DEVELOPMENT OF BRIDGE LOOP RESONATORS FOR IN VIVO L BAND STUDIES

用于体内 L 波段研究的桥环谐振器的开发

• 批准号: 6206513
• 负责人: TADEUSZ WALCZAK
• 金额: $ 0.61万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6206513
• 关键词: Mammalia; biological products; biomaterials; biomedical resource; environmental toxicology; enzymes; hematology

A new EPR-based method has been developed to obtain selective information on pO2 in a specified intracellular compartment (phagosome). The method utilises the selective incorporation of the oxygen-sensitive probe 4-(Trimethylammonium) 2,2,6,6,-tetramethylpiperidine-D17-1-Oxyl iodide (D-CAT1) into phagosomes of macrophages stimulated with zymosan. Since the signal arising from the neutral nitroxide 4-oxo-2,2,6,6,-(15N)-tetramethylpiperidine-D16-1-Oxyl (15N-PDT) does not overlap with that from the D-CAT1, these signals can be monitored simultaneously. Lipopolysaccharide (LPS) is the endotoxin from the outer membrane of Gram-negative bacteria that is associated with the high morbidity and mortality in patients with septic shock. Our previous studies have shown that LPS can influence mitochondrial oxygen consumption in a variety of cell types and alter the oxygen utilisation of organs in experimental septic shock. It is also suggested that LPS can augment the respiratory burst associated with phagocytosis in macrophages. D-CAT1 was added to cells of the murine macrophage cell lines RAW 264.7 followed by zymosan stimulation to induce phagocytosis. After washing, the probe remained in the phagosome; without stimulation of phagocytosis the probe was not incorporated and could be removed by washing. 15N-PDT was added to the same samples to give the effective extracellular oxygen concentration (as less than 5% of the PDT signal arises from inside the cells). Cells with intraphagosomal D-CAT1 and 15N-PDT were then treated with LPS and the oxygen concentrations measured for intraphagosomal and extracellular sites after callibration of the two probes in pure air and nitrogen. The results show that LPS reduces intraphagosomal oxygen concentrations by almost one half. Furthermore, EPR spin-trapping experiments (using DMPO to trap - OH and -OOH radicals) showed that LPS stimulates a sustained respiratory burst in these macrophages above that induced by a zymosan alone. These results suggest that LPS can influence macrophage phagocytosis, and in certain cases the low oxygen concentration within phagosomes can potentially limit macrophage microbicidal funtion during infections.

开发了一种新的基于 EPR 的方法来获得选择性 有关特定细胞内区室中 pO2 的信息 （吞噬体）。 该方法利用选择性掺入 氧敏探针4-(三甲基铵) 2,2,6,6,-四甲基哌啶-D17-1-氧基碘化物(D-CAT1) 用酵母聚糖刺激巨噬细胞的吞噬体。 自从信号 由中性硝基氧产生 4-氧代-2,2,6,6,-(15N)-四甲基哌啶-D16-1-Oxyl (15N-PDT) 确实 与来自 D-CAT1 的信号不重叠，可以监视这些信号 同时地。 脂多糖（LPS）是来自脂多糖的内毒素 革兰氏阴性菌的外膜，与 感染性休克患者的发病率和死亡率较高。 我们的 先前的研究表明LPS可以影响线粒体 各种细胞类型的耗氧量并改变氧气 实验性感染性休克中器官的利用。 这也是 表明 LPS 可以增强与以下疾病相关的呼吸爆发： 巨噬细胞的吞噬作用。 D-CAT1被添加到小鼠细胞中 巨噬细胞系 RAW 264.7，然后用酵母聚糖刺激 诱导吞噬作用。 清洗后，探针保留在 吞噬体；在没有刺激吞噬作用的情况下，探针不会 合并并可以通过洗涤去除。 添加 15N-PDT 相同的样品提供有效的细胞外氧 浓度（小于 5% 的 PDT 信号来自内部） 细胞）。 然后将具有吞噬体内 D-CAT1 和 15N-PDT 的细胞 用 LPS 处理并测量氧气浓度 校准后的吞噬体内和细胞外位点 探头在纯空气和氮气中。 结果表明，LPS 可以降低 吞噬体内的氧气浓度几乎降低了一半。 此外，EPR自旋捕获实验（使用DMPO捕获-OH 和-OOH自由基）表明LPS刺激持续的呼吸 这些巨噬细胞中的爆发高于单独的酵母聚糖诱导的爆发。 这些结果表明LPS可以影响巨噬细胞的吞噬作用， 在某些情况下，吞噬体内的氧气浓度较低 可能会限制巨噬细胞的杀菌功能 感染。