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)

The basic helix-loop-helix (bHLH) transcription factors NEUROD1, NEUROD2 and ATH2 are expressed during first trimester human placental development. We determined the transactivation potential of each of these factors in trophoblasts by measuring changes in the endogenous gene activity using absolute quantification by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) after transient transfection. In these assays, NEUROD1 was found to transiently transactivate NEUROD2 in trophoblast cells. Promotor truncation assays, using luciferase constructs, showed the presence of two domains in the NEUROD2 promotor, which showed increased activity after NeuroD1 transfection. Each of these NeuroD1-responsive domains contains an E-box sequence. The NEUROD2 transactivation data fit with the spatial expression pattern of NEUROD1 and NEUROD2, since they are expressed in endovascular trophoblasts. This expression pattern, as well as the present transactivation results, might suggest the presence of a NEUROD differentiation cascade during first trimester human placental development.
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PMID:NEUROD1 acts in vitro as an upstream regulator of NEUROD2 in trophoblast cells. 1473 94

Oxidative stress is one of the characteristics of diabetes and is thought to be responsible for many of the pathophysiological changes caused by the disease. We previously identified an insulin response element in the promoter of plasminogen activator inhibitor 1 (PAI-1) that was activated by an unidentified member of the forkhead/winged helix (Fox) family of transcription factors. This element mediated a 5-7-fold increase in PAI-1 transcription because of insulin. Here we report that oxidative stress also caused a 3-fold increase in PAI-1 transcription and that the effect was additive with that of insulin. Antioxidants prevent this response. Mutational analysis of the PAI-1 promoter revealed that oxidative stress acted at an AP-1 site at -60/52 of the promoter. Gel mobility shift analysis demonstrated that binding to an AP-1 oligonucleotide was increased 4-fold by oxidative stress. Jun levels were increased by oxidants as assessed by reverse transcriptase-PCR. Western blotting demonstrated that a rapid and prolonged nuclear accumulation of phospho-c-Jun followed oxidant stimulation. The nuclear c-Jun phosphorylation was not observed in cells treated with reduced glutathione. Finally, JNK/SAPK activity was found to increase in response to oxidants, and inhibition of JNK/SAP blocked TBHQ-increased PAI-1-luciferase expression. Thus, oxidative stress stimulated AP-1 and activated the PAI-1 promoter.
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PMID:Oxidative stress activates the plasminogen activator inhibitor type 1 (PAI-1) promoter through an AP-1 response element and cooperates with insulin for additive effects on PAI-1 transcription. 1506 77

There is increasing evidence implicating Transforming growth factor beta (TGF-beta) in pathological states of the lens. However, the underlying signalling mechanisms in human cells have not been fully examined. We have therefore investigated in a human lens cell line, FHL 124, the signalling characteristics of TGF-beta and Smad proteins. Moreover, we have tested the effectiveness of a fully human monoclonal anti-TGF-beta2 antibody, CAT-152, in suppressing TGF-beta2 induced changes in a number of conditions. FHL 124 cells were routinely cultured in Eagle's minimum essential medium (EMEM) supplemented with 10% FCS. Characterisation of the cell line was determined using Affymetrix gene microarrays and compared to native human lens epithelium. Cells were serum starved for 24 hr prior to exposure to TGF-beta2 in the presence and absence of CAT-152. Non-stimulated cells served as controls. Smad 4 localisation was observed by immunocytochemistry. To study Smad-dependent transcriptional activity, cells were transfected with SBE4-luc, an artificial smad-specific reporter, using Fugene-6. Transcriptional activity was determined by luciferase activity. Gene expression was assessed using reverse transcriptase-polymerase chain reaction (RT-PCR). Proliferation was determined by 3H-thymidine DNA incorporation. Growth and contraction were assessed using a scratch and patch assay. Affymettrix gene microarrays identified 99.5% homology between FHL 124 cells and the native lens epithelium with respect to expression pattern of the 22,270 genes on the chip. Moreover, FHL 124 cells expressed phenotypic markers, alphaA-crystallin and pax6 along with lens epithelial cell specific marker FoxE3. Immunocytochemical studies revealed the presence of Smad 4 which following TGF-beta2 exposure accumulated in the cell nucleus. Furthermore, Smad-dependent transcriptional activity was also stimulated. TGF-beta2 enhanced the expression of mRNA levels of alpha smooth muscle actin (alphaSMA) and connective tissue growth factor (CTGF). Exposure to TGF-beta2 resulted in a relatively small inhibition of 3H-thymidine incorporation of FHL 124 cells. However, a more marked contractile effect was also observed. In serum-supplemented medium, growth rates and TGF-beta induced contraction were enhanced. Treatment with 0.1-10 microg ml(-1) CAT-152 dose-dependently inhibited 10 ng ml(-1) TGF-beta2 induced effects in the presence and absence of serum. Exposure of FHL 124 cells to TGF-beta therefore induces Smad translocation, transcription, expression of transdifferentiation markers and induces marked contraction. Treatment with CAT-152 can effectively inhibit these responses. TGF-beta2 induced changes can also persist long after the period of exposure and when in the presence of serum TGF-beta induced contraction is enhanced. The work presented therefore demonstrates a platform technology to study TGF-beta2 signalling in human lens epithelial cells and provides evidence to show TGF-beta2 can be a potent factor in the development of posterior capsule opacification following cataract surgery.
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PMID:Characterisation of TGF-beta2 signalling and function in a human lens cell line. 1510 50

A cDNA encoding a Rel/NF-kappaB homologue was cloned from a beetle, Allomyrina dichotoma, by reverse transcriptase-polymerase chain reactions (RT-PCR) taking advantage of the conserved Rel homology domain (RHD) to synthesize primers. The Rel/NF-kappaB homologue was designated A. dichotoma (A.d.) Rel A. The amino acid sequence of the A.d. Rel A RHD was compared with those of insect RHDs. The result showed that it has 70% identity with Tribolium castaneum Dorsal, 66% with Drosophila melanogaster Dorsal, 61% with Anopheles gambiae Gambif1, and 55% with D. melanogaster Dif. A putative phosphorylation site in the RHD, RRPS, and two putative nuclear localization signals were conserved in A.d. Rel A. A recombinant fusion protein containing the A.d. Rel A RHD was confirmed to bind specifically to the NF-kappaB site of a gene encoding A.d. coleoptericin A, an antibacterial peptide from A. dichotoma. The activity of A.d. Rel A in modulating a gene construct of the A.d. coleoptericin A promoter-luciferase reporter by expressing the A.d. coleoptericin A cDNA in a Bombyx mori cell line was analyzed. The result showed that A.d. Rel A strongly activates the A.d. coleoptericin A gene construct, whereas A.d. Rel A failed to activate the gene construct containing the mutated NF-kappaB site, suggesting the importance of the interaction between the NF-kappaB site and A.d. Rel A in the signal transduction for gene expression of antibacterial peptides in A. dichotoma.
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PMID:Characterization of a homologue of the Rel/NF-kappaB transcription factor from a beetle, Allomyrina dichotoma. 1515 34

Infectious haematopoietic necrosis virus (IHNV) is a well-studied virus of salmonid fishes. A highly efficacious DNA vaccine has been developed against this virus and studies have demonstrated that this vaccine induces both an early and transient non-specific anti-viral phase as well as long-term specific protection. The mechanisms of the early anti-viral phase are not known, but previous studies noted changes in Mx gene expression, suggesting a role for type I interferon. This study used quantitative real-time reverse transcriptase PCR methodology to compare expression changes over time of a number of cytokine or cytokine-related genes in the spleen of rainbow trout following injection with poly I:C, live IHNV, the IHNV DNA vaccine or a control plasmid encoding the non-antigenic luciferase gene. The target genes included Mx-1, viral haemorrhagic septicaemia virus induced gene 8 (Vig-8), TNF-alpha1, TNF-alpha2, IL-1beta1, IL-8, TGF-beta1 and Hsp70. Poly I:C stimulation induced several genes but the strongest and significant response was observed in the Mx-1 and Vig-8 genes. The live IHN virus induced a significant response in all genes examined except TGF-beta1. The control plasmid construct and the IHNV DNA vaccine marginally induced a number of genes, but the main difference between these two groups was a statistically significant induction of the Mx-1 and Vig-8 genes by the IHNV vaccine only. The gene expression profiles elicited by the live virus and the IHNV DNA vaccine differed in a number of aspects but this study confirms the clear role for a type I interferon-like response in early anti-viral defence.
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PMID:Quantitative expression profiling of immune response genes in rainbow trout following infectious haematopoietic necrosis virus (IHNV) infection or DNA vaccination. 1531 11

Regulation of intracellular Ca(2+) mobilization has been associated with the functions of polycystin-1 (PC1) and polycystin-2 (PC2), the protein products of the PKD1 and PKD2 genes. We have now demonstrated that PC1 can activate the calcineurin/NFAT (nuclear factor of activated T-cells) signaling pathway through Galpha(q) -mediated activation of phospholipase C (PLC). Transient transfection of HEK293T cells with an NFAT promoter-luciferase reporter demonstrated that membrane-targeted PC1 constructs containing the membrane proximal region of the C-terminal tail, which includes the heterotrimeric G protein binding and activation domain, can stimulate NFAT luciferase activity. Inhibition of glycogen synthase kinase-3beta by LiCl treatment further increased PC1-mediated NFAT activity. PC1-mediated activation of NFAT was completely inhibited by the calcineurin inhibitor, cyclosporin A. Cotransfection of a construct expressing the Galpha(q) subunit augmented PC1-mediated NFAT activity, whereas the inhibitors of PLC (U73122) and the inositol trisphosphate and ryanodine receptors (xestospongin and 2-aminophenylborate) and a nonspecific Ca(2+) channel blocker (gadolinium) diminished PC1-mediated NFAT activity. PC2 was not able to activate NFAT. An NFAT-green fluorescent protein nuclear localization assay demonstrated that PC1 constructs containing the C-tail only or the entire 11-transmembrane spanning region plus C-tail induced NFAT-green fluorescent protein nuclear translocation. NFAT expression was demonstrated in the M-1 mouse cortical collecting duct cell line and in embryonic and adult mouse kidneys by reverse transcriptase-PCR and immunolocalization. These data suggest a model in which PC1 signaling leads to a sustained elevation of intracellular Ca(2+) mediated by PC1 activation of Galpha(q) followed by PLC activation, release of Ca(2+) from intracellular stores, and activation of store-operated Ca(2+) entry, thus activating calcineurin and NFAT.
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PMID:Polycystin-1 activates the calcineurin/NFAT (nuclear factor of activated T-cells) signaling pathway. 1546 61

In humans, a polymorphic gene encodes the drug-metabolizing enzyme NAT1 (arylamine N-acetyltransferase Type 1), which is widely expressed throughout the body. While the protein-coding region of NAT1 is contained within a single exon, examination of the human EST (expressed sequence tag) database at the NCBI revealed the presence of nine separate exons, eight of which were located in the 5' non-coding region of NAT1. Differential splicing produced at least eight unique mRNA isoforms that could be grouped according to the location of the first exon, which suggested that NAT1 expression occurs from three alternative promoters. Using RT (reverse transcriptase)-PCR, we identified one major transcript in various epithelial cells derived from different tissues. In contrast, multiple transcripts were observed in blood-derived cell lines (CEM, THP-1 and Jurkat), with a novel variant, not identified in the EST database, found in CEM cells only. The major splice variant increased gene expression 9-11-fold in a luciferase reporter assay, while the other isoforms were similar or slightly greater than the control. We examined the upstream region of the most active splice variant in a promoter-reporter assay, and isolated a 257 bp sequence that produced maximal promoter activity. This sequence lacked a TATA box, but contained a consensus Sp1 site and a CAAT box, as well as several other putative transcription-factor-binding sites. Cell-specific expression of the different NAT1 transcripts may contribute to the variation in NAT1 activity in vivo.
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PMID:Genomic organization of human arylamine N-acetyltransferase Type I reveals alternative promoters that generate different 5'-UTR splice variants with altered translational activities. 1548 85

Matrix metalloproteinase-2 (MMP-2) plays important roles in cancer development and aggression. Our previous studies revealed a strong association between the MMP-2 -1306C/T polymorphism and risk of several cancers. A novel -735C/T polymorphism in MMP-2 promoter has been identified but the function is undefined. This study examined our hypothesis that these two polymorphisms might have functional relevance and impact on risk of esophageal squamous cell carcinoma in the context of haplotype. Genotypes and haplotypes were analyzed in 527 cases and 777 controls and odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by logistic regression. The function of the polymorphisms was examined by electrophoretic mobility shift assays, luciferase gene expression assays, and reverse transcriptase-PCR analyses. It was found that the -735C-->T transition disrupts an Sp1 site and displays a lower promoter activity. The C(-1306)-C(-735) haplotype had 7-fold increased luciferase expression and 3.7-fold increased MMP-2 mRNA levels in esophageal tissues compared with the T(-1306)-T(-735) haplotype. A case-control analysis revealed a 1.52-fold (95% CI = 1.17-1.96) or 1.30-fold (95% CI = 1.04-1.63) excess risk of developing esophageal squamous cell carcinoma for the -1306CC or -735CC genotype carriers compared with noncarriers, respectively. A greater association was observed between elevated risk of developing esophageal squamous cell carcinoma and C(-1306) or C(-735) allele containing haplotypes, with the risk being highest for the C(-1306)-C(-735) haplotype compared with the T(-1306)-T(-735) haplotype (OR = 6.53; 95% CI = 2.78-15.33). The C(-1306)-C(-735) haplotype was also associated with increased risk for distant metastasis of esophageal squamous cell carcinoma (OR = 3.34; 95% CI = 1.16-9.63). These findings suggest that the C(-1306)-C(-735) haplotype in the MMP-2 promoter contributes to risk of the occurrence and metastasis of esophageal squamous cell carcinoma by increasing expression of MMP-2.
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PMID:Functional haplotypes in the promoter of matrix metalloproteinase-2 predict risk of the occurrence and metastasis of esophageal cancer. 1549 91

We report on the establishment of one transgenic and two endogenous reporter gene assays to determine androgenic/antiandrogenic activity. A transient transactivation assay was developed in COS-7 African green monkey kidney cells. Three plasmids were co-transfected by electroporation: the human androgen receptor expression vector pSG5AR, the reporter gene vector pMamneoLuc, expressing luciferase under the control of the mouse mammary tumor virus (MMTV) promotor containing 4 hormone responsive elements (HREs), and the control plasmid pSVbeta. Transcriptional activation was measured by luciferase-mediated chemoluminescence. In T47D human breast cancer cells two endogenous reporter gene systems were established: one based on the androgen-induced expression of prostate-specific antigen (PSA), the other based on the androgen-repressed expression of testosterone repressed message 2 (TRPM-2). PSA protein was measured by enzyme-linked immunosorbent assay (ELISA), TRPM-2 m-RNA by reverse transcriptase polymerase chain reaction (RT-PCR). All three test systems were validated using the physiological androgen dihydrotestosterone (DHT) as agonist and the established antiandrogens hydroxyflutamide and p,p'-dichlorodiphenylethene (p,p'-DDE) as antagonists. The PSA assay was the most sensitive test system with an EC50 of 0.7 nM for DHT-induced response. The transient transactivation assay in COS-7 cells was less sensitive with an EC50 of 9.7 nM DHT. In the PSA assay hydroxyflutamide was a more potent antagonist (IC30 = 0.02 microM) than p,p'-DDE (IC30 = 0.9 microM). In the transient transactivation assay in COS-7 cells, both compounds elicited about 30% of the agonistic response induced by 100 nM DHT, thus showing partial agonistic properties. In summary, three highly sensitive and complementary in vitro test systems, together achieving enhanced specificity for detection and assessment of androgenic/antiandrogenic activity have been established and validated.
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PMID:Sensitive in vitro test systems to determine androgenic/antiandrogenic activity. 1549 79

The serotonin 5-HT4 receptor (where 5-HT stands for 5-hydroxy-tryptamine) is a member of the seven transmembrane-spanning G-protein-coupled family of receptors and mediates many cellular functions both in the central nervous system and at the periphery. In the present study, we isolated and characterized the 5'-flanking region of the h5-HT4 (human 5-HT4) receptor. We demonstrate the existence of a novel exon that corresponds to the 5'-untranslated region of the h5-HT4 receptor gene. RNase protection analysis and reverse transcriptase-PCR experiments performed on human atrial RNA demonstrated that the major transcription start site of the h5-HT4 receptor gene is located at -3185 bp relative to the first ATG codon. In addition, a 1.2 kb promoter fragment which drives the transcription of the 5-HT4 receptor was characterized. The promoter region lacks TATA and CAAT canonical motifs in the appropriate location, but contains putative binding sites for several transcription factors. Transient transfection assays revealed that the (-3299/-3050) gene fragment possesses the ability to promote the expression of the luciferase reporter gene in human cell lines. In contrast, the promoter was silent in monkey COS-7 cells, indicating the requirement of specific factors to initiate transcription in human cells. In addition to the promoter element, enhancer activity was found in a region (-220/-61) located in the long 5'-untranslated region. Mutational analysis, gel shift and transfection assays identified an Nkx2.5 (NK2-transcription-factor-related 5)-like binding site as a regulatory sequence of this enhancer. Our results suggest a complex regulation of the h5-HT4 receptor gene expression involving distinct promoters and non-coding exons.
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PMID:Functional studies of the 5'-untranslated region of human 5-HT4 receptor mRNA. 1557 21


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