Yukihisa Okumura

Fusion between Jurkat cell and PEO-lipid modified liposome.

Direct fusion between Jurkat cell and a liposome modified with poly(ethylene oxide)-bearing lipid (PEO-lipid) was examined using diphtheria toxin fragment A (DTA) as the probe. Only the DTA-loaded liposome modified with PEO-lipid(n = 32) (n is the number of ethylene oxide units) exerted significant cytotoxicity against Jurkat cells, while liposomes lacking either the PEO-lipid or DTA did not. Liposomes modified by the PEO-lipid with shorter PEO chain(n = 5 or 15) did not show any cytotoxicity, irrespective of their DTA-loading. The cytotoxicity was observed even in the presence of cytochalasin B, an inhibitor of endocytosis. Judging from these results, we concluded that the PEO-lipid(n = 32)-modified liposome directly fused with plasma membrane of Jurkat cell.

Ring-selective photorearrangement of bithiazoles

Abstract Photoirradiation of methyl 2′-methyl-2,4′-bithiazole-4-carboxylate ( 1 ) leads predominantly to 4,4′-bithiazole 2 with the formation of a small amount of isothiazole 3 . The ring-selective photorearrangement is interpreted in terms of LUMO bond index.

Modification of phosphatidylcholine liposomes with poly(ethylene oxide)-substituted lipids

Modification of egg phosphatidylcholine (eggPC) and dimyristoylphosphatidylcholine liposomes with poly(ethylene oxide)-substituted lipids [PEO-lipid(m,n); m and n mean, respectively, the chain length of alkyl group and the number of PEO segments]. Morphologies of these supermolecules were investigated by means of laser dynamic light scattering and transmission electron microscopy. Reconstitution of the PEO-lipid to eggPC liposome was possible below 40–50 mol% of total lipids for PEO-lipid(12,5), and 30–40 mol% for PEO-lipid(12,13) and PEO-lipid(12,31). Effective encapsulation of FITC-dextran (1.96 kDa) into the liposome was obtained using the eggPC liposome modified with PEO-lipid. This PEO-modified liposome showed sufficient barrier function to water-soluble polymers such as dextran. Through various studies, it was found that PEO-lipid(12,13) is relatively compatible for modifying conventional eggPC liposome.

Supramolecular assembly of functionalized lipids

Abstract This article describes recent developments in the studies of Supramolecular assemblies of three types of functionalized lipids, focusing on the interactions of their liposomes with biological cells: an artificial boundary lipid, 1,2-dimyristamido-1,2-deoxyphosphatidylcholine (D 14 DPC) enhances transfer of membrane proteins from cells and tissues to the liposome; an artificial lipid bearing a poly(ethylene oxide) moiety (PEO-lipid) induces membrane fusion between cells and the liposome; and gangliosides and their artificial analogues (sialyl cholesterols), reconstituted in the liposome, directly stimulate T-lymphocytes.

Mobility of Poly(Ethylene Oxide) Segments on Surface of Lecithin Liposomes

To better understand the dynamics of PEO-lipid-modified phosphatidylcholine liposomes (“fusogenic liposomes”), the mobility of the PEO segment on the liposomal surface and the fluidity of the bilayer were investigated by measuring the spin-lattice relaxation time (T 1by 13C-NMR spectroscopy and by fluorescence depolarization. 13C-NMR studies revealed that the PEO-lipids in this work exhibit similar behavior on liposomal surfaces irrespective of the PEO chain length. Furthermore, with regard to the relationship between morphology and dynamics of the PEO-lipid and phosphatidylcholine mixture, it was found that the mobility of PEO segments are altered by the morphology; liposome < aqueous micelle < monomeric dispersion in organic solution. Fluorescence depolarization studies revealed that liposomal membranes were tighter with the addition of PEO-lipid(12,a31) than with the addition of PEO-lipid(12,a13). In contrast, the reconstitution of PEO-lipid(12,a5) into liposomes increased membrane fluidity.

Direct extraction of A and B blood group antigens from human red cells by liposomes

BACKGROUND: Some of the major blood group antigens are on lipids and proteins of the red cell membrane. Incubation of intact red cells with liposomes containing specially designed artificial lipids has been shown to result in the extraction of membrane proteins by the liposomes. The extraction of blood group structures and the retention of their antigenicity have not been reported. STUDY DESIGN AND METHODS: After the incubation of red cells with liposomes, the extraction of the antigens from human red cells by liposomes was examined by evaluation of the agglutination of the liposomes by respective antisera. RESULTS: Agglutination specific to the A and B blood group antigens was seen, which indicated that the antigenicity of the blood group antigens was retained even after the extraction by the liposomes. The presence of an artificial boundary lipid, 1,2‐dimyristamido‐1,2‐ deoxyphosphatidylcholine, in the liposome was crucial to the efficient extraction of the A and B antigens. On the other hand, the extraction of D, M, N, and P1 was not always detectable by agglutination. CONCLUSION: The A and B blood group antigens were directly extracted from red cells by liposomes without loss of antigenicity.

Enhanced Growth of Cultured Fibroblasts by a Synthetic Polyanionic Polymer

A synthetic polyanionic polymer, poly(maleic acid-alt-7,12-dioxaspiro[5,6]dodec-9-ene), exhibits mitogen-like behavior for cultured fibro-blasts. Cultures of L929 and STO fibroblasts in the presence of this polymer resulted in enhanced growth by both fibroblast cell lines. After three days, at optimum polymer concentrations, the cell numbers increased 3.7-fold for the STO cells and 1.9-fold for the L929 cells compared to the cells cultured in the same environment without the polymer. The polymer promoted this growth without any other external growth factors. This result suggests that the polymer has a direct effect on the growth of both of these fibroblast cells.

Syntheses and properties of circular dichroism active phospholipids

A group of circular dichroism (CD) active phospholipids has been synthesised, in which one or both acyl chains has been replaced with a cinnamoyl or azobenzene chromophore-containing acid. Studies on the structure, CD activity and thermodynamic property of liposome membranes composed of CD active phospholipids were carried out. CD active liposomes were found to be stable, normal liposomes of approximately 550 A diameter based on the electron micrograph and dynamic light scattering, and to have thermodynamic property similar to the conventional phospholipid membranes without serious perturbation by aromatic bulk groups based on DSC. Liposomes composed of phospholipid having two trans-azobenzene chromophores showed an extremely large CD enhancement even well above Tc. This CD enhancement was drastically changed by the presence of cis-azobenzene chromophore and cis-cis isomer content after irradiation was higher than the theoretical value, suggesting the importance of interchromophore interaction in the liposome membranes.

Transfer of Membrane Proteins from Human Erythrocytes to Liposomes

Transfer of a membrane bound enzyme acetylcholinesterase (AChE) from human erythrocyte to liposome was carefully examined. An artificial lipid 1,2-dimyristoylamido-1,2-deoxyphosphatidylcholine (D14DPC) showed predominant control over the transfer. The transfer was induced by a certain change of erythrocyte membrane during incubation with liposome, which is probably due to transfer of lipids from the liposome to the erythrocyte. The shorter induction period observed with D14DPC containing liposome can be attributed to both the faster transfer of D14DPC and the higher ability of D14DPC to induce the AChE transfer.

13C-Labeling in Methionine Methyl Groups of Glycophorin AM and Its Effect on the Secondary Structure

On the way of dynamic study of human glycophorin AM in lipid bilayer, we tried [13C]-methylation of its two methionines, Met-8 and -81, in the presence of 8 M urea. 1H, 13C and CD spectra revealed that the A, D and E helical domains of the protein were partly affected upon the methylation, while B and C domains were not disturbed. 13C signals of methyl groups for both Met-8 and -81 appeared at the same position, at 15.7 ppm. The corresponding 1H chemical shifts were assigned as 2.11 ppm by using HMQC. These results indicate that previously reported 13C chemical shift of Met-81 methyl group may be wrong, and 8 M urea which was used during the methylation procedure certainly and irreversibly affect the secondary structure of the protein.