RESUMEN
Iron deficiency, a condition currently affecting approximately 3 billion people, persists in the 21st century despite half a millennium of medical treatment. Soybean ferritin (SBFn), a large, stable protein nanocage around a mineral with hundreds of iron and oxygen atoms, is a source of nutritional iron with an unknown mechanism for intestinal absorption. Iron absorption from SBFn is insensitive to phytate, suggesting an absorption mechanism different from for the ferrous transport. Here, we investigated the mechanism of iron absorption from mineralized SBFn using Caco-2 cells (polarized in bicameral inserts) as an intestinal cell mode and analyzed binding, internalization and degradation with labeled SBFn ((131)I or fluorescent labels), confocal microscopy, and immunoanalyses to show: 1) saturable binding to the apical cell surface; dissociation constant of 7.75 +/- 0.88 nmol/L; 2) internalization of SBFn that was dependent on temperature, concentration, and time; 3) entrance of SBFn iron into the labile iron pool (calcein quenching); 4) degradation of the SBFn protein cage; and 5) assembly peptide 2 (AP2)-/clathrin-dependent endocytosis (sensitivity of SBFn uptake to hyperosmolarity, acidity, and RNA interference to the mu(2) subunit of AP2), and resistance to filipin, a caveolar endocytosis inhibitor. The results support a model of SBFn endocytosis through the apical cell membrane, followed by protein cage degradation, mineral reduction/dissolution, and iron entry to the cytosolic iron pool. The large number of iron atoms in SBFn makes iron transport across the cell membrane a much more efficient event for SBFn than for single iron atoms as heme or ferrous ions.
Asunto(s)
Complejo 2 de Proteína Adaptadora/metabolismo , Subunidades mu de Complejo de Proteína Adaptadora/metabolismo , Endocitosis/fisiología , Células Epiteliales/fisiología , Ferritinas/metabolismo , Glycine max/química , Complejo 2 de Proteína Adaptadora/genética , Subunidades mu de Complejo de Proteína Adaptadora/genética , Células CACO-2 , Ferritinas/química , Filipina/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Hierro/metabolismo , Concentración Osmolar , Interferencia de ARNRESUMEN
Alterations in brain cholesterol concentration and metabolism seem to be involved in Alzheimer's disease (AD). In fact, several experimental studies have reported that modification of cholesterol content can influence the expression of the amyloid precursor protein (APP) and amyloid beta peptide (Abeta) production. However, it remains to be determined if changes in neuronal cholesterol content may influence the toxicity of Abeta peptides and the mechanism involved. Aged mice, AD patients and neurons exposed to Abeta, show a significant increase in membrane-associated oxidative stress. Since Abeta is able to promote oxidative stress directly by catalytically producing H(2)O(2) from cholesterol, the present work analyzed the effect of high cholesterol incorporated into human neuroblastoma cells in Abeta-mediated neurotoxicity and the role of reactive oxygen species (ROS) generation. Neuronal viability was studied also in the presence of 24S-hydroxycholesterol, the main cholesterol metabolite in brain, as well as the potential protective role of the lipophilic statin, lovastatin.
Asunto(s)
Péptidos beta-Amiloides , Colesterol/metabolismo , Neuroblastoma/metabolismo , Estrés Oxidativo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/toxicidad , Animales , Anticolesterolemiantes/metabolismo , Anticolesterolemiantes/farmacología , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/metabolismo , Supervivencia Celular , Filipina/metabolismo , Humanos , Peroxidación de Lípido , Lovastatina/metabolismo , Lovastatina/farmacología , Ratones , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The distribution of membrane filipin-sterol complexes (FSC) was examined ultrastructurally in cauda epididymal sperm from normal and hypercholesterolaemic rabbits. Membrane FSC were quantitatively analysed on replicas of filipin-treated cells. We determined a significant difference in FSC concentration in the plasma membrane of the acrosome region (PMAR) of hypercholesterolaemic animals compared to normal rabbits. Hypercholesterolaemic animals had 0.56 +/- 0.05 FSC complex per micron 2 (enriched Cholesterol diet: Diet 2) in the marginal segment of PMAR; 0.62 +/- 0.05 FSC complex per micron 2 (enriched Cholesterol and fish oil diet: Diet 3) and only 0.28 +/- 0.01 FSC complex per micron 2 for normal animals (Control Diet 1). In the principal (anterior) segment we found 0.54 +/- 0.10 FSC complex per micron 2 (Diet 2), 0.56 +/- 0.03 FSC complex per micron 2 (Diet 3) and 0.30 +/- 0.04 FSC complex per micron 2 (Control Diet 1). We also counted 0.47 +/- 0.1 FSC complex per micron 2 in the equatorial segment of PMAR for Diet 2, 0.27 +/- 0.05 and 0.28 +/- 0.04 FSC complex per micron 2 in Diet 1 and Diet 3 respectively. Diet 4 (fish oil) did not differ from the control. An increase in the Cholesterol (Chol) level in biological membranes or a difference in the Chol membrane domains could cause a variation in the membrane rigidity that could modify the sperm membrane fusion capacity and functionality. The results presented in this paper are in agreement and could explain the decrease in the kinetic of the sperm acrosome reaction that we have observed in experimentally hypercholesterolaemic rabbits (Díaz-Fontdevila & Bustos-Obregón, 1992).
Asunto(s)
Membrana Celular/química , Filipina/análisis , Hipercolesterolemia/metabolismo , Espermatozoides/química , Esteroles/análisis , Acrosoma/química , Acrosoma/ultraestructura , Animales , Colesterol en la Dieta/administración & dosificación , Grasas Insaturadas en la Dieta/administración & dosificación , Filipina/metabolismo , Aceites de Pescado/administración & dosificación , Técnica de Fractura por Congelación , Masculino , Microscopía Electrónica , Conejos , Espermatozoides/ultraestructura , Esteroles/metabolismoRESUMEN
A freeze-fracture study was carried out on spermatid and spermatozoon of the mosquito Culex quinquefasciatus. In the spermatid plasma membrane few and randomly distributed intramembranous particles were observed. In the spermatozoon the density of intramembranous particles was higher on the P- than on the E-fracture face of the plasma membrane. Two populations of particles were observed. Large particles (about 15 nm in diameter) are regularly arranged in double rows as a zipper-line, longitudinally oriented in relation to the main cell axis. These strands of particles were observed in the posterior head region, mainly associated with the E-fracture face. Filipin was used to analyse the presence and distribution of cholesterol in thin sections and freeze-fracture replicas. Filipin-sterol complexes were not homogeneously distributed throughout the spermatozoon plasma membrane. They were more abundant on the P-fracture face of the membrane lining the nuclear region. The results obtained show that Culex spermatozoon differs from those of other species in that its plasma membrane exhibits only a membrane domain, the zipper-line, localized in the postacrosomal region.