| Analysis of Metals in Whole Cells, Thylakoids and Photosynthetic Protein Complexes in Synechocystis sp. PCC6803
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Author:
Date:
2018-06-20
[Abstract] Metals are essential in many biological processes, including oxygenic photosynthesis. Here we described a method to measure the metal pool in whole cells and thylakoids, including the bioactive pool in intact photosynthetic protein complexes in the model oxygenic cyanobacterium Synechocystis PCC6803. In the first part of the protocol, whole cells and thylakoid membranes are carefully prepared, in which the total metal concentrations are measured by inductively coupled plasma triple-quadrupole mass spectrometry (ICP-QQQ-MS). In the second part of the protocol, isolated thylakoids are solubilized to release the integral membrane proteins and the metal binding protein complexes. These intact photosynthetic protein complexes are subjected to size exclusion chromatography (SEC) and ...
[摘要] 金属在许多生物过程中是必不可少的，包括含氧光合作用。 在这里我们描述了测量全细胞和类囊体中金属库的方法，包括模型含氧蓝藻PCH6803中完整光合蛋白复合体中的生物活性库。 在方案的第一部分中，仔细制备全细胞和类囊体膜，其中通过电感耦合等离子体三重四极杆质谱（ICP-MS / MS）测量总金属浓度。 在该方案的第二部分，分离的类囊体被溶解以释放完整的膜蛋白和金属结合蛋白复合物。 将这些完整的光合蛋白复合物进行尺寸排阻色谱（SEC），并通过ICP-MS / MS进行连接分析尺寸分离的复合物中的金属结合。
【背景】氧光合作用的过程需要金属，因为它们在光合电子传递链中作为辅因子和催化剂的基本功能。光合装置需要Fe-S簇，血红素桥Fe和非血红素Fe形式的铁（Fe），可溶性移动电子载体蛋白质质体蓝素中的铜（Cu），叶绿素中的镁（Mg）， ...
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| Mating and Progeny Isolation in the Corn Smut Fungus Ustilago maydis
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Author:
Date:
2016-04-20
[Abstract] The corn smut pathogen, Ustilago maydis (U. maydis) (DC.) Corda, is a semi-obligate plant pathogenic fungus in the phylum Basidiomycota (Alexopoulos et al., 1996). The fungus can be easily cultured in its haploid yeast phase on common laboratory media. However, to complete its sexual cycle U. maydis strictly requires its specific plant host, maize (Zea mays). The fungus is an interesting and important model organism for the study of the interactions of fungal biotrophic pathogens with plants. In this protocol, we describe the process of plant inoculation, teliospore recovery, germination, progeny isolation and initial mating type analysis. The primary purpose of this protocol is to identify individual progeny strains of U. maydis that can be ...
[摘要] 科斯达（Corda）是在担子菌门（Basidiomycota）中的半必需的植物致病真菌（Alexopoulos等人）中的玉米黑斑病病原体（ al。，1996）。真菌可以容易地在其单倍体酵母相中在普通实验室培养基上培养。然而，为了完成其性周期，U. maydis严格要求其特定的植物宿主，玉米（ Zea mays ）。真菌是研究真菌生物营养性病原体与植物相互作用的有趣和重要的模式生物体。在这个协议中，我们描述植物接种，冬孢子恢复，萌发，后代分离和初始交配型分析的过程。本协议的主要目的是鉴定个体的子代菌株。 maydis ，可用于下游遗传分析。生成目标突变体研究各种过程是这种和许多植物致病真菌的常见方法。通过有性杂交促进产生突变组合的能力，而不需要额外的选择性标记。
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| Thioglycollate-elicited Peritoneal Macrophages Preparation and Arginase Activity Measurement in IL-4 Stimulated Macrophages
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Author:
Date:
2015-09-05
[Abstract] Macrophages are an essential cell population of innate immunity that plays important roles in inflammatory processes. Two main different phenotypes have been described with opposing activities: The classically activated macrophages (M1) and the alternatively activated macrophages (M2). Alternative activation of mouse macrophages can be induced by type 2 cytokines such as IL-4 and it is characterized by the regulation of the L-arginine metabolism. M2 macrophages convert arginine to ornithine and urea through the action of Arginase-1. Here we described a method for the isolation of peritoneal macrophages from thioglycollate-elicited mice and alternative activation by stimulation with IL-4. Intraperitoneal injection of thioglycollate elicits large numbers of macrophages into peritoneal ...
[摘要] 巨噬细胞是在炎症过程中起重要作用的先天免疫的必需细胞群体。 已经描述了具有相反活性的两种主要不同表型：经典激活的巨噬细胞（M1）和交替激活的巨噬细胞（M2）。 小鼠巨噬细胞的备选激活可以由2型细胞因子例如IL-4诱导，并且其特征在于L-精氨酸代谢的调节。 M2巨噬细胞通过精氨酸酶-1的作用将精氨酸转化为鸟氨酸和尿素。 在这里我们描述了从巯基乙酸诱发小鼠分离腹膜巨噬细胞和IL-4刺激的替代激活的方法。 腹膜内注射巯基乙酸盐引发大量巨噬细胞进入腹膜腔。
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