Nobukazu Komatsu

Mechanism of superoxide anion generation in the toxic red tide phytoplankton Chattonella marina: possible involvement of NAD(P)H oxidase

Red tide phytoplankton Chattonella marina is known to produce reactive oxygen species (ROS), such as superoxide anion (O(2)(-)), hydrogen peroxide (H(2)O(2)) and hydroxyl radical (&z.rad;OH), under normal physiological conditions. Although several lines of evidence suggest that ROS are involved in the mortality of fish exposed to C. marina, the mechanism of ROS generation in C. marina remains to be clarified. In this study, we found that the cell-free supernatant prepared from C. marina cells showed NAD(P)H-dependent O(2)(-) generation, and this response was inhibited by diphenyleneiodonium, an inhibitor of mammalian NADPH oxidase. When the cell-free supernatant of C. marina was analyzed by immunoblotting using antibody raised against the human neutrophil cytochrome b558 large subunit (gp91phox), a main band of approximately 110 kDa was detected. The cell surface localization of the epitope recognized with this antibody was also demonstrated in C. marina by indirect immunofluorescence. Furthermore, Southern blot analysis performed on genomic DNA of C. marina with a probe covering the C-terminal region of gp91phox suggested the presence of a single-copy gene coding for gp91phox homologous protein in C. marina. These results provide evidence for the involvement of an enzymatic system analogous to the neutrophil NADPH oxidase as a source of O(2)(-) production in C. marina.

Isolated Ricin B-Chain-mediated Apoptosis in U937 Cells

We have previously reported that ricin, a toxic lectin that inhibits protein synthesis induced apoptotic cell death. In this study, we have found that isolated ricin CM-B-chain, which has no effect on cellular protein synthesis, induced DNA fragmentation in U937 cells in a dose- and time-dependent manner, albeit it required a longer incubation time and higher concentration than those of holotoxin ricin. Z-Asp-CH2-DCB, a caspase family inhibitor and serine protease inhibitor, 3,4-dichloroisocoumarine (DCI) effectively inhibited the CM-B-chain-mediated DNA fragmentation as well as in ricin. Thus, like ricin, multiple proteases with different substrate specificity may also be involved in the CM-B-chain-mediated apoptotic pathway. Furthermore, BFA inhibited both ricin- and CM-B-chain-mediated DNA fragmentation, suggesting an intracellular vesicle transport system through the Golgi complex may be involved in the apoptotic induction by these proteins as a common feature. On the other hand, cycloheximide (CHA) strongly increased the CM-B-chain-mediated DNA fragmentation, but inhibited ricin-mediated DNA fragmentation. The opposite effects of CHA may reflect the difference in the apoptotic mechanism between ricin and CM-B-chain. In conclusion, our results suggest that ricin-B-chain can induce apoptosis through its lectin activity, but the underlying mechanism may be distinct from that of ricin in which the A-chain contributes profoundly to the apoptotic induction.