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)

Adrenergic regulation of the pineal enzyme serotonin N-acetyltransferase [arylalkylamine N-acetyltransferase (AA-NAT); EC 2.3.1.87] accounts for the circadian rhythm in melatonin formation. In the present study, the role of protein phosphatases in the adrenergic regulation of rat pineal AA-NAT was investigated using specific inhibitors. In cultured pineals, the serine/threonine phosphatase type 1 and type 2A inhibitors okadaic acid and calyculin A significantly decreased adrenergically or cAMP-induced AA-NAT activity, whereas the serine/threonine phosphatase type 2B inhibitor cypermethrin and tyrosine phosphatase inhibitor dephostatin were ineffective. Reverse transcriptase-polymerase chain reaction (RT-PCR) data indicate that okadaic acid exerts its effect on cAMP-dependent AA-NAT induction by downregulating the amount of AA-NAT transcript. The 'third' messengers, inducible cAMP early repressor (ICER) and Fos-related antigene-2 (Fra-2), are believed to play a negative role in pineal AA-NAT transcription. Okadaic acid increased the cAMP responsiveness of neither ICER mRNA nor Fra-2 mRNA. Therefore, the regulatory role of pineal serine/threonine phosphatases in adrenergically stimulated AA-NAT expression probably does not depend on ICER or Fra-2.
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PMID:Serine/threonine phosphatase inhibitors decrease adrenergic arylalkylamine n-acetyltransferase induction in the rat pineal gland. 1144 72

Type 2A serine/threonine protein phosphatases (PP2A) are important components in the reversible protein phosphorylation events in plants and other organisms. PP2A proteins are oligomeric complexes constituted by a catalytic subunit and several regulatory subunits that modulate the activity of these phosphatases. The analysis of the complete genome of Arabidopsis allowed us to characterize four novel genes, AtB'epsilon, AtB'zeta, AtB'eta, and AtB'theta;, belonging to the PP2A B' regulatory subunit family. Because four genes of this type had been described previously, this family is composed of eight members. Reverse transcriptase-polymerase chain reaction experiments showed that AtB'epsilon mRNAs are present in all Arabidopsis tissues analyzed, and their levels do not respond significantly to heat stress. Expressed sequence tags corresponding to AtB'zeta, AtB'eta, and AtB'theta; have been identified, indicating that the new genes are actively transcribed. The genomic organization of this family of PP2A regulatory subunits is reported, as well as its chromosomal location. An extensive survey of the family has been carried out in plants, characterizing B' subunits in a number of different species, and performing a phylogenetic study that included several B' regulatory proteins from animals. Our results indicate that the animal and plant proteins have evolved independently, that there is a relationship between the number of B' isoforms and the complexity of the organism, and that there are at least three main subfamilies of regulatory subunits in plants, which we have named alpha, eta, and kappa.
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PMID:Molecular characterization and evolution of the protein phosphatase 2A B' regulatory subunit family in plants. 1206 21

The human ov-serpin monocyte neutrophil elastase inhibitor (MNEI) is encoded by a single gene SERPINB1. It is a highly efficient inhibitor of neutrophil granule proteases. Four murine genes with high sequence identity with MNEI were identified and fully sequenced, and these were named EIA, EIB, EIC, and EID. EIA, EIB and EIC showed the same seven-exon gene structure as SERPINB1. However, EIC included an additional, alternatively spliced, exon due to the insertion of an endogenous retrovirus-like sequence. EID lacked several exons and is a pseudogene. Reverse transcriptase-PCR showed that EIA, like MNEI, is expressed at high levels in many tissues. EIB is mainly expressed in brain, and EIC was only expressed as splicing variants unlikely to encode a functional serpin. Upon incubation with serine proteases, EIA formed inhibitory covalent complexes with pancreatic and neutrophil elastases, cathepsin G, proteinase-3, and chymotrypsin, as previously shown for MNEI, whereas EIB was only able to do so with cathepsin G. According to the new serpin nomenclature, the genes encoding EIA, EIB, EIC, and EID will be called Serpinb1, Serpinb1b, Serpinb1c, and Serpinb1-ps1. These data demonstrate that the four murine homologs of MNEI have met different evolutionary fates, and that EIA is the mouse ortholog of MNEI.
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PMID:Characterization of four murine homologs of the human ov-serpin monocyte neutrophil elastase inhibitor MNEI (SERPINB1). 1218 54

Tamana bat virus (TABV, isolated from the bat Pteronotus parnellii) is currently classified as a tentative species in the genus FLAVIVIRUS: We report here the determination and analysis of its complete coding sequence. Low but significant similarity scores between TABV and member-viruses of the genus Flavivirus were identified in the amino acid sequences of the structural, NS3 and NS5 genes. A series of cysteines located in the envelope protein and the most important enzymatic domains of the virus helicase/NTPase, methyltransferase and RNA-dependent RNA polymerase were found to be highly conserved. In the serine-protease domain, the catalytic sites were conserved, but variations in sequence were found in the putative substrate-binding sites, implying possible differences in the protease specificity. In accordance with this finding, the putative cleavage sites of the TABV polyprotein by the virus protease are substantially different from those of flaviviruses. The phylogenetic position of TABV could not be determined precisely, probably due to the extremely significant genetic divergence from other member-viruses of the family FLAVIVIRIDAE: However, analysis based on both genetic distances and maximum-likelihood confirmed that TABV is more closely related to the flaviviruses than to the other genera. These findings have implications for the evolutionary history and taxonomic classification of the family as a whole: (i) the possibility that flaviviruses were derived from viruses infecting mammals rather than from mosquito viruses cannot be excluded; (ii) using the current criteria for the definition of genera in the family Flaviviridae, TABV should be assigned to a new genus.
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PMID:Genome sequence analysis of Tamana bat virus and its relationship with the genus Flavivirus. 1223 26

Human rhinoviruses (HRV) represent the single most important causative agent of the common cold. The HRV genome encodes an RNA-dependent RNA polymerase (RdRp) designated 3D polymerase that is required for replication of the HRV RNA genome. We have expressed and purified recombinant HRV-16 3D polymerase to near homogeneity from Escherichia coli transformed with an expression plasmid containing the full-length 460 amino acid HRV-16 3D sequence with a methionine at the N-terminus and a glycine-serine linker followed by a 6-histidine affinity tag at the C-terminus. The purified recombinant protein has rifampicin-resistant activity in a poly(A)-dependent poly(U) polymerase assay while corresponding fractions similarly purified from E. coli transformed with an expression plasmid without the HRV-16 3D sequence showed no activity. The optimal conditions for temperature, pH, divalent cations Mg(2+) and Mn(2+), and KCl were determined. The recombinant protein has RNA polymerase activity on homopolymeric templates poly(A) and poly(C) and heteropolymeric RNA templates primed with either RNA or DNA oligonucleotide primers or self-primed by a copy-back mechanism. A unique, secondary structureless heteropolymeric RNA template that is an efficient substrate was developed to facilitate kinetic characterizations of the enzyme. In the presence of Mg(2+), the enzyme displayed strong base and sugar specificity. However, when Mg(2+) was replaced by Mn(2+) specificity for ribonucleotides was lost, utilization of deoxynucleotides became possible and primer-independent activity was observed on the poly(C) template. Zn(2+) was found to inhibit HRV-16 3D polymerase with an IC(50) as low as 0.6 microM by a mechanism distinct from the magnesium ion stimulation. The activity of this 6His-tagged HRV-16 3D polymerase was compared with that of a recombinant HRV-16 3D polymerase expressed without the 6His-tag and was found to be identical. The availability of recombinant rhinovirus RdRp in a purified form will facilitate the structure-function analysis of this enzyme as well as the identification of specific inhibitors to the rhinovirus 3D polymerase that have therapeutic value in the treatment of the common cold.
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PMID:Biochemical characterization of rhinovirus RNA-dependent RNA polymerase. 1236 17

The phosphoprotein (P protein) of respiratory syncytial virus (RSV) is a key component of the viral RNA-dependent RNA polymerase complex. The protein is constitutively phosphorylated at the two clusters of serine residues (116, 117, and 119 [116/117/119] and 232 and 237 [232/237]). To examine the role of phosphorylation of the RSV P protein in virus replication, these five serine residues were altered to eliminate their phosphorylation potential, and the mutant proteins were analyzed for their functions with a minigenome assay. The reporter gene expression was reduced by 20% when all five phosphorylation sites were eliminated. Mutants with knockout mutations at two phosphorylation sites (S232A/S237A [PP2]) and at five phosphorylation sites (S116L/S117R/S119L/S232A/S237A [PP5]) were introduced into the infectious RSV A2 strain. Immunoprecipitation of (33)P(i)-labeled infected cells showed that P protein phosphorylation was reduced by 80% for rA2-PP2 and 95% for rA2-PP5. The interaction between the nucleocapsid (N) protein and P protein was reduced in rA2-PP2- and rA2-PP5-infected cells by 30 and 60%, respectively. Although the two recombinant viruses replicated well in Vero cells, rA2-PP2 and, to a greater extent, rA2-PP5, replicated poorly in HEp-2 cells. Virus budding from the infected HEp-2 cells was affected by dephosphorylation of P protein, because the majority of rA2-PP5 remained cell associated. In addition, rA2-PP5 was also more attenuated than rA2-PP2 in replication in the respiratory tracts of mice and cotton rats. Thus, our data suggest that although the major phosphorylation sites of RSV P protein are dispensable for virus replication in vitro, phosphorylation of P protein is required for efficient virus replication in vitro and in vivo.
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PMID:The major phosphorylation sites of the respiratory syncytial virus phosphoprotein are dispensable for virus replication in vitro. 1236 20

Microglial cells rapidly become activated in response to even minor damage of neurons, suggestive of the intimate interactions between neurons and microglial cells. Although mediators for microglia-neuron interactions have not been well identified, neurotransmitters are possible candidates transmitting signals from neurons to microglial cells. Among the neurotransmitters, we focused on the effects of norepinephrine and other adrenergic agonists on the functions of rat cultured microglial cells. Reverse transcriptase polymerase chain reaction studies revealed that microglial cells expressed mRNAs encoding alpha1A, alpha2A, beta1 and beta2 receptors. Norepinephrine and a beta2 adrenergic agonist terbutaline elevated intracellular cAMP level of microglial cells. Norepinephrine, an alpha1 agonist phenylephrine, a beta1 agonist dobutamine and terbutaline suppressed the expressions of mRNAs encoding pro-inflammatory cytokines, interleukin-6 and tumor necrosis factor alpha. Release of tumor necrosis factor alpha and nitric oxide was suppressed by norepinephrine, phenylephrine, dobutamine and terbutaline. An alpha2 agonist clonidine and dobutamine upregulated the expression of mRNA encoding catechol-O-methyl transferase, an important enzyme to degrade norepinephrine. Norepinephrine, dobutamine and terbutaline upregulated the expressions of mRNA encoding 3-phospshoglycerate dehydrogenase, an essential enzyme for synthesis of L-serine and glycine, which are amino acids necessary for neuronal survival. Clonidine upregulated the expression of mRNA encoding an anti-apoptotic factor Bcl-xL. These results suggest that norepinephrine participates in the regulation of brain function at least partly by modulating the functions of microglia.
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PMID:Effects of norepinephrine on rat cultured microglial cells that express alpha1, alpha2, beta1 and beta2 adrenergic receptors. 1242 72

The structure and distribution of PC5-A, a prohormone convertase that is thought to be involved in post-translational processing of peptide hormone and neuropeptide precursors, have not been investigated in submammalian vertebrates. In the present study, we characterized the cDNA encoding PC5-A in the European green frog Rana esculenta. The frog PC5-A cDNA encodes a 913-amino acid protein that encompasses a 28-amino acid signal peptide, the Asp/His/Ser catalytic triad found in all serine proteinases of the subtilisin family, and two potential N-linked glycosylation sites located in a C-terminal cysteine-rich domain. Reverse transcriptase polymerase chain reaction amplification showed that PC5-A mRNA is expressed in various organs including the brain, spinal cord, pituitary, lung, liver, intestine, and testis, but not in the stomach and pancreas. The distribution of PC5-A mRNA in the frog brain was studied by in situ hybridization histochemistry. Intense expression was observed in the mitral cellular layer of the olfactory bulb, the nucleus of the diagonal band of Broca, the anterior preoptic area, and the suprachiasmatic and ventral hypothalamic nuclei. The expression pattern of PC5-A mRNA in the central nervous system of anuran amphibians was consistent with the implication of this prohormone convertase in the processing of various neuropeptide precursors.
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PMID:Molecular characterization of the cDNA and localization of the mRNA encoding the prohormone convertase PC5-A in the European green frog. 1250 14

The excitatory amino acid transporters (EAAT) removes neurotransmitters glutamate and aspartate from the synaptic cleft. Most CNS glutamate uptake is mediated by EAAT2 into glia, though nerve terminals show evidence for uptake, through an unknown transporter. Reverse-transcriptase PCR identified the expression of EAAT1, EAAT2, EAAT3 and EAAT4 mRNAs in primary cultures of mouse cortical or striatal neurones. We have used synaptosomes and glial plasmalemmal vesicles (GPV) from adult mouse and rat CNS to identify the nerve terminal transporter. Western blotting showed detectable levels of the transporters EAAT1 (GLAST) and EAAT2 (Glt-1) in both synaptosomes and GPVs. Uptake of [3H]D-aspartate or [3H]L-glutamate into these preparations revealed sodium-dependent uptake in GPV and synaptosomes which was inhibited by a range of EAAT blockers: dihydrokainate, serine-o-sulfate, l-trans-2,4-pyrrolidine dicarboxylate (PDC) (+/-)-threo-3-methylglutamate and (2S,4R )-4-methylglutamate. The IC50 values found for these compounds suggested functional expression of the 'glial, transporter, EAAT2 in nerve terminals. Additionally blockade of the majority EAAT2 uptake sites with 100 micro m dihydrokainate, failed to unmask any functional non-EAAT2 uptake sites. The data presented in this study indicate that EAAT2 is the predominant nerve terminal glutamate transporter in the adult rodent CNS.
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PMID:The 'glial' glutamate transporter, EAAT2 (Glt-1) accounts for high affinity glutamate uptake into adult rodent nerve endings. 1255 72

The recent determination of the crystal structure of VP6, the major capsid protein of rotavirus, revealed a trimer containing a central zinc ion coordinated by histidine 153 from each of the three subunits. The role of the zinc ion in the functions of VP6 was investigated by site-directed mutagenesis. The mutation of histidine 153 into a serine (H153S and H153S/S339H) did not prevent the formation of VP6 trimers. At pH <7.0, about the pK of histidine, wild-type and mutated VP6 proteins display similar properties, giving rise to identical tubular and spherical assemblies. However, at pH >7.0, histidine 153 mutant proteins did not assemble into the characteristic 45-nm-diameter tubes, in contrast to wild-type VP6. These observations showed that under conditions in which histidine residues are not charged, the properties of VP6 depended on the presence of the centrally coordinated zinc atom in the trimer. Indeed, wild-type VP6 depleted of the zinc ion by a high concentration (100 mM) of a metal-chelating agent behaved like the H153 mutant proteins. The susceptibility of wild-type VP6 to proteases is greatly increased in the absence of zinc. NH(2)-terminal sequencing of the proteolytic fragments showed that they all contained the beta-sheet-rich VP6 head domain, which appeared to be less sensitive to protease activity than the alpha-helical basal domain. Finally, the mutant proteins assembled well on cores, as demonstrated by both electron microscopy and rescue of transcriptase activity. Zinc is thus not necessary for the transcription activity. All of these observations suggest that, in solution, VP6 trimers present a structural flexibility that is controlled by the presence of a zinc ion.
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PMID:A zinc ion controls assembly and stability of the major capsid protein of rotavirus. 1261 Jan 35


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