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)

Pro-UK delta GS1 was designed as a long-life and thrombin-resistant derivative of pro-urokinase (pro-UK) by deleting the growth factor domain of pro-UK and introducing a glycosylation site near the thrombin cleaving site for thrombin-resistance using site-directed mutagenesis. An expression plasmid for pro-UKDGS1, pIH1UK delta GS1SEd1-5 was constructed and introduced into Namalwa KJM-1, a lymphoblastoid cell line adapted to serum-free medium, and cells resistant to G418 and Methotrexate (MTX) were obtained. Amongst them, the highest pro-UK delta GS1 producer (resistant to 200 nM of MTX), clone 2-9, was selected and used for further studies. Under the conventional conditions, i.e. at 37 degrees C in serum-free ITPSGF medium (based on RPMI-1640 medium), the oligosaccharide structure of pro-UK delta GS1 produced by clone 2-9 mainly consisted of fucose (Fuc)-containing biantennary complex-type oligosaccharide. Addition of dexamethasone (Dex), changed the carbohydrate contents in the media, and a shift down of incubation temperature caused a change in oligosaccharide structure of pro-UK delta GS1 from mainly Fuc-containing biantennary to mainly Fuc-containing tri- and tetraantennary complex-type oligosaccharide. The modulated pro-UK delta GS1 showed superior in vivo activity for a canine femoral thrombosis formed by inserting a copper-coil.
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PMID:Modulation of oligosaccharide structure of a pro-urokinase derivative (pro-UK delta GS1) by changing culture conditions of a lymphoblastoid cell line Namalwa KJM-1 adapted to serum-free medium. 898 1

Mucopolysaccharidosis type IIID or Sanfilippo D syndrome is a lysosomal storage disorder caused by the deficiency of N-acetylglucosamine-6-sulphatase (Glc6S). In addition to human patients, a Nubian goat with this disorder has been described and the caprine Glc6S (cGlc6S) cDNA cloned. In this study, the full-length cGlc6S cDNA was inserted into the expression vector, pEFNeo, which placed the cGlc6S cDNA under the transcriptional control of the human polypeptide chain elongation factor promoter. The pEFNeo expression vector also contains the human growth hormone polyadenylation signal and the genes encoding resistance to ampicillin and G418. The cGlc6S expression construct was electroporated into Chinese hamster ovary (CHO-K1) cells, and stably transfected clones were isolated. One clone, CHOrcGlc6S.17, which secreted the highest Glc6S activity into the culture medium, was selected and cultured in cell factories. The secreted recombinant cGlc6S (rcGlc6S) precursor was purified to homogeneity from conditioned medium by a two-column procedure which consisted of a Cu2+-chelating Sepharose column followed by TSK G3000SW gel filtration. The native molecular mass of rcFlc6S was estimated to be 102 kDa and the subunit size was 94 kDa. The kinetic properties of cGlc6S were similar to those of human Glc6S isolated from liver. rcGlc6S was endocytosed by fibroblasts from patients with mucopolysaccharidosis type IIID via the mannose 6-phosphate receptor-mediated pathway resulting in correction of the storage phenotype of these cells.
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PMID:Expression, purification and characterization of recombinant caprine N-acetylglucosamine-6-sulphatase. 935 39

The ATH1 gene encoded acid trehalase in Saccharomyces cerevisiae. The gene disruption cassette combined the heterologous dominant kan(r) resistance marker with a Cre/loxP-mediated marker removal procedure. The gene disruption cassette was produced by PCR using the same long oligonucleotides comprising 50 nucleotides that annealed to sites upstream or downstream of the genomic target sequence to be deleted. After transformation of the linear disruption cassettes with a Cre/loxP-mediated marker into the cells of Saccharomyces cerevisiae BY-6, selected transformants were checked by PCR for correct the integration of the cassette and concurrent deletion of the chromosomal target sequence. The copper-resistance gene (CUP1-MT1) was cloned into pSH47, which yielded pSH-CUP. The recombinant plasmid pSH-CUP was transformed into the cells of Saccharomyces cerevisiae BY-6(delta ATH1, G418(r)), and transformants were selected for copper resistance. Upon expression of the Cre recombinase results in removal of the kan(r) gene, leaving behind a single loxP site at the chromosomal locus. Construction of the recombinant plasmid pSH-CUP avoided inserting non-yeast gene and made the loxP - kanMX - loxP gene disruption cassette more conventional for eukaryotic organism gene disruption.
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PMID:[Constructing recombinant plasmid pSH-CUP and knockout of acid trehalase gene in baker's yeast]. 1843 93

Induction of many genes encoding detoxifying enzymes and antioxidant proteins is mediated through a common mechanism, which is controlled by electrophile-responsive elements (EpRE) within the regulatory region of those genes. Copper and methyl parathion are environmental pollutants known to induce the expression of EpRE-mediated genes. In order to evaluate the molecular response triggered by these pollutants, a stable cell line was produced, which carries a transgene comprised of the green fluorescent protein (GFP) reporter gene under transcriptional control of the mouse glutathione-S-transferase (gst1) electrophile-responsive element fused to the mouse metallothionein (mt1) minimal promoter. The rat HTC hepatoma cells were transfected with the EpREmt-GFP construct and successfully selected with G418 antibiotic. EpREmt-GFP HTC cells were treated with 0.002 mg L(-1), 0.02 mg L(-1), 0.2 mg L(-1) and 2 mg L(-1) copper sulfate and 0.001 mg L(-1), 0.01 mg L(-1), 0.1 mg L(-1) and 1 mg L(-1) methyl parathion for 48 h. GFP expression was directly quantified in living cells using a microplate fluorimeter. GFP expression was significantly increased in higher concentrations of both pollutants, showing a 1.80- and 2.58-fold induction of GFP at 2mg copper L(-1) and 1mg methyl parathion L(-1), respectively (p<0.01). The results obtained in the present study demonstrate that the EpREmt-GFP HTC cell line can be an interesting model for further development for the study of the cellular response to aquatic pollutants as well as a new tool for environmental monitoring at the molecular level.
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PMID:Initial results in the development of a reporter cell line for toxicology studies at gene expression level: activation of the electrophile-responsive element by copper and methyl parathion. 1853 48

Candida glycerinogenes WL2002-5 has been used for industrial-scale fermentation of glycerol and may be a promising genetic host due to its tolerance to high osmotic pressure and fast growth. It resists many kinds of drugs, such as G418/hygromycin/cycloheximide. In previous studies, only Zeocin was used as a drug-resistant marker. But Zeocin is so expensive that it largely limits the genetic and molecular study. Here, we constructed a eukaryotic integrative vector pGAPZU, based on pGAPZB, to gain a new selectable marker of copper resistance for this strain. The results showed that the CUP1 gene of Saccharomyces cerevisiae elevated copper resistance of C. glycerinogenes. The C. glycerinogenes transformed with recombinant vector pGUC, obtained from introducing CUP1 gene into plasmid pGAPZU, could resist 21 mM copper, while the minimum inhibitory concentration (MIC) of wild type was 18 mM in solid YEPD medium. With copper resistance as a selective marker, research cost was largely reduced from 114.0 $/L with Zeocin as selective marker to 0.1 $/L. The new expression vector pGUC and selective marker of copper resistance gene establish a good foundation for further study on this industrial strain.
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PMID:The construction of a new integrative vector with a new selective marker of copper resistance for glycerol producer Candida glycerinogenes. 2223 83