| Protein Expression Protocol for an Adenylate Cyclase Anchored by a Vibrio Quorum Sensing Receptor
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Author:
Date:
2017-01-20
[Abstract] The direct regulation of a mycobacterial adenylate cyclase (Rv1625c) via exchange of its membrane anchor by the quorum sensing receptor CqsS (Vibrio harveyi) has recently been reported (Beltz et al., 2016). This protocol describes the expression and membrane preparation for these chimeric proteins.
[摘要] 最近报道了通过其群岛感应受体CqsS（Vibrio harveyi）交换其膜锚定点直接调节分枝杆菌腺苷酸环化酶（Rv1625c）（Beltz等，2016）。 该方案描述了这些嵌合蛋白的表达和膜制备。
【背景】膜分隔的哺乳动物腺苷酸环化酶（AC）是IIIa类AC。调节是间接通过第一信使细胞外刺激G蛋白偶联受体（GPCR）而在细胞内释放的刺激（或抑制性）Gα-蛋白。 AC产生使用ATP作为底物的通用第二信使cAMP。脊椎动物AC中两个六角膜结构域的大小远远超过了对简单膜锚固的要求。然而，这种内在的膜锚定/受体结构域的调节特征是未知的。为了研究潜在的功能，选择了可以容易地在细菌中表达的分类细菌的典型类IIIa AC Rv1625c（Guo等人，2001和2005）与哺乳动物AC同工型相反。我们用来自哈维氏弧菌CqsS的六价体积感知（QS）受体的受体结构域取代了Rv1625c AC的六角膜锚，以检查我们是否可以通过QS配体的霍乱自动诱导剂直接调节AC -1'，CAI-1。 QS受体和IIIa类膜锚的设计是非常相似的，即最小的跨膜α-螺旋和极短的连接环。
我们已经证明了通过细胞外信号直接调节IIIa ...
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| Neurosphere Co-culture Assay
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Author:
Date:
2016-08-05
[Abstract] The hippocampal niche is one of two areas in the brain where stem cells reside. In this neurogenic niche, stem cells can be found in close proximity to astrocytes and in contact with microvessels consisting of pericytes and endothelial cells. To study the regulatory interplay of this complex niche network in a simplified in vitro model, we established a co-culture system. We investigate the formation of neurosphere under different co-culture conditions by using primary niche cells. Here, we describe the isolation procedure for primary niche cells culture of astrocytes, endothelial cells and pericytes/smooth muscle cells from mouse brain. These niche cells are co-cultured (by hanging inserts) with freshly isolated stem and precursor cells from the adult hippocampus to study the ...
[摘要] 真菌形态发生需要细胞壁的修饰和可塑性，这意味着其组分（包括壳多糖和葡聚糖）的合成和重塑。因此几丁质酶和葡聚糖酶活性对于细胞壁生物发生和细胞分裂是至关重要的。几丁质酶活性的定量可能有助于鉴定可能负面影响一些丝状真菌如产生细胞内和分泌型几丁质酶的尖孢镰刀菌的生长和形态发生的结构缺陷。基于它们对壳多糖底物的酶作用将壳聚糖分解酶分类。内酯酶被定义为催化几丁质链中内部点的随机裂解的酶。外切球蛋白酶催化乙酰壳寡糖或N-乙酰葡糖胺从几丁质的非还原端逐步释放，因此分别称为壳聚糖酶和β-N-乙酰氨基葡糖苷酶。在这里，我们描述了一种简单的方法来轻易地纯化几丁质酶，以便比较不同的F的内切几丁质酶活性和外切几丁质酶活性。尖孢镰孢菌株。该方案可适用于任何真菌物种。...
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| Membrane Flotation Assay
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Author:
Date:
2015-04-05
[Abstract] Many postitive-stranded RNA viruses, such as Hepatitis C virus (HCV), highjack cellular membranes, including the Golgi, ER, mitchondria, lipid droplets, and utilize them for replication of their RNA genome or assembly of new virions. By investigating how viral proteins associate with cellular membranes we will better understand the roles of cellular membranes in the viral life cycle. Our lab has focused specifically on the role of lipid droplets and lipid-rich membranes in the life cycle of HCV. To analyze the role of lipid-rich membranes in HCV RNA replication, we utilized a membrane flotation assay based on an 10-20-30% iodixanol density gradient developed by Yeaman et al. (2001). This gradient results in a linear increase in density over almost the entire length of the ...
[摘要] 许多假性绞线的RNA病毒，例如丙型肝炎病毒（HCV），高压细胞膜，包括高尔基体，ER，马钱子树，脂滴，并利用它们复制其RNA基因组或组装新的病毒粒子。通过调查病毒蛋白如何与细胞膜结合，我们将更好地了解细胞膜在病毒生命周期中的作用。我们的实验室特别关注脂质滴和富含脂质的膜在HCV的生命周期中的作用。为了分析富含脂质的膜在HCV RNA复制中的作用，我们使用基于由Yeaman等人（2001）开发的10-20-30％碘克沙醇密度梯度的膜浮选测定。该梯度导致在梯度的几乎整个长度上密度的线性增加，并且膜颗粒基于其浮力特性在梯度中分离。为了保存裂解物中的膜，在缺乏洗涤剂的缓冲液中机械破碎细胞。将细胞裂解物加载在梯度的底部，用梯度覆盖，并且膜随着碘克沙醇梯度自我产生而浮起。膜的脂质含量和相关蛋白的浓度将决定梯度内不同膜的分离。离心后，可以从梯度的顶部取样级分，并使用标准SDS-PAGE和蛋白质印迹分析来分析感兴趣的蛋白质。
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