Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.1.21 (thymidine kinase)
 7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recombinant plasmid-based retroviral expression vectors were constructed using a modified spleen necrosis virus (SNV) containing the Herpes simplex virus thymidine kinase gene promoter controlling the expression of the Tn5 neomycin phosphotransferase II gene (NPTII gene). The human renin (HRn) gene (hrn) was inserted into the 5' end of the SNV sequences such that in concatemeric plasmid DNA its expression was controlled by the strong promoter in the SNV long terminal repeat (LTR). Dog cells transfected with the concatemeric plasmid DNA secreted a small amount of a HRn-like 43-kDa protein. After cotransfection of chicken cells with concatemeric plasmid DNA and proviral DNA of reticuloendotheliosis virus strain A, infectious stocks of viruses were recovered. Cells infected with the virus carrying the viral LTR-hrn gene oriented for expression secreted the 43-kDa HRn-like protein at about 100-fold higher levels than the cells transfected with the plasmid DNAs. Biological activity of secreted HRn was determined by measuring levels of angiotensin I generated by incubating culture media with either a porcine or human angiotensinogen substrate. Infected dog cells produce about 40 ng of enzymatically active HRn per 10(6) cells per 24 h. These data indicate that retroviral expression vectors provide a good system for obtaining the secretion of high levels of enzymatically active heterologous proteins from mammalian cells.
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PMID:Secretion of enzymatically active human renin from mammalian cells using an avian retroviral vector. 302 1

We transiently transfected fusion genes with the 5'-flanking region of the angiotensinogen gene linked to a bacterial chloramphenicol acetyltransferase (CAT) coding sequence as a reporter into opossum kidney (OK) cells. The addition of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) (10(-3)-10(-7) M) or forskolin (10(-9)-10(-5) M) stimulated the expression of the plasmid pOCAT [angiotensinogen nucleotide (N) -1498/+18] fusion gene in OK cells in a dose-dependent manner. The addition of dexamethasone (Dex) (10(-6) M) further enhanced the stimulatory effect of 8-BrcAMP or forskolin, whereas the addition of (R)-p-adenosine 3',5'-cyclic monophosphorothioate [(Rp)-cAMP[S], an inhibitor of cAMP-dependent protein kinase A, I and II] blocked the stimulatory effect of 8-BrcAMP. Furthermore, the addition of 8-BrcAMP (10(-3) M) or Dex (10(-6) M) or a combination of both stimulated the expression of pOCAT (angiotensinogen N -1138/+18), pOCAT (angiotensinogen N -960/+18), pOCAT (angiotensinogen N -814/+18), and pOCAT (angiotensinogen N -688/+18), but had no effect on the expression of pOCAT (angiotensinogen N -280/+18), pOCAT (angiotensinogen N -198/+18), pOCAT (angiotensinogen N -110/+18), pOCAT (angiotensinogen N -53/+18), and pOCAT (angiotensinogen N -35/+18). To further localize the putative cAMP-responsive element (CRE) in the angiotensinogen gene, we constructed fusion genes by inserting the DNA fragments angiotensinogen N -814 to N -689, angiotensinogen N -814 to N -761, and angiotensinogen N -760 to N -689 of the 5'-flanking region of the angiotensinogen gene upstream of the thymidine kinase (TK) promoter fused to a CAT gene and introduced them into OK cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Expression of the angiotensinogen gene is synergistically stimulated by 8-BrcAMP and Dex in opossum kidney cells. 784 Mar 9

Angiotensinogen is shown to be produced by the liver and the hepatoma cell line HepG2. As a first step for understanding the molecular relationship between the transcriptional regulation of the angiotensinogen gene and the pathogenesis of hypertension, we have analyzed the basal promoter of the angiotensinogen gene. Chloramphenicol acetyltransferase (CAT) assays with 5'-deleted constructs showed that the proximal promoter region from -96 to +22 of the transcriptional start site was enough to express HepG2-specific CAT activity. Electrophoretic mobility shift assay and DNase I footprinting demonstrated that the liver- and HepG2-specific nuclear factor (angiotensinogen gene-activating factor [AGF2]) and ubiquitous nuclear factor (AGF3) bound to the proximal promoter element from -96 to -52 (angiotensinogen gene-activating element [AGE2]) and to the core promoter element from -6 to +22 (AGE3), respectively. The site-directed disruption of either AGE2 or AGE3 decreased CAT expression, and the sequential titration of AGF3 binding by in vivo competition remarkably suppressed HepG2-specific CAT activity. Finally, the heterologous thymidine kinase promoter assay showed that AGE2 and AGE3 synergistically conferred HepG2-specific CAT expression. These results suggest that the synergistic interplay between AGF2 and AGF3 is important for the angiotensinogen promoter activation.
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PMID:Molecular mechanism of transcriptional activation of angiotensinogen gene by proximal promoter. 816 41

We transiently co-transfected opossom kidney (OK) cells with the plasmid containing the cDNA for beta 1-adrenoceptor (pBC-beta 1 AR) or beta 2-adrenoceptor (pBC-beta 2 AR) and a fusion gene with the 5'-flanking region of the angiotensinogen (ANG) gene linked to a bacterial chloramphenicol acetyl transferase (CAT) coding sequence as a reporter, pOCAT (ANG N-1498/ +18). Co-transfection of plasmid pBC-beta 1 AR or pBC-beta 2 AR alone enhanced the expression of pOCAT (ANG N-1498/+18). The addition of isoproterenol further stimulated the expression of pOCAT (ANG N-1498/ +18) when co-transfected with pBC-beta 1AR, but not with pBC-beta 2AR. Moreover, the addition of a combination of dexamethasone and isoproterenol synergistically stimulated the expression of pOCAT (ANG N-1498/+18) when co-transfected with pBC-beta 1AR, but not when cotransfected with pBC-beta 2AR. The synergistic effect of dexamethasone and isoproterenol was inhibited by the presence of RU 486 (an antagonist of glucocorticoid) or Rp-cAMP (an inhibitor of cAMP-dependent protein kinase A I and II). To localize the putative cAMP-responsive element (CRE) and glucocorticoid responsive element (GRE) in the ANG gene, we constructed the fusion gene by inserting the DNA fragment, ANG N-806 to N-465 upstream of the thymidine kinase (TK) promoter fused to a CAT gene and introduced them with pBC-beta 1AR into OK cells. The addition of dexamethasone or isoproterenol alone stimulated the expression of pTKCAT (ANG N-806/-465). The addition of isoproterenol and dexamethasone synergistically stimulated the transcriptional activity of pTKCAT (N-806/-465). These studies demonstrate that the beta 1-adrenoceptor and dexamethasone act synergistically to stimulate the expression of the ANG gene in OK cells via the putative CRE and GREs in the 5'-flanking region of the rat ANG gene. These data should aid in the understanding of the molecular mechanism(s) of the stimulatory effect of catecholamines/glucocorticoid induced expression of the ANG gene in the kidney.
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PMID:beta-Adrenoceptors and dexamethasone synergistically stimulate the expression of the angiotensinogen gene in opossum kidney cells. 880 77

It has been reported previously that the addition of isoproterenol or forskolin stimulates the expression of the angiotensinogen (ANG) gene in opossum kidney (OK) 27 cells, an OK cell line with a fusion gene containing the 5'-flanking regulatory sequence of the rat ANG gene fused with a human growth hormone (hGH) gene as a reporter, pOGH (ANG N-1498/+18), permanently integrated into their genomes. To investigate whether the effect of isoproterenol or forskolin on the expression of the ANG gene is mediated via the nuclear 43-kD cAMP-responsive element binding protein (CREB), OK 27 cells were transiently transfected with an expression plasmid containing the cDNA for the 43-kD CREB (pRSV/CREB). The level of expression of the pOGH (ANG N-1498/+18) in OK 27 cells was estimated by the amount of immunoreactive hGH secreted into the culture medium. Transfection of pRSV/CREB alone stimulated the expression of pOGH (ANG N-1498/+18). The addition of isoproterenol or forskolin further enhanced the stimulatory effect of pRSV/ CREB on the expression of pOGH (ANG N-1498/+18). The enhancing effect of isoproterenol was inhibited by the presence of propranolol (an inhibitor of beta-adrenoceptors) and (R)-p-adenosine 3'5'-cyclic monophospho-orthioate (Rp)-cAMP (an inhibitor of cAMP-dependent protein kinase A I and II). Transfection of pRSV/CREB had no effect on the expression of thymidine kinase growth hormone in OK 13 cells, an OK cell line with a fusion gene containing the promoter/enhancer DNA sequence of the viral thymidine-kinase gene fused with an hGH gene as a reporter, thymidine kinase growth hormone, permanently integrated into their genomes. These studies demonstrate that isoproterenol stimulates the expression of ANG gene via the cAMP-dependent protein kinase A and probably via the interaction of the 43-kD CREB with the 5'-flanking region of the ANG gene. Our data indicate that the nuclear 43-kD CREB may have a modulatory role on the expression of the ANG gene in OK cells.
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PMID:Angiotensinogen gene expression is stimulated by the cAMP-responsive element binding protein in opossum kidney cells. 921 56