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)

Factor I is an active serine proteinase in plasma that regulates both the classical and alternative complement pathways by cleaving C3b and C4b thereby preventing the assembly of C3 and C5 convertase enzymes. In this study, a full-length human factor I cDNA was cloned into the pMT2 expression vector and the pMT2-fI construct was expressed transiently in COS-1 cells and stably in CHO-K1 cells. The transfected COS-1 cells secreted large amounts of recombinant pro-factor I (85 kD). Co-transfection of COS-1 cells with pMT2-fI and the cDNA expression plasmid for PACE (paired basic amino acid cleaving enzyme), resulted predominantly in the secretion of a proteolytically processed form of recombinant factor I (heavy chain, 47 kD; light chain, 35 kD). Following co-transfection of pMT2-fI and pSVNeo.1 into CHO-K1 cells and selection in medium containing G418, a stably transfected clone was isolated that secreted pro-factor I (85 kd) and proteolytically processed factor I (heavy chain, 48 kD; light chain, 37 kD) in approximately equal amounts. The molecular sizes of the subunit chains of the expressed factor I were generally slightly smaller than those of human plasma factor I. The activity of recombinant factor I present in the culture supernatants of transfected COS-1 and CHO-K1 cells was assayed by its ability to cleave 125I-C3b in the presence of factor H and was found to be low when compared with factor I purified from human plasma. However, since the functional activity of purified factor I was reduced approximately 50% in the presence of conditioned medium from non-transfected cells, it is suggested that the cold C3b present in the factor I-deficient serum used to supplement the culture medium probably competed with the 125I-C3b tracer, thereby decreasing the sensitivity of the assay for the recombinant factor I proteins.
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PMID:Processing of human factor I in COS-1 cells co-transfected with factor I and paired basic amino acid cleaving enzyme (PACE) cDNA. 773 77

C3 convertase regulatory proteins, decay accelerating factor (DAF, CD55) and membrane cofactor protein (MCP, CD46), have complementary function and transfected into non-human cells might confer protection against human complement. This may be an effective strategy to alleviate C-mediated cell damage by combining the two activities. In this study, we constructed a dicistronic mammalian expression vector pcDNA3-MCPIRESDAF using the internal ribosomal entry sites (IRES) of the encephalomyocarditis virus (EMCV), and stable cell lines were obtained by G418 screening. Integration of extraneous genes was identified by PCR. RT-PCR and Western blotting analysis demonstrated that the EMCV IRES allowed for efficient co-expression of hMCP and hDAF in NIH3T3 cells stably transfected with pcDNA3-MCPIRESDAF. Human complement-mediated cytolysis assays showed that co-expressed DAF and MCP proteins could provide more significant protection against complement-mediated cytolysis than either hMCP or hDAF alone. These results suggest that DAF and MCP synergize the actions of each other, and the IRES-mediated polycistronic vector should improve the efficiency and effectiveness of multi-gene delivery. The pcDNA3-MCPIRESDAF vector has potential therapeutic value for effectively controlling complement activation, thereby increasing the possibility of inter-species transplantation.
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PMID:Co-expression of human complement regulatory proteins DAF and MCP with an IRES-mediated dicistronic mammalian vector enhances their cell protective effects. 1897 20