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

人小脑提取物中β淀粉样蛋白聚集抑制活性的表征

• 批准号: 09670638
• 负责人: TAMAOKA Akira
• 金额: $ 1.79万
• 依托单位: University of Tsukuba
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09670638/
• 关键词: Alzheimer's disease; amyloid β protein; cerebellum; insulin degrading enzyme; diffuse plaque; aggregation; Thioflavin T; 瀰漫性老人斑; Alzheimer's disease; amyloid β protein; cerebellum; insulin degrading enzyme; diffuse plaque; aggregation; Thioflavin T; 瀰漫性老人斑

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

为了比较阿尔茨海默氏病（AD）和正常对照大脑之间的降解和聚集活性，我们用Tris-saline缓冲液从AD和对照大脑中匀浆了脑皮质，它们提取了固体馏分，这些固体馏分在37 c co孵育，并在37 c co孵育，并与添加的合成淀粉样amibyidβ（Aβ）（Aβ）1-42 Peperides孵育。在不同的孵育时间之后，通过ECL方法将这些样品用抗Aβ1-42抗体进行免疫印迹。在对照样品中孵育12小时后，通常降低了约4KDA分子量的Aβ单体，但仅在AD大脑的样品中24小时后才降低。此外，与对照组相比，在AD样品中，具有较高分子量的涂片模式变得越来越出色。这些发现表明，与对照相比，AD大脑中的Aβ降解和凝聚活化的活性可能分别增加，并且在研究Aβ降解活性的情况下，我们测量了胰岛素降解酶（IDE）活性，此前报告的胰岛素降解酶（IDE）的活性，以前报道，在可溶性FRA中降低了Aβ的AB，在可溶性的FRA中……更多的AD和对照室中的更多t。在可溶性部分中加入II D1125 D1标记的胰岛素，并用三氯乙酸（TCA）沉淀。 TCA降水后在上清液中恢复的放射性活性被测量为IDE活性，这并未显示AD和对照大脑之间的任何显着差异，表明IDE与AD.的发病机理无关紧要，以评估来自人类小脑和夹心的Elios corebellar cortios corteb and cortios corte the corteb and cortebellar corte the cortep corte corte的aβ物质，我们对它们进行了测量。全长Aβ1-42的较低，因为总Aβ量较小。这些发现表明，在弥漫斑块中，代表小脑皮层中的主要斑块，AβX-42含有氨基末端截短的片段沉积在较早的阶段，然后是Aβ1-42和Aβ40落日，当总AβAmounts增加时，sun会积聚。从刚果红染色没有显示出脑部切片中的双折射的事实，Aβ沉积在小脑皮层中，几乎不形成淀粉样蛋白原纤维。在本文中分析了针对Aβ-凝集的抑制活性，我们使用了脑部的脑部corperaties corterication，我们用脑部的corterations sypofite corteration sypofited sypoftion sypofter sypofter sypoftion sypopent offraption syplopent offraption softers offrapt和超离心以获得上清液。将合成的Aβ1-42胡椒溶解在去离子水中以制成Aβ溶液。将小脑上清液和Aβ溶液混合并在不同的持续时间内孵育，并使用硫素T结合测定法评估Aβ-聚集，这证实小脑提取含有抑制Aβ-聚集的抑制作用。较少的