hymecromone and 5-bromo-4-chloro-3-indolyl-beta-galactoside

Brain inflammation in GM2 gangliosidosis has been recently realized as a key factor in disease development. The aim of this study was to investigate the effects of a FIV beta-hexosaminidase vector in the brain of HexB-deficient (Sandhoff disease) mice following intraperitoneal administration to pups of neonatal age. Since brain inflammation, lysosomal storage and neuromuscular dysfunction are characteristics of HexB deficiency, these parameters were employed as experimental outcomes in our study. The ability of the lentiviral vector FIV(HEX) to infect murine cells was initially demonstrated with success in normal mouse fibroblasts and human Tay-Sachs cells in vitro. Furthermore, systemic transfer of FIV(HEX) to P2 HexB-/- knockout pups lead to transduction of peripheral and central nervous system tissues. Specifically, beta-hexosaminidase expressing cells were immunolocalized in periventricular areas of the cerebrum as well as in the cerebellar cortex. FIV(HEX) neonatal treatment resulted in reduction of GM2 storage along with attenuation of the brain inflammation and amelioration of the attendant neuromuscular deterioration. In conclusion, these results demonstrate the effective transfer of a beta-hexosaminidase lentiviral vector to the brain of Sandhoff mice and resolution of the GM2 gangliosidosis after neonatal intraperitoneal administration.

Topics: Animals; Animals, Newborn; Behavior, Animal; beta-N-Acetylhexosaminidases; Cell Count; Cell Line; Central Nervous System; Cricetinae; Fibroblasts; G(M2) Ganglioside; Galactosides; Gene Expression Regulation; Genetic Vectors; Glial Fibrillary Acidic Protein; Hexosaminidase B; Histocompatibility Antigens Class II; Histocytochemistry; Humans; Hymecromone; Immunodeficiency Virus, Feline; Indoles; Lentivirus; Lentivirus Infections; Mice; Mice, Knockout; Peripheral Nerves; Reverse Transcriptase Polymerase Chain Reaction; Rotation; Transduction, Genetic

A comparison was made with different chromogenic and fluorogenic substrates, 4-methylumbelliferyl-beta-D-glucuronide (MUG), 4-nitrophenyl-beta-D-glucuronide (PNPG), 4-methylumbelliferyl-beta-D-galactopyranoside (MUGA), 2-nitrophenyl-beta-D-galactopyranoside (ONPG), 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-GAL), for the rapid and simultaneous enumeration of total coliforms and E. coli in water samples, based on 2 commercially available culture-media. The combination of the chromogenic compound X-GAL (for detecting coliforms) and of the fluorogenic compound MUG (for detecting E. coli) incorporated either into ECD agar or into lauryl sulfate broth proved to be most useful. The optimum concentration of the X-GAL/MUG supplement was (50 micrograms/ml/70 micrograms/ml) for the solid medium (EMX agar) and (60 micrograms/ml/70 micrograms/ml) for the fluid medium (LMX broth). As a result of the examination of 244 Enterobacteriaceae strains isolated from water samples and clinical material, it was shown that the use of EMX agar (LMX broth) had several advantages over conventional methods. A routine method for the analysis of water samples was proposed involving the EMX agar and the LMX broth.

Topics: Chromogenic Compounds; Culture Media; Enterobacteriaceae; Escherichia coli; Fluorescent Dyes; Galactosides; Glucuronates; Hymecromone; Indoles; Nitrophenylgalactosides; Water Microbiology