Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.69 (APC)
 16,337 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Plasmid vectors pHSG293 and pHSG747, suitable for in vitro gene amplification for subsequent animal-cell expression, were developed. A cosmid vector pHSG293 confers Km resistance to Escherichia coli host cells and G418 resistance to animal cells and contains a single BstXI recognition/cleavage site, CCACGGGG/CTGG, near the cos site (the recognition site is underlined). The cassette vector plasmid pHSG747 contains a multiple cloning site (MCS) between the simian virus 40 early promoter and the poly(A) signal sequence flanked by the same BstXI sites and confers Cm resistance to E. coli host cells. After inserting a coding fragment for human protein C or its derivative in the appropriate orientation in the MCS of pHSG747, the BstXI expression unit fragment was purified, mixed with BstXI-digested pHSG293 DNA at a molecular ratio of 20 to 40:1 and ligated. This allowed for tandem gene amplification due to asymmetric cohesive ends. Ligation products were packaged in lambda phage particles, amplified in E. coli cells as large cosmid molecules, and then introduced into CHO cells. G418R transformants were found to produce and secrete recombinant protein molecules at a high level. The plasmid vectors developed in this work will provide a rapid screening system useful for protein engineering in animal cells.
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PMID:Tandem gene amplification in vitro for rapid and efficient expression in animal cells. 306 15

The aim of our study was to examine the potential usefulness of transducing the protein kinase C-gamma (PKC-gamma) cDNA gene into tumor-specific T cells as a technique for facilitating the generation of large numbers of functional Ag-specific T for tumor therapy. Murine CD8+, F-MuLV gag-specific CTL clones, and CD4+, F-MuLV env-specific Th clones, as well as bulk-cultured T cell lines with defined Ag specificity to FBL-3, a Friend murine leukemia virus (F-MuLV)-induced tumor, were transduced with a retroviral vector pZipNeoPKC-gamma and selected in G418. The results demonstrated that PKC-gamma-transduced clones remained activated in culture, as evidenced by continued expression of up-regulated levels of IL-2R, which were as high after 6 mo in culture without Ag restimulation as 24 h after Ag stimulation. In vitro functional studies demonstrated that PKC-gamma-transduced CD8+ T cell clones maintained specific cytolytic activity to FBL-3, and PKC-gamma-transduced CD4+ T cell clones maintained specific proliferative activity to FBL-3 or F-MuLV Ag presented by irradiated syngeneic APC. Short-term bulk-cultured T cells specific to FBL-3 were also transduced and could be grown long term in vitro with maintenance of functional specificity. In vivo study showed that PKC-gamma-transduced CD4+ T cells were able to proliferate in response to Ag plus IL-2 stimulation in vivo in a similar pattern as the parental T cells. Therapy with adoptively transferred PKC-gamma-transduced T cell clones and lines into syngeneic mice, with or without FBL-3 tumor, showed that the PKC-gamma-transduced T cells were not tumorigenic and were effective in curing mice with disseminated FBL-3.
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PMID:Retroviral transduction of protein kinase C-gamma into tumor-specific T cells allows antigen-independent long-term growth in IL-2 with retention of functional specificity in vitro and ability to mediate tumor therapy in vivo. 793 May 83

Nonsense mutations, generating premature termination codons (PTCs), account for 10% to 30% of the mutations in tumor suppressor genes. Nonsense translational suppression, induced by small molecules including gentamicin and G418, has been suggested as a potential therapy to counteract the deleterious effects of nonsense mutations in several genetic diseases and cancers. We describe here that NB124, a synthetic aminoglycoside derivative recently developed especially for PTC suppression, strongly induces apoptosis in human tumor cells by promoting high level of PTC readthrough. Using a reporter system, we showed that NB124 suppressed several of the PTCs encountered in tumor suppressor genes, such as the p53 and APC genes. We also showed that NB124 counteracted p53 mRNA degradation by nonsense-mediated decay (NMD). Both PTC suppression and mRNA stabilization contributed to the production of a full-length p53 protein capable of activating p53-dependent genes, thereby specifically promoting high levels of apoptosis. This new-generation aminoglycoside thus outperforms the only clinically available readthrough inducer (gentamicin). These results have important implications for the development of personalised treatments of PTC-dependent diseases and for the development of new drugs modifying translation fidelity.
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PMID:Characterization of new-generation aminoglycoside promoting premature termination codon readthrough in cancer cells. 2814 97