Takehiko Ueda

Molecular assemblies of bis- and tris-adenine derivatives

Abstract Molecular assemblies of bis- and tris-adenine derivatives were studied by means of NMR, IR, and DSC. Mixtures of the adenine derivatives and 1-hexadecylthymine showed multiple phase transitions in the temperature range of the solid phase. A difference of the NMR, IR, and DSC data between bis-adenine and tris-adenine derivatives may be attributable to a formation of intramolecular hydrogen bonds.

Surface Pressure Analysis of Poly(ethylene oxide)-Modified Fusogenic Liposomes Incorporated into a Phospholipid Monolayer

Fusogenic liposomes have a wide-range of applications as DDS and gene/protein delivery into living cells. A variety of surface modifications of drug carriers, to enable fusion with cells, have been proposed, however, the mechanism of fusion has still not been determined. To further improve the efficiency of drug carriers, a simple and easily examinable model of a living cell surface is needed. In this study, the time-course of a fusion phenomena was made by measuring the surface pressure increase of a phospholipid monolayer spread at the air/water interface due to the fusion of liposomes carrying PEO-lipid (dialkyl-terminated polyethylene oxide) reconstituted on their outer surface. The kinetics of the surface pressure change appeared to be bimodal, indicating the coexistence of different fusion pathways. It was found that the presence of the PEO-lipid on the liposome surface led to a faster lipid transfer compared to non-modified DMPC liposomes. This indicated that the reconstitution of PEO-lipid provided an alternative transfer pathway to that for non-fusogenic liposomes that show only a slow lipid transfer to phospholipid monolayers. The relation between the rate of fusion and the surface pressure of the host membrane is discussed.

Identification of a mutagenic chlorination by-product produced from (E)-1, 3-dichloropropene (a component of nematocide DD) by using high resolution LC/MS and multivariate analysis

The estimated domestic usage of DD (a typical nematocide) has been the greatest among all the agricultural chemicals in Japan. DD is involved in a document which is used to establish tap water quality standard in Japan. The document indicates that DD is potentially detectable in raw water for tap waters. DD in raw water will be treated with chlorine at waterworks, which raises concerns about formation of mutagenic chlorination by-products through the treatment. The objective of this work was to identify a mutagenic chlorination by-product by using high resolution mass spectrometers and multivariate analyses. Mutagenicity of the chlorination by-product was evaluated with the Ames Salmonella mutagenicity assay. (E)-1,3-Dichloropropene (DCP) was used as a model compound of DD. The mutagenicity of chlorine treated model aqueous solutions of DD increased with increasing chlorine dosages up to 3.00 mol-Cl2/mol-DCP. From the chlorine treated aqueous solutions of DD, 1,3-dichloroacetone (DCA) was identified as a major mutagen by a cochromatography with an authentic standard, precursor ion analyses and first-generation product ion analyses with the high resolution mass spectrometers. The mutagenicity of DCA against TA100 strain without microsomal activation was 160,000 net revertant colony/μmol (the toxicity equivalent factor was 0.14). Based on the mutagenicity of the chlorine treated DCP sample, the specific mutagenicity of DCA, and the DCA concentration in the sample, the mutagenic contribution of DCA to the chlorine treated DCP sample was calculated. High contribution (98%) clearly shows that DCA is a major mutagen in the chlorine treated DCP sample. Because the exploration of DCA was performed by using the unrealistic high concentration samples of DCP and chlorine, formation of DCA in the practical concentration samples (0-200 μg-DCP/L, 0.10 or 1.27 mg-Cl2/L) was examined. It was proven that DCA formation reaction takes place in the practical concentrations of DCA and chlorine.

COMPLEXATION OF NEOCARZINOSTATIN CHROMOPHORE WITH A HYDROPHOBIZED POLYSACCHARIDE AS AN APOPROTEIN MODEL

Interaction between neocarzinostatin chromophore (NCS-chr) and a self-aggregate of cholesterol-bearing pullulan (CHP) was investigated. NCS-chr was isolated by gel chromatography (solvent: dimethylsulfoxide (DMSO)) after NCS-chr was released from neocarzinostatin (NCS) in DMSO. The amphiphilic CHP nanoparticle bound NCS-chr in the hydrophobic binding site. The chemical stability of the chromophore in water increased upon the complexation.

Reconstitution of Bovine Placental Insulin Receptor on Artificial Vesicles by Using Direct Protein Transfer Techniques

Direct protein transfer with artificial boundary lipid, DDPC, has been used as a powerful isolation procedure for membrane proteins. In contrast to other methods using detergents as protein solubilizer, the target membrane proteins can be transferred directly from biomembrane to liposomes by this direct protein transfer method, without severe denaturation in the course of isolation. We have investigated the transfer of insulin receptor proteins from bovine placental plasma membrane into liposomes. All the subunits of insulin receptor was found reconstituted in liposomes. The reconstituted insulin receptor retained almost full activity to bind to insulin, leading to autophosphorylation as a result of signal transduction activity. It is quite important that the reconstituted insulin receptor can be isolated in liposomes, which can be served to further investigation of insulin receptor without any interferences from other enzymes in cells. Then we focused on the insulin binding activity. The reconstituted insulin receptor showed much higher binding affinity than that on plasma membrane or that solubilized in micelles, suggesting that insulin receptor protein isolated by a conventional method might catch a significant denaturation. A fully active and complete insulin receptor was isolated by this direct protein transfer method. This method is applicable extensively to other membrane proteins.