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)

Biochemical methods for single muscle fibre analysis provide sensitive measures for elucidating muscle fibre heterogeneity. The understanding of the complexity of skeletal muscle fibres, initially based on qualitative histochemistry and immunohistochemistry, has been greatly expanded by quantitative micromethods, such as microphotometry and microbiochemical assays. Assessment of metabolic enzyme activity levels has revealed pronounced scattering within and between different fibre types and has highlighted the use of specific enzyme activity ratios as discriminative measures. With the exception of type I fibres, metabolic properties are loosely coupled with molecular properties of the myofibrillar apparatus. As such, myosin heavy chain (MHC) isoforms appear to be the best choice for fibre type delineation. Among the two available methods for MHC-based fibre type distinction, single fibre electrophoresis appears to be superior to immunohistochemistry. The electrophoretic separation of MHC isoforms in single fibres is quantitative and, as opposed to immunohistochemistry, yields important information on MHC isoform proportions in hybrid fibres. Histochemical staining for myofibrillar ATPase activity can, thus, be correlated in most cases with specific MHC isoform profiles. Single fibre studies have demonstrated a relationship between ATP phosphorylation potential and MHC isoform complement. This relationship corresponds to different tension costs and provides an additional rationale for the MHC-based fibre type diversity and transitions. The combination of reverse transcriptase (RT) with polymerase chain reaction (PCR) has proved to be a highly sensitive tool and has extended single fibre analysis to the level of MHC mRNA isoforms. Application of RT-PCR techniques to single fibre fragments identified by their MHC protein isoform profile, provides insights at two levels of expression and, thus, has extended our knowledge on the plasticity of muscle and the dynamical state of muscle fibres.
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PMID:The impact of biochemical methods for single muscle fibre analysis. 1046 63

Dideoxynucleosides, which are potent inhibitors of HIV reverse transcriptase and other viral DNA polymerases, are a common component of highly active anti-retroviral therapy (HAART) (ref. 1). Six reverse transcriptase inhibitors have been approved for human use: azidothymidine; 2'3'-dideoxycytidine; 2'3'-dideoxyinosine; 2', 3'-didehydro-3'deoxythymidine; 2',3'-dideoxy-3'-thiacytidine; and 4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-++ +metha nol. Although drug-resistant HIV strains resulting from genetic mutation have emerged in patients treated with HAART (ref. 1), some patients show signs of drug resistance in the absence of drug-resistant viruses. In our study of alternative or additional mechanisms of resistance operating during antiviral therapy, overexpression and amplification of the MRP4 gene correlated with ATP-dependent efflux of PMEA (9-(2-phosphonylmethoxyethyl)adenine) and azidothymidine monophosphate from cells and, thus, with resistance to these drugs. Overexpression of MRP4 mRNA and MRP4 protein severely impaired the antiviral efficacy of PMEA, azidothymidine and other nucleoside analogs. Increased resistance to PMEA and amplification of the MRP4 gene correlated with enhanced drug efflux; transfer of chromosome 13 containing the amplified MRP4 gene conferred resistance to PMEA. MRP4 is the first transporter, to our knowledge, directly linked to the efflux of nucleoside monophosphate analogs from mammalian cells.
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PMID:MRP4: A previously unidentified factor in resistance to nucleoside-based antiviral drugs. 1047 83

Bladder filling depends upon the coordinated control of a storage chamber, the bladder body, and its outlet, the bladder base and urethra. Bladder emptying results from development of force in the bladder body and relaxation of the outlet. Muscle strips from bladder body reveal phasic characteristics, whereas the strips from urethral wall are tonic. To determine whether the compositions of myosin heavy chain (MHC) isoforms and the level of myosin light chain (MLC) phosphorylation contribute to the regional variation in the contractile states of the bladder smooth muscle, we analyzed the levels of MLC phosphorylation and the expression of myosin isoforms in smooth muscle tissues from different regions of the urinary bladder. Strips of bladder from the dome, mid body, base of the bladder and urethra were removed and analyzed for the levels of MLC phosphorylation at the resting tone. The expression of MHC isoforms that differ in the C-terminus (SM1 and SM2) and in the N-terminal region (SM-A and SM-B), formed by alternative splicing of the pre-mRNA at either the 3' end or the 5' end, respectively, was analyzed. The expression of these isoforms was characterized at the mRNA and protein levels using reverse transcriptase-polymerase chain reaction (RT-PCR), SDS-PAGE, and Western blotting. The levels of MLC phosphorylation were 35.5 +/- 4.6, 24.7 +/- 2.2, 13.6 +/- 2.1, and 12.8 +/- 2.7 for dome, mid bladder body, base and urethra respectively. Almost 100% of the MHC mRNA in the dome, mid bladder body, and base contains a 7-amino acid insert near the ATP-binding region, whereas the MHC in the urethral smooth muscle is only 81% inserted. Prior studies have shown that inserted myosin has a two-fold higher actin-activated ATPase activity compared to the myosin isoform that lacks the insert, and the maximum velocity of shortening of smooth muscle containing this insert is high compared to muscle that do not contain the insert. The expression of SM1 and SM2 were not significantly different. Our data suggests the presence of a high degree of inserted myosin and LC20 phosphorylation in the bladder dome and mid-body helps to facilitate rapid force development and emptying. Non-inserted myosin and the low level of MLC phosphorylation in the urethra may contribute to slowly or non-cycling myosin cross bridges and the maintenance of a tonic or contracted state during bladder filling.
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PMID:Myosin light chain phosphorylation at resting level and the composition of myosin isoforms in the bladder body and urethra. 1057 76

Previous studies have indicated the expression of multiple P2Y receptors by rat hepatocytes although they have not been identified. Here we show by reverse transcriptase-polymerase chain reaction (RT - PCR) that rat hepatocytes express mRNA encoding all of the four cloned rat P2Y receptors (P2Y(1), P2Y(2), P2Y(4) and P2Y(6)). The effects of UTP have been examined on single aequorin-injected rat hepatocytes. The [Ca(2+)](i) transients induced by UTP were indistinguishable from those induced by ATP in the same cell. The modulatory effects of elevated intracellular cyclic AMP concentration were the same on both UTP- and ATP-induced [Ca(2+)](i) transients. UDP, an agonist at the P2Y(6) receptor, failed to induce transients in hepatocytes, indicating that functional P2Y(6) receptors coupled to increased [Ca(2+)](i) are not expressed. The transients evoked by ADP were more sensitive to inhibition by suramin than those induced by either ATP or UTP. Within an individual cell, the transients induced by ATP and UTP were inhibited by the same concentration of suramin. This sensitivity of ATP and UTP responses to suramin suggests action through P2Y(2) rather than P2Y(4) receptors. Co-application of 30 microM pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) caused a decrease in frequency and amplitude of transients induced by ADP. ATP- and UTP-induced transients also displayed a decrease in amplitude in response to addition of PPADS, but this was accompanied by an increase in frequency of transients. In conclusion the data presented here are consistent with the co-expression of P2Y(1) and P2Y(2) receptors by rat hepatocytes.
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PMID:Evidence that rat hepatocytes co-express functional P2Y1 and P2Y2 receptors. 1068 1

ATP-induced Ca2+ transients were examined in individual PC12 cells of a well defined clone, before and after treatment with nerve growth factor (NGF) to induce a neurone-like phenotype. Using reverse transcriptase PCR these cells were found to express mRNA for several P2 receptors. In undifferentiated cells the ATP-induced Ca2+ response was entirely dependent on Ca2+ influx, could not be mimicked by UTP, alpha,beta-methylene ATP or dibenzoyl ATP or be blocked by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). ATP had no significant effect on levels of cyclic AMP or inositol 1,4,5-trisphosphate (InsP3). These results suggest that in undifferentiated PC12 cells ATP mainly acts on a P2X receptor, possibly the P2X4 subtype. After treatment with NGF for 7 days the ATP response was increased and partially sensitive to PPADS. A component of the ATP-induced Ca2+ increase was due to mobilisation of intracellular Ca2+ stores and another to capacitative Ca2+ entry. UTP caused an increase in intracellular Ca2+, and InsP3 formation could be stimulated by ATP and UTP. ATP also caused a small increase in cyclic AMP, but this was abolished in the presence of indomethacin. Thus, after NGF treatment ATP acts partially via a P2Y receptor, possibly the P2Y2 subtype.
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PMID:P2Y receptors contribute to ATP-induced increases in intracellular calcium in differentiated but not undifferentiated PC12 cells. 1069 14

Extracellular ATP is a neurotransmitter and mediates a variety of responses. In the endocrine system, there are data suggesting a physiological role for ATP in Ca(2+) signalling and hormone secretion. However, the ATP receptor subtype involved has not been clearly elucidated in GH3 cells, a rat anterior pituitary cell line. BzATP- and ATP-induced [Ca(2+)](i) responses had EC(50) values of 18 and 651 microM, respectively. The maximal response to ATP was only 59+/-8% of that for BzATP. The BzATP-induced [Ca(2+)](i) increase was dependent upon the extracellular Ca(2+) concentration. Preincubation with oxidized ATP (oATP) nearly abolished the ATP- and BzATP-induced [Ca(2+)](i) increases. Both BzATP and ATP induced depolarization in GH3 cells, with EC(50) values of 31 microM and 1 mM, respectively. The maximal depolarization to BzATP and ATP were 152+/-21 and 146+/-16% of that elicited by 30 mM KCl. The rank order of agonist potency for [Ca(2+)](i) and depolarization responses was BzATP > > ATP >2-MeSATP and purine derivatives such as ADP, AMP, adenosine were ineffective. Neither UTP nor alpha, beta-methylene ATP showed any effect. In low-divalent conditions BzATP evoked non-desensitizing inward currents, which were reversed at approximately 0 mV. This nonselective cationic conductance was increased by repeated applications of BzATP and the cells became very permeable to NMDG. Longer applications (30 min) of BzATP stimulated ethidium bromide influx in low divalent conditions, suggesting increased permeability to larger molecules. We also identified the existence of P2X(7) mRNA on GH3 cells by using reverse transcriptase (RT)-polymerase chain reaction (PCR). These results suggest that the GH3 cells have an endogenous P2X(7) receptor and purinergic stimulation may play a potential role in neuroendocrine modulation on these cells.
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PMID:ATP-induced [Ca(2+)](i) changes and depolarization in GH3 cells. 1095 73

To investigate a cDNA encoding cation current, we isolated an alternatively spliced form of a rat Trp3, designated Trp3sv. Trp3sv encodes 736 amino acids with a unique N terminus and six transmembrane segments. Expression of the cRNA in Xenopus oocytes was successfully performed. The cation selective current appeared after the addition of ionomycin or induced by prolonged depolarization but not by hyperpolarization. This induction was not observed by a treatment with thapsigargin, phorbol ester, or ATP. Na(+), K(+), tetraethylammonium, and divalent cations were permeable, while N-methylglucamine and chloride were nominally impermeable ions. The currents were not inhibited by flufenamate ruthenium red but nonspecifically by 2 mm Gd(3+). Northern as well as Western blot suggested lower levels of the expression observed in some organs, while reverse transcriptase-polymerase chain reaction suggested that it widely spread among various organs. Therefore, we may conclude that N-terminal spliced valiant of Trp3, Trp3sv, encodes a calcium-activated cation channel in various organs.
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PMID:A calcium-activated cation current by an alternatively spliced form of Trp3 in the heart. 1098 75

The findings presented here originally arose from the suggestion that the synthesis of dinucleoside polyphosphates (Np(n)N) may be a general process involving enzyme ligases catalyzing the transfer of a nucleotidyl moiety via nucleotidyl-containing intermediates, with release of pyrophosphate. Within this context, the characteristics of the following enzymes are presented. Firefly luciferase (EC 1.12. 13.7), an oxidoreductase with characteristics of a ligase, synthesizes a variety of (di)nucleoside polyphosphates with four or more inner phosphates. The discrepancy between the kinetics of light production and that of Np(n)N synthesis led to the finding that E*L-AMP (L = dehydroluciferin), formed from the E*LH(2)-AMP complex (LH(2) = luciferin) shortly after the onset of the reaction, was the main intermediate in the synthesis of (di)nucleoside polyphosphates. Acetyl-CoA synthetase (EC 6.2.1.1) and acyl-CoA synthetase (EC 6.2.1. 8) are ligases that synthesize p(4)A from ATP and P(3) and, to a lesser extent, Np(n)N. T4 DNA ligase (EC 6.5.1.1) and T4 RNA ligase (EC 6.5.1.3) catalyze the synthesis of Np(n)N through the formation of an E-AMP complex with liberation of pyrophosphate. DNA is an inhibitor of the synthesis of Np(n)N and conversely, P(3) or nucleoside triphosphates inhibit the ligation of a single-strand break in duplex DNA catalyzed by T4 DNA ligase, which could have therapeutic implications. The synthesis of Np(n)N catalyzed by T4 RNA ligase is inhibited by nucleoside 3'(2'),5'-bisphosphates. Reverse transcriptase (EC 2.7.7.49), although not a ligase, catalyzes, as reported by others, the synthesis of Np(n)ddN in the process of removing a chain termination residue at the 3'-OH end of a growing DNA chain.
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PMID:Synthesis of dinucleoside polyphosphates catalyzed by firefly luciferase and several ligases. 1100 93

The distribution of human sulfonylurea receptor-2 (SUR2)-containing K(ATP) channels was investigated using reverse transcriptase-polymerase chain reaction (RT-PCR). mRNA for SUR2B was detected in a variety of tissues including brain, skeletal, cardiac and smooth muscle, whereas SUR2A message was restricted to cardiac and skeletal muscle. An additional splice variant of SUR2 that lacked exon 17 was also identified by RT-PCR in tissues expressing both SUR2A and SUR2B or SUR2B alone. Quantification of RNA for SUR2 exon 17+ and SUR2 exon 17- splice variants using real-time Taqman PCR indicated differential levels of expression in brain, kidney, skeletal muscle, heart and small intestine. Interestingly, the SUR2 exon 17+ variant is the major species expressed in all tissues examined in this study. Each of the SUR2 splice variants transiently expressed with the inward rectifier Kir 6.2 formed functional K(ATP) channels in HEK 293 cells as assessed either by changes in DiBAC(4)(3) fluorescence responses or glyburide-sensitive whole cell currents. Collectively, our findings demonstrate that various SUR2 splice variants have distinct expression patterns and can form functional K(ATP) channels.
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PMID:Molecular characterization of human SUR2-containing K(ATP) channels. 1105 56

RUSH proteins are SWI/SNF-related transcription factors with RING finger signatures near their COOH termini. Long suspected of mediating protein-protein interactions, the RING motif was used to clone a binding partner. The RING finger binding protein (RFBP) is a Type IV P-type ATPase, a putative phospholipid pump, with conserved sequences for two loop segments, an ATP-binding site, a phosphorylation domain, and transmembrane passes potentially involved in substrate binding and translocation. However, RFBP differs from all other Type IV P-type ATPases in three ways. It has only three of four highly conserved NH(2)-terminal transmembrane passes, it is located in the inner nuclear membrane, and it binds the RING domain. Topographically the orientation of the adjacent hydrophilic domains and the determinants of transport specificity are altered. As a result, the small, hydrophilic loop extends into the perinuclear space that is contiguous with the lumen of the endoplasmic reticulum. The large, conformationally flexible loop extends into the nucleoplasm to contact euchromatin. Competitive reverse transcriptase-polymerase chain reaction and high performance liquid chromatography analysis revealed that endometrial RFBP mRNA expression is hormonally regulated. The physical association of a hormone-dependent RING finger-binding protein with transcriptionally active chromatin supports the speculation that RFBP plays a role in the subnuclear trafficking of transcription factors with RING motifs.
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PMID:Cloning and characterization of an atypical Type IV P-type ATPase that binds to the RING motif of RUSH transcription factors. 1105 86


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