| Drosophila Endurance Training and Assessment of Its Effects on Systemic Adaptations
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Author:
Date:
2018-10-05
[Abstract] Exercise induces beneficial systemic adaptations that reduce the incidence of age-related diseases. However, the molecular pathways that elicit these adaptations are not well understood. Understanding the molecular mechanisms that underlie the exercise response can lead to widely beneficial therapies. Large populations, relatively short lifespan, and easily modifiable genetics make Drosophila a well-suited model system for complex, longitudinal studies. We have developed an enforced climbing apparatus for Drosophila, known as the Power Tower, for the study of systemic exercise adaptations. The Power Tower takes advantage of the fly’s natural instinct for negative geotaxis, an innate behavior to run upwards after being tapped to the bottom of their vial. Flies will ...
[摘要] 运动诱导有益的系统适应，减少与年龄有关的疾病的发生率。然而，引起这些适应的分子途径还不是很清楚。了解作为运动反应基础的分子机制可以产生广泛有益的疗法。大群体，相对较短的寿命和易于修改的遗传学使得 Drosophila 成为复杂纵向研究的一个非常适合的模型系统。我们开发了一种用于 Drosophila 的强制攀爬装置，称为Power Tower，用于研究系统性运动适应性。 Power Tower利用苍蝇的天然本能来消极地质，这是一种天生的行为，在被轻敲到他们的小瓶底部之后向上运行。苍蝇将持续运行至耗尽点或直至机器关闭，以先到者为准。运动3周后，雄性 Drosophila 适应训练，具有许多类似于哺乳动物模型和人类所见的保守，易于量化的生理改善。在这里，我们描述了一个有用的耐力训练协议和一套有效量化训练效果的训练后评估。
【背景】耐力运动可以降低几乎所有与年龄相关的疾病（Ciolac，2013）。耐力训练对心血管功能，能量代谢和活动有很强的作用，从苍蝇到人类都是高度保守的（Piazza et al。，2009; Booth et al。，2015 ; Wilson et al。，2015）。更好地理解运动的遗传介质可能会导致治疗方法，这些治疗方法可以使因疾病或受伤而无法运动的个体受益。 Drosophila ...
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| Striatal Synaptosomes Preparation from Mouse Brain
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Author:
Date:
2018-09-05
[Abstract] The striatum is located in the subcortical region of the forebrain, it contains medium spiny neurons, cholinergic interneurons and GABAergic interneurons, and receives dopaminergic projection in the nigrostriatal pathway. This protocol provides a method to collect synaptosomes from mouse brain dorsal stratum. The synaptosomes can be used to study dopamine uptake activity, dopaminergic terminal endocytosis/trafficking using biochemical and microscopy methods, and protein analysis (Sorkina et al., 2018).
[摘要] 纹状体位于前脑的皮质下区域，它包含中等刺状神经元，胆碱能中间神经元和GABA能中间神经元，并在黑质纹状体通路中接受多巴胺能投射。 该方案提供了从小鼠脑背侧层收集突触体的方法。 突触体可用于研究多巴胺摄取活性，使用生物化学和显微镜方法的多巴胺能末端内吞作用/运输，以及蛋白质分析（Sorkina 等，，2018）。
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| The RiboPuromycylation Method (RPM): an Immunofluorescence Technique to Map Translation Sites at the Sub-cellular Level
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Author:
Date:
2018-01-05
[Abstract] While isotopic labeling of amino acids remains the reference method in the field for quantifying translation rate, it does not provide any information on spatial localization of translation sites. The rationale behind developing the ribopuromycylation method (RPM) was primarily to map translation sites at the sub-cellular level while avoiding detection of newly synthesized proteins released from ribosomes. RPM visualizes actively translating ribosomes in cells via standard immunofluorescence microscopy in fixed and permeabilized cells using a puromycin-specific monoclonal antibody to detect puromycylated nascent chains trapped on ribosomes treated with a chain elongation inhibitor.
[摘要] 尽管氨基酸的同位素标记仍然是用于定量翻译速率的领域中的参考方法，但是其不提供关于翻译位点的空间定位的任何信息。 开发ribopuromycylation方法（RPM）的基本原理主要是在亚细胞水平上绘制翻译位点，同时避免检测从核糖体释放的新合成的蛋白质。 RPM通过使用嘌呤霉素特异性单克隆抗体的固定的和透化的细胞中的标准免疫荧光显微镜可视化主动翻译细胞中的核糖体以检测捕获在用链延长抑制剂处理的核糖体上的嘌呤化新生链。
【背景】几十年来，氨基酸的同位素标记被认为是研究蛋白质翻译的金标准。虽然这种方法已被证明是非常准确的评估翻译率，它没有提供翻译核糖体的位置信息。最近，氨基酸类似物使荧光检测新生链（Dieterich等人，2007）。然而，几乎所有检测到的信号都来自核糖体释放的多肽。我们最初的想法是开发一种方法来标记新生链，同时还束缚于翻译核糖体。
嘌呤霉素（PMY）是一种氨基糖苷类抗生素，模拟带电荷的tRNA Tyr并掺入核糖体A位点。因此，PMY通过核糖体催化 - ...
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