Taxus cell culture may be an alternative source of paclitaxel and related taxane production. Significantly increased amounts of paclitaxel and baccatin III were observed in cultured cells of Taxus species after exposure to methyl jasmonate. Among the three species of Taxus tested, Taxus media showed the highest paclitaxel content while Taxus baccata showed the highest baccatin III content when 100 microM of methyl jasmonate was added to the culture media. Furthermore, the activities of methyl jasmonate and related substances for inducing paclitaxel production were compared in cell suspension cultures of T. media. Methyl jasmonate and its free acid showed the strongest promoting activity. Reduction of the keto group at the C-3 position greatly reduced this activity. cis-Jasmone, which does not have a carboxyl group at the C-1 position, had almost no activity. These results suggest that these two regions of methyl jasmonate are important for promoting the production of paclitaxel and related taxanes in Taxus cell cultures.

Purple membrane (PM), which is a membrane patch formed by the self-assembly of the membrane protein bacteriorhodopsin (bR) with archaeal lipids, is a good subject for studying the mechanism for the supramolecular structural formation of membrane proteins. Several studies have suggested that PM is not simply planar but that it has a curvature. Atomic force microscopy (AFM) studies also indicate the presence of dome-like structures (bumps) on the cytoplasmic surface of PM. PM must have a curvature to form the bump structures; therefore, bump formations will be related to a mechanism for supramolecular structural formation via self-assembly. To elucidate the effect of an asymmetric distribution of charged residues between two aqueous domains on the bump curvature, AFM topography of identical PM sheets were examined with variation of the solvent ionic strength and pH using a newly constructed solvent circulation system. The radius and height distributions of the bumps on the identical PM sheets indicated a linear correlation. The bump curvature, which was simply estimated by the slope of the distribution, became smaller with increasing KCl concentration, which suggests that tension at the cytoplasmic surface caused by electrostatic repulsive force between negatively charged amino acid residues becomes weaker by the electrostatic shielding effect. AFM observations revealed that the bump curvature remained even at high KCl concentration where the Debye length is within a few Angstroms; therefore, the contribution of the intrinsic difference between the domain sizes of bR between two sides was confirmed. Interestingly, the bump curvature was significantly increased by the addition of CaCl2 and then decreased with a similar dependency to KCl at higher CaCl2 concentration. The effect of pH on the bump curvature was also examined, where the curvature increased and reached a maximum at pH 9, while it decreased above pH 10, at which point the two-dimensional crystalline lattice of bR began to disassemble. These experimental results indicate that the bump curvature is strongly influenced by electrostatic interactions. A plausible model for bump structure formation by electrostatic repulsive force is presented based on these results.

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Surface Enhance Infrared Absorption (SEIRAS) have unique properties that enhances signals at vicinity (in an order of several nm's) of a substrate metal. This property is useful to determine solely the surface chemical process distinguished from that occurred in bulk phase. Therefore when a biological sample of interest is confined to such surface, one can selectively monitor a chemical reaction of the target regardless of the complex ensemble biological process in the bulk phase. We present an application of SEIRAS to in-situ investigation for a folding process of membrane protein (bacteriorhodopsin) during cell free expression on the nano-disc artificial lipid bilayer. 2P104等温滴定型熱量計による多剤輸送担体 EmrE の基質結合様 EmrE is a substrate/proton antiporter, which extrudes a wide range of lipophilic cations. Here, we had demonstrated with ITC how tetraphenyl phosphonium ion (TPP + ) binds to the wild-type and mutants (E14Q, D, E25Q). As pH increase, the driving force of EmrE/TPP + binding shifted from enthalpy-driven to entropy-driven and the binding affinity was more potent, inferring that EmrE has a more open conformation at acidic pH, and a closed conformation at neutral and alkaline pH. Especially, Glu25 might be one of determinant residues to drive the substrate binding, because in E25Q, at pH 7.4, the contribution of enthalpy to interaction energy remained a little, but significantly. The mechanism of proton coupled substrate transport will be discussed here. 式の解析 2P105多剤排出トランスポーター The multidrug transporter AcrB actively exports a wide variety of noxious compounds using the proton-motive force as an energy source in Gramnegative bacteria. AcrB adopts an asymmetric structure comprising three protomers with different conformations that are sequentially converted during drug export. These cyclic conformational changes for drug export are called the functional rotation. The Motion Tree is a new method to describe the structural change between two states of proteins in a hierarchical manner. This method can detect the rigid body motions and clarify the relations among them. In the present study, we applied the Motion Tree analysis to the cyclic conformational changes of AcrB. As a result, the key motions for the functional rotation were observed. Purple membrane (PM) is a membrane patch which is formed by self-assembly of membrane protein bacteriorhodopsin with archaeal lipids. Atomic force microscopy (AFM) studies for PM supported onto mica substrate shows dome-like "bump" structures on the surface. Since PM must have a curvature to form the bump structure, bump formations will be related to a mechanism for structural formations via self-assembly. To elucidate effects of asymmetric distribution of charged residues between two aqueous domains on bump curvature, AFM observations were carried out toward identical PM sheets with varying solvent pH and ionic strength. The bump curvature studies indicated large influence of electrostatic interaction. The model for the bump structure will be discussed. 2P106 ABC トランスポーターにおける薬剤結合の影響...