| Analytical Gel Filtration for Probing Heavy Metal Transfer between Proteins
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Author:
Date:
2016-08-05
[Abstract] Heavy metals can cause damage to biomolecules such as proteins and DNA in multiple ways. Cells therefore strive for keeping intracellular (heavy) metal ions bound to specific proteins that are capable of handling detoxification, export or integration as cofactors. Metal binding proteins usually provide specific coordination sites that bind certain ions with ultrahigh affinity, with the thermodynamic driving force being the stability of organometallic complexes. However, the metal binding properties of these proteins can be highly variable. Therefore the transfer of specific ions between separate proteins or even between distinct binding sites located on one and the same protein does not always follow affinity gradients, but depends on particular protein interactions that are difficult to ...
[摘要] 重金属可以以多种方式对生物分子例如蛋白质和DNA造成损害。因此，细胞努力保持细胞内（重）金属离子结合到能够处理解毒，输出或整合作为辅因子的特定蛋白质。金属结合蛋白通常提供结合某些离子的特异性配位位点，具有超高的亲和力，热力学驱动力是有机金属配合物的稳定性。然而，这些蛋白质的金属结合性质可以是高度可变的。因此，在分开的蛋白质之间或甚至在位于同一蛋白质上的不同结合位点之间的特异性离子的转移不总是遵循亲和梯度，而是取决于难以预测的特定蛋白质相互作用。我们建立了一种适合探测两种蛋白质之间的金属转移的方法，只要这些蛋白质可以进行纯化和体外处理。它由金属负载，共孵育和金属交换蛋白的分离，随后确定结合金属含量。该方法通过我们探测膜 - 外在金属结合结构域MBD2和来自大肠杆菌的铜输出ATP酶的跨膜结构域的膜 - 外部金属结合结构域MBD2之间的铜（I）转移的实验数据来举例说明（Drees < em=""> 。，2015）。
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| ACE-score-based Analysis of Temporal miRNA Targetomes During Human Cytomegalovirus Infection Using AGO-CLIP-seq
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Author:
Date:
2016-04-20
[Abstract] Although temporal regulation of gene expression during the course of infection is known to be critical for determining the outcome of host-virus interactions, systematic temporal analysis of the miRNA targetomes during productive viral infection has been technically challenging due to the large range of miRNA-mRNA cross-talks at the host-virus interface. High-confidence quantifying models of the suppression efficacy in targeting sites by integrating bioinformatics with Argonaute-crosslinking and immunoprecipitation followed by high-throughput sequencing (AGO-CLIP-seq) (Chi et al., 2009) data have been poorly developed. To accurately identify miRNA target sites and calculate the targeting efficacy of miRNA-target interactions, we developed a new bioinformatic quantitation method, ...
[摘要] 尽管已知在感染过程中基因表达的时间调节对于确定宿主 - 病毒相互作用的结果是至关重要的，但是在生产性病毒感染期间对miRNA targetomes的系统时间分析在技术上是具有挑战性的，因为大范围的miRNA- mRNA在主机 - 病毒接口交叉对话。数据通过将生物信息学与Argonaute-交联和免疫沉淀接着高通量测序（AGO-CLIP-seq）数据（Chi等人，2009）数据结合，已经不发达。为了准确地鉴定miRNA靶位点并计算miRNA-靶相互作用的靶向效果，我们开发了新的生物信息学定量方法，即AGO-CLIP-seq富集（ACE） - 评分算法（Kim等， 2015）。在我们的AGO-CLIP-seq分析中包括未感染的对照可以显着提高病毒或人miRNA的真实靶位点识别的准确性，并且在我们的ACE评分方法中提取生产性人巨细胞病毒（HCMV）感染期间的生理学显着变化。因此，我们建议我们新的基于ACE评分的方法可以应用于各种miRNA targetome研究，这将在其他类型的时间背景下进行，如发展阶段，细胞因子或病原体的免疫刺激和其他病毒。
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| Detection of Alternative Oxidase Expression in Arabidopsis thaliana Protoplasts Treated with Aluminium
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Author:
Date:
2015-07-05
[Abstract] Aluminium (Al), a non-essential metal widespread in the environment that is known to be toxic to humans as well as to plants, can cause damage not only to the roots but also to the aerial parts of plants. Its toxicity has been recognized as one of the major factors that limit crop production on acid soil. Alternative oxidase, the respiratory terminal oxidase in plants, which contributes to maintain the electron flux and reduce mitochondrial ROS levels, is often dramatically induced to make plants to adapt better to stress conditions like Al stress. In this protocol, the expression of alternative oxidase induced by Al treatment was detected in Arabidopsis protoplasts using an adaptation of previous methods (Yamamoto et al., 2002; Li et al., 2011; Liu et al. ...
[摘要] 铝（Al），一种已知对人类以及对植物有毒的环境中普遍存在的非必需金属，不仅对根而且对植物的地上部分都造成损害。 其毒性已被认为是限制作物在酸性土壤上生产的主要因素之一。 替代氧化酶，植物中的呼吸末端氧化酶，有助于维持电子通量和降低线粒体ROS水平，经常被显着诱导，使植物更好地适应应力条件，如铝胁迫。 在该方案中，使用先前方法的改编，在拟南芥原生质体中检测由A1处理诱导的替代氧化酶的表达（Yamamoto等人，2002; et al。，2011; Liu et al。，2014），它们有助于研究Al处理中替代氧化酶的机制。
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