, 2000; Naim et al., 2001). Mature forms of TDH and TRH consist of 165 amino acids with a pair of intramolecular disulfide bonds between cysteine moieties in positions 151 and 161 (Iida & Honda, 1997). TDH-positive V. parahaemolyticus is hemolytic on Wagatsuma agar, which is a special type of blood agar; this effect is known as the Kanagawa phenomenon (Miwatani et al., 1972; Okuda & Nishibuchi, 1998). Electron microscopic observations indicated that TDH formed pore-like structures on the surface of erythrocyte membranes (Honda et al., 1992). Furthermore, when lipid bilayers were treated with TDH, single channel pore formation was observed (Hardy et al., 2004). In addition, Miwatani reported

that heating crude TDH at 60 °C inactivated its hemolytic activity but the activity was restored by rapid cooling from the denatured state at 90 °C (Miwatani

et al., 1972). This paradoxical phenomenon Forskolin is known as PI3K Inhibitor Library the Arrhenius effect, which was originally reported with the α-hemolysin of Staphylococcus aureus by S.A. Arrhenius in 1907 (Arrhenius, 1907). We have previously determined that the underlying molecular mechanism mediating the Arrhenius effect in TDH is the reversibility of amyloid fibril formation upon heating of TDH (Fukui et al., 2005). On the other hand, TRH lost its hemolytic activity upon heating at 90 °C, suggesting that TRH activity is not associated with the Arrhenius effect in the same way as TDH (Honda et al., 1988). We have also previously identified the C4-symmetric tetrameric structure of TDH and its model in low solutions using

small-angle X-ray scattering, ultracentrifugation, and transmission electron microscopy (Hamada et al., 2007), and presented the crystal structure of TDH tetramers with a central pore at a 1.5 Å resolution (Yanagihara et al., 2010). Single amino acid substitutions of TDH showed that π-cation interactions between R46 and Y140 played an important role in maintaining the tetrameric structure, whereas the monomeric mutant, R46E, lost its hemolytic activity (Yanagihara et al., 2010). TRH shares antigenicity in part with TDH. Hybridization tests with trh gene-specific DNA ligase probes showed that trh gene had nucleotide sequence variations, trh1 and trh2 gene, in clinical strains (Nishibuchi et al., 1989; Kishishita et al., 1992). The trh1 gene is 84% homologous to the trh2 gene, and its nucleotide sequence analysis indicated that it shares 68% homology with tdh gene. The amino acid sequence of trh1 gene also shares 63% homology with that of tdh gene (Nishibuchi et al., 1989). However, detailed structural analysis and the association state of native TRH remain unclear. Protein aggregation and amyloid formation are related to many protein conformational diseases, including Alzheimer’s, Huntington’s, and Parkinson’s disease (Bucciantini et al., 2002; Quist et al., 2005).