RESUMEN
Vacuolation in cellular organelles within the central nervous system is a common manifestation of oxidative injury. We found that the spongiform vacuolation observed in PVC-211 murine leukemia virus (PVC-MuLV) neurodegeneration was associated with oxidative damage as detected by immunoreactivity for 3-nitrotyrosine and protein carbonyl groups. This oxidative injury was present in brain before or concomitant with the appearance of activated microglia, vacuolation, and gliosis that characterize PVC-MuLV neuropathology. Treatment of infected F344 rat pups with the antioxidant vitamin E transiently protected and prolonged the latency of PVC-MuLV neurodegeneration. Taken together, these findings implicate oxidative damage and lipid peroxidation in the pathogenesis of PVC-MuLV neurodegeneration. This animal model may be useful for studies of mechanisms and potential therapies for progressive neurodegeneration following a well-defined insult.
Asunto(s)
Enfermedades Virales del Sistema Nervioso Central/patología , Virus de la Leucemia Murina/patogenicidad , Enfermedades Neurodegenerativas/patología , Estrés Oxidativo , Células 3T3 , Animales , Biomarcadores/análisis , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/virología , Enfermedades Virales del Sistema Nervioso Central/tratamiento farmacológico , Enfermedades Virales del Sistema Nervioso Central/metabolismo , Enfermedades Virales del Sistema Nervioso Central/virología , Suplementos Dietéticos , Ratones , Microglía/metabolismo , Microglía/patología , Enfermedades Neurodegenerativas/dietoterapia , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/virología , Oxidación-Reducción/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Ratas , Ratas Endogámicas F344 , Vacuolas/patología , Vitamina E/administración & dosificaciónRESUMEN
Phagocytic cells possess a tightly regulated multicomponent enzyme complex, the NADPH oxidase, which produces superoxide, a reactive oxygen molecule that is an essential component of host defense against infection. Upon stimulation, a functional NADPH oxidase is assembled when the cytosolic proteins, Rac, p67phox, p47phox, and possibly p40phox, associate with the gp91phox and p22phox transmembrane proteins. Rac is a GTPase that in the GTP-bound state binds p67phox to activate NADPH oxidase. The function of p40phox is not known; it is believed to have a regulatory function in sequestering p67phox and p47phox in a cytosolic complex. We investigated binding interactions between p40phox, p67phox, and Rac and found that Rac1-GTP displaced p67phox bound to p40phox. In contrast, Cdc42, a GTPase homologous to Rac, did not displace p67phox from p40phox. A synthetic peptide corresponding to p67phox amino acids 170-199, a region identified previously as a Rac binding domain, significantly reduced the ability of Rac1-GTP to disrupt p67phox/p40phox binding. We hypothesize that Rac-GTP binds the p67phox N-terminal domain encompassing amino acids 170-199 that transmits a conformational change which causes p40phox to dissociate from its binding site in the p67phox C-terminus.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , NADPH Oxidasas/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Activación Enzimática , Humanos , Técnicas In Vitro , NADPH Oxidasas/química , NADPH Oxidasas/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fosfoproteínas/química , Fosfoproteínas/genética , Unión Proteica , Conformación Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteína de Unión al GTP cdc42 , Proteínas de Unión al GTP racRESUMEN
OBJECTIVE: To identify and characterize 4 components of the NADPH oxidase complex, gp91-phox, p22-phox, p67-phox, and p47-phox, in common laboratory animal species. ANIMALS: 2 clinically normal animals from each of the following species: rabbit, sheep, cow, pig, and macaque (Macaca nemistrina). A pool of 8 rats. PROCEDURE: Neutrophils were harvested from blood, Membrane and cytosol fractions were isolated and separated by sodium dodecyl sulfate-polyacrylamide gels. Gels were transferred, and immunoblots were probed with antibodies directed against individual human NADPH oxidase proteins. Human neutrophil membrane and cytosol fractions served as controls. RESULTS: Immunoreactive bands were observed in all species for gp91-phox, p67-phox, and p47-phox proteins. Immunoreactive bands for p22-phox protein were observed in cells from rats, rabbits, pigs, and macaques. CONCLUSIONS: The NADPH oxidase and its component proteins have been highly conserved across mammalian species. Lack of immunoreactivity to p22-phox in sheep and cows can be explained by sequence divergence and epitope variability at the p22-phox C-terminus. CLINICAL RELEVANCE: The high degree of NADPH oxidase protein conservation may allow the existing knowledge of the human neutrophil NADPH oxidase to be applied to the study of animal disease.
Asunto(s)
Mamíferos/sangre , NADH NADPH Oxidorreductasas/sangre , Neutrófilos/enzimología , Animales , Bovinos , Fraccionamiento Celular , Membrana Celular/enzimología , Membrana Celular/ultraestructura , Separación Celular/veterinaria , Citosol/enzimología , Citosol/ultraestructura , Electroforesis en Gel de Poliacrilamida/veterinaria , Immunoblotting/veterinaria , Macaca nemestrina , NADH NADPH Oxidorreductasas/química , NADPH Oxidasas , Neutrófilos/citología , Neutrófilos/ultraestructura , Conejos , Ratas , Ovinos , PorcinosRESUMEN
The phagocyte NADPH oxidase complex is an unusual electron transfer system. Its principal component, cytochrome b558, is a heme-containing integral membrane protein consisting of two subunits, gp91-phox and p22-phox. We used a novel method to measure precisely the gp91-phox:p22-phox stoichiometry. Cytochrome b558 was isolated in high purity from human neutrophil membrane preparations using a novel affinity purification method. We performed direct peptide sequencing of purified cytochrome b558 and detected two amino acid sequences which matched predicted sequences for gp91-phox and p22-phox. We quantitated amounts of both amino acids released from p22-phox and gp91-phox in each sequencing cycle. Averaging over 25 cycles, the mean p22-phox:gp91-phox ratio of released amino acids was 0.93 +/- 0.01. To correct for recovery differences between individual amino acids, we measured individual p22-phox:gp91-phox ratios for the eight different amino acids common to both p22-phox and gp91-phox in the first 25 positions. The mean of individual p22-phox:gp91-phox ratios for the eight common amino acids was 0.96 +/- 0.05. The p22-phox:gp91-phox ratios for each of the eight common amino acids varied from 0.81 to 1.20. Taken together, measured ratios for total and individual amino acids are consistent with a predicted ratio of 1.0 for 1:1 p22-phox:gp91-phox stoichiometry in cytochrome b558.