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)

3T3 Swiss mouse fibroblast cell lines expressing tyrosinase, the critical enzyme in melanin synthesis, have been established by co-transfection of a mouse tyrosinase cDNA and a G418-resistance gene. Of sixty-three clones isolated, four are brown in colour, presumably due to synthesis of melanin. Expression of both the tyrosine hydroxylase and dopa oxidase activities of tyrosinase by these pigmented clones has been demonstrated directly by enzyme assays. Electron microscopic studies suggest that the brown pigment is located in membrane-bound cytoplasmic vesicles.
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PMID:Expression of a mouse tyrosinase cDNA in 3T3 Swiss mouse fibroblasts. 190 37

Melanocyte cell lines, with characteristic dendritic morphology and melanosomes, were established from young mice of wild-type (C57BL/6) and of two albino (C57BL/6-c2J/c2J and BALB/c) inbred strains. The albino cells were cotransfected with two plasmids: pMTtyr1, containing the full-length tyr1 cDNA for tyrosinase encoded by the c locus, under the control of the inducible mouse metallothionein-I (MT-I) promoter; and pSVneo beta, allowing selection of transformants by G418 resistance. The intrinsic albino defect was corrected by the tyr1 cDNA in transfected cells, thereby validating the coding capability of tyr1 for tyrosinase. Black melanin was formed in the genetically black (B/B) C57BL/6-c2J/c2J cells and brown melanin in the genetically brown (b/b) BALB/c cells. Pigment was produced even without adding heavy metal (for induction of the MT-I promoter), thus obviating the need for adding it, but was formed more rapidly upon addition of ZnSO4 up to 100 microM. Stable transfected albino melanocyte lines with active tyrosinase and melanization were obtained. Addition of ZnSO4 at 200 microM was lethal to the cells. However, this toxicity--attributable at least in part to melanin precursors--was prevented if the cells sojourned at 100 microM ZnSO4 for two weeks before being exposed to the 200 microM level. Adaptation was lost when the cells were removed from 200 microM ZnSO4 for one week and then returned to it. Avoidance of toxicity under these conditions is thus the result of physiological detoxification mechanisms rather than selection for a genetic change.
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PMID:Pigmented cell lines of mouse albino melanocytes containing a tyrosinase cDNA with an inducible promoter. 212 Jul 78

Many genes mapping to pigmentation loci are involved in the regulation of melanin synthesis in the mouse. The brown (b) locus controls black/brown coat coloration, and its product has significant homology to the key melanogenic enzyme tyrosinase. This has led to suggestions that the b-protein is itself a melanogenic enzyme. In order to investigate its function, we have established lines of mouse fibroblasts stably expressing the b-protein by co-transfection of a b-protein expression vector and a plasmid conferring resistance to the antibiotic G418. The b-protein synthesised by these cells has the expected molecular mass of 75 kDa and reacts with three different anti-b-protein antibodies. We were unable to confirm previous reports that the b-protein has tyrosinase or catalase activity, but detected stereospecific dopachrome tautomerase activity in b-protein-expressing fibroblasts. This dopachrome tautomerase binds to Concanavalin A-Sepharose, and the major product of its action on L-dopachrome is 5,6-dihydroxyindole-2-carboxylic acid. Since this activity is not present in untransfected cells we conclude that the b-protein has dopachrome tautomerase activity. Fibroblasts do not contain melanosomes, the specialised organelles in which the b-protein is located in melanocytes. Nevertheless, indirect immunofluorescence localisation of the b-protein in transfected fibroblasts produces a distinctive pattern of intense juxtanuclear staining combined with punctate cytoplasmic staining. Double-labelling shows co-localisation of the b-protein with the late endosomal/lysosomal markers beta-glucuronidase and LAMP-1, both in transfected fibroblasts and in mouse melanoma cells. These findings are consistent with the hypothesis that melanosomes are closely related to lysosomes.
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PMID:The mouse brown (b) locus protein has dopachrome tautomerase activity and is located in lysosomes in transfected fibroblasts. 827 Jun 21

Normal melanosome biogenesis requires the association of structural proteins with tyrosinase. 3T3 Swiss fibroblasts transfected with mouse tyrosinase cDNA (line 13.4, clone c) are a unique system in which melanogenesis takes place in the absence of melanosomal structural proteins. Our study confirmed that transfected fibroblasts displayed tyrosinase activity and some of them produced pigment granules. In the absence of melanosomal structural proteins the granules failed both to show a typical ultrastructure and to undergo the usual melanosome ontogenesis. The differentiating agent--dimethyl sulfoxide--increased phaeomelanin production. Pigment was localized in membrane-bound vesicles which were identified as lysosomes by means of immunogold electron microscopy. Cell line 13.4 had higher levels of lysosomal enzymes (beta-hexosaminidase, alpha-mannosidase) than both parental 3T3 cells and clone pKG4 (fibroblasts transfected with the G418 resistance plasmid). Melanosomal proteins act as scavengers of toxic products of melanogenesis, and our results suggest that in their absence cells may employ an alternative mechanism to sequester injurious products.
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PMID:Melanogenesis in transfected fibroblasts induces lysosomal activation. 912 62

Tyrosinase is the key enzyme in melanin biosynthesis in pigmented cells. We transfected 9L rat glioma cells with human tyrosinase cDNA that had been cloned in a high expression vector. Stable transfectants were selected by their resistance to the antibiotic G418. More than a dozen G418-resistant clones were isolated and were screened for tyrosinase expression using dopa-oxidase activity. The clone with the highest tyrosinase activity was selected and expanded for further studies. Northern blot analyses of total RNA from cells showed that the transfected cells had relatively more tyrosinase transcript than SK-MEL-23 human melanotic melanoma cells. The melanin content of the transfected cells was dependent on the concentration of L-tyrosine in the culture medium. In addition, the growth of transfected cells was inhibited when grown in a medium containing high concentrations of L-tyrosine. These results suggest that tyrosinase activity is cytotoxic in a substrate-dependent manner. This may have far reaching therapeutic use for glioma tumours.
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PMID:Tyrosinase transfection produces melanin synthesis and growth retardation in glioma cells. 991 10

Cells from a lung metastasis, arising from Cloudman S91 melanoma cells implanted s.c. in the tail of a BALB/c nu/nu mouse, were comprised chiefly of host x tumor hybrids. These lung metastasis cells showed: (a) 30-40% increased DNA content; (b) resistance to 10(-4) M hypoxanthine, 4 x 10(-7) M aminopterin, and 1.6 x 10(-5) M thymidine (HAT) + G418; and (c) the presence in genomic DNA of genes for both wt and albino tyrosinase, reflecting the DBA/2J (Cloudman S91) and BALB/c mouse genotypes, respectively. Individual clones of lung metastasis cells expressed enhanced pigmentation, motility, and responsiveness to MSH/IBMX, a behavior similar to that recently reported for artificially generated melanoma x macrophage fusion hybrids. These similarities suggested that the host fusion partner generating the lung metastasis hybrids might have been a macrophage, although formal proof for this was not possible. The results provide the first direct evidence that host x tumor hybridization could serve as an initiating mechanism for melanoma metastasis.
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PMID:A spontaneous murine melanoma lung metastasis comprised of host x tumor hybrids. 1081 Nov 33