| Mouse Liver Mitochondria Isolation, Size Fractionation, and Real-time MOMP Measurement
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Author:
Date:
2016-08-05
[Abstract] The mitochondrial pathway of apoptosis involves a complex interplay between dozens of proteins and lipids, and is also dependent on the shape and size of mitochondria. The use of cellular models in past studies has not been ideal for investigating how the complex multi-factor interplay regulates the molecular mechanisms of mitochondrial outer membrane permeabilization (MOMP). Isolated systems have proven to be a paradigm to deconstruct MOMP into individual steps and to study the behavior of each subset of MOMP regulators. In particular, isolated mitochondria are key to in vitro studies of the BCL-2 family proteins, a complex family of pro-survival and pro-apoptotic proteins that directly control the mitochondrial pathway of apoptosis (Renault et al., 2013).
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[摘要] 凋亡的线粒体途径涉及数十种蛋白质和脂质之间的复杂相互作用，并且还依赖于线粒体的形状和大小。在过去的研究中使用细胞模型不是理想的调查如何复杂的多因素相互作用调节线粒体外膜透化（MOMP）的分子机制。分离系统已被证明是将MOMP解构成各个步骤并研究每个子集的MOMP调节剂的行为的范例。特别地，分离的线粒体是BCL-2家族蛋白的体外研究的关键，BCL-2家族蛋白是直接控制凋亡的线粒体途径的促存活和促凋亡蛋白的复合家族（Renault > et al 。，2013）。
在这个协议，我们描述三个补充程序用于实时调查使用孤立的线粒体MOMP调节器的影响。第一种方法是"肝线粒体分离"，其中从小鼠中分离肝脏以获得线粒体。 "用JC-1和大小分级分离的线粒体标记"是描述标记，按大小分级并标准化线粒体亚群的方法的第二个方法。最后，"实时MOMP测量"协议允许在孤立的线粒体上实时跟踪MOMP。上述程序用于在体外确定线粒体膜形状在分离的细胞和分离的线粒体水平上的作用（Renault等人，2015）。
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| Expression and Partial Purification of His-tagged Proteins in a Plant System
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Author:
Date:
2015-09-05
[Abstract] Plant protein expression can be a challenging enterprise in any biochemical or molecular biology research project. Several heterologous systems like bacteria, yeast, insect cells and cell free systems have been used to produce plant proteins for in vitro experiments and structural characterization. However, due to particularities of plant proteins, for example the specific type and abundance of post-translational modifications (e.g. glycosylation), a plant system to express plant proteins is extremely desirable. The use of Nicotiana benthamiana (N. benthamiana) plants for protein expression has proven to be quick and reliable. To illustrate the robustness and rapidity of this system, recent efforts to produce the first protein based drug against the ...
[摘要] 植物蛋白表达可以是任何生物化学或分子生物学研究项目中的挑战性企业。几种异源系统如细菌，酵母，昆虫细胞和无细胞系统已被用于生产植物蛋白用于体外实验和结构表征。然而，由于植物蛋白的特殊性，例如翻译后修饰（例如糖基化）的特异性类型和丰度，表达植物蛋白的植物系统是非常需要的。已经证明使用本氏烟草（本生烟草）植物进行蛋白质表达是快速和可靠的。为了说明该系统的稳健性和快速性，最近在N中进行了生产针对埃博拉病毒的第一种基于蛋白质的药物的努力。本草案蛋白质表达系统（Choi等人，2015）。
该方案描述了一种简单的系统，用于表达和富集（亲和纯化） > N。本生烟草叶，其被成功地用于拟南芥果胶乙酰酯酶，PAE8和PAE9的表征中（de Souza等人，2014）。
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| Evaluation of Nodulation Speed by Sinorhizobium Strains
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Author:
Date:
2015-08-05
[Abstract] Rhizobia interact symbiotically with legumes to form root nodules, where by rhizobia fix atmospheric dinitrogen into ammonia in exchange for carbon produced via photosynthesis. The symbiotic interaction is agriculturally important by reducing the need for fertilizer containing nitrogen. The root and stem nodule bacteria commonly include bacteria in the genera Rhizobium, Mesorhizobium, Sinorhizobium (Ensifer), and Bradyrhizobium, although other genera of bacteria have now been shown to form root nodule symbioses with several legume species (Weir, 2012). Different rhizobial strains form different numbers of nodules on specific legume plant varieties (or cultivars), and the nitrogen fixing effectiveness of each rhizobial strain, its ability to fix ...
[摘要] 根瘤菌与豆科植物共生相互作用形成根瘤，其中根瘤菌将大气氮固定为氨，以交换通过光合作用产生的碳。共生相互作用在农业上是重要的，通过减少对含氮肥的需要。根和茎结节细菌通常包括根瘤菌属，中生根瘤菌，中华根瘤菌属（ Ensifer ）和 Bradyrhizobium >，虽然其他属的细菌现在已经显示形成根瘤与多种豆科物种的共生（Weir，2012）。不同的根瘤菌菌株在特定豆科植物品种（或品种）上形成不同数目的结节，并且每种根瘤菌菌株的氮固定效率，其固定氮并将其转移到植物的能力也是高度可变的（Toro，1996）。一些天然根瘤菌在固氮但在所生长的植物中形成大多数结节方面无效。这被称为结瘤问题的竞争（Triplett和Sadowsky，1992）。当评估大量不同的天然菌株时，竞争研究是不可行的。然而，单个菌株的结瘤速度与总体竞争性良好相关，并且可用于鉴定克服竞争问题的天然菌株（De Oliveira和Graham，1990; Bhuvaneswari等人，1980）。
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