Conformational Changes and Association of Membrane-Interacting Peptides in Myelin Membrane Models: A Case of the C-Terminal Peptide of Proteolipid Protein and the Antimicrobial Peptide Melittin.

Model membranes composed of various lipid mixtures can segregate into liquid-ordered (Lo) and liquid-disordered (Ld) phases. In this study, lipid vesicles composed of mainly Lo or Ld phases as well as complex lipid systems representing the cytosolic leaflet of the myelin membrane were characterized by fluorescence resonance energy transfer with a donor/acceptor pair that preferentially partitioned into Lo or Ld phases, respectively. The fluidity of the lipid systems containing >30% cholesterol was modulated in the presence of the amphipathic peptide melittin. With all the studied lipid systems, melittin attained an α-helical conformation as determined by CD spectroscopy and attained varying degrees of membrane association and penetration as determined by intrinsic Trp fluorescence. The other protein domain utilized was a putative amphipathic helical peptide derived from the cytosolic C-terminal sequence of proteolipid protein (PLP) which is the most abundant protein in the myelin membrane. The C-terminal PLP peptide transitioned from a random coil to an α-helix in the presence of trifluoroethanol. Upon interacting with each of lipid vesicle system, the PLP peptide also folded into a helix; however, at high concentrations of the peptide with fluid lipid systems, associated helices transmuted into a ß-sheet conformer. The membrane-associated aggregation of the cytosolic C-termini could be a mechanism by which the transmembrane PLP multimerizes in the myelin membrane.

Dynamic turn conformation of a short tryptophan-rich cationic antimicrobial peptide and its interaction with phospholipid membranes.

Cationic antimicrobial peptides are promising sources for novel therapeutic agents against multi-drug-resistant bacteria. HHC-36 (KRWWKWWRR) is a simple but effective antimicrobial peptide with similar or superior activity compared with several conventional antibiotics. In this biophysical study, unique conformational properties of this peptide and some of its analogs as well as its interaction with lipid membranes are investigated in detail. Circular dichroism (CD) and molecular dynamics modeling studies of HHC-36 in different environments reveal a dynamic amphipathic structure composed of competing turn conformations with free energies lower than that of the unfolded state, implying a strong influence of tryptophan interactions in formation of the turns. CD spectra and gel electrophoresis also show strong evidence of self-association of this peptide in aqueous milieu and interaction with both neutrally and negatively charged lipid membrane systems. Isothermal titration calorimetry and acrylamide fluorescence quenching experiments emphasize the preference of HHC-36 for negatively charged vesicles. In addition, dye leakage experiments suggest that this peptide functions through a surface-associated mechanism with weak lytic activity against bacterial model membranes.

White matter rafting--membrane microdomains in myelin.

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.

Partitioning of myelin basic protein into membrane microdomains in a spontaneously demyelinating mouse model for multiple sclerosis.

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.

N-hydroxyarylamine O-acetyltransferase-deficient Escherichia coli strains are resistant to the mutagenicity of nitro compounds.

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.

Perspectives on the chemical etiology of breast cancer.

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.