Reactive oxygen species (ROS) are not only a cause of oxidative stress in a range of disease conditions but are also important regulators of physiological pathways in vivo. One mechanism whereby ROS can regulate cell function is by modification of proteins through the reversible oxidation of their thiol groups. An experimental challenge has been the relative lack of techniques to probe the biological significance of protein thiol oxidation in complex multicellular tissues and organs. We have developed a sensitive and quantitative fluorescence labeling technique to detect and localize protein thiol oxidation in histological tissue sections. In our technique, reduced and oxidized protein thiols are visualized and quantified on two consecutive tissue sections and the extent of protein thiol oxidation is expressed as a percentage of total protein thiols (reduced plus oxidized). We tested the application of this new technique using muscles of dystrophic (mdx) and wild-type C57Bl/10Scsn (C57) mice. In mdx myofibers, protein thiols were consistently more oxidized (19 ± 3%) compared with healthy myofibers (10 ± 1%) in C57 mice. A striking observation was the localization of intensive protein thiol oxidation (70 ± 9%) within myofibers associated with necrotic damage. Oxidative stress is an area of active investigation in many fields of research, and this technique provides a useful tool for locating and further understanding protein thiol oxidation in normal, damaged, and diseased tissues.

译文

活性氧 (ROS) 不仅是一系列疾病条件下氧化应激的原因,而且还是体内生理途径的重要调节剂。ROS调节细胞功能的一种机制是通过蛋白质的巯基可逆氧化来修饰蛋白质。实验挑战是相对缺乏技术来探索复杂的多细胞组织和器官中蛋白质硫醇氧化的生物学意义。我们已经开发了一种灵敏且定量的荧光标记技术来检测和定位组织学组织切片中的蛋白质硫醇氧化。在我们的技术中,还原和氧化的蛋白质硫醇在两个连续的组织切片上进行可视化和定量,并且蛋白质硫醇氧化的程度表示为总蛋白质硫醇的百分比 (还原加氧化)。我们使用营养不良 (mdx) 和野生型C57Bl/10Scsn (C57) 小鼠的肌肉测试了这种新技术的应用。在mdx肌纤维中,与C57小鼠的健康肌纤维 (10 ± 1%) 相比,蛋白硫醇始终被氧化 (19 ± 3%)。一个引人注目的观察是与坏死损伤相关的肌纤维内蛋白质巯基氧化 (70 ± 9%) 的定位。氧化应激是许多研究领域中积极研究的领域,该技术为定位和进一步了解正常,受损和患病组织中的蛋白质硫醇氧化提供了有用的工具。

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