Tooru Taga

Effect of DNA/liposome mixing ratio on the physicochemical characteristics, cellular uptake and intracellular trafficking of plasmid DNA/cationic liposome complexes and subsequent gene expression

In order to identify the important factors involved in cationic liposome-mediated gene transfer, in vitro transfection efficiencies by plasmid DNA complexed with DOTMA/DOPE liposomes at different DNA/liposome mixing ratios were evaluated using four types of cultured cells with respect to their physicochemical properties. Significant changes were observed in the particle size and zeta potential of the complexes as well as in their structures, assessed by atomic force microscopy, which depended on the mixing ratio. In transfection experiments, except for RAW 264.7 cells (mouse macrophages), efficient gene expression was obtained in MBT-2 cells (mouse bladder tumor), NLH3T3 cells (mouse fibroblasts) and HUVEC (human umbilical vein endothelial cells) at an optimal ratio of 1:5, 1:7.5 or 1:5, respectively. On the other hand, cellular uptake of the [32P]DNA/liposome complexes increased in all cell types with an increase in the mixing ratio, which was not reflected by the transfection efficiency. The cellular damage determined by MTT assay was minimal even at the highest DNA/liposome ratio (1:10), indicating that the lower gene expression level at the higher ratio was not due to cytotoxicity induced by the complex. An ethidium bromide intercalation assay showed that the release of plasmid DNA from the complex, following the addition of negatively charged liposomes, was restricted as the mixing ratio increased. Furthermore, confocal microscopic studies using HUVEC showed that the 1:5 complexes exhibited a dispersed distribution in the cytoplasm whereas a punctuate intracellular distribution was observed for the 1:10 complexes. This suggests that there was a significant difference in intracellular trafficking, probably release from the endosomes or lysosomes, of the plasmid DNA/cationic liposome complexes between these mixing ratios. Taken together, these findings suggest that the DNA/liposome mixing ratio significantly affects the intracellular trafficking of plasmid DNA complexed with the cationic liposomes, which is an important determinant of the optimal mixing ratio in cationic liposome-mediated transfection.

Interaction between DNA–cationic liposome complexes and erythrocytes is an important factor in systemic gene transfer via the intravenous route in mice: the role of the neutral helper lipid

Recent studies have indicated that there are many barriers to successful systemic gene delivery via cationic lipid vectors using the intravenous route. The purpose of this study was to investigate the effect of binding and interaction between erythrocytes, a major constituent of blood cells, and the complexes, in relation to the role of the helper lipid, on the in vivo gene delivery to the lung following intravenous injection. We used three types of cationic lipid vectors, DNA–DOTMA/Chol liposome complexes, DNA–DOTMA liposome complexes, and DNA–DOTMA/DOPE liposome complexes. Although the three types of vectors bind to murine blood cells in vivo and in vitro, DOTMA/Chol and DOTMA complexes with a higher in vivo transfection activity do not induce fusion between erythrocytes, whereas DOTMA/DOPE complexes, a less efficient vector in vivo, induce fusion between the erythrocytes after a short incubation period. Pre-incubation of DOTMA/DOPE complexes with erythrocytes significantly reduced the transfection efficiency while DOTMA/Chol- and DOTMA complexes were more resistant to such treatment. The differences in the physicochemical and structural properties of these complexes could explain the differences in interaction with erythrocytes and subsequent gene expression. Lipids in DOTMA/Chol and DOTMA complexes have a stable lamellar structure. However, lipids in DOTMA/DOPE complexes have a highly curved structure with high fluidity. These results indicate that the interaction with erythrocytes depends on the properties of the cationic lipid vectors and this is an important factor for intravenous gene delivery using cationic lipid vectors.

Experimental charge density and electrostatic potential in nicotinamide

The accurate crystal structure of nicotinamide, 3-pyridinecarboxamide, was determined from X-ray and neutron diffraction experiments: C(6)H(6)N(2)O, M(r) = 122.13, monoclinic, P2(1)/c, Z = 4. The electron distribution at 150 K was determined by the maximum entropy method and the electrostatic potential in the crystal was calculated by Fourier convolution of the electron distribution. The electrostatic properties of the nicotinamide molecule depend on the molecular conformation. The asymmetric electrostatic potential field observed above and below the pyridine-ring plane is related to the rotation of the carboxamide group with respect to the pyridine plane. The positive potential peak at the C4 atom of the pyridine ring extends to the C=O-group side of the plane. The asymmetry of the potential on the C4 atom is consistent with the stereospecificity of hydride transfer in NAD(+)/NADH oxidoreduction.

Differentiation of enantiotopic carbonyl groups by the horner-wadsworth-emmons reaction

Abstract A chiral phosphonoacetate 2 differentiates the enantiotopic carbonyl groups in an α-diketone 5 to afford the Z-olefin 6 in high yield and ee. The absolute stereochemistry of 6 was determined by X-ray analysis of 8 derived from 6.

Structural studies of copper(I) complexes with ethylene. Crystal structures of [Cu(2,2′-bipyridine)(ethylene)]ClO4 and [Cu(1,10-phenanthroline)(ethylene)]ClO4

Abstract The crystal structures of the complexes [Cu(2,2′-bipyridine)(ethylene)]ClO4 (I) and [Cu(1,10-phenanthroline)(ethylene)]ClO4 (II) have been determined from X-ray diffraction studies. Complex I crystallizes in the triclinic space group, P 1 , with four molecules in a unit cell of dimensions a 10.657(1), b 6.998(1), c 18.251(2) A, α 91.05(1), β 93.35(1), and γ 84.97(1)°. Complex II crystallizes in the monoclinic space group, P21/a, with four molecules in a unit cell of dimensions a 19.981(5), b 10.904(2), c 6.955(1) A, and β 103.90(2)°. Both structures were solved by heavy-atom methods, and refined by block-diagonal least-squares methods. The final R-values for I and II were 0.044 for 2225 observed reflections for I and 0.066 for 2005 observed reflections for II. In both crystals, the CuI ion is coordinated to two nitrogen atoms of 2,2′-bipyridine or 1,10-phenanthroline, and two carbon atoms of ethylene in an approximately planar form. The CC bond distances of the coordinated ethylene molecules, 1.360(13) and 1.346(18) A for I and 1.361(22) A for II, do not show marked lengthening compared with that for free ethylene, and may be explained by dπ(Cu) → dπ (ethylene) back-bonding between the Cu 3dπ orbital and the Rydberg 3dπ orbital of the ethylene molecule.

SYNTHESIS OF CHIRAL AMINO ALLENES VIA AN ORGANOCYANOCUPRATE-MEDIATED RING-OPENING REACTION OF ENANTIOPURE ETHYNYLAZIRIDINES

Amino allenes have been synthesized from 2-ethynylaziridines via organocopper-mediated reactions. Whereas treatment of enantiomerically pure (2 R ,3 S )-2,3- trans -3-alkyl-2-ethynylaziridines with RCu(CN)M (M = Li or MgX) yield exclusively (S,S) -allenylamines in high yields, isomeric (2 S ,3 S )-2,3- cis -3-alkyl-2-ethynylaziridines afford (S,R) -allenylamines in comparable high yields.

Structures of nine new diterpenoids from Taxus chinensis

Abstract The structures of nine new diterpenoids from the leaves and stems of Taxus chinensis were elucidated by means of NMR spectroscopy. Some of the new diterpenes were subjected to X-ray crystallographic analysis. Taxchinins D, G, E, H, I, J, K have an 11(15 → 1)-abeotaxane skeleton. Taxchins A and B possess an ordinary taxane skeleton, and belong to the first example of taxoids without an oxygen functionality or the sp 2 -hybridized carbon at C-4. Taxchinins D, E, I, and J exist as a mixture of conformational isomers in solution, whose behavior was investigated by variable-temperature NMR spectra and compared with the conformation obtained from X-ray analysis.

A new access to chiral aziridines by enzymatic transesterification of meso-bis(acetoxymethyl)aziridines

(2R, 3S)-2-Acetoxymethyl-3-hydroxymethylazirizines of high enantiomeric purity were prepared through enzymatic transesterification of meso-bis(acetoxymethyl)aziridines.

SN2′ Ring opening of aziridines bearing an α,β-unsaturated ester group with organocopper reagents. A new stereoselective synthetic route to (E)-alkene dipeptide isosteres

Regio- and stereo-selective synthesis of (E)-alkene dipeptide isosteres has been successfully achieved by exposing both (E)- and (Z)-N-(4-methylphenyl)sulfonyl-γ,δ-epimino-α,β-enoates to organocopper reagents at –78 °C for 30 min.

Taxchinin a : a diterpenoid from Taxus chinensis

Abstract A diterpenoid with a novel basic skeleton has been isolated from Taxus chinensis , the structure of which was elucidated by spectral means and unambiguously determined by an X-ray analysis.