ABSTRACT
The myelin membrane comprises a plethora of regions that are compositionally, ultrastructurally, and functionally distinct. Biochemical dissection of oligodendrocytes, Schwann cells, and central and peripheral nervous system myelin by means such as cold-detergent extraction and differential fractionation has led to the identification of a variety of detergent-resistant membrane assemblies, some of which represent putative signalling platforms. We review here the different microdomains that have hitherto been identified in the myelin membrane, particularly lipid rafts, caveolae, and cellular junctions such as the tight junctions that are found in the radial component of the CNS myelin sheath.
Subject(s)
Membrane Microdomains/chemistry , Myelin Sheath/chemistry , Animals , Caveolae/chemistry , Detergents/pharmacology , Drug Resistance , Humans , Intercellular Junctions/chemistry , Protein Processing, Post-Translational , Tight Junctions/chemistryABSTRACT
We have characterized the lipid rafts in myelin from a spontaneously demyelinating mouse line (ND4), and from control mice (CD1 background), as a function of age and severity of disease. Myelin was isolated from the brains of CD1 and ND4 mice at various ages, and cold lysed with 1.5% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulphonate). The lysate was separated by low-speed centrifugation into supernatant and pellet fractions, which were characterized by Western blotting for myelin basic protein (MBP) isoforms and their post-translationally modified variants. We found that, with maturation and with disease progression, there was a specific redistribution of the 14-21.5 kDa MBP isoforms (classic exon-II-containing vs exon-II-lacking) and phosphorylated forms into the supernatant and pellet. Further fractionation of the supernatant to yield detergent-resistant membranes (DRMs), representing coalesced lipid rafts, showed these to be highly enriched in exon-II-lacking MBP isoforms, and deficient in methylated MBP variants, in mice of both genotypes. The DRMs from the ND4 mice appeared to be enriched in MBP phosphorylated by MAP kinase at Thr95 (murine 18.5 kDa numbering). These studies indicate that different splice isoforms and post-translationally modified charge variants of MBP are targeted to different microdomains in the myelin membrane, implying multifunctionality of this protein family in myelin maintenance.
Subject(s)
Disease Models, Animal , Membrane Microdomains/chemistry , Multiple Sclerosis/pathology , Myelin Basic Protein/chemistry , Aging , Animals , Cell Fractionation , Cholic Acids/pharmacology , Detergents/pharmacology , Mice , Mice, Inbred Strains , Mice, Transgenic , Myelin Basic Protein/genetics , Phosphorylation , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Processing, Post-Translational , Severity of Illness Index , Silver Staining , Subcellular Fractions/chemistry , Threonine/chemistryABSTRACT
In Salmonella typhimurium, a single enzyme catalyzes both the acetyl CoA-dependent O-acetylation of hydroxylamines (a key step in the activation of mutagenic nitroaromatic compounds and related aromatic and heterocyclic amines) and the N-acetylation of aromatic amines. S. typhimurium Ames test mutants lacking this activity are highly resistant to the genotoxic effects of nitro compounds. However, such mutants have not yet been obtained in Escherichia coli. We used a PCR-based method to engineer a null mutation (deletion) of the nhoA gene encoding the enzyme in E. coli and we transduced this mutation into a lacZ strain background suitable for use in mutation assays. In E. coli, as in S. typhimurium, nhoA mutants show marked resistance to nitro compound mutagenicity. The new strains provide a clean background for expression of recombinant N-acetyltransferases.
Subject(s)
Acetyltransferases/deficiency , Escherichia coli/enzymology , Escherichia coli/genetics , Mutagens/toxicity , Nitro Compounds/toxicity , Alleles , Anti-Bacterial Agents/pharmacology , Drug Resistance, Bacterial , Frameshift Mutation/genetics , Kanamycin/pharmacology , Lac Operon/genetics , Mutagenicity Tests , Plasmids/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sp1 Transcription Factor/genetics , Transduction, Genetic , rhoA GTP-Binding Protein/geneticsABSTRACT
Multiple factors, known and unknown, contribute to human breast cancer. Hereditary, hormonal, and reproductive factors are associated with risk of breast cancer. Environmental agents, including chemical carcinogens, are modifiable risk factors to which over 70% of breast cancers have been attributed. Polymorphisms of drug-metabolizing enzymes may influence risk of breast cancer from environmental chemicals, dietary agents, and endogenous steroids. The environmental factors discussed in this review include pollutants, occupational exposures, tobacco smoke, alcohol, and diet. Aromatic amines are discussed as potential mammary carcinogens, with a focus on heterocyclic amine food pyrolysis products. These compounds are excreted into the urine after consumption of meals containing cooked meats and have recently been detected in the breast milk of lactating women.
