Observation of Pneumococcal Phase Variation in Colony Morphology
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Author:
Date:
2017-08-05
[Abstract] Streptococcus pneumoniae (pneumococcus) is an important human pathogen that causes pneumonia, meningitis, sepsis, and otitis media. This bacterium normally resides in the nasopharynx as a commensal, but sometimes disseminates to sterile sites of humans and causes local or systemic inflammation. This biphasic behavior of S. pneumoniae is correlated with a reversible switch between the opaque and transparent colony forms on agar plates, a phenomenon referred to as phase variation. The opaque variants appear to be more virulent in animal models of bacteremia but are deficient in nasopharyngeal colonization animal models. In contrast, the transparent variants display higher levels of nasopharyngeal colonization but relatively lower virulence in animal models. We have ...
[摘要] 肺炎链球菌(肺炎球菌)是导致肺炎,脑膜炎,败血症和中耳炎的重要人类病原体。这种细菌通常作为共生体存在于鼻咽中,但有时会传播到人类的无菌部位并导致局部或全身炎症。这种双相行为。肺炎支原体与琼脂平板上的不透明和透明集落形式之间的可逆转换相关,这称为相变。不透明变体在菌血症的动物模型中似乎更具毒性,但在鼻咽定殖动物模型中是缺陷的。相比之下,透明变体在动物模型中显示较高水平的鼻咽定植,但相对较低的毒力。我们最近证实,这两种菌落类型之间的肺炎球菌相变是由基因组DNA甲基化(或表观遗传)模式的可逆转换引起的,由DNA甲基转移酶基因的DNA反转驱动。菌落形态的观察是区分具有不同特征(如大小,颜色和不透明度)的菌落的简单且有用的方法。该方案描述了如何利用解剖显微镜研究集落形态的肺炎球菌相变。 【背景】肺炎链球菌是全球儿童细菌性肺炎,脑膜炎和败血症的主要原因(Walker等人,2013)。这种病原体在适应人类宿主的各种生态环境中的成功取决于其显着的表型可塑性(Croucher等人,2013; Johnston等人,,2014a) ,其已被荚膜多糖和表面蛋白质中的菌株间抗原变异反映(Croucher等人,2013和2011),获得新的毒力因子(Park等人, ...
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Determination of Hydrodynamic Radius of Proteins by Size Exclusion Chromatography
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Author:
Date:
2017-04-20
[Abstract] Size exclusion chromatography (SEC) or gel filtration is a hydrodynamic technique that separates molecules in solution as a function of their size and shape. In the case of proteins, the hydrodynamic value that can be experimentally derived is the Stokes radius (Rs), which is the radius of a sphere with the same hydrodynamic properties (i.e., frictional coefficient) as the biomolecule. Determination of Rs by SEC has been widely used to monitor conformational changes induced by the binding of calcium (Ca2+) to many Ca2+-sensor proteins. For this class of proteins, SEC separation is based not just on the variation in protein size following Ca2+ binding, but likely arises from changes in the hydration shell structure.
This ...
[摘要] 尺寸排阻色谱法(SEC)或凝胶过滤是一种流体动力学技术,它将溶液中的分子作为其尺寸和形状的函数。在蛋白质的情况下,可以通过实验得出的流体动力学值是斯托克斯半径(R s),它是具有相同流体力学性质的球体的半径(即, >摩擦系数)作为生物分子。通过SEC测定R s已经被广泛用于监测由钙(Ca 2+)与许多Ca 2+连接引起的构象变化传感器蛋白。对于这类蛋白质,SEC分离不仅基于Ca 2 + 结合后的蛋白质尺寸变化,而且可能来自水合壳结构的变化。 该方案旨在使用快速蛋白质液相色谱(FPLC)系统对预填充柱进行凝胶过滤实验,以确定蛋白质的R 1,其中一些适用于Ca 2 + 传感器蛋白。
凝胶过滤基于其相对的能力分离不同大小和形状的分子,以穿透具有明确孔径的多孔珠床,其识别分馏范围。大于完全排除进入孔隙的分馏范围的分子快速流过色谱柱,首先以空间体积(V 0 O)(其为支持颗粒外的间隙体积)洗脱。能够扩散到珠的孔中的小于分级范围的分子具有可用于流动相的总体积,因此它们更缓慢地移动通过床并最后洗脱。具有中等维度的分子将以包含在流动相可利用的空隙体积和总体积之间的洗脱体积(V e e e e)被洗脱(分子越小,其进入孔隙越大矩阵,因此其V e e越大)。 蛋白质的分子量可以通过比较其洗脱体积参数K ...
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Direct Visualization and Quantification of the Actin Nucleation and Elongation Events in vitro by TIRF Microscopy
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Author:
Date:
2017-03-05
[Abstract] Total internal reflection fluorescence (TIRF) microscopy is a powerful tool for visualizing the dynamics of actin filaments at single-filament resolution in vitro. Thanks to the development of various fluorescent probes, we can easily monitor all kinds of events associated with actin dynamics, including nucleation, elongation, bundling, fragmentation and monomer dissociation. Here we present a detailed protocol regarding the visualization and quantification of actin nucleation and filament elongation events by TIRF microscopy in vitro, which is based on the methods previously reported (Liu et al., 2015; Yang et al., 2011).
[摘要] 全内反射荧光(TIRF)显微镜是用于在体外单丝分辨率下可视化肌动蛋白丝的动力学的强大工具。由于各种荧光探针的发展,我们可以轻松监测与肌动蛋白动力学相关的各种事件,包括成核,伸长,捆扎,碎裂和单体解离。在这里,我们提供了一个关于通过TIRF显微镜在体外可视化和定量肌动蛋白成核和细丝伸长事件的详细方案,其基于先前报道的方法(Liu等人, ,2015; Yang等人,2011)。
背景 ...
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