Extraction and Quantification of Polyphosphate (polyP) from Gram-negative Bacteria
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Author:
Date:
2018-09-20
[Abstract] Polyphosphate (polyP), a universally conserved biomolecule, is composed of up to 1,000 phosphate monomers linked via phosphoanhydride bonds. Reaching levels in bacteria that are in the high nmoles per mg protein range, polyP plays important roles in biofilm formation and colonization, general stress protection and virulence. Various protocols for the detection of polyP in bacteria have been reported. These methods primarily differ in the ways that polyP is extracted and/or detected. Here, we report an improved method, in which we combine polyP extraction via binding to glassmilk with a very sensitive PolyP kinase/luciferase-based detection system. By using this procedure, we significantly enhanced the sensitivity of polyP detection, making it potentially applicable for mammalian tissues.
[摘要] 多磷酸盐(polyP)是一种普遍保守的生物分子,由多达1,000个通过磷酸酐键连接的磷酸盐单体组成。 达到每毫克蛋白质高纳摩尔细菌的水平,polyP在生物膜形成和定植,一般应力保护和毒力中起重要作用。 已经报道了用于检测细菌中polyP的各种方案。 这些方法主要在于提取和/或检测polyP的方式不同。 在这里,我们报告了一种改进的方法,其中我们结合polyP提取通过结合到玻璃奶与非常敏感的PolyP激酶/荧光素酶检测系统。 通过使用该程序,我们显着增强了polyP检测的灵敏度,使其可能适用于哺乳动物组织。
【背景】聚磷酸盐(polyP)是一种由多达1,000种无机磷酸盐单体的直链组成的生物聚合物,存在于生命的所有三个领域的细胞中。然而,细菌是唯一已经充分研究了polyP代谢酶的生物。将ATP转化为polyP的细菌polyP激酶(PPK)催化正向和反向反应。虽然polyP的合成显然是细胞中有利的反应,但通过在体外提供足够量的ADP ,该酶可用于从polyP产生ATP,使得基于荧光素酶的ATP检测成为可能(Ault -Riché et al。,1998)。缺乏PPK的细菌在生物膜形成,运动性,持久性和各种应激反应方面存在缺陷,并显示出对次卤酸(即,漂白)应激或磷酸盐饥饿的显着增加的敏感性(图1)(Rao et al。,2009; Gray et ...
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In vitro Analysis of Ubiquitin-like Protein Modification in Archaea
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Author:
Date:
2018-05-20
[Abstract] The ubiquitin-like (Ubl) protein is widely distributed in Archaea and involved in many cellular pathways. A well-established method to reconstitute archaeal Ubl protein conjugation in vitro is important to better understand the process of archaeal Ubl protein modification. This protocol describes the in vitro reconstitution of Ubl protein modification and following analysis of this modification in Haloferax volcanii, a halophilic archaeon serving as the model organism.
[摘要] 泛素样(Ubl)蛋白广泛分布于古细菌中并参与许多细胞途径。 为了更好地理解古细菌Ub1蛋白质修饰的过程,重建体外古细菌Ubl蛋白质缀合物的完善方法是很重要的。 该协议描述了Ubl蛋白质修饰的体外重建以及在作为模型生物的嗜盐古细菌Haloferax volcanii 中对这种修饰进行分析。
【背景】泛素(Ub)与靶蛋白共价连接的过程被称为泛素化,其控制真核细胞中大量的细胞过程(Glickman和Ciechanover,2002; Komander和Rape,2012)。遍在蛋白化由一系列酶(包括Ub激活酶(E1),Ub结合酶(E2s)和Ub连接酶(E3s))催化。泛素化的体外重建是确定酶之间或E3与蛋白质底物之间特异性的有用测定法(Zhao等人,2012)。在古细菌中,Ubl蛋白SAMP采用Ub折叠,并且与E1样酶UbaA催化的蛋白靶标异肽连接[Maupin-Furlow,(2014)综述]。尽管E1同系物在古细菌中广泛存在,但基于一级序列比较,在大多数古细菌中未预测经典E2或E3酶。我们最近对Haloferax volcanii的研究表明甲硫氨酸亚砜还原酶A(MsrA)是Ubl蛋白质修饰(sampylation)与UbaA一起在体内温和的氧化条件下和< (体外)(fu="">
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Intravenous Labeling and Analysis of the Content of Thymic Perivascular Spaces
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Author:
Date:
2018-03-05
[Abstract] Following development in the thymus, T cells are thought to exit into the periphery predominantly through perivascular spaces (PVS). This exit route is used by conventional T cells, and likely also applies to unconventional T cell subsets, such as precursors of CD8αα and TCRγδ intraepithelial lymphocytes, regulatory T cells and natural killer T cells. Additional cell types might also be found in the PVS and initiate interactions with exiting T cells. The exact content of the PVS, and the processes within, are not well studied. To distinguish vascular from resident cells within various tissues by flow cytometry, intravenous (i.v.) labeling is becoming a commonly employed method. We recently used anti-CD45.2 antibodies and magnetic enrichment to further evaluate this technique, and compared ...
[摘要] 在胸腺发育后,T细胞被认为主要通过血管周围间隙(PVS)进入周边。 这种退路由常规T细胞使用,也可能适用于非常规T细胞亚群,例如CD8αα和TCRγδ上皮内淋巴细胞的前体,调节性T细胞和天然杀伤T细胞。 其他细胞类型也可能在PVS中发现并启动与退出T细胞的相互作用。 PVS的确切内容及其内部过程尚未得到充分研究。 为了通过流式细胞术将血管与各种组织中的驻留细胞区分开,静脉内(静脉内)标记正在成为常用方法。 我们最近使用抗CD45.2抗体和磁性富集来进一步评估这种技术,并比较胸腺和血液中的标记和未标记的细胞。 该测定可用于特异性研究胸腺PVS内的造血细胞亚群。
【背景】未成熟的胸腺细胞经历一系列成熟步骤,包括正向和负向选择,其消除了大部分发育中的T细胞。由此产生的成熟T细胞库因此形成朝向更高比例的有益克隆和减少比例的危险自反应克隆。胸腺还产生较少丰富的T细胞亚群,其通常用于维持免疫系统,组织和代谢稳态,包括:TCRγδ细胞,调节性T细胞(Treg),自然杀伤T细胞(NKT),上皮内淋巴细胞(IEL)和粘膜相关不变T(MAIT)细胞。成熟的胸腺细胞准备迁移到外周,上调表达识别鞘氨醇-1磷酸(S1P)的受体(S1PR1)的表达,S1P是血液中高浓度存在的脂质分子。 S1PR1 + T细胞沿着S1P梯度迁移并卷入血管循环中。
胸腺血管周围间隙(PVS)是实质和脉管系统之间的基膜分隔室。它们被认为促进了细胞的运输,特别是从胸腺移出的成熟T细胞(Mori等人,2007; ...
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