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

已知的单细胞海洋浮游植物Chattonella marina具有很强的杀鱼活性。已有研究表明，滨海梭子藻能产生活性氧(ROS)，并推测了ROS介导的鱼毒机制。事实上，已有多项研究表明，查顿菌属(Chattonella spp.)产生活性氧物种(ROS)。此外，我们对自旋陷阱5，5-二甲基-1-吡咯啉-N-氧化物和N-叔丁基-α-苯基硝酮的电子自旋共振(ESR)谱研究表明，滨海梭子藻产生羟基自由基(OH)。由于ROS通常被认为对生物有毒性，所以由Chattonella spp.产生的ROS。可能至少部分参与了捕杀鱼类的机制。此外，我们还发现，滨海梭子藻对海洋细菌溶藻弧菌具有ROS介导的毒性作用。除Chattonella spp.外，另一种鞭毛藻赤潮异弯藻(Heterosigma Akashiwo)也表现出ROS-Medium…对虹鲑鱼有更多的毒性。因此，ROS的产生似乎是石藻鞭毛虫的一个共同特征。然而，确切的毒性机制仍存在争议。在这项研究中，我们获得了在正常生长条件下滨海梭子藻产生一氧化氮(NO)的证据。我们利用NO与鲁米诺-H_2O_2的化学发光(CL)反应来检测滨海对虾细胞悬浮液中的NO。在本实验中，以细胞数量依赖的方式在滨海梭子藻中观察到显著的化学发光，并且这种显著的化学发光被添加一种特定的NO清除剂--羧基-PTIO所减弱。以重氮反应阳性物质(NOx)的测定为基础的分光光度测定法和使用NO的高特异性荧光指示剂的荧光测定法也证实了滨海梭子藻产生了NO。一氧化氮合酶抑制剂L-NAME能显著降低近海对虾体内的NO水平。加入L精氨酸后，NO水平升高，而NaNO_2对NO水平无影响。这些结果表明，一种类一氧化氮合酶是滨海梭子蟹产生一氧化氮的主要原因。较少

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