusion and extensive morphological differentiation following their treatment with effectors of cyclic AMP metabolism. Forskolin incubation caused a dose-dependent increase in intracellular and secreted cyclic AMP and a coordinate fusion of cells which yielded syncytia containing hundreds of nuclei per cytoplasm and a mature dense "placental-like" brush border. These fused cells also synthesized and secreted large amounts of both subunits of chorionic gonadotropin. However, they continued to synthesize several other placenta-specific proteins--placental-like alkaline phosphatase, placental lactogen, and SP1--at rates similar to those in control cells. Other reported effectors of cyclic AMP metabolism also induced cell fusion, although theophylline, an inhibitor of phosphodiesterase, induced fusion by a cyclic AMP-independent mechanism. Additionally, unlike the case with forskolin, treatment of BeWo cells with theophylline did not induce other morphological features of mature syncytiotrophoblasts. Thus, this system will allow one to examine the biochemical mechanism of placental cell fusion in the absence of other variables of cell differentiation.
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PMID:Modulators of cyclic AMP metabolism induce syncytiotrophoblast formation in vitro. 215 59

To characterize the transcriptional effects of human (h)FSH and hCG on the POMC gene, primary rat granulosa cells were transiently transfected with a chloramphenicol acetyltransferase (CAT) reporter plasmid under the control of the POMC promoter and 5' region. POMC-CAT contains a fragment of the rat POMC gene, extending from nucleotide -704 to nucleotide +63, fused to the CAT gene. Treatment of POMC-CAT-transfected cells with either hFSH (20 ng/ml) or hCG (10 ng/ml) significantly increased CAT enzyme activity; however, neither hCG nor hFSH increased CAT enzyme activity in cells transfected with pSV2-CAT, a reporter plasmid under the control of the SV40 virus promoter and 5' region. The phosphodiesterase inhibitor isobutylmethylxanthine or the nonhydrolyzable cAMP analog cAMP-chlorothiophenyl significantly increased CAT activity in POMC-CAT-transfected granulosa cells. Human FSH stimulated transcription 10, 20, and 40 h after treatment, but FSH stimulation at the two earlier time points was 2.5- to 5.5-fold greater than that at 40 h. Gonadotropin-stimulated steroidogenesis was equivalent in POMC-CAT-transfected granulosa cells, untransfected, and mock-transfected cells. This indicates that transfection left the physiological hormone response intact. These data demonstrate the following. 1) 767 basepairs of the rat POMC gene are enough to confer gonadotropin stimulation on the CAT marker gene in granulosa cells. 2) Although the POMC promotor lacks a well conserved cAMP response element, either of two different pharmacological manipulations of granulosa cells that raise intracellular cAMP can also stimulate POMC-driven CAT expression. 3) Transfected primary cultures of granulosa cells provide a nontransformed, physiologically relevant model with which to study hormone-regulated gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Gonadotropin regulation of the rat proopiomelanocortin promoter: characterization by transfection of primary ovarian granulosa cells. 246 53

Hybrid nucleases consisting of an oligonucleotide fused to a unique site on the relatively nonspecific phosphodiesterase staphylococcal nuclease have been shown to sequence specifically cleave DNA. We have introduced mutations into the binding pocket of the nuclease which lower the kcat/Km of the enzyme. Hybrid nucleases generated from these mutants sequence selectively hydrolyze single-stranded DNA in a catalytic fashion, and under a much wider range of conditions than was previously possible. One such hybrid nuclease (Y113A, K116C) was able to site selectively cleave single-stranded M13mp7 DNA (7214 nt), primarily at one phosphodiester bond. Another hybrid nuclease (Y113A, L37A, K116C) catalyzed the hydrolysis of a 78-nt DNA substrate with a kcat of 1.2 min-1 and a Km of 120 nM. The effects of variations in the length and sequence of the oligonucleotide binding region were examined, as were changes in the length of the tether between the oligonucleotide and the enzyme. Cleavage specificity was also assayed as a function of substrate DNA primary and secondary structure and added poly(dA).
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PMID:Generation of a catalytic sequence-specific hybrid DNase. 260 84

1. The effects of caffeine (0.2-20 mmol l-1) have been examined on calcium transients (measured with aequorin) and isometric force in intact bundles of fibres from soleus (slow-twitch) and extensor digitorum longus (EDL; fast-twitch) muscles of the rat. 2. At 25 degrees C, threshold caffeine concentration for an observable increase in resting [Ca2+]i was 0.2 and 1.0 mmol l-1 for soleus and EDL muscles respectively. Increases in resting force were first detectable at about 0.5 mmol l-1 caffeine for soleus muscles and 5.0 mmol l-1 caffeine for EDL muscles and occurred in the range 0.2-0.4 mumol l-1 [Ca2+]i for soleus and 0.7-0.9 mumol l-1 for EDL. 3. Caffeine potentiated the twitch responses of soleus and EDL in a dose-related manner. The soleus was more sensitive in this respect, with 50% potentiation occurring at 1 mmol l-1 caffeine compared with 3.5 mmol l-1 for the EDL. Concentrations of caffeine below 2 mmol l-1 potentiated Ca2+ transients associated with twitches in both soleus and EDL muscles with no apparent change in the decay rate constant. 4. High concentrations of caffeine (greater than 2 mmol l-1) further potentiated peak Ca2+ in the EDL but depressed it in the soleus. The rate of decay of the Ca2+ transient in high caffeine was significantly prolonged in the soleus but remained unaffected in the EDL. 5. The phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX) had little effect on force or [Ca2+]i at concentrations known to significantly increase intracellular cyclic AMP levels. 6. The Ca2+ transient during fused tetani was characterized by an initial peak, a decline to a plateau level and sometimes a gradual rise towards the end of the stimulus train. Peak [Ca2+]i during normal tetani ranged between 1.1 and 2.4 mumol l-1 in the soleus and 1.9 and 4.0 mumol l-1 in the EDL. 7. Caffeine potentiated both force and [Ca2+]i during tetanus. Since the increase of the Ca2+ transient was significantly greater than potentiation of force, it is likely that saturation of myofilaments occurs. The primary effect of caffeine on the Ca2+ transient was an elevation of the plateau phase. 8. Caffeine concentrations below 5 mmol l-1 potentiate twitch and tetanic force in both fast- and slow-twitch mammalian skeletal muscles primarily by increasing both the basal and stimulus-evoked release of Ca2+ from the sarcoplasmic reticulum.
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PMID:Actions of caffeine on fast- and slow-twitch muscles of the rat. 260 58

The adenosine cyclic 3',5'-monophosphate (c-AMP) phosphodiesterase (PDE) inhibitory activity of a series of mesoionic 1,3,4-thiadiazolopyrimidines and of a group of benz-fused mesoionic xanthine analogs are found to be significantly correlated with the van der Waals volume (VW) of the substituents or the first order valence connectivity index (1 chi V) of the molecule. From the correlating equations it is observed that the size of the substituents at certain positions, of pyrimidine ring particularly, in the molecule are determinative to the activity. Further based on these equations it may be suggested that PDE inhibition by this class of drugs involves either hydrophobic interaction or van der Waals type of interaction. In certain cases steric and electronic factors are also indicated to affect the inhibition.
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PMID:Correlation of biological activities of mesoionic and benz-fused mesoionic xanthine analogs with van der Waals volume and molecular connectivity. 609 Jun 34

1. Proteins of fat-globule membrane from bovine milk were solubilized with the non-ionic detergent Triton X-100 in the presence of protease inhibitors. Approximately 25% of the total membrane protein was solubilized and the extracts were shown to contain a sample of most of the major membrane proteins and glycoproteins. 2. The solubilized proteins were separated in flat-beds of Ultrodex by electrofocusing and the pI values for the major proteins, glycoproteins and certain enzymes determined. Several of the proteins displayed marked heterogeneity indicating the existence of protein variants and isoenzymes. Principal pI values for the enzymes assayed were as follows: xanthine oxidase, 7.35--7.55; NADH2: iodonitrotetrazolium reductase, less than 4.5; 5'-nucleotidase, 7.15--7.4; alkaline phosphatase, 5.4--5.7; phosphodiesterase, 4.6--4.8; gamma-glutamyl transpeptidase, 4.4--4.55. 3. Fractions after electrofocusing were analyzed by 'fused rocket' immunoelectrophoresis and crossed immunoelectrophoresis after separation in polyacrylamide gels containing sodium dodecyl sulphate. Major antigens of the membrane include xanthine oxidase and glycoproteins of apparent molecular weights 67 000, 49 500 and 46 000. The latter two components share common antigenic determinants and could not be separated by gel filtration, ion-exchange chromatography, lectin-affinity chromatography or preparative electrofocusing.
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PMID:Separation of the proteins of bovine milk-fat-globule membrane by electrofocusing with retention of enzymatic and immunological activity. 610 13

The spleen cells of a Balb/c mouse immunized with purified bovine calmodulin-dependent cyclic nucleotide phosphodiesterase were fused with nonsecreting mouse myeloma cells (P3-X63-Ag8-653). Antibody producing hybridomas were screened by the enzyme-linked immunosorbent assay using purified phosphodiesterase as the antigen. One monoclonal cell line, CR-B1, was found to produce antibodies which showed positive enzyme-linked immunosorbent assay reactions with bovine brain calcineurin and rabbit muscle phosphorylase kinase in addition to phosphodiesterase. The antibody was purified and characterized. It was shown to immunoprecipitate the calmodulin (CaM)-dependent phosphodiesterase and phosphorylase kinase activities but not those of CaM itself, CaM-independent phosphodiesterase and the catalytic unit of cAMP-dependent protein kinase. The immunoprecipitation of phosphodiesterase could be inhibited by calcineurin and phosphorylase kinase. These results suggest that the antibody interacts at a common site on these calmodulin-dependent proteins. The antigenic determinant in phosphodiesterase does not appear to reside in the calmodulin-binding domain of the enzyme since the antibody and phosphodiesterase interaction is not inhibited by calmodulin, and the calmodulin activation of phosphodiesterase is not affected by CR-B1 antibody. It is therefore suggested that the structural similarity among the three calmodulin-dependent proteins extends beyond the calmodulin-binding domains.
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PMID:A monoclonal antibody showing cross-reactivity toward three calmodulin-dependent enzymes. 631 38

A novel plasmid was generated which allowed the expression of the cytosolic bacterial enzyme chloramphenicol acetyl transferase (CAT) in COS-7 cells. Upon transfection, the majority of the novel CAT activity was found in the cytosol fraction of COS cells. Chimeric molecules were made between N-terminal portions of the type IVA cyclic AMP-specific rat 'dunce-like' phosphodiesterase (RD1) (RNPDE4A1A; rPDE-IVA1) fused to CAT at its N-terminus. Expression in COS-7 cells of chimeras formed from 1-100RD1-CAT and 1-25RD1-CAT now showed CAT activity associated with the membrane fraction. In contrast, a chimera formed from 26-100RD1-CAT showed an identical expression pattern to native CAT, with the major fraction of CAT activity occurring in the cytosol fraction. Membrane-bound CAT activity provided by 1-100RD1-CAT and 1-25RD1-CAT was not released by either high-salt or washing treatments but was solubilized in a dose-dependent fashion by the non-ionic detergent Triton X-100. Subcellular fractionation of COS-7 cells showed that, as with RD1, the membrane-bound activity of the RD1-CAT chimera followed that of the plasma membrane marker 5'-nucleotidase. Plasmids containing chimeric cDNAs were exposed to a coupled transcription-translation system that, in addition to the full-length chimeras, was found to generate a range of N-terminal truncated species due to initiation at different methionine residues. Incubation of the mature protein products formed in this system with a COS cell membrane fraction showed that only those chimeric CAT constructs containing the first 25 amino acids of RD1 became membrane-associated. The unique 25 amino acid N-terminal domain of RD1 contains structural information that can confer membrane association upon an essentially soluble protein.
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PMID:Chimeric constructs show that the unique N-terminal domain of the cyclic AMP phosphodiesterase RD1 (RNPDE4A1A; rPDE-IVA1) can confer membrane association upon the normally cytosolic protein chloramphenicol acetyltransferase. 777 57

Dictyostelium discoideum secretes a cyclic nucleotide phosphodiesterase to control cAMP levels during development. Three promoters control expression of the gene--one during vegetative growth, one during aggregation, and one which constrains phosphodiesterase synthesis to prestalk cells. In this report we show that the expression of phosphodiesterase (PDE) in prestalk cells is necessary for morphogenesis. A gene that codes for a specific glycoprotein inhibitor of the phosphodiesterase (Kd = 0.1 nM) was fused to the prestalk-specific promoter of the PDE gene. Transformants carrying multiple copies of this construct secreted inhibitor in 100-fold excess after the aggregation process had occurred. The first effect seen was an elongated tip, followed by a block in slug formation and an inability to culminate. Stalk and spores cells are produced but morphogenesis is uncoupled from cellular differentiation. Overproduction of inhibitor during earlier stages delayed aggregation, but did not affect fruiting body formation. A phosphodiesterase mutant was transformed with a plasmid that expresses PDE only during aggregation and not in prestalk cells. The defect in aggregation was rescued, but the defect in later development was not. The combined results indicate that PDE expression in prestalk cells is critical to morphogenesis. To ask whether the inhibitor gene under its normal regulation had a role in aggregation or later morphogenesis, it was destroyed by homologous recombination. The loss of the gene did not prevent development under the conditions used.
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PMID:The phosphodiesterase secreted by prestalk cells is necessary for Dictyostelium morphogenesis. 785 34


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