| Budget Amount *help |
¥3,830,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥330,000)
Fiscal Year 2007: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
Fiscal Year 2006: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Research Abstract |
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
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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
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