RESUMEN
Oxidative stress markers as well as high concentrations of copper are found in the vicinity of Abeta amyloid deposits in Alzheimer's disease. The neurotoxicity of Abeta in cell culture has been linked to H(2)O(2) generation by an unknown mechanism. We now report that Cu(II) markedly potentiates the neurotoxicity exhibited by Abeta in cell culture. The potentiation of toxicity is greatest for Abeta1-42 > Abeta1-40 >> mouse/rat Abeta1-40, corresponding to their relative capacities to reduce Cu(II) to Cu(I), form H(2)O(2) in cell-free assays and to exhibit amyloid pathology. The copper complex of Abeta1-42 has a highly positive formal reduction potential ( approximately +500-550 mV versus Ag/AgCl) characteristic of strongly reducing cuproproteins. These findings suggest that certain redox active metal ions may be important in exacerbating and perhaps facilitating Abeta-mediated oxidative damage in Alzheimer's disease.
Asunto(s)
Péptidos beta-Amiloides/toxicidad , Encéfalo/efectos de los fármacos , Cobre/farmacología , Peróxido de Hidrógeno/metabolismo , Animales , Células Cultivadas , Simulación por Computador , Cobre/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Oxidación-Reducción , RatasRESUMEN
Oxidative stress markers characterize the neuropathology both of Alzheimer's disease and of amyloid-bearing transgenic mice. The neurotoxicity of amyloid A beta peptides has been linked to peroxide generation in cell cultures by an unknown mechanism. We now show that human A beta directly produces hydrogen peroxide (H2O2) by a mechanism that involves the reduction of metal ions, Fe(III) or Cu(II), setting up conditions for Fenton-type chemistry. Spectrophotometric experiments establish that the A beta peptide reduces Fe(III) and Cu(II) to Fe(II) and Cu(I), respectively. Spectrochemical techniques are used to show that molecular oxygen is then trapped by A beta and reduced to H2O2 in a reaction that is driven by substoichiometric amounts of Fe(II) or Cu(I). In the presence of Cu(II) or Fe(III), A beta produces a positive thiobarbituric-reactive substance (TBARS) assay, compatible with the generation of the hydroxyl radical (OH.). The amounts of both reduced metal and TBARS reactivity are greatest when generated by A beta 1-42 >> A beta 1-40 > rat A beta 1-40, a chemical relationship that correlates with the participation of the native peptides in amyloid pathology. These findings indicate that the accumulation of A beta could be a direct source of oxidative stress in Alzheimer's disease.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Peróxido de Hidrógeno/metabolismo , Metales Pesados/metabolismo , Péptidos beta-Amiloides/química , Animales , Cobre/química , Cobre/metabolismo , Compuestos Férricos/química , Compuestos Férricos/metabolismo , Compuestos Ferrosos/química , Compuestos Ferrosos/metabolismo , Humanos , Peróxido de Hidrógeno/química , Sustancias Macromoleculares , Metales Pesados/química , Oxidación-Reducción , Ratas , Especificidad de la Especie , Superóxidos/química , Superóxidos/metabolismo , Sustancias Reactivas al Ácido Tiobarbitúrico/químicaRESUMEN
The cortical deposition of Abeta is an event that occurs in Alzheimer's disease, Down's syndrome, head injury, and normal aging. Previously, in appraising the effects of different neurochemical factors that impact upon the solubility of Abeta, we observed that Zn2+ was the predominant bioessential metal to induce the aggregation of soluble Abeta at pH 7.4 in vitro and that this reaction is totally reversible with chelation. We now report that unlike other biometals tested at maximal biological concentrations, marked Cu2+-induced aggregation of Abeta1-40 emerged as the solution pH was lowered from 7.4 to 6.8 and that the reaction was completely reversible with either chelation or alkalinization. This interaction was comparable to the pH-dependent effect of Cu2+ on insulin aggregation but was not seen for aprotinin or albumin. Abeta1-40 bound three to four Cu2+ ions when precipitated at pH 7.0. Rapid, pH-sensitive aggregation occurred at low nanomolar concentrations of both Abeta1-40 and Abeta1-42 with submicromolar concentrations of Cu2+. Unlike Abeta1-40, Abeta1-42 was precipitated by submicromolar Cu2+ concentrations at pH 7.4. Rat Abeta1-40 and histidine-modified human Abeta1-40 were not aggregated by Zn2+, Cu2+, or Fe3+, indicating that histidine residues are essential for metal-mediated Abeta assembly. These results indicate that H+-induced conformational changes unmask a metal-binding site on Abeta that mediates reversible assembly of the peptide. Since a mildly acidic environment together with increased Zn2+ and Cu2+ are common features of inflammation, we propose that Abeta aggregation by these factors may be a response to local injury. Cu2+, Zn2+, and Fe3+ association with Abeta explains the recently reported enrichment of these metal ions in amyloid plaques in Alzheimer's disease.
Asunto(s)
Acidosis/metabolismo , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Cobre/metabolismo , Fragmentos de Péptidos/metabolismo , Animales , Humanos , Concentración de Iones de Hidrógeno , Unión Proteica , Ratas , Zinc/metabolismoRESUMEN
The heterogeneous precipitates of Abeta that accumulate in the brain cortex in Alzheimer's disease possess varying degrees of resistance to resolubilization. We previously found that Abeta1-40 is rapidly precipitated in vitro by physiological concentrations of zinc, a neurochemical that is highly abundant in brain compartments where Abeta is most likely to precipitate. We now present evidence that the zinc-induced precipitation of Abeta is mediated by a peptide dimer and favored by conditions that promote alpha-helical and diminish beta-sheet conformations. The manner in which the synthetic peptide is solubilized was critical to its behavior in vitro. Zinc-induced Abeta aggregation was dependent upon the presence of NaCl, was enhanced by alpha-helical-promoting solvents, but was abolished when the peptide stock solution was stored frozen. The Abeta aggregates induced by zinc were reversible by chelation, but could then be reprecipitated by zinc for several cycles, indicating that the peptide's conformation is probably preserved in the zinc-mediated assembly. In contrast, Abeta aggregates induced by low pH (5.5) were not resolubilized by returning the pH milieu to 7.4. The zinc-Abeta interaction exhibits features resembling the gelation process of zinc-mediated fibrin assembly, suggesting that, in events such as clot formation or injury, reversible Abeta assembly could be physiologically purposive. Such a mechanism is contemplated in the early evolution of diffuse plaques in Alzheimer's disease and suggests a possible therapeutic strategy for the resolubilization of some forms of Abeta deposit in the disease.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Fragmentos de Péptidos/metabolismo , Zinc/farmacología , Péptidos beta-Amiloides/aislamiento & purificación , Encéfalo/metabolismo , Cromatografía en Gel , Humanos , Fragmentos de Péptidos/aislamiento & purificación , Unión Proteica , Conformación ProteicaRESUMEN
We have used an immunofluorescent technique to localize type II insulin-like growth factor (IGF) receptors in rat liver, rat hepatocytes and three rat hepatoma cell lines (HTC, H-35 and 5123) using a polyclonal antibody (C-1) raised to purified rat liver type II IGF receptor. Specificity of the antiserum was confirmed by Western blotting of microsomal membranes prepared from hepatocytes and hepatoma cells which showed a single class of receptor in all cells, of Mr approximately 210,000 for hepatocytes, HTC and H-35 cells and approximately 220,000 for 5123 cells, on non-reduced, 4-15% polyacrylamide gradient gels. The specificity of the immunofluorescent technique was also verified by abolition of labelling after preincubation of antiserum with purified type II IGF receptor. Rat liver cryosections contained areas of juxtanuclear labelling in hepatocytes, consistent with the presence of type II IGF receptor in the Golgi region. Brightest immunofluorescence was seen in sections from fetal and neonatal rats with adult rat hepatocytes staining brightly only around central veins. Areas of labelling were also seen in connective tissue surrounding larger veins. Cultured adult rat hepatocytes and rat hepatoma cell lines also showed bright areas of juxtanuclear nuclear immunofluorescence, with HTC and H-35 cells staining more than 5123 and adult hepatocytes. Fetal rat hepatocytes in culture also labelled very brightly both in a juxtanuclear location and in small clusters over the cell, possibly on the cell surface. These observations indicate that type II IGF receptors are located predominantly on intracellular membranes and are most abundant in rapidly growing cells and tissues (such as fetal liver and hepatoma cells).
Asunto(s)
Factor II del Crecimiento Similar a la Insulina/análisis , Neoplasias Hepáticas Experimentales/análisis , Hígado/análisis , Receptores de Superficie Celular/análisis , Somatomedinas/análisis , Envejecimiento , Animales , Western Blotting , Células Cultivadas , Feto , Técnica del Anticuerpo Fluorescente , Ratas , Receptores de SomatomedinaRESUMEN
The response of megakaryocytes to thrombin (1-10 U/ml) has been examined by time-lapse cinemicrography and electron microscopy. The study was confined to mature megakaryocytes which had developed processes following incubation in vitro. The initial response of all cells was to undergo retraction of processes, behaviour thought to be linked with the depolymerization of microtubules which extend longitudinally through the processes. The majority of cells completely withdrew their processes, but about 30% responded differently and underwent only limited retraction, followed by secretion. Analysis of time-lapse film showed that processes from the latter group of cells had formed attachments with the coverslip prior to exposure to thrombin. Within the partially retracted processes of these cells, secretory granules were found to be clustered centripetally and enveloped by a microfilamentous structure in the form of a cylinder. Vacuoles appeared, some of which were located outside the microfilamentous structure. Microtubules were present, but many appeared disorientated. The shape of the microfilamentous structure suggests that the cytoplasm is not organized into putative platelets at the time of process formation.
Asunto(s)
Megacariocitos/efectos de los fármacos , Trombina/farmacología , Citoesqueleto de Actina/ultraestructura , Animales , Gránulos Citoplasmáticos/ultraestructura , Técnicas In Vitro , Masculino , Megacariocitos/ultraestructura , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica , Microtúbulos/ultraestructura , Factores de Tiempo , Vacuolas/ultraestructuraRESUMEN
We have examined megakaryocyte process fragments that migrate out of bone marrow explants after a short period of incubation and assume a beaded form, consisting of 2 or more putative platelets. The fragmentation appears to occur in vivo and supports the proposal that platelet liberation does not always occur in a sequential manner from the distal ends of megakaryocyte processes. Transmission electron microscopy revealed that microtubules were generally oriented longitudinally in the process fragments. Rarely, a microtubule coil was found in a terminally located putative platelet. The observations favour the view that the marginal coil of microtubules, which is a characteristic of circulating platelets, does not usually form until after platelets have been liberated.