Separation and Purification of Glycosaminoglycans (GAGs) from Caenorhabditis elegans
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Author:
Date:
2017-08-05
[Abstract] The nematode Caenorhabditis elegans is a popular model organism for studies of developmental biology, neurology, ageing and other fields of basic research. Because many developmental processes are regulated by glycosaminoglyans (GAGs) on cell surfaces and in the extracellular matrix, methods to isolate and analyze C. elegans GAGs are needed. Such methods have previously been optimized for other species such as mice and zebrafish. After modifying existing purification protocols, we could recently show that the nematodes also produce chondroitin sulfate, in addition to heparan sulfate, thus challenging the view that only non-sulfated chondroitin was synthesized by C. elegans. We here present our protocol adapted for C. elegans. Since the purification ...
[摘要] 线虫秀丽隐杆线虫是研究发育生物学,神经学,衰老等基础研究领域的流行模型生物。 因为许多发育过程由细胞表面和细胞外基质中的糖胺聚糖(GAG)调节,分离和分析C的方法。 线虫需要GAG。 此类方法先前已针对其他物种如小鼠和斑马鱼进行了优化。 在修改现有的纯化方案后,我们最近可以显示除了硫酸乙酰肝素外,线虫也产生硫酸软骨素,因此挑战了仅通过C合成非硫酸软骨素的观点。线虫。 我们在这里介绍我们适用于C的协议。线虫。 由于净化策略涉及非硫酸化和硫酸化GAG的分离,所以对于其他可能有利的方法也是有用的。 【背景】糖胺聚糖(GAG)是重复二糖单元的直链多糖链,其通常被硫酸根取代。除了透明质酸,其是在质膜上合成而不被锚定到任何蛋白质的非硫酸化GAG,所有其他GAG都与核心蛋白共价连接,从而形成蛋白聚糖(PG)。在PG上发现的最常见的GAG是硫酸乙酰肝素(HS)和硫酸软骨素(CS)/硫酸皮肤素,含有N-乙酰基 - 葡糖胺和N,N-乙酰基 - 半乳糖胺,分别(张,2010)。在高尔基隔室的生物合成过程中,它们是非模板驱动的过程,它们经过多种修饰,包括将葡萄糖醛酸差向异构化成艾杜糖醛酸,并在不同位置硫酸化(Bulow和Hobert,2006; ...
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In vivo Mitophagy Monitoring in Caenorhabditis elegans to Determine Mitochondrial Homeostasis
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Author:
Date:
2017-04-05
[Abstract] Perturbation of mitochondrial function is a major hallmark of several pathological conditions and ageing, underlining the essential role of fine-tuned mitochondrial activity (Lopez-Otin et al., 2013). Mitochondrial selective autophagy, known as mitophagy, mediates the removal of dysfunctional and/or superfluous organelles, preserving cellular and organismal homeostasis (Palikaras and Tavernarakis, 2014; Pickrell and Youle, 2015; Scheibye-Knudsen et al., 2015). In this protocol, we describe a method for assessing mitophagy in the nematode Caenorhabditis elegans.
[摘要] 线粒体功能的扰动是几种病理状况和衰老的主要标志,强调了线粒体活性调控的重要作用(Lopez-Otin等,2013)。 线粒体选择性自噬,称为嗜中性细胞因子介导功能障碍和/或多余的细胞器的去除,保留细胞和有机体内稳态(Palikaras和Tavernarakis,2014; Pickrell和Youle,2015; Scheibye-Knudsen等,2015)。 在这个协议中,我们描述了一种评估线虫秀丽隐杆线虫线粒体的方法。 【背景】线粒体被认为是真核细胞的细胞动力,因为它们是通过氧化磷酸化(OXPHOS)和ATP生成的主要能量提供者。此外,它们在细胞稳态中的关键作用突出表现在它们对几种基本细胞过程(包括钙缓冲,代谢物合成和凋亡等)调节的贡献。线粒体功能的放松规律与几种病理状况(包括衰老和年龄相关的神经变性疾病)的发病有关(Vafai和Mootha,2012; Palikaras和Tavernarakis,2014)。因此,真核生物已经发展了几种复杂和高度专门化的分子途径来保护能量稳态(Pickrell和Youle,2015; Scheibye-Knudsen等,2015)。 Mitophagy是一种选择性类型的自噬,促进线粒体受损的消除,以及细胞调节线粒体内源物质和环境信号的主要降解途径(Palikaras等,2015; Schiavi et al。,2015; ...
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Penetration Assays, Fungal Recovery and Pathogenicity Assays for Verticillium dahliae
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Author:
Date:
2017-02-20
[Abstract] Verticillium dahliae is a soil-borne phytopathogenic fungus that infects host roots and proliferates in vascular tissues. The great loss of economically important crop caused by V. dahliae has raised worldwide concern, however, little is known about the mechanism of its pathogenicity (Klosterman et al., 2011; Yadeta and Thomma, 2013). Our recent work has shown that V. dahliae develops hyphopodium as an infection structure to breach plant root cell wall (Zhao et al., 2016). Here, we provide a detailed protocol to analyze the penetration ability and the pathogenicity of V. dahliae as well as recover fungal hyphae from infected cotton stems developed from our previous studies (Zhang et al., 2016a and 2016b; Zhao et al., ...
[摘要] 大丽轮枝菌属是土壤传播的植物病原真菌,其感染宿主根并在血管组织中增殖。经济上重要的作物造成的巨大损失。大丽花已经引起了全世界的关注,然而,其致病性机制知之甚少(Klosterman等人,2011; Yadeta和Thomma,2013)。我们最近的工作表明,大丽花开发hyphopodium作为感染结构以破坏植物根细胞壁(Zhao等人,2016)。在这里,我们提供了一个详细的方案来分析渗透能力和致病性。大丽花,以及从以前的研究(Zhang等人,2016a和2016b; Zhao等人,2016年)开发的感染棉花茎中恢复真菌菌丝)。玻璃纸膜已被用于诱导叶面病原体的发育,而不是根系病原体(Bourett和Howard,1990)。我们采用玻璃纸膜的方法诱导和评估hyphopodium的发展。希望能大力促进调控传染性发育的宿主的分子生物学研究。该协议还有助于识别控制V的致病性的关键成分。大丽花,并扩大了我们对植物与微生物相互作用机理的理解。
背景 玻璃纸膜被广泛用于研究叶病原体感染结构的发展(Bourett和Howard,1990; Kleemann等人,2012; Gu等人, ...
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