| Separation of Free and Bound cAMP in Mycobacteria
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Author:
Date:
2016-07-20
[Abstract] Mycobacterial genomes encode a plethora of genes that are involved in the synthesis, utilization and degradation of cAMP. The genome of M. tuberculosis H37Rv, for example, encodes 16 adenylyl cyclases and 10 genes harbouring the cyclic nucleotide-binding (CNB) domain (Shenoy and Visweswariah, 2006). Cyclic AMP is efficiently secreted by mycobacteria, and cytosolic as well as extracellular levels of cAMP can reach hundreds of micromolar. We have recently reported that an abundantly expressed universal stress protein (USP; Rv1636 in M. tuberculosis H37Rv and MSMEG_3811 in M. smegmatis, respectively) binds cAMP (Banerjee et al., 2015). Given the number of cAMP-binding proteins present in mycobacteria, it is expected that a significant fraction of ...
[摘要] 分枝杆菌基因组编码涉及cAMP的合成，利用和降解的大量基因。例如，结核分枝杆菌H37Rv的基因组编码16个腺苷酸环化酶和10个携带环核苷酸结合（CNB）结构域的基因（Shenoy和Visweswariah，2006）。循环AMP由分枝杆菌有效分泌，细胞溶质以及细胞外cAMP水平可达数百微摩尔。我们最近报道，大量表达的普遍应激蛋白（USP; Rv1636在结核分枝杆菌H37Rv和MSMEG_3811分别在耻垢分枝杆菌中）分别结合cAMP（Banerjee等，2015）。鉴于存在于分枝杆菌中的cAMP结合蛋白的数量，预期细胞内cAMP的显着部分可能与蛋白质结合。通常用于测量cAMP的方法是放射免疫测定（RIA）和ELISA。然而，这些方法包括将cAMP“结合”解离成蛋白质的样品的预先酸化，因此代表样品中存在的“总”cAMP。在本协议中，我们描述了一种将cAMP'结合'蛋白质与蛋白质“自由”分离或与蛋白质不相关的方法。这通过使细胞溶质级分或培养物上清液通过具有3kDa截止值的膜过滤来进行。只有'自由'cAMP才能通过膜。因此，滤液中的cAMP浓度代表样品中的“游离”cAMP。原始细胞溶质级分或培养上清液中的环AMP水平代表“总”cAMP浓度。从“总”中减去“自由”提供了与蛋白质结合的cAMP量。
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| Analysis of Mycobacterial Protein Secretion
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Author:
Date:
2014-06-20
[Abstract] Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis. Analysis of proteins secreted by Mtb has been of interest to the field of tuberculosis research since certain secreted proteins interact with the host to promote virulence, while others may be important antigens or serve as biomarkers of infection. Here, we describe a protocol to prepare whole cell extracts (WCE) and short term culture filtrate (CF) from Mtb or the vaccine strain Mycobacterium bovis- bacillus Calmatte- Guérin (BCG) (Mehra et al., 2013). These are both slow growing mycobacteria, but the same basic procedure can easily be adapted to analyze secreted proteins from rapidly growing mycobacteria, such as Mycobacterium smegmatis (Msmeg), a non-pathogenic species commonly ...
[摘要] 结核分枝杆菌（Mtb）是结核病的致病因子。由Mtb分泌的蛋白质的分析已经对结核病研究领域感兴趣，因为某些分泌的蛋白质与宿主相互作用以促进毒力，而其他可能是重要的抗原或用作感染的生物标志物。在这里，我们描述了从Mtb或疫苗菌株牛分枝杆菌 - 卡介苗（BCG）制备全细胞提取物（WCE）和短期培养物滤液（CF）的方案（Mehra等人， et al。，2013）。这些都是缓慢生长的分枝杆菌，但是相同的基本程序可以容易地适于分析来自快速生长的分枝杆菌的分泌蛋白，例如耻垢分枝杆菌（Msmeg），其是实验室中常用的非致病物种。可以通过蛋白质印迹分析获得的级分，以检查感兴趣的蛋白质，或者如果抗体不可获得或通过质谱法检查整个分泌蛋白质组。所关注基因的遗传敲除突变体用作阴性对照。另外，应当在CF部分中评估胞质蛋白如分子伴侣GroEL或丙酮酸脱氢酶E2组分sucB（Rv2215/dlaT）的水平，以排除CF中的阳性信号是由于细菌裂解导致的可能性（参见图1）。通过改变菌株的生长条件，这种体外分泌测定法可用于检查改变分泌物组织的条件。我们感谢Magnus Stiegedal提供有关TCA（三氯乙酸）沉淀的有用提示。
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| DNA Damage Sensitivity Assays with Arabidopsis Seedlings
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Author:
Date:
2014-04-05
[Abstract] We describe fast and reproducible sensitivity assays to quantify the response of Arabidopsis seedlings of different genotypes to a wide range of DNA damaging agents. We apply (1) γ-irradiation, which produces DNA breaks, (2) bleocin, a radiomimetic drug, (3) mitomycin C, a DNA intrastrand cross-linker, (4) hydroxyurea, an inhibitor of DNA synthesis and (5) UV-C, which causes mainly photoproducts. The “true leaf assay” and the “UV resistance assay” are based on easily determined phenotypes as readouts. Using a set of diverse damaging agents combined with different readouts allows establishing relative sensitivity/resistance compared to a reference line, e.g. wild type, determining the most effective type of induced damage and the potential repair pathway affected.
[摘要] 我们描述快速和可重复的灵敏度测定以量化不同基因型的拟南芥幼苗对广泛的DNA损伤剂的反应。 我们应用（1）γ辐射，其产生DNA断裂，（2）bleocin，放射性模拟药物，（3）丝裂霉素C，DNA intrastrand交联剂，（4）羟基脲，DNA合成抑制剂， UV-C，其主要产生光产物。 "真叶试验"和"抗UV试验"基于容易确定的表型作为读数。 使用一组不同的损伤剂结合不同的读数允许相对于参考线（例如野生型）建立相对灵敏度/电阻，确定最有效类型的诱导损伤和潜在的修复途径受影响。
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