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)

A retroviral vector (N2-SV-GC) was constructed by inserting a normal human glucocerebrosidase (GC) cDNA under control of the SV40 early region promoter into the Moloney murine leukemia virus-derived N2 vector. N2-SV-GC produced human GC in murine 3T3 fibroblasts at levels in the range of the endogenous murine GC as determined by enzymatic assay and Western blot analysis. The N2-SV-GC retroviral vector was used for studies of gene transduction of murine hematopoietic progenitor cells (HPC). Infection of bone marrow cultured for 2 to 10 days in medium containing hematopoietic growth factors was significantly more efficient than infection of freshly isolated marrow cells (24% to 32% G418-resistant CFU-GM v 15%, respectively). The marrow infected by N2-SV-GC was maintained in long-term bone marrow culture (LTBMC) and had a stable level of G418-resistant HPC over 2 months of serial assays. The human GC gene of the vector was persistently expressed in the nonadherent cell fraction of the murine LTBMC as determined by Northern blotting, Western blotting, and immunohistochemical staining using a monoclonal antibody specific for human GC. N2-SV-GC also expressed the human GC gene in day 12 CFU-S. LTBMC represents a novel system for retroviral vector-mediated gene transduction of HPC and may accurately predict the activities of vectors in vivo.
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PMID:Expression of human glucocerebrosidase in murine long-term bone marrow cultures after retroviral vector-mediated transfer. 229 79

Retroviral gene transfer has been used successfully to correct the glucocerebrosidase (GCase) deficiency in primary hematopoietic cells from patients with Gaucher disease. For this model of somatic gene therapy, we developed a high-titer, amphotropic retroviral vector designated NTG in which the human GCase gene was driven by the mutant polyoma virus enhancer/herpesvirus thymidine kinase gene (tk) promoter (Py+/Htk). NTG normalized GCase activity in transduced Gaucher fibroblasts and efficiently infected human monocytic and erythroleukemic cell lines. RNA blot-hybridization (Northern blot) analysis of these hematopoietic cell lines showed unexpectedly high-level expression from the Moloney murine leukemia virus long terminal repeat (Mo-MLV LTR) and levels of Py+/Htk enhancer/promoter-initiated human GCase RNA that approximated endogenous GCase RNA levels. Furthermore, NTG efficiently infected human hematopoietic progenitor cells. Detection (by means of the polymerase chain reaction) of the provirus in approximately one-third of NTG-infected progenitor colonies that had not been selected in G418-containing medium indicates that relative resistance to G418 underestimated the actual gene transfer efficiency. Northern blot analysis of NTG-infected, progenitor-derived cells showed expression from both the Mo-MLV LTR and the Py+/Htk enhancer/promoter. NTG-transduced hematopoietic progenitor cells from patients with Gaucher disease generated progeny in which GCase activity had been normalized.
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PMID:Correction of glucocerebrosidase deficiency after retroviral-mediated gene transfer into hematopoietic progenitor cells from patients with Gaucher disease. 231 24

Glucocerebrosidase cDNA and the neomycin-resistance gene (neo) were cloned into a retrovirus vector. Mouse fibroblasts infected with this vector expressed human glucocerebrosidase, which was readily distinguished from the mouse enzyme using mouse monoclonal anti-glucocerebrosidase antibodies. Cultured fibroblasts and transformed lymphoblasts from patients with type I Gaucher disease were infected with the retrovirus rescued from the mouse fibroblasts by a helper virus. Transformed cells were selected with the antibiotic G418. The enzyme activity of cells infected with virus containing glucocerebrosidase cDNA was restored to normal, while uninfected cells or cells infected with virus containing only the neo gene did not produce glucocerebrosidase.
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PMID:Complete correction of the enzymatic defect of type I Gaucher disease fibroblasts by retroviral-mediated gene transfer. 354 1

Gaucher disease (GD) is a disorder of glycosphinglipid metabolism caused by deficiency of lysosomal acid beta-glucosidase (GC), resulting in progressive deposition of glucosylceramide in macrophages. The glucose analogue, N-butyl-deoxynojirimycin (NB-DNJ, Miglustat), is an inhibitor of the ceramide-specific glucosyltransferase (CSG) which catalyzes the first step of glycosphingolipids biosynthesis and is currently approved for the oral treatment of type 1 GD. Using site-directed mutagenesis, we constructed plasmids containing wild-type and several mutations in glucocerebrosidase (GBA) gene. The plasmids were transfected into COS-7 cells and stable transfected cell lines were obtained by geneticin (G418) selection. Cells were cultured during 6 days with medium with or without 10 microM NB-DNJ. The addition of NB-DNJ to COS-7 cell medium leads to 1.3-, 2.1-, 2.3-, 3.6-, and 9.9-fold increase in the activity of S364R, wild-type, N370S, V15M, and M123T GC, respectively. However, no significant changes were observed in the activity of the L444P, L336P, and S465del mutated proteins, but a small decrease in the rare P266L variant was observed. These results suggest that NB-DNJ, in addition to the inhibitory effect on CSG, also works as a "chemical chaperone", increasing the activity of acid beta-glucosidase of wild-type and several GC mutated proteins, including the most frequent N370S mutation. The specific location of the Miglustat binding site in GC is unknown. Potential binding sites in the enzyme have been searched for using computational molecular docking. The searching strategy identified three potential GC binding sites for Miglustat, one being the substrate-binding site of the enzyme, which was the best-ranked site by AutoDock program. Therefore, it is possible that Miglustat exerts its chaperoning activity on acid beta-glucosidase by acting as an inhibitor bound at the active site. This increase on the activity of the acid beta-glucosidase would imply that Miglustat is not only a substrate reducer but also an inhibitor of the GC degradation, with very promising clinical implications for the treatment of GD patients.
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PMID:Miglustat (NB-DNJ) works as a chaperone for mutated acid beta-glucosidase in cells transfected with several Gaucher disease mutations. 1603 81