Single cell analysis and selection of living retrovirus vector-corrected mucopolysaccharidosis VII cells using a fluorescence-activated cell sorting-based assay for mammalian beta-glucuronidase enzymatic activity.
AuthorsLorincz MC, Parente MK, Roederer M, Nolan GP, Diwu Z, Martin DI, Herzenberg LA, Wolfe JH
JournalJ Biol Chem
PubMed ID9872999
'Mutations in the acid beta-glucuronidase gene lead to systemic accumulation of undegraded glycosaminoglycans in lysosomes and ultimately to clinical manifestations of mucopolysaccharidosis VII (Sly disease). Gene transfer by retrovirus vectors into murine mucopolysaccharidosis VII hematopoietic stem cells or fibroblasts ameliorates glycosaminoglycan accumulation in some affected tissues. The efficacy of gene ... More
Gene expression imaging by enzymatic catalysis of a fluorescent probe via membrane-anchored beta-glucuronidase.
AuthorsSu YC, Chuang KH, Wang YM, Cheng CM, Lin SR, Wang JY, Hwang JJ, Chen BM, Chen KC, Roffler S, Cheng TL,
JournalGene Ther
PubMed ID17235292
'Development of nonimmunogenic and specific reporter genes to monitor gene expression in vivo is important for the optimization of gene therapy protocols. We developed a membrane-anchored form of mouse beta-glucuronidase (mbetaG) as a reporter gene to hydrolyze a nonfluorescent glucuronide probe (fluorescein di-beta-D-glucuronide, (FDGlcU) to a highly fluorescent reporter to ... More
AuthorsLorincz M, Roederer M, Diwu Z, Herzenberg LA, Nolan GP
JournalCytometry
PubMed ID8866216
'We report the development of a new fluorescence-activated cell sorter (FACS)-based reporter gene system utilizing the enzymatic activity of the E. coli beta-glucuronidase (gus) gene. When loaded with the Gus substrate fluorescein-di-beta-D-glucuronide (FDGlcu), individual mammalian cells expressing and translating gus mRNA liberate sufficient levels of intracellular fluorescein for quantitative analysis ... More
Direct influence of S9 liver homogenate on fluorescence signals: impact on practical applications in a bacterial genotoxicity assay.
AuthorsDreier J, Breitmaier EB, Gocke E, Apfel CM, Page MG
JournalMutat Res
PubMed ID11719102
Assays based on the bacterial SOS-response offer the possibility of automatization of genotoxicity testing for screening of large compound libraries. While existing assays use colorimetric detection or luminescence read-out, we describe here the use of a fluorescence-based system to achieve high sensitivity of detection required for assay miniaturization. Three commonly ... More
Critical elements of oligosaccharide acceptor substrates for the Pasteurella multocida hyaluronan synthase.
AuthorsWilliams KJ, Halkes KM, Kamerling JP, DeAngelis PL
JournalJ Biol Chem
PubMed ID16361253
Three-dimensional structures are not available for polysaccharide synthases and only minimal information on the molecular basis for catalysis is known. The Pasteurella multocida hyaluronan synthase (PmHAS) catalyzes the polymerization of the alternating beta1,3-N-acetylglucosamine-beta1,4-glucuronic acid sugar chain by the sequential addition of single monosaccharides to the non-reducing terminus. Therefore, PmHAS possesses ... More
Reversible glycosidic switch for secure delivery of molecular nanocargos.
Authors
JournalNat Commun
PubMed ID29748577
Extracellular vesicles protect glucuronidase model enzymes during freeze-drying.