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)

Expression of the biologically active beta-subunit of mouse nerve growth factor (beta-NGF) was conferred onto cultured AtT-20 mouse pituitary cells via a replication-defective retroviral vector. The retroviral LTR promoter was used for expression of a cDNA for beta-NGF corresponding to the shorter mRNA species produced by most tissues that receive sympathetic innervation. The vector included the TU5 gene conferring resistance to the neomycin analogue G418 under the control of an SV40 early promoter. AtT-20 cells, which produce essentially no endogenous beta-NGF, were infected and then cloned under G418 selection. Clones were evaluated for release into the medium of biologically active beta-NGF using a bioassay for neurite extension from PC-12 cells. The biological activity was equivalent to 1 to 10 ng of beta-NGF per mg cell protein over 24 hours. Immune precipitation and SDS/polyacrylamide gel electrophoresis of labelled proteins in the medium showed that the major form of immunoreactive beta-NGF secreted from cells comigrated with authentic mature beta-NGF, apparent Mr 13,000. Release of this beta-NGF from cells was stimulated by addition of 1 mM-8-bromocyclic AMP or 10 nM-corticotropin releasing factor, suggesting that at least some of the processed factor is stored in secretory vesicles. These studies, together with those on other cultured cells, which produce beta-NGF and lack secretory granules, e.g. L cells, suggest that the beta-NGF precursor synthesized from the shorter mRNA species can be processed and secreted through either the regulated or constitutive route. This retroviral vector provides a potential means of conferring beta-NGF expression onto a number of different cell types in culture and in vivo.
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PMID:Retrovirus-mediated gene transfer of beta-nerve growth factor into mouse pituitary line AtT-20. 283 91

A recombinant plasmid containing the human proenkephalin gene ligated to pBR322 was introduced into a mouse pituitary cell line (AtT-20D16v) that normally expresses pro-opiomelanocortin but not proenkephalin. The plasmid was introduced by co-transformation with the G418-selectable plasmid, pRSVneo. Stable transformants were isolated and analyzed for the presence of the human proenkephalin gene. AtT-20 transformants which had one or more copies of the human proenkephalin gene integrated stably into the mouse chromosomal DNA expressed a 1.45 kb mRNA identical in size to human proenkephalin mRNA. Primer extension analysis indicated that the human proenkephalin gene was accurately and efficiently transcribed from its own promoter. AtT-20 transformants that expressed the 1.45 kb human proenkephalin mRNA also expressed proenkephalin protein and cleaved the protein to form free Met-enkephalin. This is of particular interest because these cells do not cleave all of the available pairs of basic amino acids in the endogenous protein, pro-opiomelanocortin, the precursor to ACTH, beta-endorphin and melanocyte stimulating hormones. The release of both ACTH and Met-enkephalin from these cells is stimulated by corticotropin releasing factor, a natural secretagogue for ACTH, indicating that the two classes of peptide share a related secretory pathway.
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PMID:Expression of the human proenkephalin gene in mouse pituitary cells: accurate and efficient mRNA production and proteolytic processing. 300 33