Nitric oxide synthase(NOS) like enzyme mediated nitric oxide(NO) generation by harmful red tide phytoplankton, Chattonella marina

一氧化氮合酶（NOS）样酶介导有害赤潮浮游植物、查托氏菌码头产生一氧化氮（NO）

基本信息

批准号：
18580205
负责人：
ODA Tatsuya
金额：
$ 2.45万
依托单位：
Nagasaki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18580205/
关键词：
Red tide phytoplankton Chattonella marina reactive oxygen species nitric oxide NO synthase toxicity flagellate

项目摘要

The unicellular marine phytoplankton Chattonella marina is known to exhibit potent fish-killing activity. Previous studies have demonstrated that C. marina produces reactive oxygen species (ROS), and ROS-mediated ichthyotoxic mechanism has been postulated. In fact, several studies have demonstrated that Chattonella spp. generate reactive oxygen species (ROS). Furthermore, our studies using electron spin resonance (ESR) spectroscopy with the spin traps 5,5-dimethyl-1-pyrroline-N-oxide and N-t-butyl-a-phenylnitrone showed that C. marina generates hydroxyl radical (OH). Since ROS are generally considered to be toxic against living organisms, the ROS generated by Chattonella spp. may at least partly involve in the fish-kill mechanism. Furthermore, we have found that C. marina exhibited ROS-mediated toxic effect on a marine bacterium, Vibrio alginolyticus. In addition to Chattonella spp., it has been reported that another raphidophycean flagellate, Heterosigma akashiwo also showed ROS-media … More ted toxicity on rainbow trout. Thus, it seems likely that the production of ROS is a common feature of raphidophycean flagellates. However, the exact toxic mechanism is still controversial. In this study, we obtained evidence that C. marina produces nitric oxide (NO)under normal growth conditions. We utilized chemiluminescence (CL)reaction between NO and luminol-H_2O_2 to detect NO in C. marina cell suspensions. In this assay, significant CL was observed in C. marina in a cell-number dependent manner, and this was diminished by the addition of carboxy-PTIO, a specific NO scavenger. The NO generation by C. marina was also confirmed by a spectrophotometric assay based on the measurement of the diazo-reaction positive substances (NO_x)and by fluorometric assay using highly specific fluorescent indicator of NO. The NO level in C. marina was significantly decreased by L-NAME, a specific NO synthase (NOS)inhibitor. The addition of L-arginine resulted in the increase in NO level, whereas NaNO_2 had no effect. These results suggest that a NOS-like enzyme is mainly responsible for NO generation in C. marina. Less

已知单细胞海洋浮游植物查托氏菌 (Chattonella marina) 具有强大的杀鱼活性，此前的研究表明，查托氏菌 (Chatonella marina) 会产生活性氧 (ROS)，并且已经推测了 ROS 介导的鱼类毒性机制。 Chattonella spp. 此外，我们的研究使用电子自旋共振 (ESR) 光谱和自旋陷阱。 5,5-二甲基-1-吡咯啉-N-氧化物和N-叔丁基-a-苯基硝酮表明C. marina 会产生羟基自由基(OH) 由于ROS 通常被认为对生物体有毒，因此查托氏菌产生的ROS。此外，我们发现 C. marina 对海洋细菌弧菌具有 ROS 介导的毒性作用。除了溶藻菌外，据报道，另一种针藻类鞭毛虫 Heterosigma akashiwo 也对虹鳟鱼表现出 ROS 介质毒性，因此，ROS 的产生似乎是针藻类鞭毛虫的一个共同特征。然而，确切的毒性机制仍然存在争议，在这项研究中，我们获得了 C. marina 在正常生长下产生一氧化氮 (NO) 的证据。我们利用NO和鲁米诺-H_2O_2之间的化学发光（CL）反应来检测C. marina细胞悬液中的NO。在该测定中，在C. marina中以细胞数量依赖性方式观察到显着的CL，并且这种情况被减少。添加羧基-PTIO，一种特定的 NO 清除剂，C. marina 产生的 NO 也通过基于重氮反应阳性物质 (NO_x) 的测量的分光光度测定法得到证实。使用高度特异性的 NO 荧光指示剂进行荧光测定。 L-NAME（一种特异性 NO 合酶 (NOS) 抑制剂）显着降低了 C. marina 中的 NO 水平。L-精氨酸的添加导致 NO 水平增加，而 NaNO_2 则增加了 NO 水平。这些结果表明 NOS 样酶主要负责 C. marina 中 NO 的产生。