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)

The double-stranded RNA (dsRNA)-activated protein kinase PKR (protein kinase dsRNA-dependent) plays an important role in the regulation of protein synthesis by phosphorylating the alpha-subunit of eukaryotic initiation factor 2. Through this activity, PKR is thought to mediate the antiviral and antiproliferative actions of interferon. Here, we show that the human T cell leukemia Jurkat cells express an alternatively spliced form of PKR with a deletion of exon 7 (PKRDeltaE7), resulting in a truncated protein that retains the two dsRNA-binding motifs. PKRDeltaE7 exhibits a dominant negative function by inhibiting both PKR autophosphorylation and eukaryotic initiation factor 2 alpha-subunit phosphorylation in vitro and in vivo. Reverse transcriptase-polymerase chain reaction assays showed that PKRDeltaE7 is expressed in a broad range of human tissues at variable levels. Interestingly, expression of PKRDeltaE7 is higher in Jurkat cells than in normal peripheral blood mononuclear cells, raising the possibility of a role in cell proliferation and/or transformation. Thus, expression of alternatively spliced forms of PKR may represent a novel mechanism of PKR autoregulation with important implications in the control of cell proliferation.
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PMID:Dominant negative function by an alternatively spliced form of the interferon-inducible protein kinase PKR. 1127 90

Subtraction hybridization after the exposure of keratinocytes to ultraviolet radiation identified a differentially expressed cDNA that encodes a protein of 630 amino acid residues possessing significant similarity to the catalytic domain of the sucrose-non-fermenting protein kinase (SNF1)/AMP-activated protein kinase (AMPK) family of serine/threonine protein kinases. Northern blotting and reverse-transcriptase-mediated PCR demonstrated that mRNA transcripts for the SNF1/AMPK-related kinase (SNARK) were widely expressed in rodent tissues. The SNARK gene was localized to human chromosome 1q32 by fluorescent in situ hybridization. SNARK was translated in vitro to yield a single protein band of approx. 76 kDa; Western analysis of transfected baby hamster kidney (BHK) cells detected two SNARK-immunoreactive bands of approx. 76-80 kDa. SNARK was capable of autophosphorylation in vitro; immunoprecipitated SNARK exhibited phosphotransferase activity with the synthetic peptide substrate HMRSAMSGLHLVKRR (SAMS) as a kinase substrate. SNARK activity was significantly increased by AMP and 5-amino-4-imidazolecarboxamide riboside (AICAriboside) in rat keratinocyte cells, implying that SNARK might be activated by an AMPK kinase-dependent pathway. Furthermore, glucose deprivation increased SNARK activity 3-fold in BHK fibroblasts. These findings identify SNARK as a glucose- and AICAriboside-regulated member of the AMPK-related gene family that represents a new candidate mediator of the cellular response to metabolic stress.
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PMID:Identification and characterization of a novel sucrose-non-fermenting protein kinase/AMP-activated protein kinase-related protein kinase, SNARK. 1128 15

Double-stranded RNAs (dsRNAs) are widespread in plant pathogenic fungi, but their functions in fungal hosts remain mostly unclear, with a few exceptions. We analyzed dsRNAs from Nectria radicicola, the causal fungus of ginseng root rot. Four distinct sizes of dsRNAs, 6.0, 5.0, 2.5, and 1.5 kbp, were detected in 24 out of the 81 strains tested. Curing tests of individual dsRNAs suggested that the presence of 6.0-kbp dsRNA was associated with high levels of virulence, sporulation, laccase activity, and pigmentation in this fungus. The 6.0-kbp dsRNA-cured strains completely lost virulence-related phenotypes. This 6.0-kbp dsRNA was reintroduced by hyphal anastomosis to a dsRNA-cured strain marked with hygromycin resistance, which resulted in the restoration of virulence-related phenotypes. These results strongly suggest that 6.0-kbp dsRNA up regulates fungal virulence in N. radicicola. Sequencing of several cDNA clones derived from 6.0-kbp dsRNA revealed the presence of a RNA-dependent RNA polymerase (RDRP) gene. Phylogenetic analysis showed that this gene is closely related to those of plant cryptic viruses. Biochemical analyses suggested that the 6.0-kbp dsRNA may regulate fungal virulence through signal-transduction pathways involving cyclic AMP-dependent protein kinase and protein kinase C.
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PMID:A viral double-stranded RNA up regulates the fungal virulence of Nectria radicicola. 1131 Jul 37

In the serum-free culture medium of bovine odontoblasts we detected active gelatinolytic metalloproteinases, matrix metalloproteinase (MMP)-2 and MMP-9 (gelatinases A and B). The activity of MMP-2, in particular, appeared suddenly around day 21 in the culture, coinciding with the development of odontoblastic cell processes and the loss of alkaline phosphatase. Reverse transcriptase-polymerase chain reaction analysis of these odontoblasts demonstrated that messages of MMP-2 but not MMP-9 increased significantly between day 15 and day 21. The in vitro observation indicates that medium conditioned by these odontoblasts and containing significant amounts of MMP-2 degrades not only the collagenous substrates but also purified dentin phosphophoryn as well. We have also observed that dephosphorylated dentin phosphoprotein becomes a better substrate for casein kinase II after limited proteolysis with MMP-2. These results support our working hypothesis that MMP-2-mediated proteolytic processing is an important step in accelerating the process of dentin matrix maturation, which includes phosphorylation and subsequent mineralization. As has been suggested previously, extracellular phosphorylation of matrix proteins is an important step in biomineralization both in bone and in dentin (Mikuni-Takagaki et al., J Bone Miner Res 1995;10:231-42; Zhu et al., Biochem J 1997; 323:637-43). Our present histochemical analysis in MMP-2 knockout mice confirms the concept with the delayed formation of mineralized tissues, dentin, and bone.
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PMID:Matrix metalloproteinase-2 in dentin matrix mineralization. 1150 97

Phosphodiesterase 4D (PDE4D), part of the complex cAMP-specific PDE4 family, plays a pivotal role in the regulation of airway smooth muscle relaxation by catalyzing the hydolysis of cAMP. Its gene on chromosome 5q12 encodes 5 splice variants, which show tissue-dependent expression and regulation. The genomic arrangement of PDE4D was determined using in silico methods, and a putative promoter of one of the protein kinase A-activated, long isoforms, PDE4D5 was identified. Promoter-luciferase constructs, transiently transfected into a beta(2) adrenoreceptor-expressing CHO-K1 cell line, were used to demonstrate that the PDE4D5 promoter up-regulated reporter gene expression in response to increased cell cAMP. Site-directed mutagenesis of the cAMP-response element (CRE) at position -201 identified this as the principal component of the mechanism underlying this cAMP responsiveness. In the second part of this study, cAMP-dependent induction of PDE4D5 transcript in primary cultured human airway smooth muscle cells (hASMs) was demonstrated using both qualitative reverse-transcriptase PCR and quantitative real-time PCR. Isolated PDE4D5 isoenzyme activity, measured after selective immunoprecipitation from hASMs, confirmed that this increase in expression led to an up-regulation of functional activity. We present evidence for cAMP-driven PDE4D5 up-regulation in hASMs and suggest a CRE-containing, isoform-specific promoter as the primary mechanism.
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PMID:Cyclic AMP-dependent transcriptional up-regulation of phosphodiesterase 4D5 in human airway smooth muscle cells. Identification and characterization of a novel PDE4D5 promoter. 1212 97

We previously isolated, from the earliest population of CD34+ hematopoietic progenitors that form in the aorta of the human embryo, a partial DNA complementary to RNA (cDNA) sequence that was later identified as the human homologue of rat sucrose non-fermenting protein (SNF-1) related kinase (rSNRK), a novel SNF-1-related kinase previously characterized in the rat. In the present study we report the cloning of the complete human SNF-1 related kinase (hSNRK) cDNA and show that the gene spans 39.8 kb at region 3p21 and contains six exons. Recombinant expression of the hSNRK coding sequence in Escherichia coli led to the production of a functional protein kinase of 85 kDa. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of hSNRK expression in fetal CD34+ hematopoietic progenitors revealed its continuous expression throughout human development with higher levels in highly dividing CD34+ CD38+ cells compared to quiescent CD34+ CD38- cells. This observation, together with the expression of hSNRK in numerous human leukemic cell lines, may reflect an implication of hSNRK protein in hematopoietic cell proliferation or differentiation. In the mouse, the SNRK cDNA is 4.6-kb-long and encodes a protein of 748 amino acids with a predicted molecular mass of 81,930 Da. The proteins from human, rat and mouse are strongly conserved and are characterized by the presence of a serine/threonine kinase catalytic domain, a bipartite nuclear targeting signal and an ubiquitin-associated domain. In situ hybridization and RT-PCR analysis of the pattern of mSNRK expression in the mouse reveals that it is temporally and spatially regulated during embryogenesis, and widespread expressed in adult tissues.
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PMID:Cloning and characterization of human and mouse SNRK sucrose non-fermenting protein (SNF-1)-related kinases. 1223 63

Previous in vivo studies in cardiomyopathic hamsters suggested that the expression of vasopressin (AVP) V2 mRNA is up- regulated by angiotensin II. The present study was performed to determine whether angiotensin II plays a role in regulating the expression of AVP V2 mRNA and aquaporin-2 (AQP2) mRNA in the inner medullary collecting duct (IMCD) of the male Wistar rat. The expression of AVP V2 mRNA and AQP2 mRNA in the IMCD was measured by competitive reverse-transcriptase polymerase chain reaction (RT-PCR). Six groups of experiments were performed. In the first group, we incubated IMCD with 3 different doses of angiotensin II (10(-11), 10(-9) and 10(-7) mol/L). Angiotensin II caused a significant increase in the AVP V2 mRNA in a dose-dependent manner but its effect on AQP2 mRNA was modest. This effect of angiotensin II was inhibited by angiotensin II receptor antagonist, [Sar1,Ile8]-angiotensin II. To examine the role of PKA in mediating an increase in AVP V2 mRNA expression, we incubated IMCD with 10(-7) and 10(-11) M of angiotensin II in the presence of a specific protein kinase A (PKA) inhibitor, Rp diasteroisomer of adenosine 3'-5'-cylic monophosphothionate (Rp-cAMPS). The angiotensin II-induced upregulation of V2 mRNA was abolished. In the fourth group, we examined the effect of protein kinase C (PKC) inhibition on V2 mRNA expression. The upregulation of V2 mRNA induced by angiotensin II was greatly exaggerated when IMCD was incubated with angiotensin II and RO-31-8220 (PKC inhibitor). In the fifth and sixth groups of studies, we determined the direct effect of PKA and PKC on regulating the expression of V2 mRNA and AQP2 mRNA in the IMCD, respectively. Dibutryl cAMP stimulated an upregulation in the expression of V2 mRNA and AQP2 mRNA, whereas phorbol esters suppressed the expression of V2 mRNA. These results suggested that PKA stimulates and PKC suppresses the expression of V2 mRNA in the IMCD of the kidney.
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PMID:Angiotensin II upregulates the expression of vasopressin V2 mRNA in the inner medullary collecting duct of the rat. 1264 65

The EVI1 proto-oncogene encodes a nuclear zinc finger protein that acts as a transcription repressor factor. In myeloid leukemia it is often activated by chromosomal rearrangements involving band 3q26, where the gene has been mapped. Here we report two leukemia cases [a chronic myeloid leukemia blast crisis (CML-BC) and an acute myeloid leukemia (AML) M4] showing a t(3;7)(q26;q21) translocation in a balanced and unbalanced form, respectively. Fluorescent in situ hybridization (FISH) analysis revealed that both patients showed a breakpoint on chromosome 3 inside the clone RP11-33A1 containing the EVI1 oncogene and, on chromosome 7, inside the clone RP11-322M5, partially containing the CDK6 oncogene which is a D cyclin-dependent kinase gene, observed to be overexpressed and disrupted in many hematological malignancies. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed overexpression of EVI1 in both cases, but excluded the presence of any CDK6/ EVI1 fusion transcript. CDK6 expression was also detected. Together, these data indicate that EVI1 activation is likely due not to the generation of a novel fusion gene with CDK6 but to a position effect dysregulating its transcriptional pattern.
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PMID:A novel chromosomal translocation t(3;7)(q26;q21) in myeloid leukemia resulting in overexpression of EVI1. 1455 38

Nitric oxide (NO)-mediated relaxation of colonic smooth muscle is crucial for the maintenance of human gut function. The molecular mechanisms of NO-dependent smooth muscle relaxation involve cyclic GMP-mediated inhibition of store-dependent calcium signaling. Recently, IRAG (inositol 1,4,5-trisphophate receptor-associated cGMP kinase substrate) has been characterized as a novel target molecule of cGMP-dependent protein kinase (cGKI) mediating NO-/cGMP-dependent inhibition of inositol 1,4,5-trisphosphate (InsP(3))-dependent calcium release in transfected COS cells. The aim of the present study was to characterize IRAG expression and its functional role in NO-dependent signaling in human colonic smooth muscle. Reverse transcriptase-PCR revealed IRAG mRNA expression in human colon, rectum, and cultured colonic smooth muscle cells. In cultured human colonic smooth muscle cells, bradykinin (BK) elicited InsP(3)-dependent calcium transients that were repeatable and independent of extracellular calcium. The NO donor sodium nitroprusside and the specific cGK activator 8-(4-chlorophenylthio)guanosine-3',5'-cyclic-monophosphate (8-pCPT-cGMP) significantly inhibited BK-induced increase in intracellular calcium. Cells transfected with antisense oligonucleotides raised against IRAG (IRAG-AS) showed strongly decreased IRAG protein expression. In these cells, sodium nitroprusside and 8-pCPT-cGMP both failed to modulate BK-induced calcium transients. Thus, endogenous IRAG appears to be essentially involved in the NO/cGK-dependent inhibition of InsP(3)-dependent Ca(2+)-signaling in colonic smooth muscle.
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PMID:InsP3R-associated cGMP kinase substrate (IRAG) is essential for nitric oxide-induced inhibition of calcium signaling in human colonic smooth muscle. 1472 8

Post-transcriptional gene silencing is a powerful tool to reveal gene function. In this chapter, we have used gene-array technology to analyze changes in gene expression after specific targeting of the protein kinase Calpha isoform with small interfering RNAs (siRNAs). Expression profiles were investigated on a human gene array of 588 key genes involved in DNA synthesis, apoptosis, cell-cell communication, intracellular signal-transduction pathways, cell-surface molecules and transcription factors. The data indicate that siRNAs are an ideal tool for target validation, and show that RNA-dependent RNA polymerase is not required for RNAi in mammalian cells.
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PMID:Gene-array analysis of glioma cells after treatment with an anti-PKCalpha siRNA: a general protocol. 1501 74


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