Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.48 (transcriptase)
 9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cells of two human follicular thyroid carcinoma cell lines (FTC133, FTC236) were stably transfected with a cDNA encoding the PDE4A cAMP-specific phosphodiesterase (PDE) splice variant RD1 (RNPDE4A1A) so as to generate the cloned cell lines, FTC133A and FTC236A. This allowed the expression of a novel rolipram-inhibited cAMP-specific PDE activity in these cells. Unlike the parent cell lines in which Ca2+/calmodulin caused a profound activation (approx. 3-4-fold) of homogenate PDE activity, no such stimulation was evident in the RD1-expressing cell lines, indicating loss of PDE1 activity. Reverse transcriptase-PCR analysis indicated that this was due to the down-regulation of the PDE1C isoform. The novel PDE4 activity in transfected cells was located exclusively in the membrane fraction, as was immunoreactive RD1. Low concentrations of the detergent Triton X-100, but not high NaCl concentrations, allowed RD1 to be solubilized. Laser scanning confocal immunofluorescence analyses identified RD1 immunoreactivity in a discrete perinuclear region of these RD1-expressing transfected cell lines. A similar pattern of labelling was observed using the antiserum Tex1, which specifically identified the Golgi apparatus. Treatment of FTC133A cells with the Golgi-perturbing agents monensin and brefeldin A led to a similar redistribution of immunoreactive species detected using both the Tex1 and anti-RD1 antisera. It is suggested that the PDE4A splice variant RD1 contains a membrane-association signal which allows the targeted expression of RD1 within the Golgi complex of these human follicular thyroid carcinoma cell lines.
 ...
PMID:Intracellular localization of the PDE4A cAMP-specific phosphodiesterase splice variant RD1 (RNPDE4A1A) in stably transfected human thyroid carcinoma FTC cell lines. 900 17

The HSPDE4A gene spans 50 kb, consists of at least 17 exons and is orientated 5'-3', telomere to centromere. It is located at chromosome 19p13.2, being 350 kb proximal to the gene encoding TYK2 and 850 kb distal to the gene encoding the low-density lipoprotein receptor. Its structure is consistent with the production of active 'long' and 'short' isoenzymes as the result of alternative mRNA splicing at two splice junctions. Identified is the single alternatively spliced 5' exon encoding the unique N-terminal region of the long isoenzyme HSPDE4A4B (pde46). The upstream conserved regions, UCR1 and UCR2, which form characteristic domains of PDE4 long forms are each encoded by three exons. The PDE4A-subfamily-specific linker region LR1, which joins UCR1 and UCR2, is encoded by two exons, whereas LR2, which joins UCR2 to the catalytic unit, is encoded by a single exon. Identification of exons encoding an enzymically inactive product of this gene, HSPDE4A8A (2el), indicates that this is an authentic gene product. The 5' exon encoding the unique N-terminal region of the human homologue of the rodent isoform RNPDE4A1A (RD1) was located, and the splice junction used to produce this short PDE4A isoform shown to occur at a different position from that seen in both the rat PDE4B and PDE4D genes. Reverse transcriptase PCR analysis indicates that RD1 homologues are conserved across species, having a conserved membrane-targeting region and a hypervariable LR2 region. Human RD1 was expressed transiently in COS-7 cells and detected as an 83 kDa species primarily associated with the high-speed membrane fraction. Human RD1 exhibited a Km for cAMP of about 3 microM, an IC50 value for inhibition by the PDE4-selective inhibitor rolipram of about 0.3 microM and was considerably more thermostable than rat RD1. Human RD1 was generated as a mature 80 kDa species in an in vitro transcription-translation system and shown to be capable of binding to membranes. Knowledge of the gene structure and the associated sequence information should facilitate analysis of the involvement of PDE4A in hereditary disorders that may result from alterations in enzyme expression, activity, regulation and intracellular targeting and serve as a resource for determining authenticity of cloned PDE4A species.
 ...
PMID:Identification and characterization of the human homologue of the short PDE4A cAMP-specific phosphodiesterase RD1 (PDE4A1) by analysis of the human HSPDE4A gene locus located at chromosome 19p13.2. 967 30

Theophylline, a drug known to inhibit several classes of adenosine 3'5' cyclic monophosphate (cAMP) phosphodiesterases (PDEs), induces apoptosis in chronic lymphocytic leukemia (CLL) cells. Because the PDE target for theophylline in CLL remains unknown, we examined the ability of isoform-specific PDE inhibitors to increase cAMP levels and induce apoptosis in primary CLL cells. Reverse transcriptase-polymerase chain reaction of purified CLL cDNA amplified transcripts for PDE1B, 4A and 4B. The type 4 PDE inhibitor rolipram but not the type 1 inhibitor vinpocetine increased CLL cAMP levels. Rolipram-inhibitable (type 4) but not calcium-calmodulin augmented (type 1) PDE enzyme activity was detected in CLL samples. In samples from 13 of 14 CLL patients, rolipram induced apoptosis in a dose-dependent fashion over a 48-hour period. Interleukin-2 (IL-2)-cultured whole mononuclear cells (WMC) and anti-Ig stimulated CD19(+) B cells were resistant to the induction of apoptosis by rolipram while unstimulated CD19(+) B cells, which had a high basal apoptotic rate, were more sensitive. Rolipram stimulated elevations in cAMP levels in all four of these cell populations, suggesting that they differed in sensitivity to cAMP-induced apoptosis. Consistent with this hypothesis, incubation with the cell permeable cAMP analog dibutyryl-cAMP induced apoptosis in CLL cells and unstimulated B cells but not in IL-2-cultured WMC or anti-Ig stimulated B cells. These data identify PDE4 as a family of enzymes whose inhibition induces apoptosis in CLL cells.
 ...
PMID:Type 4 cyclic adenosine monophosphate phosphodiesterase as a therapeutic target in chronic lymphocytic leukemia. 974 89

Phosphodiesterase 4D (PDE4D), part of the complex cAMP-specific PDE4 family, plays a pivotal role in the regulation of airway smooth muscle relaxation by catalyzing the hydolysis of cAMP. Its gene on chromosome 5q12 encodes 5 splice variants, which show tissue-dependent expression and regulation. The genomic arrangement of PDE4D was determined using in silico methods, and a putative promoter of one of the protein kinase A-activated, long isoforms, PDE4D5 was identified. Promoter-luciferase constructs, transiently transfected into a beta(2) adrenoreceptor-expressing CHO-K1 cell line, were used to demonstrate that the PDE4D5 promoter up-regulated reporter gene expression in response to increased cell cAMP. Site-directed mutagenesis of the cAMP-response element (CRE) at position -201 identified this as the principal component of the mechanism underlying this cAMP responsiveness. In the second part of this study, cAMP-dependent induction of PDE4D5 transcript in primary cultured human airway smooth muscle cells (hASMs) was demonstrated using both qualitative reverse-transcriptase PCR and quantitative real-time PCR. Isolated PDE4D5 isoenzyme activity, measured after selective immunoprecipitation from hASMs, confirmed that this increase in expression led to an up-regulation of functional activity. We present evidence for cAMP-driven PDE4D5 up-regulation in hASMs and suggest a CRE-containing, isoform-specific promoter as the primary mechanism.
 ...
PMID:Cyclic AMP-dependent transcriptional up-regulation of phosphodiesterase 4D5 in human airway smooth muscle cells. Identification and characterization of a novel PDE4D5 promoter. 1212 97

Cyclic nucleotide PDEs (phosphodiesterases) are important enzymes that regulate intracellular levels of cAMP and cGMP. In the present study, we identify and characterize novel PDEs in the genetic model, Drosophila melanogaster. The Drosophila genome encodes five novel PDE genes in addition to dunce. Predicted PDE sequences of Drosophila show highly conserved critical domains when compared with human PDEs. Thus PDE-encoding genes of D. melanogaster are CG14940-PDE1C, CG8279-PDE6beta, CG5411-PDE8A, CG32648-PDE9 and CG10231-PDE11. Reverse transcriptase-PCRs of adult tissues reveal widespread expression of PDE genes. Drosophila Malpighian (renal) tubules express all the six PDEs: Drosophila PDE1, dunce (PDE4), PDE6, PDE8, PDE9 and PDE11. Antipeptide antibodies were raised against PDE1, PDE6, PDE9 and PDE11. Verification of antibody specificity by Western blotting of cloned and expressed PDE constructs allowed the immunoprecipitation studies of adult Drosophila lysates. Biochemical characterization of immunoprecipitated endogenous PDEs showed that PDE1 is a dual-specificity PDE (Michaelis constant Km for cGMP: 15.3+/-1 microM; Km cAMP: 20.5+/-1.5 microM), PDE6 is a cGMP-specific PDE (Km cGMP: 37+/-13 microM) and PDE11 is a dual-specificity PDE (Km cGMP: 6+/-2 microM; Km cAMP: 18.5+/-5.5 microM). Drosophila PDE1, PDE6 and PDE11 display sensitivity to vertebrate PDE inhibitors, zaprinast (IC50 was 71+/-39 microM for PDE1, 0.65+/-0.015 microM for PDE6 and 1.6+/-0.5 microM for PDE11) and sildenafil (IC50 was 1.3+/-0.9 microM for PDE1, 0.025+/-0.005 microM for PDE6 and 0.12+/-0.06 microM for PDE11). We provide the first characterization of a cGMP-specific PDE and two dual-specificity PDEs in Drosophila, and show a high degree of similarity in structure and function between human and Drosophila PDEs.
 ...
PMID:Cyclic nucleotide phosphodiesterases in Drosophila melanogaster. 1567 86

PDE4A11 is a novel cAMP-specific phosphodiesterase that is conserved in humans, mouse, rat, pig, and bat. Exon-1(4A11) encodes its unique, 81 amino acid N-terminal region. Reverse-transcriptase polymerase chain reaction performed across the splice junction, plus identification of expressed sequence tags, identifies PDE4A11 as a long isoform possessing UCR1 and UCR2 regulatory domains. Transcript analysis shows that PDE4A11 is widely expressed compared with PDE4A10 and PDE4A4B long isoforms. Truncation analysis identifies a putative promoter in a 250-base pair region located immediately upstream of the start site in Exon-1(4A11). Recombinant PDE4A11, expressed in COS-7 cells, is a 126-kDa protein localized predominantly around the nucleus and in membrane ruffles. PDE4A11 exhibits a K(m) for cAMP hydrolysis of 4 microM, with relative V(max) similar to that of PDE4A10 and PDE4A4B. PDE4A11 is dose-dependently inhibited by rolipram, 4-[(3-butoxy-4-methoxyphenyl)-methyl]-2-imidazolidinone (Ro 20-1724), cilomilast, roflumilast, and denbufylline, with IC(50) values of 0.7, 0.9, 0.03, 0.004, and 0.3 microM, respectively. Soluble and particulate PDE4A11 exhibit distinct rates of thermal inactivation (55 degrees C; T((0.5)) = 2.5 and 4.4 min, respectively). Elevating cAMP levels in COS-7 cells activates PDE4A11 concomitant with its phosphorylation at Ser119 by protein kinase A (PKA). PDE4A11 differs from PDE4A4 in sensitivity to cleavage by caspase-3, interaction with LYN SH3 domain, redistribution upon long-term rolipram challenge, and sensitivity to certain PDE4 inhibitors. PDE4A11, PDE4A10, and PDE4A4 all can interact with betaarrestin. PDE4A11 is a novel, widely expressed long isoform that is activated by PKA phosphorylation and shows a distinct intracellular localization, indicating that it may contribute to compartmentalized cAMP signaling in cells in which it is expressed.
 ...
PMID:Identification and characterization of PDE4A11, a novel, widely expressed long isoform encoded by the human PDE4A cAMP phosphodiesterase gene. 1573 10