Asunto(s)
Adyuvantes Inmunológicos/uso terapéutico , Aminoquinolinas/uso terapéutico , Leucoplasia Bucal/tratamiento farmacológico , Infecciones por Papillomavirus/tratamiento farmacológico , Anciano , Femenino , Humanos , Imiquimod , Leucoplasia Bucal/virología , Persona de Mediana Edad , Prevención Secundaria , Resultado del TratamientoRESUMEN
Apolipoprotein E (apoE) 4 and aging are risk factors for Alzheimer's disease (AD). Mice expressing human apoE4 and aged wild-type mice show a similarity in the transbilayer distribution of cholesterol in synaptic plasma membranes (SPMs) but differ markedly compared with apoE3 mice and young mice. The largest changes in cholesterol distribution were observed in the SPM exofacial leaflet where there was a doubling of cholesterol. Lipid rafts are thought to be associated with the exofacial leaflet, and we proposed that lipid raft protein and lipid composition would be associated with apoE genotype and age. Lipid rafts were isolated from synaptosomes of different age groups (2, 12, 24 months) of mice expressing human apoE3 and apoE4. Lipid raft markers, alkaline phosphatase (ALP), flotillin-1, cholesterol and sphingomyelin (SM) were examined. Lipid rafts of young apoE4 mice were more similar to older mice as compared with young apoE3 mice in reductions in alkaline phosphatase activity and flotillin-1 abundance. Lipid raft cholesterol and sphingomyelin levels were not significantly different between the young apoE3 and apoE4 mice but cholesterol levels of lipid rafts did increase with age in both genotypes. Results of the present study demonstrate that the two risk factors for Alzheimer's disease, apoE4 genotype and increasing age have similar effects on brain lipid raft protein markers and these findings support the notion that the transbilayer distribution of cholesterol is associated with lipid raft function.
Asunto(s)
Envejecimiento/metabolismo , Apolipoproteínas E/metabolismo , Microdominios de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Sinaptosomas/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Apolipoproteína E3 , Apolipoproteína E4 , Colesterol/sangre , Glicosilfosfatidilinositoles/metabolismo , Humanos , Técnicas In Vitro , Luz , Ratones , Ratones Transgénicos , Dispersión de Radiación , Esfingomielinas/sangreRESUMEN
A polysaccharide consisting of mainly 1,4-linked glucose units was found associated with prion rods, which are composed mainly of insoluble aggregates of the N-terminally truncated prion protein (PrP 27-30) exhibiting the ultrastructural and tinctorial properties of amyloid. The polysaccharide differs in composition from the Asn-linked oligosaccharides and the GPI-anchor of the prion protein. Prion rods were prepared from scrapie-infected hamster brains using two different purification protocols. Prolonged digestion of rods with proteinase K reduced PrP by a factor of at least 500, leaving about 10% (w/w) of the sample as an insoluble remnant. Only glucose was obtained by acid hydrolysis of the remnant and methylation analysis showed 80% 1,4-, 15% 1,6- and 5% 1,4,6-linked glucose units. The physical and chemical properties as well as the absence of terminal glucose units indicate a very high molecular mass of the polysaccharide. No evidence was found for covalent bonds between PrP and the polysaccharide. The polysaccharide certainly contributes to the unusual chemical and physical stability of prion rods, acting like a scaffold. A potential structural and/or functional relevance of the polysaccharide scaffold is discussed.
Asunto(s)
Polisacáridos/química , Priones/química , Animales , Asparagina/química , Bioquímica/métodos , Conformación de Carbohidratos , Secuencia de Carbohidratos , Cricetinae , Endopeptidasa K/química , Endopeptidasa K/metabolismo , Glucanos/análisis , Glucanos/química , Glucosa/análisis , Glicosilfosfatidilinositoles/química , Datos de Secuencia Molecular , Polisacáridos/análisis , Polisacáridos/aislamiento & purificación , Priones/metabolismoRESUMEN
The N-terminally truncated form of the prion protein, PrP 27-30, and the corresponding recombinant protein, rPrP, were solubilized in 0.2% SDS, and the transitions induced by changing the conditions from 0.2% SDS to physiological conditions, i.e. removing SDS, were characterized with respect to solubility, resistance to proteolysis, secondary structure and multimerization. Circular dichroism, electron microscopy and fluorescence correlation spectroscopy were used to study the structural transitions of PrP. Within one minute the alpha-helical structure of PrP was transformed into one that was enriched in beta-sheets and consisted mainly of dimers. Larger oligomers were found after 20 minutes and larger multimers exhibiting resistance to proteolysis were found after several hours. It was concluded that the monomeric alpha-helical conformation was stable in SDS or when attached to the membrane; however, the state of lowest free energy in aqueous solution at neutral pH seems to be the multimeric, beta-sheet enriched conformation.