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Middle brook 7H9 broth

中间肉7H9肉汤

Company: Difco International
Catalog#: 271310
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Separation of Free and Bound cAMP in Mycobacteria
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。

Extraction and Purification of Mycobacterial Mycolic Acids
Author:
Date:
2014-10-20
[Abstract]  Mycolic acids are major long-chain fatty acids, containing up to 80-90 carbon atoms that represent essential components of the mycobacterial cell wall (Pawelczyk and Kremer, 2014). Each mycobacterial species possesses a specific mycolic acid profile characterized by various chemical modifications that decorate the lipid. Mycolic acids play a critical role in the architecture and impermeability of the cell envelope, hence the natural resistance of mycobacteria to most antibiotic treatments. They are also key determinants of virulence in pathogenic species, including Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis. In addition, they are known as the primary target of several first-line and second-line antitubercular drugs. Thus, the unique ... [摘要]  霉菌酸是主要的长链脂肪酸,含有高达80-90个碳原子,代表分枝杆菌细胞壁的基本组分(Pawelczyk和Kremer,2014)。每种分枝杆菌物种具有特征性霉菌酸分布,其特征在于装饰脂质的各种化学修饰。霉菌酸在细胞包膜的结构和不可渗透性中起关键作用,因此分枝杆菌对大多数抗生素治疗的天然抗性。它们也是在致病物种中的毒力的关键决定因素,包括结核分枝杆菌(结核分枝杆菌),结核病的致病因子。此外,它们被认为是几种一线和二线抗结核药物的主要靶点。因此,参与霉菌酸生物合成途径的独特的酶代表了用于未来化学疗法的靶的有吸引力的储库,其发展在M的多药耐药和广泛耐药菌株的背景下是特别保证的。肺结核。在这里,我们描述了从分枝杆菌中提取霉菌酸的方案。各种亚种的纯化对于涉及质谱或NMR的后续结构研究可能是特别有用的。通过薄层色谱的霉菌酸模式的定性和定量生物化学表征可用于解决药物如何改变霉菌酸生物合成(Alahari等人,2007,Hartkoorn等人, (Bhatt等人,2007)或解开新的霉菌酸调节机制(Vilcheze等人,2012),以研究在该代谢途径中受影响的遗传修饰的突变体的表型(Bhatt等人, ,2014)。相同的方案可以应用于所有分枝杆菌,包括环境和致病物种。

Phagolysosomal Trafficking Assay
Author:
Date:
2014-07-05
[Abstract]  Phagolysosomal trafficking is an important innate defense pathway that clears microbes by delivering them to lysosomes, the degradative compartment of the cell. Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, subverts this host defense mechanism by arresting maturation of the phagosome. The ability of Mtb to arrest its delivery to the lysosome can be demonstrated by the prolonged co-localization of bacteria containing phagosomes/vacuole with early phagosomal markers [such as, Ras- related proteins in the brain 5 (Rab5) and Transferrin receptor (TfR)], and a failure to acquire late phagosomal and lysosomal markers (such as Rab7 and LAMP1) (Deretic and Fratti, 1999, Mehra et al., 2013). Here, a protocol is outlined for infection of macrophages with ... [摘要]  吞噬溶酶体运输是重要的先天防御途径,通过将其递送至溶酶体,即细胞的降解区室来清除微生物。结核病的致病因子结核分枝杆菌(Mtb)通过阻滞吞噬体的成熟来破坏这种宿主防御机制。 Mtb阻止其递送至溶酶体的能力可以通过含有吞噬体/液泡的细菌与早期吞噬体标记物[例如,脑5中的Ras相关蛋白(Rab5)和转铁蛋白受体(TfR) )],以及未获得晚期吞噬体和溶酶体标记物(例如Rab7和LAMP1)(Deretic和Fratti,1999,Mehra等人,2013)。在这里,概述了用分枝杆菌物种感染巨噬细胞的方案,所述分枝杆菌物种如致病性Mtb疫苗菌株牛分枝杆菌 - 卡介苗(BCG)和快速分裂的非致病性耻垢分枝杆菌(Msmeg),然后间接免疫荧光显微镜观察宿主液泡标记。此后,通过使用数学工具处理细菌的二值图像来进行分枝杆菌和宿主液泡标记如TfR和LAMP1之间的共定位程度的自动定量。这导致直接在细菌/细菌簇周围的这些宿主标志物的平均荧光强度(MFI)的定量,相对于手动完成时具有增加的灵敏度。通过操纵宿主或病原体,该测定可用于评价细胞内运输的宿主或细菌决定簇。基本方法可以应用于研究其他细菌或颗粒状珠子的运输,尽管感染和吞噬体成熟的动力学将取决于吞噬性货物。数学分析工具可用于许多标准成像分析程序。然而,对于类似分析的任何适应性应由个体用户利用其成像和分析平台来确认。

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