| Highly Accurate Real-time Measurement of Rapid Hydrogen-peroxide Dynamics in Fungi
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Author:
Date:
2016-12-20
[Abstract] Reactive oxygen species (ROS) are unavoidable by-products of aerobic metabolism. Despite beneficial aspects as a signaling molecule, ROS are principally recognized as harmful agents that act on nucleic acids, proteins and lipids. Reactive oxygen species, and, in particular, hydrogen peroxide (H2O2), are deployed as defense molecules across kingdoms, e.g., by plants in order to defeat invading pathogens like fungi. Necrotrophic plant pathogenic fungi themselves secrete H2O2 to induce host cell death and facilitate infection. Hydrogen ...
[摘要] 活性氧（ROS）是有氧代谢的副产物。尽管作为信号分子的有利方面，ROS主要被认为是作用于核酸，蛋白质和脂质的有害物质。反应性氧物质，特别是过氧化氢（H 2 O 2 O 2），作为防御分子跨越诸如，通过植物为了击败入侵病原体如真菌。营养不良的植物致病真菌本身分泌H 2 O 2 O 2以诱导宿主细胞死亡并促进感染。过氧化氢在一定程度上可以通过膜自由扩散。为了能够监测真菌中的细胞内过氧化氢动力学，我们最近在丝状植物病原体禾谷镰刀菌（Mentges and Bormann，2015）中建立了多功能的HyPer成像技术。 HyPer由插入到原核H 2 O 2 O 2 - 感觉蛋白OxyR的调节结构域（RD）中的循环置换的黄色荧光蛋白（cpYFP）组成。 OxyR域使传感器高度特异于H 2 O 2 O 2。 HyPer的氧化增加了在488nm激发的cpYFP的荧光，并降低了在405nm激发的荧光，从而促进了比例式读数（Belousov等人，2006）。 HyPer原来是对pH敏感。 HyPer的H 2 O 2 O 2 - 感官域中的单个氨基酸突变使传感器对H 2 O 2不敏感。该记者称为SypHer，作为pH变化的对照。
＆nbsp; ＆nbsp;通过使用HyPer成像技术，我们可以证明：i）HyPer成像能够特异和准确地检测细胞内H 2 O 2 O 2平衡的快速变化，ii）。 ...
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| High-throughput Quantification of Ammonium Content in Arabidopsis
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Author:
Date:
2015-08-20
[Abstract] This protocol is a simple colorimetric assay for internal ammonium quantification in aqueous extracts from plant tissues. The method is based on the phenol hypochlorite assay (Berthelot reaction):
NH4+ + hypochlorite + OH- + phenol → indophenol
The oxidation of indophenol caused by phenol oxidation is a blue dye that is quantified at 635 nm in a spectrophotometer. Per ammonium molecule one molecule of indophenol is formed. The protocol described here is for Arabidopsis thaliana (A. thaliana) leaves and roots, although ...
[摘要] 该方案是一种简单的比色测定法，用于在植物组织的水性提取物中进行内部铵定量。 该方法基于苯酚次氯酸盐测定（Berthelot反应）：
NH 4+ sup + +次氯酸盐+ OH - +苯酚→靛酚
由酚氧化引起的靛酚的氧化是在分光光度计中在635nm处定量的蓝色染料。 每个铵分子形成一个靛酚分子。 这里描述的方案是用于拟南芥（拟南芥）叶和根，虽然它也适用于其他植物物种。
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| In vitro Colorimetric Method to Measure Plant Glutamate Dehydrogenase Enzyme Activity
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Author:
Date:
2015-08-20
[Abstract] Glutamate dehydrogenase (GDH) is an NAD(H) dependent enzyme that catalyzes, in vitro, the reversible amination of glutamate. Here we describe how to determine spectrophotometrically GDH activity monitoring NADH evolution. This protocol is described here for Arabidopsis thaliana (A. thaliana) although it is also valid for other plant species. GDH protein is a hexamer composed, in the case of Arabidopsis, of a combination of GDHα, GDHβ and GDHγ subunits. Every combination of subunits is possible; however, it is still barely known whether different ...
[摘要] 谷氨酸脱氢酶（GDH）是NAD（H）依赖性酶，其在体外催化谷氨酸盐的可逆胺化。在这里我们描述如何确定分光光度法GDH活动监测NADH进化。此协议在这里描述为拟南芥（ A thaliana ），虽然它也适用于其他植物物种。 GDH蛋白是在拟南芥属的情况下由GDHα，GDHβ和GDHγ亚基的组合组成的六聚体。每个亚基的组合是可能的;然而，仍然几乎不知道不同的组合是否影响六聚体的酶性质。在其他物种中，六聚体是GDHα和GDHβ的组合，但不能丢弃其它基因的存在，因为例如在Fontaine等人（2012）中描述了拟南芥中的GDHγ亚基。
谷氨酸盐+ NAD + + H + →2-氧代戊二酸+ NADH + NH4 +
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