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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To explore the possible role of purinergic receptors in thymocyte development and in pathogenesis of adenosine deaminase SCID, we studied effects of extracellular adenosine triphosphate (ATP(ext)) and adenosine on TCR- and steroid hormone-triggered processes in mouse thymocytes. Reverse transcriptase-PCR analysis confirms the mRNA expression of several types of purinergic receptors, while the functioning of ATP receptors in thymocytes is reflected by ATP(ext)-induced intracellular calcium increases and by thymocyte subset-specific sensitivity to the effects of ATP(ext) and adenosine. Only ATP(ext), but not the ATP catabolites, adenosine, dexamethasone, or TCR cross-linking, was efficient in triggering rapid protein synthesis independent lysis of CD4+8- thymocytes and peripheral CD4+ T cells. In contrast, extracellular adenosine specifically induced the apoptosis of CD4+8+ thymocytes. ATP(ext) also induced a slower process of DNA fragmentation and protein synthesis-dependent apoptosis in all thymocyte subsets. ATP(ext) had an additive effect with TCR cross-linking in the induction of thymocyte death, but, unexpectedly, the effects of ATP(ext) at high concentration were antagonistic to steroid-induced apoptosis. Described here, the properties of ATP(ext) and adenosine are consistent with their involvement in the regulation of T cell development due to differential expression and signaling through purinergic receptors in different thymocyte subsets. The possible role of purinergic receptor signaling in T cell differentiation and adenosine deaminase SCID is discussed.
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PMID:Effects of extracellular ATP and adenosine on different thymocyte subsets: possible role of ATP-gated channels and G protein-coupled purinergic receptor. 916 24

In the last decade, as a result of molecular cloning and the reverse-transcriptase polymerase chain reaction, numerous isoforms of the contractile protein myosin have been discovered. What lags behind their discovery is knowledge of their functions. This review focuses on some of my recent work on the structure, function and regulation of isoforms of the heavy chain of vertebrate smooth muscle and nonmuscle myosin II. Reference to related work in the field is included where appropriate. The particular isoforms discussed are those that are generated by alternative splicing near the 5' end of the pre-mRNA, resulting in either an insertion or a deletion of a cassette of amino acids near the amino-terminus of the myosin heavy chain (MHC) protein. In both the smooth muscle and nonmuscle MHCs, this splicing occurs in the exact same region, which begins at amino acid 212 in the primary sequence. In the three-dimensional structure of the molecule, these inserts are located near the ATP-binding pocket in a region of the MHC that was not resolved in the crystal structure and therefore is believed to represent a flexible loop. In the smooth muscle MHC, the insertion of seven amino acids in this loop confers a higher enzymatic activity on the myosin. The potential mechanism by which this occurs and the significance to smooth muscle contractile diversity is discussed. In the nonmuscle MHC, the insert in this region is a different size and sequence of amino acids than that in the smooth muscle MHC. A serine residue (Ser-214) in the nonmuscle loop is phosphorylated by p34cdc2 kinase in Xenopus during meiotic maturation of oocytes to eggs and is dephosphorylated in interphase egg extracts that are equivalent to the interphase after fertilization of the egg. Thus, MHC-B phosphorylation by cdc2 kinase correlates with the cortical reorganization that occurs during meiosis, and dephosphorylation correlates with the cortical contraction that occurs at fertilization, which aids in pronuclear fusion. In summary, these inserts in the MHC molecule, in a flexible loop near the ATP-binding pocket, appear to be important in determining differences in function or regulation among myosin II isoforms.
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PMID:Characterization of isoform diversity among smooth muscle and nonmuscle myosin heavy chains. 918 13

The complete nucleotide sequence of major core protein gene (segment S3) of rice dwarf virus (RDV) Chinese isolate was determined after cDNA cloning from the viral genomic RNA. Sequence analysis showed that the cloned fragment is 3195 bp in length and contains a single open reading frame (ORF), encoding the major core protein (P3) which M(r) of 114 K. The nucleotide and deduced amino acid sequences of S3 of this isolate share significant homology (94.1% and 97%, respectively) with those of S3 of the Japanese isolate. At the amino acid level, P3 of RDV Chinese isolate shares significant homology with P3 of rice gall dwarf virus (RGDV), significant regional homology with the rotavirus penetration, and homology with spheroidin of amsacta entomopoxvirus (SPH), which is the major protein of the occlusion body, with clp-like ATP-dependent protease binding subunit and with ATP-dependent protease ATP-binding subunit. Amino acid sequence analysis also showed that P3 contains RNA-dependent RNA polymerase (RDRP) motif-like elements such as DXXXD, SGXXXXXXN, GDD and ENXXXY. These results may suggest that P3 is a multifunctional protein which plays very important roles in the virus structure formation, virus replication and penetration processes. The full length cDNA sequence of RDV S3 and a partial one which covers nt 1004-3195 were cloned into bacterial expression vector pTrcHisB for expression. The full length cDNA sequence failed to be expressed in E. coli, but the partial sequence was successfully expressed there as confirmed by the Western blot analysis. Further analysis of RDV P3 is under way.
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PMID:Molecular cloning, sequencing, functional analysis and expression in E. coli of major core protein gene (S3) of rice dwarf virus Chinese isolate. 938 5

1. We have used the patch-clamp technique to study modulation of the inwardly rectifying K+ current (IK(IR)) in cultured bovine pulmonary artery endothelial cells (CPAE cells). In whole-cell mode, IK(IR) was defined as the Ba(2+)-sensitive current. In single channel recordings, we observed a strongly inwardly rectifying and K(+)-selective channel with a conductance of 31 +/- 3 pS. 2. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and functional data suggest that the endothelial IRK is most probably Kir2.1. 3. Intracellular ATP is required to prevent run-down of IRK in whole-cell mode. Single channel activity disappeared in inside-out patches exposed to ATP-free solution and in cell-attached patches on cells exposed to metabolic inhibition (KCN, 2-deoxyglucose). 4. The non-hydrolysable ATP analogues, ATP gamma S and adenylyl imidodiphosphate (AMP-PNP), did not prevent run-down. Run-down did not occur in the presence of okadaic acid, a phosphatase inhibitor, but was enhanced in the presence of protamine, an activator of phosphatase 2A (PP2A). 5. GTP gamma S and AlF4- inhibited IRK, also in the presence of ATP. GTP beta S antagonized the GTP gamma S effect. Pretreatment of the cells with PTX did not affect the GTP gamma S-induced inhibition. Okadaic acid, however, slowed this inhibition. 6. Neither activation of protein kinase A (PKA) nor activation of protein kinase C (PKC) affected IRK. Additionally, neither cytochalasin B nor a high concentration of intracellular Ca2+ affected the time course of IRK run-down. 7. We conclude that run-down of IRK is probably due to dephosphorylation by PP2A. Activation of a PTX-insensitive G protein inhibits this current by a mechanism that is neither mediated via the PKA and PKC pathways nor by intracellular Ca2+, but supposedly by a G protein-dependent activation of a phosphatase.
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PMID:Modulation of inwardly rectifying potassium channels in cultured bovine pulmonary artery endothelial cells. 940 63

The ATP requirement of influenza A virus RNA-dependent RNA polymerase was studied during in vitro transcription reactions. In complete transcription reactions, the Km for ATP was 10-fold higher than the Km values for the other NTPs. However, during transcription elongation the Km for ATP was as low as the Km values for the other NTPs, suggesting a special requirement for ATP during transcription initiation. Gel analysis of RNA products of transcription initiation reactions showed that the incorporation of AMP into nascent RNA was more efficient at positions 4, 6 and 7 relative to the template RNA than at position 5. The polymerase produced short, abortive transcripts with lengths corresponding to positions 3 and 4 relative to the template but never to position 5 or longer. These results suggest that incorporation of AMP at position 5 induces the influenza A virus polymerase to go through a transition from a transcription initiation to an elongation complex. This functional change of the polymerase complex rather than a requirement for ATP beta-gamma bond hydrolysis is the most likely reason for the particularly high Km for ATP during the early phase of transcription. This conclusion is supported by the fact that the ATP analogue ATPgammaS [adenosine 5'-O-(3-thiotriphosphate)] can efficiently replace ATP in in vitro transcription reactions and shows a comparable drop of Km between transcription initiation and elongation.
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PMID:Variation in ATP requirement during influenza virus transcription. 960 18

We have previously investigated, in studies of rat distal colonic mucosa, the effect of ATP added to the basolateral side on ion transport and [Ca2+]i. It was demonstrated that ATP acts via a P2Y1 receptor to increase [Ca2+]i and NaCl secretion. In the present study we investigated the effect of luminally added nucleotides (ATP, UTP) on transepithelial voltage (Vte) and resistance (Rte) in Ussing chamber experiments on rat distal colonic mucosa. Both nucleotides induced a rapid and transient (within 30 s) change of Vte to lumen-positive values (resting Vte: -2+/-1 mV; peak Vte after 100 micromol/l ATP: +2.4+/-1.1 mV) and a decrease of Rte from 89. 9+/-10.3 to 83.8+/-9.1 Omegacm2 (n=10). Similar values were obtained with luminal UTP (n=15). The estimated EC50 values for both nucleotides were approximately 6 micromol/l. The ATP-induced Vte effect was nearly completely sensitive to Ba2+. Addition of the K+ channel blocker Ba2+ (1 mmol/l) to the luminal solution reversibly inhibited 77+/-4% (n=5) of the ATP-induced Vte effect. Experiments to identify the respective P2 receptor subtype revealed the following rank order of potency at 500 micromol/l agonist: UTP>/=ATP>>2-methylthio-ATP=ADP>>adenosine> AMP>beta, gamma-methylene-ATP (n=5). This closely resembles the published rank order for the P2Y2 receptor. Using the reverse-transcriptase polymerase chain reaction (RT-PCR) technique P2Y2 receptor-specific mRNA was detected in total RNA extracted from isolated crypts. In summary these data indicate that luminal ATP and UTP act via a P2Y2 receptor in the luminal membrane of colonic mucosa to elicit a transient K+ secretion.
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PMID:Luminal ATP induces K+ secretion via a P2Y2 receptor in rat distal colonic mucosa. 971 4

The biochemical properties of the RNA-dependent RNA polymerase (RdRp) of the hepatitis C virus were analyzed. A hexahistidine affinity-tagged NS5B fusion protein was expressed with recombinant baculoviruses in insect cells and purified to near homogeneity. Enzymatic activity of the purified protein was inhibited by KCl or high concentrations of NaCl and was absolutely dependent on Mg2+, which could be replaced by Mn2+. NS5B was found to be processive and able to copy long heteropolymeric templates with an elongation rate of 150-200 nucleotides/min at 22 degreesC. Kinetic constants were determined for all four nucleoside triphosphates and different templates. In case of a heteropolymeric RNA template corresponding to the last 319 nucleotides of the hepatitis C virus genome, Km values for UTP, GTP, ATP, and CTP were approximately 1.0, approximately 0.5, approximately 10, and approximately 0.3 microM, respectively. The profile of several inhibitors of RdRp activity and substrate analogs indicated that the enzyme has a strong preference for ribonucleoside 5'-triphosphates and that it closely resembles 3Dpol of picornaviruses.
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PMID:Biochemical and kinetic analyses of NS5B RNA-dependent RNA polymerase of the hepatitis C virus. 974 Jul 82

Two maize cDNAs were isolated and sequenced that had open reading frames with approximately 37% amino acid identity to mammalian pyruvate dehydrogenase kinases. Both maize kinase sequences contain the five domains with conserved signature residues typical of procaryotic two-component histidine kinases. Sequence comparisons identified six other highly conserved motifs that are proposed to be specific to pyruvate dehydrogenase kinases. In addition, specific Trp and Cys residues are also invariant in these sequences. The maize cDNAs are 1332 (PDK1) and 1602 (PDK2) nucleotides in length, encoding polypeptides with calculated molecular masses of 38,867 and 41,327 Da that share 77% amino acid identity. Reverse transcriptase-polymerase chain reaction analysis with oligonucleotide-specific primers revealed a differential expression pattern for the two isoforms. PDK1 and PDK2 were expressed in Escherichia coli with N-terminal His6 tags to facilitate purification. The recombinant proteins migrated at 44 and 48 kDa, respectively, during SDS-polyacrylamide gel electrophoresis. Anti-PDK1 antibodies immunoprecipitated 75% of pyruvate dehydrogenase kinase activity from a maize mitochondrial matrix fraction, and recognized a matrix protein of 43 kDa. Recombinant PDK2, expressed as a fusion with the maltose-binding protein, inactivated kinase-depleted maize pyruvate dehydrogenase complex when incubated with MgATP, coincident with incorporation of 32P from [gamma-32P]ATP into the alpha subunit of pyruvate dehydrogenase.
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PMID:Molecular analysis of two pyruvate dehydrogenase kinases from maize. 975 1

CHO-K1 cells were examined for their cellular responses to the P2 receptor agonist, 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (DbATP), and for the presence of mRNA for P2X receptors. Reverse transcriptase-polymerase chain reactions, using primers directed against the rat P2X subunits, detected the presence of P2X7 but not P2X1-P2X6 subunits. DbATP (EC50 approximately equal to 100 microM) evoked non-desensitizing inward currents which reversed at approximately equal to 0 mV, suggesting activation of a non-selective cation channel. ATP also evoked inward currents but was less potent than DbATP. DbATP also stimulated the accumulation of 45calcium (45Ca2+) and the DNA binding dye, YO-PRO-1, in CHO-KI cells. Both responses were inhibited by NaCl and MgCl2. In 280 mM sucrose buffer, 45Ca2+ accumulation was measurable within 10-20 s of agonist addition, whereas YO-PRO-1 accumulation was only detectable after 8 min. ATP and ATPgammaS were also agonists but were less potent than DbATP, while UTP, 2-methylthio ATP, ADP and (alphabeta)methylene ATP were inactive at concentrations up to 100 microM. DbATP increased lactate dehydrogenase release from CHO-K1 cells, suggesting cell lysis, although this effect was only pronounced after 60-90 min. These data suggest that CHO-K1 cells express an endogenous P2X7 receptor which can be activated by DbATP to cause a rapid inward current and accumulation of 45Ca2+. Prolonged receptor activation results in a delayed, increased permeability to larger molecules such as YO-PRO-1 and ultimately leads to cell lysis. Importantly, the presence of an endogenous P2X7 receptor should be considered when these cells are used to study recombinant P2X receptors.
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PMID:Identification and characterization of an endogenous P2X7 (P2Z) receptor in CHO-K1 cells. 986 47

NS5B of the hepatitis C virus is an RNA template-dependent RNA polymerase and therefore the key player of the viral replicase complex. Using a highly purified enzyme expressed with recombinant baculoviruses in insect cells, we demonstrate a stimulation of RNA synthesis up to 2 orders of magnitude by high concentrations of GTP but not with ATP, CTP, UTP, GDP, or GMP. Enhancement of RNA synthesis was found with various heteropolymeric RNA templates, with poly(C)-oligo(G)12 but not with poly(A)-oligo(U)12. Several amino acid substitutions in polymerase motifs B, C, and D previously shown to be crucial for RdRp activity were tested for GTP stimulation of RNA synthesis. Most of these mutations, in particular those affecting the GDD motif (motif C) strongly reduced or completely abolished activation by GTP, suggesting that the same NTP-binding site is used for stimulation and RNA synthesis. Since GTP did not affect the overall RNA binding properties or the elongation rate, high concentrations of GTP appear to accelerate a rate-limiting step at the level of initiation of RNA synthesis. Finally, enhancement of RNA synthesis by high GTP concentrations was also found with NS5B of the pestivirus classical swine fever virus, but not with the 3D polymerase of poliovirus. Thus, stimulation of RdRp activity by GTP is evolutionarily conserved between the closely related hepaciviruses and pestiviruses but not between these and the more distantly related picornaviruses.
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PMID:Selective stimulation of hepatitis C virus and pestivirus NS5B RNA polymerase activity by GTP. 1019 56


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