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Full Description
Discovered and first isolated in 1966 in C. De Duve's laboratory, peroxisomes - organelles which are present in nearly all eukaryotic cells - are still not fully understood. More than 40 peroxisomal enzymes catalyzing a variety of reactions have been characterized. Moreover, peroxisomes can be regarded as toxicological indicators: several compounds, including hypolipemic drugs, plasticizers or pesticides trigger their proliferation. This proliferation may lead to hepatocarcinogenesis in rodents. Interest in peroxisomes stems not only fromtheir biology, but also because there is a deficiency of peroxisomal functions in several genetic diseases. Some genes involved in inborn errors of peroxisomal function have recently been identified.
In this manual, based on a FEBS Advanced Course on peroxisomes, protocols on the following topics are described in detail: Isolation and characterization of peroxisomes by ultracentrifugationand immunoblotting; gene regulation studied by mRNA isolation, hybridizationand DNA cell transfection; use of cell lines as peroxisome proliferator targets; transformation with retrovirus; peroxisomes as toxicological markers; cytochrome P450 induction; drug design and computer analysis of ligand/receptor interaction involved in peroxisomal gene expression.
Contents
I: Isolation and Characterization of Peroxisomes.- General Indroduction to Isolation and Characterization of Peroxisomes.- Experiment 1. Preparation and Purification of Peroxisomes; Subfractionation of Purified Peroxisomes; Acyl-CoA Oxidase Activity Measurements.- Experiment 2. Immunoblotting of Peroxisomal Proteins with Monospecific Antibodies.- Experiment 3. Morphology of Peroxisomes in Light- and Electron Microscopy.- II: Molecular Biology.- Experiment 4. Northern Blotting Analysis of Rat Liver mRNA Encoding for Peroxisomal Acyl-CoA Oxidase.- Experiment 5. Transient Co-transfection Assay: Measure of the Chloramphenicol Acetyl Transferase (CAT) Activity in Cytosol Extracts from Transfected Cells.- Experiment 6. Synthesis of Oligonucleotides Used as Probes; Purification by HPLC.- Experiment 7. Computer Analysis of DNA and Protein Sequences.- III: Toxicology and Pharmacology.- Experiment 8. Cytochrome P450 Isoenzyme Induction Pattern in Rats Treated with Peroxisome Proliferators and Classical Inducers.- Experiment 9. Measurement of the in Vitro Glucuronidation of Peroxisome Proliferator Carboxylic Acids by Liver Microsomes and by Genetically Modified V 79 Cell Line.- Experiment 10. Molecular Modeling and Drug Design: Application to Some Peroxisome Proliferator Agents.- IV: Cell Culture and Genetic Diseases.- Experiment 11. Fao Cell Line as a Model for the Study of the Effect of Peroxisome Proliferators on Cellular Functions.- Experiment 12. Measurement of Dihydroxyacetone-Phosphate Acyl-Transferase (DHAP-AT) Activity in Liver Peroxisomes and Cell Lines.- Experiment 13. In Vitro Transformation of Human Fibroblasts with SV40 T Antigen (Lipofection).- Experiment 14. Somatic Cell Hybridization as a Tool for Genetic Analysis of Peroxisomal Activities.- Supplement.
