Use of Optogenetic Amyloid-β to Monitor Protein Aggregation in Drosophila melanogaster, Danio rerio and Caenorhabditis elegans
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Author:
Date:
2020-12-05
[Abstract] Alzheimer’s Disease (AD) has long been associated with accumulation of extracellular amyloid plaques (Aβ) originating from the Amyloid Precursor Protein. Plaques have, however, been discovered in healthy individuals and not all AD brains show plaques, suggesting that extracellular Aβ aggregates may play a smaller role than anticipated. One limitation to studying Aβ peptide in vivo during disease progression is the inability to induce aggregation in a controlled manner. We developed an optogenetic method to induce Aβ aggregation and tested its biological influence in ...
[摘要] [摘要]Alzheimer'sdisease(AD)长期以来与淀粉样前体蛋白产生的细胞外淀粉样斑块(Aβ)的积聚有关。然而,在健康人身上发现了斑块,并不是所有的AD大脑都有斑块,这表明细胞外Aβ聚集体的作用可能比预期的要小。在疾病进展过程中研究Aβ肽的一个局限性是无法以可控的方式诱导聚集。我们开发了一种诱导Aβ聚集的光遗传学方法,并在三种模式生物中测试了其生物学效应:D.melanogaster、C.elegans和D.rerio。我们产生了一个荧光标记的,光生的
一种β肽,在所有生物体内,在蓝光存在下迅速寡聚。在这里,我们详细介绍了在动物模型中表达该融合蛋白的程序,使用延时光片显微镜研究对神经系统的影响,并进行代谢分析来测量由于细胞内Aβ聚集而引起的变化。这种方法利用光遗传学来研究AD的病理学,实现了目前任何其他方法都无法实现的体内时空控制。
[背景]阿尔茨海默病(AD)是一种衰弱的、与年龄相关的神经退行性疾病(Zhang等人,2011年;De ...
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Protease Activity Assay in Fly Intestines
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Author:
Date:
2017-09-20
[Abstract] The intestine is a central organ required for the digestion of food, the absorption of nutrients and for fighting against aggressors ingested along with the food. Impairment of gut physiology following mucosal damages impacts its digestive capacities that consequently will affect growth, wellbeing or even survival of the individual. Hence, the assessment of intestinal functions encompasses, among others, the monitoring of its integrity, its cellular renewing, its immune defenses, the production of enteroendocrine hormones and its digestive capacities. Here, we describe in detail how to assess ...
[摘要] 肠是消化食物所需的中枢器官,吸收营养物质,并与食物一起摄入的侵略者作斗争。 粘膜损伤后的肠道生理损伤影响其消化能力,从而影响个体的生长,健康甚至生存。 因此,肠功能的评估包括监测其完整性,细胞更新,免疫防御,肠内分泌激素的产生及其消化能力。 在这里,我们详细描述如何评估分泌在成年果蝇的肠腔中的蛋白酶的活性。 这种方法也可以用于幼虫肠。 本协议由“蛋白酶荧光检测试剂盒”(产品代码PF0100)中提出的Sigma-Aldrich公司的方案进行了改进和改进。 【背景】肠道受到诸如斋戒,禁食,化学物质,病原体,损伤等诸多压力的影响。肠道能够通过保持其体内平衡的生理平衡来克服这种压力。为了感知进入的压力并产生适应性的维持肠功能的答案,肠已经开发出强健和保守的机制,如局部先天免疫防御和组织再生(Royet和Charroux,2013; Bonfini等,2016)。然而,在某些情况下,肠道稳态的维持可能受到影响。例如,在老化期间,随着许多未成熟或分化不良的细胞的存在,组织稳态维持总体下降(Jasper,2015; ...
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Fluorometric Estimation of Glutathione in Cultured Microglial Cell Lysate
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Author:
Date:
2017-06-05
[Abstract] Glutathione is one of the major antioxidant defense components present in cells. It is predominantly present as reduced glutathione (GSH) and converted into oxidized glutathione (GSSG) while reducing the free radicals like hydroxyl ions (OH-). For the measurement of GSH and GSSG, o-phthalaldehyde (OPT) has been used as a fluorescent reagent. O-phthalaldehyde has an ability to react specifically with GSH at pH 8 and GSSG at pH 12 respectively. N-ethylmaleimide (NEM) has been used to prevent auto-oxidation of GSH during measurement of GSSG in the present protocol. The ...
[摘要] 谷胱甘肽是细胞中存在的主要抗氧化防御成分之一。它主要作为还原型谷胱甘肽(GSH)存在并转化为氧化型谷胱甘肽(GSSG),同时还可以减少自由基,如羟基离子(OH)。对于GSH和GSSG的测定,邻苯二甲醛(OPT)已被用作荧光试剂。邻苯二甲醛具有与pH8的GSH和pH12的GSSG特异性反应的能力。已经使用N-甲基马来酰亚胺(NEM)来在本方案中测量GSSG期间防止GSH的自动氧化。 Hissin和Hilf的原始方案被开发用于大鼠肝组织中的谷胱甘肽估计。目前的方案已经在Hissin和Hilf(1976)之后被标准化,用于估计培养的小胶质细胞裂解物中的谷胱甘肽,但它也可以用于其它哺乳动物细胞裂解物。在我们的实验中,相同的方案已被用于估计小鼠神经母细胞瘤细胞N2a的全细胞裂解物中的谷胱甘肽。
背景 该方法于1976年由Hissin和Hilf在分析生物化学方面出版(Hissin和Hilf,1976)。然而,有可用于准确检测GSH的方法;由于GSH容易氧化转化为GSSG,所以大多数方法给出过高的GSSG。 ...
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