| Pit Assay to Measure the Bone Resorptive Activity of Bone Marrow-derived Osteoclasts
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Author:
Date:
2016-06-20
[Abstract] Although it is possible to use a tartrate-resistant acid phosphatase (TRAP) stain to assist in identifying osteoclasts, a separate method is needed to determine the bone resorption activity of osteoclasts. Since osteoclasts leave “pits” after bone matrix resorption (Charles et al., 2014), it is possible to stain pits as a method of measuring osteoclast bone resorption activity. The pit assay protocol enables researchers to stain bony slices that were co-cultured with osteoclasts with toluidine blue in order to allow the visualization, capture, and analysis of osteoclast resorptive ...
[摘要] 尽管可以使用抗酒石酸盐的酸性磷酸酶（TRAP）染色来辅助鉴定破骨细胞，但是需要单独的方法来确定破骨细胞的骨吸收活性。 由于破骨细胞在骨基质吸收之后离开"凹陷"（Charles等人，2014），因此可以将斑点染色作为测量破骨细胞骨吸收活性的方法。 坑测定方案使研究人员能够将与破骨细胞与甲苯胺蓝共培养的骨切片染色，以便基于坑的数量，尺寸和深度可视化，捕获和分析破骨细胞再吸收活性（Zhou等人， et al。，2015）。 坑测定方案分为三个连续阶段：骨切片的制备（1）; 制备破骨细胞前体（Ross等人，2006; Teitelbaum等人，2000）（2）和骨吸收坑测定（3）。
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| Assays of Polyphenol Oxidase Activity in Walnut Leaf Tissue
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Author:
Date:
2014-08-20
[Abstract] Polyphenol oxidase (PPO) is an enzyme that catalyzes the hydroxylation of monophenols into ortho-diphenols (cresolase activity) and the oxidation of o-diphenols into quinones (catecholase activity) (Figure 1). These quinones spontaneously polymerize to form dark-colored phytomelanins, most often seen in the browning of damaged plant tissue. PPO activity can be easily assayed in crude protein extracts from English walnut (Juglans regia) leaves and from many other plant tissue extracts. PPO activity is most commonly measured by spectrophotometric assay, in which the rate of ...
[摘要] 多酚氧化酶（PPO）是催化单酚羟基化成邻二酚（甲酚酶活性）和将β-二酚氧化成醌（儿茶酚酶活性）（图1）的酶。这些醌自发聚合形成深色的植物色素，最常见于受损的植物组织的褐变。 PPO活性可以容易地在来自英国胡桃（Juglans regia）叶和来自许多其它植物组织提取物的粗蛋白提取物中测定。 PPO活性最通常通过分光光度测定法测量，其中植物肉豆蔻毒素产生的速率被量化，或通过氧电极测定，其中酶对氧的消耗量化（图1）。尽管更简单，分光光度测定的效用受到从不同酚类底物产生的植物角蛋白的最大吸收的变化的限制。氧电极测定通常被认为是用于测量PPO活性的"金标准"，但是与单酚底物一起实施是更费时和困难的，因为与儿茶酚酶活性相比，甲酚水解酶活性通常相当低。该方案将描述从核桃叶的粗蛋白质提取，分光光度测定法和用于测定PPO活性的氧电极测定法。
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| Determination of Rifampicin-resistance Mutation Frequency and Analysis of Mutation Spectra in Mycobacteria
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Author:
Date:
2014-07-05
[Abstract] Understanding the genetic safeguarding mechanism of Mycobacterium tuberculosis (Mtb) may help us to explain i), how Mtb survive the genetic assaults elicited by both reactive oxygen species (ROS) and reactive nitrogen species (RNS) produced by host macrophages and ii), why some strains of Mtb, e.g., Mtb strains from East Asian lineage and Beijing sublineage, exhibit high mutation rate and are more likely to acquire drug resistant mutations (e.g., rifampicin-resistance mutation) during infection. Mutation frequency analysis is a basic methods to study the genetic ...
[摘要] 了解结核分枝杆菌（Mtb）的遗传保护机制可以帮助我们解释i）如何Mtb存活由活性氧（ROS）和活性氮（RNS）产生的遗传攻击 宿主巨噬细胞和ii），为什么一些来自东亚谱系和北京亚系的Mtb菌株（例如eg）具有高突变率，并且更可能获得耐药突变（例如，利福平 - 抗性突变）。 突变频率分析是研究遗传保护机制的基本方法。 此外，为了研究突变的分子机制，有必要分析突变谱（例如，氧化的胞嘧啶可能诱导CG到TA突变）。 该协议描述了确定耻骨分枝杆菌（Msm）和Mtb中的突变频率和了解突变谱的方法。
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