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4 results on '"Glycerol/pharmacology"'

1. Catabolite Repression and Induction of the Mg 2+ -Citrate Transporter CitM of Bacillus subtilis

Author

Bastiaan P. Krom, Christian Magni, Wil N. Konings, Jessica B. Warner, Juke S. Lolkema, and Preventive Dentistry

Subjects
Glycerol, ATP-Binding Cassette Transporters/genetics, Succinic Acid, Catabolite repression, Glutamic Acid/pharmacology, Bacillus subtilis, Bacillus subtilis/drug effects, chemistry.chemical_compound, Genes, Reporter, Phosphoproteins/genetics, Phosphoenolpyruvate Sugar Phosphotransferase System/genetics, Beta-galactosidase, Promoter Regions, Genetic, Organometallic Compounds/metabolism, Symporters, biology, Glucose/pharmacology, DNA-Binding Proteins, Biochemistry, Succinic Acid/pharmacology, CCPA, Recombinant Fusion Proteins/metabolism, Physiology and Metabolism, Recombinant Fusion Proteins, Glutamic Acid, Repressor, Phosphocarrier protein, Microbiology, Citric Acid, Promoter Regions, Reporter/genetics, Bacterial Proteins, Genetic, Citric Acid/metabolism, Inositol/pharmacology, Genes, Reporter/genetics, Organometallic Compounds, beta-Galactosidase/genetics, Phosphoenolpyruvate Sugar Phosphotransferase System, Molecular Biology, Psychological repression, Repressor Proteins/genetics, Reporter gene, Bacterial Proteins/biosynthesis, Phosphoproteins, beta-Galactosidase, biology.organism_classification, Repressor Proteins, Glucose, Genes, chemistry, Mutation, biology.protein, ATP-Binding Cassette Transporters, Carrier Proteins/biosynthesis, Glycerol/pharmacology, Carrier Proteins, Inositol, DNA-Binding Proteins/genetics
Abstract

In Bacillus subtilis the citM gene encodes the Mg 2+ -citrate transporter. A target site for carbon catabolite repression ( cre site) is located upstream of citM . Fusions of the citM promoter region, including the cre sequence, to the β-galactosidase reporter gene were constructed and integrated into the amyE site of B. subtilis to study catabolic effects on citM expression. In parallel with β-galactosidase activity, the uptake of Ni 2+ -citrate in whole cells was measured to correlate citM promoter activity with the enzymatic activity of the CitM protein. In minimal media, CitM was only expressed when citrate was present. The presence of glucose in the medium completely repressed citM expression; repression was also observed in media containing glycerol, inositol, or succinate-glutamate. Studies with B. subtilis mutants defective in the catabolite repression components HPr, Crh, and CcpA showed that the repression exerted by all these medium components was mediated via the carbon catabolite repression system. During growth on inositol and succinate, the presence of glutamate strongly potentiated the repression of citM expression by glucose. A reasonable correlation between citM promoter activity and CitM transport activity was observed in this study, indicating that the Mg 2+ -citrate uptake activity of B. subtilis is mainly regulated at the transcriptional level.

Published
2000
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4. Transfer of high copy number plasmid into mammalian cells by calcium phosphate transfection

Author

Pascal Batard, Florian M. Wurm, and Martin Jordan

Subjects
Calcium Phosphates, Glycerol, Azides, Gene Expression Regulation/drug effects, Endosomes, CHO Cells, Biology, Transfection, Lysosomes/metabolism, Plasmids/*drug effects/genetics/pharmacokinetics, Sensitivity and Specificity, Flow cytometry, Cell Line, chemistry.chemical_compound, Plasmid, Cricetinae, Calcium chloride transformation, Genetics, medicine, Animals, Humans, Microscopy, Microscopy, Confocal, medicine.diagnostic_test, Rhodamines, Chinese hamster ovary cell, General Medicine, DNA, Endosomes/metabolism, Cell sorting, Calcium Phosphates/*pharmacology, Molecular biology, Transfection/*methods, Cell biology, DNA/drug effects/genetics/pharmacokinetics, chemistry, Gene Expression Regulation, Cell culture, Confocal, Fluorescein, Glycerol/pharmacology, Lysosomes, Plasmids
Abstract

Using flow cytometry, single cell sorting, confocal microscopy and fluorescent plasmids, a thorough study of DNA uptake, DNA fate and DNA expression in mammalian cells transfected with the widely used calcium-phosphate precipitation method was executed. We show for the first time that up to 100,000 plasmid molecules can be delivered into individual cells, but also that DNA transfer into cells is a dynamic process that follows a defined kinetics of uptake and intracellular processing. Analyses by flow cytometry and confocal microscopy have also supported results suggesting endocytosis during Ca-Pi transfection. We also demonstrate that expression-enhancing treatment with glycerol during transfection did not result in increased DNA uptake. While cells with maximal DNA load appear to express the highest level of the transgene, these cells are negatively impacted in terms of growth and survival.

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