Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:EXPT01586 (G418)
 2,237 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human renin is synthesized as a 406-amino acid preprorenin protein that is processed by a signal peptidase during secretion, to release prorenin as a 386-amino acid zymogen. The 46-amino acid "pro" domain is removed by a renin-processing enzyme, to produce enzymatically active renin, by cleavage at an Arg-Leu bond. The effects of the renin-processing enzyme can be mimicked by trypsin activation, where high concentrations of trypsin are incubated with prorenin for brief periods of time, followed by excess trypsin inhibitor to minimize secondary proteolytic processing by trypsin. In order to study the role of the pro segment in the secretion, folding, and activity of human renin, we engineered a construct where the pro domain from the preprorenin cDNA was deleted. This construct was introduced into mammalian cells and its expression was assayed in transient and stable systems. In COS-1 cells transfected with the prerenin expression vector pREN3, active renin was secreted with a specific activity of 1360 micrograms of angiotensin l/min/mg, compared with trypsin-activated prorenin, which has a specific activity of 818 micrograms of angiotensin l/min/mg. The active renin secreted in this system had a significantly reduced potency for the renin inhibitor SQ 32,970. These results demonstrate that the pro segment is dispensable for the folding and secretion of renin. A permanent cell line expressing the active form of renin was obtained by co-transfection of NRP cells with pREN3 and pHyg. A colony designated B/1 was identified, subcloned, and shown to secrete active renin (110 pg of renin/10(6) cells) optimally when maintained in both G418 and hygromycin.
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PMID:Stable expression, secretion, and characterization of active human renin in mammalian cells. 173 22

Studies of adrenal steroidogenesis have been facilitated by the availability of immortalized mouse adrenocortical Y-1 cells. We sought to make new, alternative mouse steroidogenic cell lines by genetically targeted tumorigenesis. Transgenic mice were constructed expressing both the SV40 T-antigen and a bacterial neomycin-resistance gene under the control of the promoter for the human P450 cholesterol side-chain cleavage (P450scc) gene, which encodes the first and rate-limiting enzyme in steroidogenesis. Two female transgenic mice expressed T-antigen in various nonsteroidogenic tissues but generated tumors only in the adrenals, suggesting adrenal tumor formation was an early event. Ovarian tissues, which, unlike the adrenal, do not make steroids in fetal or early postnatal life, did not develop tumors. Cell lines derived from the adrenal tumors were resistant to the neomycin analog G418. Clonal sublines are stable, growing easily in monolayers with a doubling time of 24-60 h. The cell lines secrete progesterone and 11-deoxycorticosterone, indicating these cells express the P450scc system, 3 beta-hydroxysteroid dehydrogenase, and 21-hydroxylase activity. However the 21-hydroxylase activity was not mediated by P450c21, as the cells lacked P450c21 mRNA. The cells did not secrete any 11-hydroxylated steroids, although they contained P450c11 beta mRNA. Both the secretion of progesterone and the abundance of P450scc mRNA increase in response to 8-bromo-cAMP, but not to ACTH or angiotensin II. In addition to expression of steroidogenic enzyme mRNAs, one cell line also expresses mouse renin-1 mRNA, making these cells useful for studies of the role of adrenal renin in regulating adrenal steroidogenesis. These findings represent an approach in transgenic mice to develop highly differentiated adrenal cell lines.
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PMID:Steroidogenic adrenocortical cell lines produced by genetically targeted tumorigenesis in transgenic mice. 815 34

Using transgenic mice, we targeted SV40 T antigen and the bacterial neomycin resistance gene to steroidogenic tissues using a human P450 cholesterol side-chain cleavage promoter. Expression of SV40 T antigen resulted in adrenocortical tumors. Adrenocortical cell lines from one of these tumors (ST5R) was previously characterized. We have now obtained clonal lines from the second more differentiated tumor. After dispersion of the left adrenal tumor, ST5L parental cells were selected with G418 and subcloned. The resulting adrenocortical subcloned cell lines are more highly differentiated than those cell lines resulting from the right adrenal tumor (ST5R). ST5L cell lines secrete progesterone and corticosterone to varying degrees, whereas ST5R cells secrete only progesterone. One of the clonal cell lines, ST5Lc16, expresses both P450c11 beta and P450c11AS mRNAs, which normally are regionally distributed in different zones of the adrenal cortex. Thus, ST5Lc16 cells may be progenitor cells for both glomerulosa and fasciculata cells and may provide clues to the cellular and molecular events leading to the differentiation of the glomerulosa and the fasciculata-reticularis. Other ST5Lc cell lines are more representative of the fasciculata-reticularis, because they express P450c11 beta mRNA and secrete corticosterone, and they neither express P450c11AS mRNA nor do they secrete aldosterone. All cell lines also have 21-hydroxylase activity, but none express P450c21, indicating that some other, as yet unidentified, enzyme has this activity. In all cell lines, steroid secretion is regulable by cAMP stimulation but not by ACTH stimulation. All ST5L cell lines also express mouse renin-1 mRNA. In addition to their utility in studies of adrenal steroidogenesis, these cell lines may also be useful in studying the etiology of adrenocortical tumors.
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PMID:Characterization of adrenocortical cell lines produced by genetically targeted tumorigenesis in transgenic mice. 905 83