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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An adenylyl cyclase gene (cyaA) present upstream of an osmosensor protein gene (mokA) was isolated from Myxococcus xanthus. cyaA encoded a polypeptide of 843 amino acids with a predicted molecular mass of 91,187 Da. The predicted cyaA gene product had structural similarity to the receptor-type adenylyl cyclases that are composed of an amino-terminal sensor domain and a carboxy-terminal catalytic domain of adenylyl cyclase. In reverse transcriptase PCR experiments, the transcript of the cyaA gene was detected mainly during development and spore germination. A cyaA mutant, generated by gene disruption, showed normal growth, development, and germination. However, a cyaA mutant placed under conditions of ionic (NaCl) or nonionic (sucrose) osmostress exhibited a marked reduction in spore formation and spore germination. When wild-type and cyaA mutant cells at developmental stages were stimulated with 0.2 M NaCl or sucrose, the mutant cells increased cyclic AMP accumulation at levels similar to those of the wild-type cells. In contrast, the mutant cells during spore germination had mainly lost the ability to respond to high-ionic osmolarity. In vegetative cells, the cyaA mutant responded normally to osmotic stress. These results suggested that M. xanthus CyaA functions mainly as an ionic osmosensor during spore germination and that CyaA is also required for osmotic tolerance in fruiting formation and sporulation.
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PMID:An adenylyl cyclase, CyaA, of Myxococcus xanthus functions in signal transduction during osmotic stress. 1205 52

Accumulating evidence indicates that TNFalpha plays an important role in the pathogenesis of periodontitis, but the effect of TNFalpha on the degradation of the periodontal ligament is not well understood. This study used reverse transcriptase-PCR to investigate the effects of TNFalpha on matrix metalloproteinase (MMP) mRNA expression in human periodontal ligament fibroblasts. TNFalpha increased MMP-1, MMP-3 and MMP-13 mRNA levels in both a time-dependent (0-24 h) and a dose-dependent (0.1-10 ng/ml) manner. TNFalpha also increased COX-2 mRNA levels. Because elevation of COX-2 mRNA levels enhances the production of prostaglandins, we therefore investigated whether endogenous prostaglandins are involved in the MMP mRNA expression that is enhanced by TNFalpha. Pretreatment with the selective COX-2 inhibitor, NS-398, increased MMP-13 mRNA levels, while prostaglandin E2 and dibutyryl cyclic AMP decreased MMP-13 mRNA levels. Neither MMP-1 nor MMP-3 mRNA levels were affected by these chemicals. These findings indicate that prostaglandin E2 has a lowering effect on TNFalpha-enhanced MMP-13 mRNA levels, and that this effect is dependent on cAMP. Our results suggest that TNFalpha participates in periodontal ligament destruction by stimulating the production of MMPs (MMP-1, MMP-3 and MMP-13), while endogenous prostaglandin E2 has a negative feedback role in TNFalpha-enhanced MMP-13 production.
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PMID:Effects of TNFalpha and prostaglandin E2 on the expression of MMPs in human periodontal ligament fibroblasts. 1211 50

Since macrophages are a source of increased PGE(2) in AIDS, we investigated the role of PGE(2) in the replication of HIV-1 in these cells. PGE(2) inhibited HIV-1 replication measured by reverse transcriptase in human monocyte-derived macrophage (MDM). Treatment of MDM with the PGE(1) analog misoprostol, the adenylate cyclase activator forskolin, and the cyclic AMP analog dibutyryl-cyclic AMP (db-cAMP) suppressed HIV replication. The protein kinase A (PKA) activator 8-bromo-cyclic AMP also inhibited HIV-1 replication. Similar results were observed with the entry-independent, latently HIV-infected U1 cells. There was a parallel decrease in HIV-1 mRNA levels following PGE(2) treatment. Co-transfection of the HIV-1 promoter LTR.luciferase, with the vector CMV.Calpha, which expresses the PKA catalytic unit increasing PKA activity, reduced HIV-1 promoter activity. Inhibition of PKA activity with the pMT.RAB vector, a mutant regulatory unit of PKA, augmented HIV-1 promoter activity. In summary, PGE(2) inhibits HIV-1 gene expression in MDM through a PKA-dependent mechanism.
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PMID:Prostaglandin E(2) inhibits replication of HIV-1 in macrophages through activation of protein kinase A. 1214 37

Corticotropin-releasing hormone (CRH) is a 41 amino acid neuropeptide which plays an important role in the stress response in the hypothalamus. We describe the development of an immortalized hypothalamic cell line which expresses CRH. We hypothesized that this cell line would possess the relevant characteristics of parvocellular CRH-expressing neurones such as glucocorticoid receptor (GR) expression and vasopressin (VP) coexpression. For production of hypothalamic cells, embryonic day 19 rat pup hypothalami were dissected and dissociated into tissue culture dishes. They were immortalized by retrovirus-mediated transfer of the SV40 large T antigen gene at 3 days of culture and then screened for expression of CRH following dilution cloning. One cell line was chosen (IVB) which exhibited CRH-like immunoreactivity (CRH-LI) and expressed CRH, VP and CRH1 receptor RNA via the reverse transcriptase-polymerase chain reaction. In addition, the cell line expressed the neuronal marker, microtubule-associated protein-2. We verified that the CRH-LI from IVB cell lysates coeluted with CRH standard via reversed-phase high-performance liquid chromatography (HPLC). Furthermore, oxidation of the lysate converted its HPLC profile to that identical with oxidized CRH standard. In addition, IVB cells exhibited high affinity binding to CRH. Incubation of IVB cells with CRH lead to increases in cAMP levels and protein kinase A activity in a concentration-dependent manner. Incubation of IVB cells with CRH also resulted in increases in phospho-cyclic-AMP response element binding protein (CREB) immunostaining as detected by immunocytochemical analysis. Finally, CRH treatment of IVB cell lines has been linked to CREB-mediated gene expression as determined via the PathDetect CREB trans-reporting system. The characteristics of IVB cells, such as CRH and VP coexpression, GR expression and a biologically active CRH-R1-mediated signalling pathway, suggest that this neuronal cell line may serve as model of parvocellular CRH neurones.
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PMID:Corticotropin-releasing hormone (CRH) expression and protein kinase A mediated CRH receptor signalling in an immortalized hypothalamic cell line. 1269 78

A novel salicylate-degrading Streptomyces sp., strain WA46, was identified by UV fluorescence on solid minimal medium containing salicylate; trace amounts of gentisate were detected by high-pressure liquid chromatography when strain WA46 was grown with salicylate. PCR amplification of WA46 DNA with degenerate primers for gentisate 1,2-dioxygenase (GDO) genes produced an amplicon of the expected size. Sequential PCR with nested GDO primers was then used to identify a salicylate degradation gene cluster in a plasmid library of WA46 chromosomal DNA. The nucleotide sequence of a 13.5-kb insert in recombinant plasmid pWD1 (which was sufficient for the complete degradation of salicylate) showed that nine putative open reading frames (ORFs) (sdgABCDEFGHR) were involved. Plasmid pWD1 derivatives disrupted in each putative gene were transformed into Streptomyces lividans TK64. Disruption of either sdgA or sdgC blocked salicylate degradation; constructs lacking sdgD accumulated gentisate. Cell extracts from Escherichia coli DH5 alpha transformants harboring pUC19 that expressed each of the sdg ORFs showed that conversions of salicylate to salicylyl-coenzyme A (CoA) and salicylyl-CoA to gentisyl-CoA required SdgA and SdgC, respectively. SdgA required CoA and ATP as cofactors, while NADH was required for SdgC activity; SdgC was identified as salicylyl-CoA 5-hydroxylase. Gentisyl-CoA underwent spontaneous cleavage to gentisate and CoA. SdgA behaved as a salicylyl-CoA ligase despite showing amino acid sequence similarity to an AMP-ligase. SdgD was identified as a GDO. These results suggest that Streptomyces sp. strain WA46 degrades salicylate by a novel pathway via a CoA derivative. Two-dimensional polyacrylamide gel electrophoresis and reverse transcriptase-PCR studies indicated that salicylate induced expression of the sdg cluster.
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PMID:Novel pathway of salicylate degradation by Streptomyces sp. strain WA46. 1500 46

Adrenergic drugs acting through the beta(2)-adrenoceptor (beta(2)-AR) adenylate cyclase (AC) signal transduction system elicit a variety of responses within the mammalian airway epithelium; however, its composition of multiple phenotypically differentiated cell types complicates the understanding of the regulation cascades within this tissue. The present study evaluates beta(2)-AR mRNA level, number, subtype and the cyclic adenosine-3',5'-monophosphate (cyclic AMP) response to isoproterenol (iso) in the human airway epithelial cell lines 16HBE14o(-), Calu-3 and A549, using reverse transcriptase polymerase chain reaction (RT-PCR), radioligand binding studies, [(3)H]-radioimmunoassay and immunocytochemical staining. After 4-5 days in culture, all three cell types produced beta(2)-AR mRNA and protein at a magnitude of gene expression levels Calu-3>or=16HBE14o(-)>A549, whereas control cells Cos-1 and Caco-2 were negative. The beta(2)-AR adenylate cyclase system was highly expressed and functional in the human airway epithelial cells Calu-3 and 16HBE14o(-). The mean beta(2)-AR density (B(max)), equilibrium dissociation constant (K(D)), and the percentage of beta-AR subtypes assessed by radioligand binding were approximately 9908+/-1127 and 6423+/-895 binding sites/cell, 32+/-2.7 pM and 25+/-1.1 pM, and approximately 100% in Calu-3 and 16HBE14o(-)cells, respectively. However, in the alveolar cell type A549 the cell surface beta(2)-AR was virtually undetectable by (-)-[(125)I]-iodocyanopindolol (ICYP) binding. Stimulation of cultured cells with (-)-isoproterenol enhanced the basal cyclic AMP accumulation only in Calu-3 and 16HBE14o(-) cells, which was blocked by the beta(2)-selective antagonist ICI 118,551, but not by the beta(1)-selective antagonist CGP 20712A, confirming functional coupling of the beta(2)-AR to adenylate cyclase in these cells. Immunocytochemical staining localised the receptor on the cell membrane and the cytoplasm in Calu-3 and 16HBE14o(-) cells, while it was confined to the cytoplasm only in A549 cells. In conclusion, the beta(2)-AR expression and its functional coupling to adenylyl cyclase was very high in the human airway epithelial cells Calu-3 and 16HBE14o(-), but not in A549, suggesting that the cell lines Calu-3 and 16HBE14o(-) present suitable models to study function and regulation of the beta-adrenoceptor signalling in the respiratory system.
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PMID:Expression of functional beta2-adrenergic receptors in the lung epithelial cell lines 16HBE14o(-), Calu-3 and A549. 1511 Sep 97

The mouse corticotropin-releasing factor (CRF) type 2a receptor (CRF2(a)) splice variant was cloned by a PCR-based approach. The corresponding cDNA was found to encode a 411-amino acid polypeptide with highest sequence homology to the rat CRF2(a) receptor. By semiquantitative reverse transcriptase PCR (RT-PCR) analysis, the CRF2(b) mRNA was mainly found in the heart and skeletal muscle with only low level expression in the brain. In contrast, CRF2(a) mRNA was restricted to the brain with major expression sites in the cortex, hippocampus, hypothalamus and telencephalon. Binding and cyclic AMP stimulation studies showed a similar ligand selective profile for both mCRF2 receptor splice variants. A notable exception however, was urotensin I which displayed a approximately 3-fold higher affinity for the CRF2(a) receptor and also stimulated cyclic AMP production in mCRF2(a)-transfected cells with a approximately 3-fold higher potency than in mCRF2(b)-transfected cells. These data show that the mouse like other mammalian species expresses two ligand-selective CRF2 receptor splice variants and that the mCRF2(a) receptor is the predominant central CRF2 receptor in the mouse.
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PMID:Molecular cloning and functional expression of the mouse CRF2(a) receptor splice variant. 1525 78

Previous NMR relaxation studies of the isolated RNase H domain of HIV-1 reverse transcriptase at low pH have revealed that it is substantially more dynamic and less ordered than the relatively stable and catalytically active E. coli RNase HI. Using more recently developed techniques, we have investigated the dynamic behavior of the RNase H domain of HIV-1 reverse transcriptase at a more physiological pH (6.8), under a variety of solution conditions: no Mg(2+), 80 mM Mg(2+), and 80 mM Mg(2+) plus AMP ligand. In addition, we have repeated the previous measurements on a sample containing 100 mM sodium acetate, pH 5.4. Under all conditions studied, the order parameters from NMR relaxation analysis are uniformly high (>0.8) for most of the domain with the exception of the C-terminal region. Subtle differences can be found among the conditions studied, although the statistical significance of the differences is marginal. Residues 71-114 show a slight increase in order parameter with the addition of 5'-AMP. Conformational exchange, measured with CPMG relaxation dispersion experiments in the presence of Mg and AMP, were detected for some NH sites, predominantly located in the N-terminal region of the protein near strands beta2 and beta3 and helix alpha(A) (residues 28-69). In contrast with earlier studies indicating pathologically extreme dynamic behavior that apparently correlated with inactivity of the isolated domain, the relaxation analysis under the conditions of the present study yielded parameters that are more similar to those of the active E. coli RNase HI. A comparison of the order parameters obtained from a model-free analysis of the relaxation data with the B-factors in the crystal structures of the RNase H domain, both for the isolated domain and for the full HIV-1 reverse transcriptase structure, suggests that the dynamic behavior is similar in all cases.
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PMID:Backbone dynamics of the RNase H domain of HIV-1 reverse transcriptase. 1526 Apr 76

HIV-1 reverse transcriptase can remove chain terminators from blocked DNA ends through a nucleotide-dependent mechanism. We show that the catalytic efficiency of the removal reaction can vary several hundred-fold in different sequence contexts and is most strongly affected by the nature of the base pair at the 3'-primer terminus and the six base pairs upstream of it. Similar effects of the upstream sequence were observed with primer-templates terminated with 2',3'-dideoxy-AMP, 2',3'-dideoxy-CMP, or 2',3'-dideoxy-GMP. However, the removal of 2',3'-dideoxy-TMP or 3'-azido-2',3'-dideoxy-TMP was much less influenced by upstream primer-template sequence, and the rate of excision of these thymidylate analogues was greater than or equal to that of the other chain-terminating residues in each sequence context tested. These results strongly indicate that the primer terminus and adjacent upstream base pairs interact with reverse transcriptase in a sequence-dependent manner that affects the removal reaction. We conclude that primer-template sequence context is a major factor to consider when evaluating the removal of different chain terminators by HIV-1 reverse transcriptase.
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PMID:Effects of primer-template sequence on ATP-dependent removal of chain-terminating nucleotide analogues by HIV-1 reverse transcriptase. 1530 46

AMPK (AMP-activated protein kinase) is a key sensor of energy status within the cell. Activated by an increase in the AMP/ATP ratio, AMPK acts to limit cellular energy depletion by down-regulating selective ATP-dependent processes. The purpose of the present study was to determine the role of AMPK in regulating intestinal glucose transport. [3H]3-O-methyl glucose fluxes were measured in murine jejunum in the presence and absence of the AMPK activators AICAR (5-aminoimidazole-4-carboxamide riboside) and metformin and the p38 inhibitor, SB203580. To differentiate between a sodium-coupled (SGLT1) and diffusive (GLUT2) route of entry, fluxes were measured in the presence of the SGLT1 and GLUT2 inhibitors phloridzin and phloretin. Glucose transporter mRNA levels were measured by reverse transcriptase-PCR, and localization by Western blotting. Surface-expressed GLUT2 was assessed by luminal biotinylation. Activation of p38 mitogen-activated protein kinase was analysed by Western blotting. We found that treatment of jejunal tissue with AICAR resulted in enhanced net glucose uptake and was associated with phosphorylation of p38 mitogen-activated protein kinase. Inhibition of p38 abrogated the stimulation of AICAR-stimulated glucose uptake. Phloretin abolished the AICAR-mediated increase in glucose flux, whereas phloridzin had no effect, suggesting the involvement of GLUT2. In addition, AICAR decreased total protein levels of SGLT1, concurrently increasing levels of GLUT2 in the brush-border membrane. The anti-diabetic drug metformin, a known activator of AMPK, also induced the localization of GLUT2 to the luminal surface. We conclude that the activation of AMPK results in an up-regulation of non-energy requiring glucose uptake by GLUT2 and a concurrent down-regulation of sodium-dependent glucose transport.
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PMID:5-aminoimidazole-4-carboxamide riboside (AICAR) enhances GLUT2-dependent jejunal glucose transport: a possible role for AMPK. 1536 3


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