Characterization of the inhibitory activity against aggregation of amyloid β protein in the extract of human cerebellum

为了比较阿尔茨海默氏病（AD）和正常控制脑之间的退化和聚合活动，我们从AD和Tris-saline缓冲液中同源的晶体管和控制脑中提取的溶解碎片，在37 ° C中孵化了附加的合成淀粉样β （Aβ） 1-42肽。在不同的孵化时间之后，这些样本是用ECL方法对抗-A β1-42抗体免疫的。带有~ 4 kDa分子重量的Aβ单体在12小时后在控制样品中通常减少，但在24小时后在样品中从AD大脑中减少。在添加中，较小的模式与较高的分子重量在AD样本中变得更引人注目，与控件相比。这些发现建议，Aβ-退化和聚合活性可能被分解和增加在与控制比较的AD脑中适当地进行比较。为了调查Aβ-退化活性，我们测量了胰岛素退化酶（IDE）活性，以前曾报道过退化Aβ，在解决方案中。 ... More 来自广告和控制大脑的策略。I-D1125 I-D1-labeled insulin was added in soluble fractions and precipitated with trichloroacetic acid （TCA）。在TCA预防措施被测量为IDE活动后，在显性植物中恢复的放射活性，在AD和控制大脑之间没有显示任何明显的差异，指示IDE不能在AD的病理发生中发挥作用。要评估从人类Cerebellum提取的片段中提取的Aβ物种的数量，我们用sandwich ELISA测量它们，并证实，Cerebellar Cortices包含比A β42更多的Aβ40和全长度Aβ1-42比Aβ 40更小的比率。这些发现包括在分歧片中，在晶粒细胞中表现出的主片，在早期阶段含有Aβ x-42的氨基末端断裂片段沉积物，然后在Aβ1-42和Aβ40的情况下，在总Aβ剂量增加时，充分吸收Aβ x-42。来自刚果红染色没有显示出在cerebellar sections中的birefringence， Aβ Deposited in cerebellar cortices scarcely form amyloid fibrils.最后到分析抑制剂活动against Aβ-聚合，我们已部署的cerebellar cortices，其中表明主要包含非纤维Aβ Deposition。Cerebellar Cortices与正常人类自动化的大脑、与磷酸盐缓冲区同源化，并被超饱和吸收。合成Aβ1-42肽在电离水中溶解，以产生Aβ溶液。Cerebellar Supernatant和Aβ溶液在不同的时间混合并孵化，Aβ-聚合使用Thiflavin T绑定试验进行了评估，证实了Cerebellar提取物包含抑制剂活动反对Aβ-聚合。Less（低）

To compare the degrading and the aggregating activity between Alzheimer's disease (AD) and normal control brains, we homogenized cerebral cortices from AD and control brains with Tris-saline buffer, extracted soluble fractions, which were incubated at 37 ゜C with added synthetic amyloid β (Aβ) 1-42 peptides. After different incubation times, these samples were immunoblotted with anti-Aβ1-42 antibody by ECL method. Aβ monomer with 〜4kDa molecular weight was usually decreased after 12 hours incubation in control samples, but only after 24 hours in samples from AD brains. In addition, smear patterns with higher molecular weights were getting more remarkable in AD samples as compared with controls. These findings suggest that Aβ-degrading and -aggregating actives may be decreased and increased respectively in AD brains in comparison with controls.To investigate Aβ-degradating activities, we measured insulin degrading enzyme (IDE) activities, previously reported to degrade Aβ, in soluble fra … More ctions from AD and control brains. IィイD1125ィエD1-labeled insulin was added in soluble fractions and precipitated with trichloroacetic acid (TCA). Radioactivities recovered in supernatants after TCA precipitation were measured as IDE activities, which did not show any significant differences between AD and control brains, indicating that IDE could not be involved in the pathogenesis of AD.To evaluate amounts of Aβ species in extracted fractions from human cerebellum, we measured them using sandwich ELISA, and revealed that cerebellar cortices contain more Aβ42 than Aβ40 and the ratios of full-length Aβ1-42 were lower as the total Aβ amounts were smaller. These findings suggest that in diffuse plaques, which represent main plaques in cerebellar cortices, Aβx-42 containing amino-terminal truncated fragments deposits in earlier stage and then Aβ1-42 and Aβ40 sunsequantly accumulate when total Aβamounts increase. From the fact that Congo red staining did not show birefringence in cerebellar sections, Aβ deposited in cerebellar cortices scarcely form amyloid fibrils.Finally to analyze inhibitory activities against Aβ-aggregation, we employed cerebellar cortices, which has been revealed to mainly contain non-fibrillar Aβ deposition. Cerebellar cortices were separated from normal human autopsied brains, homogenized with phosphate buffer, and ultracentrifuged to get supernatants. Synthetic Aβ1-42 peptides were solubilized in deionized water to make Aβ solution. Cerebellar supernatant and Aβ solution were mixed and incubated for different durations, and Aβ-aggregation were assessed using Thiflavin T binding assay, which confirmed that cerebellar extractions contain inhibitory activities against Aβ-aggregation. Less