ESR DETECTION OF NITRIC OXIDE PRODUCED IN VIVO BY PROKARYOTIC CELLS

原核细胞体内一氧化氮的 ESR 检测

• 批准号: 7723985
• 负责人: Rosemary Loria
• 金额: $ 0.13万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7723985
• 关键词: Appearance; Arginine; Bacillus anthracis; Binding; Biology; Characteristics; Chemistry; Citrulline; Complex; Computer Retrieval of Information on Scientific Projects Database; Data; Deinococcus radiodurans; Detection; Diffuse; Doctor of Philosophy; Electrons; Funding; Gram-Positive Bacteria; Grant; Heme; Homologous Gene; Institution; Iron; Mammals; N-terminal; Nitric Oxide; Nitric Oxide Synthase; Organism; Oxidoreductase; Oxygenases; Paper; Production; Prokaryotic Cells; Proteins; Research; Research Personnel; Resources; Rosemary; Sampling; Sarcosine; Signal Transduction; Source; Spin Trapping; Staphylococcus aureus; Students; United States National Institutes of Health; Work; in vivo; interest; mutant; research study

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Prof Rosemary Loria is interested in detection, via ESR, of nitric oxide produced in vivo by prokaryotic cells. Nitric oxide synthases (NOSs) in mammals are dimeric proteins containing N-terminal oxygenase domains that convert arginine into citrulline and NO, and a reductase domain that shuttles electrons to the heme of the oxygenase domain. Homologs to NOSs exist in prokaryots, but direct evidence for NO production by these proteins has been lacking. The aim of current work was to demonstrate that NOS also produces diffusible NO. To demonstrate NO production, the spin trap dithiocarboxy-sarcosine (DTCS) was used. DTCS binds iron to form the active spin trap Fe(DTCS)2 that lacks an ESR signal. After NO binds to the Fe(DTCS)2 complex, the spin trap produces a characteristic 3-peak signal. Incubation of Fe(DTCS)2 with the wild type culture of S. turgidiscabies resulted in the appearance of the characteristic 3-peak signal of NO-Fe(DTCS)2 identical to that produced in control experiments by addition of artificial NO-donor. This NO-specific 3-peak signal was not observed in samples from NOS-deletion mutant cultures. These results confirmed that NOS-dependent free-diffusing NO is released by thaxtomin-producing streptomycetes and were the first data to document in vivo production of NO by prokaryotic NOSs. Structural conservation of bacterial NOSs predicts NO production by other Gram-positive bacteria including medically and environmentally important organisms such as Bacillus anthracis, Staphylococcus aureus, and Deinococcus radiodurans. A paper on this work was submitted to Chemistry & Biology. This work also represents a substantial part of the PhD thesis of a graduate student of Prof. Loria: Evan Johnson.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 罗斯玛丽·洛里亚（Rosemary Loria）教授对核细胞在体内产生的一氧化氮的检测感兴趣。哺乳动物中的一氧化氮合酶（NOSS）是含有N末端氧酶结构域的二聚体蛋白，这些蛋白将精氨酸转化为瓜氨酸和NO，以及将电子将电子延伸到氧化加酶域的血红素的还原酶结构域。原始人存在于NOSS的同源物，但是缺乏这些蛋白质生产的直接证据。当前工作的目的是证明NOS也会产生可扩散的NO。 为了证明未产生的生产，使用了自旋陷阱二硫代辅助辅助 - sarcosine（DTC）。 DTC结合铁以形成缺乏ESR信号的活动自旋陷阱Fe（DTC）2。 在与Fe（DTCS）2复合物结合后，自旋陷阱会产生一个特征性的3峰信号。 Fe（DTCS）2与毛刺链球菌的野生型培养物的孵育导致出现Noto-Fe（DTCS）2的特征性3峰信号（DTCS）2通过添加人工Notormal notoral notoy-Donor所产生的与对照实验中产生的相同。 在NOS-删除突变培养物的样品中未观察到这种无特异性的3峰信号。 这些结果证实，thaxtomin的产生链霉菌释放了NOS依赖性的自由扩散NO，并且是第一个记录促脑化NOSS体内生产NO的数据。 细菌NOSS的结构保守性预测其他革兰氏阳性细菌的产生不可能，包括医学和环境重要的生物，例如炭疽芽孢杆菌，金黄色葡萄球菌和放射性甲状腺菌。 有关这项工作的论文已提交化学与生物学。 这项工作也代表了洛里亚教授的研究生：埃文·约翰逊（Evan Johnson）的博士学位论文的重要组成部分。