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)

Telomerase is a specialized reverse transcriptase that contains its own RNA template for synthesis of telomeric DNA [Greider, C. W., & Blackburn, E. H. (1989) Nature 337, 331-337; Shippen-Lentz, D., & Blackburn, E. H. (1990) Science 247, 546-552]. The activity of this ribonucleoprotein enzyme has been associated with cancer cells [Kim et al. (1994) Science 266, 2011-2015] and is thus a potential target for anticancer chemotherapy. Telomeric DNA.RNA hybrids are important intermediates in telomerase function and form after extension of the growing telomere on the telomerase RNA template. Translocation is a critical step in telomerase function and consists of unwinding of the telomeric DNA.telomerase RNA hybrid followed by repositioning of the 3'-end of the extended telomere. A central question in telomerase function is how translocation of the extended telomere occurs in the absence of ATP or GTP. It has been hypothesized that unwinding of the telomeric hybrid may be facilitated by the formation of stable hairpins or G-quadruplexes by the telomere product (i.e., a hybrid to G-quadruplex transition) and that this may provide at least part of the driving force for translocation [Shippen-Lentz & Blackburn, 1990; Zahler et al. (1991) Nature 350, 718-720]. However, so far there has been no effort aimed at examining the possibility that a hybrid/G-quadruplex equilibrium can occur and to what extent this equilibrium depends on buffer and concentration conditions. Examination of these transitions may provide insight into telomerase function and may also provide clues for the development of anti-telomerase agents. Using a model system consisting of the DNA.RNA hybrid d(GGTTAGGGTTAG).r(cuaacccuaacc), we present evidence that a thermally induced transition of telomeric DNA.RNA hybrid to G-quadruplex can occur under certain conditions. These results provide support for the hypothesis that G-quadruplex formation by the telomere product may in fact regulate telomerase function at the translocation step (Zahler et al., 1991) and suggest an Achilles' heel for indirectly targeting telomerase. Thus, on the basis of the insight gained from the present studies and the results of Zahler et al. (1991), we propose that ligands that selectively bind or cleave G-quadruplex structures may modulate telomerase processivity.
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PMID:Thermally induced DNA.RNA hybrid to G-quadruplex transitions: possible implications for telomere synthesis by telomerase. 897 82

Assembly of hepadnaviruses depends on the formation of a ribonucleoprotein (RNP) complex comprising the viral polymerase polypeptide and an RNA segment, epsilon, present on pregenomic RNA. This interaction, in turn, activates the reverse transcription reaction, which is primed by a tyrosine residue on the polymerase. We have shown recently that the formation of this RNP complex in an avian hepadnavirus, the duck hepatitis B virus, depends on cellular factors that include the heat shock protein 90 (Hsp90). We now report that RNP formation also requires ATP hydrolysis and the function of p23, a recently identified chaperone partner for Hsp90. Furthermore, we also provide evidence that the chaperone complex is incorporated into the viral nucleocapsids in a polymerase-dependent reaction. Based on these findings, we propose a model for hepadnavirus assembly and priming of viral DNA synthesis where a dynamic, energy-driven process, mediated by a multi-component chaperone complex consisting of Hsp90, p23 and, potentially, additional factors, maintains the reverse transcriptase in a specific conformation that is competent for RNA packaging and protein priming of viral DNA synthesis.
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PMID:Hepadnavirus assembly and reverse transcription require a multi-component chaperone complex which is incorporated into nucleocapsids. 900 68

The gene product affected in cystic fibrosis, the cystic fibrosis transmembrane conductance regulator (CFTR), is a chlorideselective ion channel that is regulated by cAMP-dependent protein kinase-mediated phosphorylation, ATP binding and ATP hydrolysis. Mutations in the CFTR gene may result in cystic fibrosis characterized by severe pathology (e.g. recurrent pulmonary infection, male infertility and pancreatic insufficiency) involving organs expressing the CFTR. Interestingly, in the kidney, where expression of the CFTR has been reported, impaired ion transport in patients suffering from cystic fibrosis could not be observed. To understand the role of the CFTR in chloride transport in the kidney, we attempted to identify an epithelial cell line that can serve as a model. We demonstrate that the CFTR is expressed constitutively in Madine-Darby canine kidney (MDCK) type I cells, which are thought to have originated from the distal tubule of the dog nephron. We show expression at the mRNA level, using reverse transcriptase-PCR, and at the protein level, using Western blot analysis with three different monoclonal antibodies. Iodide efflux measurements indicate that CFTR expression confers a plasma membrane anion conductance that is responsive to stimulation by cAMP. The cAMP-stimulated iodide release is sensitive to glybenclamide, diphenylamine carboxylic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid, but not to 4,4'-di-isothiocyanostilbene-2,2'-disulphonic acid, an inhibitor profile characteristic of the CFTR chloride channel. Finally, the polarized localization of the CFTR to the apical plasma membrane was established by iodide efflux measurements and cell-surface biotinylation on MDCK I monolayers. Interestingly, MDCK type II cells, which are thought to have originated from the proximal tubule of the kidney, lack CFTR protein expression and cAMP-stimulated chloride conductance. In conclusion, we propose that MDCK type I and II cells can serve as convenient model systems to study the physiological role and differential expression of CFTR in the distal and proximal tubule respectively.
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PMID:Functional expression and apical localization of the cystic fibrosis transmembrane conductance regulator in MDCK I cells. 907 71

The P-type ATPases (e.g., Na+-K+-ATPase and Ca2+-ATPase) occur widely in living cells of fungi, Protozoa, plants, and animals. These ion pumps show a high degree of divergence in their primary structures but share a limited number of common amino acid residues for their ATP-catalytic function. Particularly, the amino acid sequences for the phosphorylation site (DKTGTLT) and the binding site for ATP (and its analogs; GDGVND) are conserved throughout evolution. Using two degenerate oligonucleotides corresponding to these regions, we applied a polymerase chain reaction (PCR) technique to the search for P-type ATPase isoforms, which will provide a clue to the evolutionary mechanisms of ion pumps in Tetrahymena thermophila. A total of 12 distinct P-type ATPase genes were identified. Sequence comparisons revealed that seven of them can be compiled into a multigene family, which is similar to animal Na+-K+- and H+-K+-ATPase genes. One of them is close to the sarco(endo)plasmic reticulum Ca2+-ATPase gene, and the other four share a significant homology with the gene encoding Plasmodium ATPase-1 whose function is unknown. A Northern blot analysis and reverse transcriptase-PCR demonstrated that all identified genes are expressed, but the expression levels vary widely under different culture conditions. A Southern blot analysis after pulse-field gel electrophoresis showed that all of these genes exist in T. thermophila macronuclei. The Na+-K+- and H+-K+-ATPase gene family has a high multiplicity (at least 10 different genes detected on genomic Southern blot analysis) and is distributed on four different macronuclear chromosomes. On the basis of a calculation with the amino acid sequences of the cloned cytoplasmic loop region (between the phosphorylation and the gamma-[4-(N-2-chloroethyl-N-methylamino)]-benzylamido ATP sites), the genes with >80% identity form a cognate linkage group within the same macronuclei chromosome, whereas the genes with <70% identity are separated in different chromosomes. The phylogenetic analysis showed that this multigene family is the result of a series of gene duplications.
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PMID:Primary structure and evolution of the ATP-binding domains of the P-type ATPases in Tetrahymena thermophila. 912 16

1. We have investigated the mechanism by which L-arginine stimulates membrane depolarization, an increase of intracellular calcium ([Ca2+]i) and insulin secretion in pancreatic beta-cells. 2. L-Arginine failed to affect beta-cell metabolism, as monitored by NAD(P)H autofluorescence. 3. L-Arginine produced a dose-dependent increase in [Ca2+]i, which was dependent on membrane depolarization and extracellular calcium. 4. The cationic amino acids L-ornithine, L-lysine, L-homoarginine (which is not metabolized) and NG-monomethyl-L-arginine (L-NMMA, a nitric oxide synthase inhibitor) produced [Ca2+]i responses similar to that produced by L-arginine. The neutral nitric oxide synthase inhibitors NG-nitro-L-arginine (L-NNA) and N omega-monomethyl-L-arginine (L-NAME) also increased [Ca2+]i. D-Arginine was ineffective. 5. L-Arginine did not affect whole-cell Ca2+ currents or ATP-sensitive K+ currents, but produced an inward current that was carried by the amino acid. 6. The reverse transcriptase-polymerase chain reaction demonstrated the presence of messenger RNA for the murine cationic amino acid transporters mCAT2A and mCAT2B within the beta-cell. 7. L-Arginine did not affect beta-cell exocytosis as assayed by changes in cell capacitance. 8. Our data suggest that L-arginine elevates [Ca2+]i and stimulates insulin secretion as a consequence of its electrogenic transport into the beta-cell. This uptake is mediated by the mCAT2A transporter.
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PMID:Electrogenic arginine transport mediates stimulus-secretion coupling in mouse pancreatic beta-cells. 913 Jan 59

cDNA clones encoding two new Arabidopsis thaliana peroxidases, ATP 1a and ATP 2a, have been identified by searching the Arabidopsis database of expressed sequence tags (dbEST). They represent a novel branch of hitherto uncharacterized plant peroxidases which is only 35% identical in amino acid sequence to the well characterized group of basic plant peroxidases represented by the horseradish (Armoracia rusticana) isoperoxidases HRP C, HRP E5 and the similar Arabidopsis isoperoxidases ATP Ca, ATP Cb, and ATP Ea. However ATP 1a is 87% identical in amino acid sequence to a peroxidase encoded by an mRNA isolated from cotton (Gossypium hirsutum). As cotton and Arabidopsis belong to rather diverse families (Malvaceae and Crucifereae, respectively), in contrast with Arabidopsis and horseradish (both Crucifereae), the high degree of sequence identity indicates that this novel type of peroxidase, albeit of unknown function, is likely to be widespread in plant species. The atp 1 and atp 2 types of cDNA sequences were the most redundant among the 28 different isoperoxidases identified among about 200 peroxidase encoding ESTs. Interestingly, 8 out of totally 38 EST sequences coding for ATP 1 showed three identical nucleotide substitutions. This variant form is designated ATP 1b. Similarly, six out of totally 16 EST sequences coding for ATP 2 showed a number of deletions and nucleotide changes. This variant form is designated ATP 2b. The selected EST clones are full-length and contain coding regions of 993 nucleotides for atp 1a, and 984 nucleotides for atp 2a. These regions show 61% DNA sequence identity. The predicted mature proteins ATP 1a, and ATP 2a are 57% identical in sequence and contain the structurally and functionally important residues, characteristic of the plant peroxidase superfamily. However, they do show two differences of importance to peroxidase catalysis: (1) the asparagine residue linked with the active site distal histidine via hydrogen bonding is absent; (2) an N-glycosylation site is located right at the entrance to the heme channel. The reverse transcriptase polymerase chain reaction (RT-PCR) was used to identify mRNAs coding for ATP 1a/b and ATP 2a/b in germinating seeds, seedlings, roots, leaves, stems, flowers and cell suspension culture using elongation factor 1alpha (EF-1alpha) for the first time as a positive control. Both mRNAs were transcribed at levels comparable to EF-1alpha in all plant tissues investigated which were more than two days old, and in cell suspension culture. In addition, the mRNA coding for ATP 1a/b was found in two day old germinating seeds. The abundant transcription of ATP 1a/b and ATP 2a/b is in line with their many entries in dbEST, and indicates essential roles for these novel peroxidases.
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PMID:Sequence and RT-PCR expression analysis of two peroxidases from Arabidopsis thaliana belonging to a novel evolutionary branch of plant peroxidases. 913 61

We demonstrate, using reverse transcriptase-polymerase chain reaction, that, whereas abdominal aorta from rabbit consists almost entirely of myosin heavy chain (MHC) mRNA with no insert at the 5'-terminal coding region, the distributing arteries (femoral and saphenous) begin to show MHC mRNA with the 21-nucleotide insert that encodes seven amino acids in the ATP-binding region located in the myosin head. The femoral/iliac artery contains > 50% inserted mRNA, whereas the more distal saphenous artery contains > 80% inserted mRNA. This insert is also present in the smooth muscle from rat tail artery but is absent in the smooth muscle from rat aorta. The actin-activated ATPase activity of myosin from the rabbit femoral/saphenous artery is 1.7-fold higher than that of the myosin from the aorta. A concomitant increase (about twofold) in the maximum shortening velocity of the saphenous artery, compared with that of the aorta, indicates that the preponderance of the inserted myosin is associated with both an increase in the actin-activated ATPase activity and a larger maximum velocity of shortening. Furthermore, analysis of the 17-kDa essential light chain from the aorta reveals near equal quantities of the 17-kDa light chain isoforms a and b, whereas the myosin from the femoral/ saphenous artery contains predominantly the 17-kDa light chain a isoform. Together, these data indicate that the smooth muscle cells from the small distributing arteries are similar to those of visceral smooth muscle with respect to the expression of myosin isoforms, actin-activated myosin ATPase activity and contractility.
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PMID:NH2-terminal-inserted myosin II heavy chain is expressed in smooth muscle of small muscular arteries. 917 44

There is growing evidence that extracellular ATP causes a dramatic change in the membrane conductance of a variety of inflammatory cells. In the present study, using the nystatin perforated patch recording configuration, we found that ATP (0.3-30 microM) induced a transient outward current in a concentration-dependent manner and that the reversal potential of the ATP-induced outward current was close to the K+ equilibrium potential, indicating that the membrane behaves like a K+ electrode in the presence of ATP. The first application of ATP to alveolar macrophages perfused with Ca2+-free external solution could induce the outward current, but the response to ATP was diminished with successive applications. Intracellular perfusion with a Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid, also diminished the response. When cyclic ADP-ribose (cADPR) was applied to the macrophage cytoplasm, a transient outward current was elicited. Thereafter, the successive outward current was inhibited, suggesting the involvement of cADPR in the response. Intracellular perfusion with inositol 1,4, 5-trisphosphate also induced a transient outward current, but the successive current was not inhibited. The ATP-induced outward current was abolished when 8-amino-cADPR (as a blocker of cADPR, 10(-6)-10(-5) M) was introduced into the cytoplasm. Homogenates of alveolar macrophages showed both ADP-ribosyl cyclase and cADPR hydrolase activities, and CD38 (ADP-ribosyl cyclase/cADPR hydrolase) expression was confirmed by reverse transcriptase-polymerase chain reaction and Western blot analyses. These results indicate that ATP activates K+ currents by releasing Ca2+ from cADPR-sensitive internal Ca2+ stores.
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PMID:Role of cyclic ADP-ribose in ATP-activated potassium currents in alveolar macrophages. 918 6

Mechanisms contributing to altered heterotrimeric G-protein expression and subsequent signaling events during cholesterol accretion have been unexplored. The influence of cholesterol enrichment on G-protein expression was examined in cultured smooth muscle cells that resemble human atherosclerotic cells by exposure to cationized LDL (cLDL). cLDL, which increases cellular free and esterified cholesterol 2-fold and 10-fold, respectively, reduced the cell membrane content of Galphai-1, Galphai-2, Galphai-3, Gq/11, and Galphas. The following evidence supports the premise that the mechanism by which this occurs is due to reduced isoprenylation of the Ggamma-subunit. First, the inhibitory effect of cholesterol enrichment on the membrane content of Galphai subunits was found to be post-transcriptional, since the mRNA steady-state levels of Galphai(1-3) were unchanged following cholesterol enrichment. Second, the membrane expression of alpha and beta subunits was mimicked by cholesterol and 17-ketocholesterol, both of which inhibit HMG-CoA reductase. Third, inhibition of Galphai and Gbeta expression in cholesterol-enriched cells was overcome by mevalonate, the immediate product of HMG-CoA reductase. Fourth, pulse-chase experiments revealed that cholesterol enrichment did not reduce the degradation rate of membrane-associated Galphai subunits. Fifth, cholesterol enrichment also reduced membrane expression of Ggamma-5, Ggamma-7upper; these gamma subunits are responsible for trafficking of the heterotrimeric G-protein complex to the cell membrane as a result of HMG-CoA reductase-dependent post-translational lipid modification (geranylgeranylation) and subsequent membrane association. Cholesterol enrichment did not alter expression of G-gamma-5 mRNA, as assessed by reverse transcriptase polymerase chain reaction, supporting a post-transcriptional defect in Ggamma subunit expression. Fifth, cholesterol enrichment also reduced the membrane content of p21ras (a low molecular weight G-protein requiring farnesylation for membrane targeting) but did not alter the membrane content of the two proteins that do not require isoprenylation for membrane association&sbd;PDGF-receptor or p60-src. Reduced G-protein content in cholesterol-laden cells was reflected by reduced G-protein-mediated signaling events, including ATP-induced GTPase activity, thrombin-induced inhibition of cyclic AMP accumulation, and MAP kinase activity. Collectively, these results demonstrate that cholesterol enrichment reduces G-protein expression and signaling by inhibiting isoprenylation and subsequent membrane targeting. These results provide a molecular basis for altered G-protein-mediated cell signaling processes in cholesterol-enriched cells.
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PMID:G-protein-mediated signaling in cholesterol-enriched arterial smooth muscle cells. 1. Reduced membrane-associated G-protein content due to diminished isoprenylation of G-gamma subunits and p21ras. 923 98

Airway goblet cells secrete mucin in response to ATP and uridine 5'-triphosphate (UTP), but the underlying signal transduction pathways are poorly understood. Cultures of SPOC1 cells (L. H. Abdullah, S. W. Davis, L. Burch, M. Yamauchi, S. H. Randell, P. Nettesheim, and C. W. Davis. Biochem. J. 316: 943-951, 1996) secreted mucin on exposure to phorbol 12-myristate 13-acetate (PMA) [apparent affinity (K0.5) approximately 100 nM] and ionomycin (K0.5 approximately 5 microM) almost fivefold over baseline. Thapsigargin also elicited secretion (K0.5 approximately 20 nM). Ionomycin and PMA together elicited approximately twice the secretion of either agent alone. Overnight exposure to half-maximal PMA abolished the response to maximal doses of UTP and PMA, whereas ionomycin was fully effective. Protein kinase C (PKC) activity in the membrane fraction was increased by maximal doses of PMA and UTP, whereas ionomycin had no effect. PKC inhibitors were relatively ineffective against PMA- and UTP-induced mucin secretion. Human and canine goblet cells in epithelial explants, by video microscopy, underwent exocytosis with ionomycin (1 microM) and PMA (0.1 or 1 microM). SPOC1 cell mucin secretion was not stimulated by forskolin, 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate, or 8-bromoguanosine 3',5'-cyclic monophosphate. Cystic fibrosis transmembrane conductance regulator was not detected in SPOC1 cells by Western blotting, and its mRNA was detected by reverse transcriptase polymerase chain reaction (PCR) only as a very weak band and after 55 PCR cycles. Multidrug resistance (MDR1), however, was readily detected by Western blotting, and its mRNA was detected as a major band after 35 PCR cycles. Thus airway goblet cell mucin secretion, distal to receptor activation, may be regulated independently by Ca(2+)- and PKC-dependent pathways. Cystic fibrosis transmembrane conductance regulator and cyclic nucleotides, however, may not play a major role in this secretion.
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PMID:Protein kinase C and Ca2+ activation of mucin secretion in airway goblet cells. 925 57


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