| Analysis of TORC1-body Formation in Budding Yeast
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Author:
Date:
2021-04-05
[Abstract] The Target of Rapamycin kinase Complex I (TORC1) is the master regulator of cell growth and metabolism in eukaryotes. In the presence of pro-growth hormones and abundant nutrients, TORC1 is active and drives protein, lipid, and nucleotide synthesis by phosphorylating a wide range of proteins. In contrast, when nitrogen and/or glucose levels fall, TORC1 is inhibited, causing the cell to switch from anabolic to catabolic metabolism, and eventually enter a quiescent state. In the budding yeast Saccharomyces cerevisiae, TORC1 inhibition triggers the movement of TORC1 from its position around the vacuole to a single focus/body on the edge of the vacuolar membrane. This relocalization depends on the activity of numerous key TORC1 regulators and thus analysis of TORC1 localization can be used to ...
[摘要] [摘要]雷帕霉素激酶复合体I（TORC1）的靶标是真核生物中细胞生长和代谢的主要调节剂。在促生长激素和丰富营养素的存在下，TORC1活跃并通过磷酸化多种蛋白质来驱动蛋白质，脂质和核苷酸合成。相反，当氮和/或葡萄糖水平下降时，TORC1被抑制，导致细胞从合成代谢转换为分解代谢，并最终进入静止状态。在发芽酵母中酿酒酵母 抑制TORC1会触发TORC1从其在液泡周围的位置移动到液泡膜边缘的单个焦点/物体。这种重新定位取决于许多关键的TORC1调节子的活动，因此可以使用TORC1定位分析来跟踪通过TORC1途径发出的信号。在这里，我们提供了使用荧光显微镜测量TORC1（特别是Kog1-YFP）重新定位/信号传递的详细协议。重点放在确保以下方面的程序上：（1）尽管葡萄糖和氮饥饿期间它们相对较低的荧光和自发荧光灶的积累，但仍正确地识别（并计数）TORC1抗体；（2）在每次实验开始时，使细胞保持对数生长期，以便正确监测TORC1体形成的动力学；（3）使用适当的荧光标签来避免检查错误定位的TORC1。
[背景]雷帕霉素的靶激酶复合物I（TORC1 ）是真核生物的细胞生长控制网络中的中央轮毂（Loewith和Hall，2011;冈萨雷斯和Hall，2017; Liu和萨巴蒂尼，2020 ...
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| Quantification of Ethylene Production in Tomato Leaves Infected by Xanthomonas euvesicatoria
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Author:
Date:
2016-02-05
[Abstract] Ethylene is a gaseous plant hormone controlling fruit ripening, flower opening, leaf senescence as well as abscission, and disease symptom development. Ethylene plays a critical role in the bacterial pathogen Xanthomonas euvesicatoria (X. euvesicatoria)-elicited symptom development in tomato. This protocol describes the measurement of ethylene gas produced by tomato leaves infected with X. euvesicatoria. Infected leaflets are placed in a glass tube for 30 min without sealing. The glass tubes are then capped with a septa stopper, and incubated for an hour. A 1 ml gas sample is removed from the tube using a syringe and then injected into a gas chromatograph to quantify ethylene gas levels. This protocol will be applicable for other plants with other pathogens with ...
[摘要] 乙烯是控制果实成熟，开花，叶衰老以及脱落和疾病症状发展的气态植物激素。 乙烯在细菌病原体黄单胞菌属（Xanthomonas euvesicatoria）（ X。euvesicatoria ）中起到关键作用 - 引起番茄中的症状发展。 该方案描述了用感染了X的番茄叶产生的乙烯气体的测量。 euvesicatoria 。 将感染的小叶放置在玻璃管中30分钟而不密封。 然后将玻璃管用隔膜塞子盖上，并孵育1小时。 使用注射器从管中移出1ml气体样品，然后注入气相色谱仪中以定量乙烯气体水平。 该协议将适用于其他具有修改的其他病原体的植物。
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| Establishing a Biofilm Co-culture of Pseudomonas and Aspergillus for Metabolite Extraction
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Author:
Date:
2015-12-05
[Abstract] Filamentous fungi and bacteria form mixed-species biofilms in nature and diverse clinical contexts (Frey-Klett et al., 2011; Peleg et al., 2010). The interactions between fungi and bacteria, often mediated by secreted metabolites, have important ramifications for the biology of the interacting partners (Frey-Klett et al., 2011). This is particularly true for the bacterium Pseudomonas aeruginosa (P. aeruginosa) and the fungus Aspergillus fumigatus (A. fumigatus) which often reside in the same niche such as lungs of cystic fibrosis (CF) patients. Some studies have reported that co-infection with P. aeruginosa and A. fumigatus could lead to a decrease in lung function relative to their respective single species ...
[摘要] 丝状真菌和细菌在自然界和不同的临床背景中形成混合物种生物膜（Frey-Klett等人，2011; Peleg等人，2010）。真菌和细菌之间的相互作用，通常由分泌的代谢物介导，对相互作用的伴侣的生物学具有重要的影响（Frey-Klett等人，2011）。对于绿脓假单胞菌（铜绿假单胞菌）和真菌烟曲霉（ A。fumigatus >），其常常位于相同的位置，例如囊性纤维化（CF）患者的肺。一些研究报道了与p的共感染。铜绿和 烟曲霉可能导致相对于它们各自的单一物种感染的肺功能降低（Amin等人，2010; Peleg等人，2010）。代谢物提取和分析允许表征多微生物生物膜中的特定微生物代谢物。该方案描述了如何在固体培养基上制备假单胞菌属 - 曲霉菌共培养生物膜以准备代谢物提取。
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