Shoko Yokoyama

Membrane Incorporation, Channel Formation, and Disruption of Calcium Homeostasis by Alzheimer's β-Amyloid Protein.

Oligomerization, conformational changes, and the consequent neurodegeneration of Alzheimers β-amyloid protein (AβP) play crucial roles in the pathogenesis of Alzheimers disease (AD). Mounting evidence suggests that oligomeric AβPs cause the disruption of calcium homeostasis, eventually leading to neuronal death. We have demonstrated that oligomeric AβPs directly incorporate into neuronal membranes, form cation-sensitive ion channels (“amyloid channels”), and cause the disruption of calcium homeostasis via the amyloid channels. Other disease-related amyloidogenic proteins, such as prion protein in prion diseases or α-synuclein in dementia with Lewy bodies, exhibit similarities in the incorporation into membranes and the formation of calcium-permeable channels. Here, based on our experimental results and those of numerous other studies, we review the current understanding of the direct binding of AβP into membrane surfaces and the formation of calcium-permeable channels. The implication of composition of membrane lipids and the possible development of new drugs by influencing membrane properties and attenuating amyloid channels for the treatment and prevention of AD is also discussed.

Preparation and physicochemical properties of various soybean lecithin liposomes using supercritical reverse phase evaporation method

Abstract Three kinds of soybean lecithin liposomes composed of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidic acid (PA), were prepared by using the previously developed supercritical reverse phase evaporation method (Langmuir 17 (2001) 3898). The effect of phospholipid composition on the formation of liposomes and their physicochemical properties were examined by means of trapping efficiency measurements, transmission electron microscopy, dynamic light scattering and zeta potential measurements. The trapping efficiency of liposomes for d -(+)-Glucose made of Lecinol S-10EX which contains approximately 95% PC is higher than that of Lecinol S-10 and SLP white SP which contain approximately 31% PC. However there is not any difference between the trapping efficiency of liposomes for d -(+)-Glucose made of Lecinol S-10 which has saturated hydrocarbons tails and that of liposomes made of SLP white SP which has unsaturated hydrocarbon chains. The electron micrographs of liposomes made of Lecinol S-10 and SLP white SP show small spherical liposomes with diameter of 0.1–0.25 μm, while that of Lecinol S-10EX shows large unilamellar liposomes (LUV) with diameter of 0.2–1.2 μm. These results clearly show that phospholipid structure of PC allows an efficient preparation of LUV and a high trapping efficiency for water-soluble substances. Liposomes made of Lecinol S-10 and SLP white SP remained well-dispersed for at least 14 days, while liposome suspension made of Lecinol S-10EX separated in two phase at 14 days due to aggregation and fusion of liposomes. The dispersibility of liposomes made of Lecinol S-10EX is lower than that of Lecinol S-10 and SLP white SP due to the smaller zeta potential of Lecinol S-10EX.

Effect of adsorption of bovine serum albumin on liposomal membrane characteristics

The effect of adsorption of bovine serum albumin (BSA) on the membrane characteristics of liposomes at pH 7.4 was examined in terms of zeta potential, micropolarity, microfluidity and permeability of liposomal bilayer membranes, where negatively charged L-alpha-dipalmitoylphosphatidylglycerol (DPPG)/L-alpha-dipalmitoylphosphatidylcholine (DPPC), negatively charged dicetylphosphate (DCP)/DPPC and positively charged stearylamine (SA)/DPPC mixed liposomes were used. BSA with negative charges adsorbed on negatively charged DPPG/DPPC mixed liposomes but did not adsorb on negatively charged DCP/DPPC and positively charged SA/DPPC mixed liposomes. Furthermore, the adsorption amount of BSA on the mixed DPPG/DPPC liposomes increased with increasing the mole fraction of DPPG in spite of a possible electrostatic repulsion between BSA and DPPG. Thus, the adsorption of BSA on liposomes was likely to be related to the hydrophobic interaction between BSA and liposomes. The microfluidity of liposomal bilayer membranes near the bilayer center decreased by the adsorption of BSA, while the permeability of liposomal bilayer membranes increased by the adsorption of BSA on liposomes. These results are considered to be due to that the adsorption of BSA brings about a phase separation in liposomes and that a temporary gap is consequently formed in the liposomal bilayer membranes, thereby the permeability of liposomal bilayer membranes increases by the adsorption of BSA.

Effects of lysozyme and bovine serum albumin on membrane characteristics of dipalmitoylphosphatidylglycerol liposomes

The effects of adsorption of two kinds of proteins on the membrane characteristics of liposomes were examined at pH 7.4 in terms of adsorption amounts of proteins on liposomes, penetrations of proteins into liposomal bilayer membranes, phase transition temperature, microviscosity and permeability of liposomal bilayer membranes, using positively charged lysozyme (LSZ) and negatively charged bovine serum albumin (BSA) as proteins and negatively charged L-alpha-dipalmitoylphosphatidylglycerol (DPPG) liposomes. The saturated adsorption amount of LSZ was 720 g per mol of liposomal DPPG, while that of BSA was 44 g per mol of liposomal DPPG. The penetration of LSZ into DPPG lipid membranes was greater than that of BSA. The microviscosity in the hydrophobic region of liposomal bilayer membranes increased due to adsorption (penetration) of LSZ or BSA, while the permeability of liposomal bilayer membranes increased. The gel-liquid crystalline phase transition temperature of liposomal bilayer membranes was not affected by adsorption of LSZ or BSA, while the DSC peak area (heat of phase transition) decreased with increasing adsorption amount of LSZ or BSA. It is suggested that boundary DPPG makes no contribution to the phase transition and that boundary DPPG and bulk DPPG are in the phase-separated state, thereby increasing the permeability of liposomal bilayer membranes through adsorption of LSZ or BSA. A possible schematic model for the adsorption of LSZ or BSA on DPPG liposomes was proposed.

Preparation of liposomes containing Ceramide 3 and their membrane characteristics.

Liposomes composed of Ceramide 3, [2S,3S,4R-2-stearoylamide-1,3,4-octadecanetriol], and L-alpha-dipalmitoylphosphatidylcholine (DPPC) were prepared by varying the amount of Ceramide 3, and the effects of Ceramide 3 on the liposome formation, particle size, dispersibility, microviscosity and phase transition temperature were examined by means of a microscopy, a dynamic light scattering method, a fluorescence polarization method, a differential scanning calorimetry (DSC) and so on. All the DPPC was able to contribute to the formation of liposomes up to 0.130 mol fraction of Ceramide 3. The particle size of liposomes was almost unaffected by the addition of Ceramide 3. The dispersibility of liposomes containing Ceramide 3 was maintained for at least 15 days. The microviscosity of liposomal bilayer membranes in the liquid crystalline state was increased with increasing the mole fraction of Ceramide 3, while that in the gel state was independent of the mole fraction of Ceramide 3. The phase transition temperature from gel to liquid crystalline states of DPPC bilayer membranes was shifted upwards with the addition of Ceramide 3, indicating a cooperative interaction between DPPC and Ceramide 3 molecules. However, a sharp DSC peak became broad and split at higher mole fractions of Ceramide 3, suggesting a phase separation in the mixed DPPC/Ceramide 3 liposomal bilayer membranes. These phenomena were suggested to be related to the previously observed fact for the mixed DPPC/Ceramide 3 monolayers that Ceramide 3 interacts with DPPC in the liquid-expanded phase with consequent phase separation accompanied with domain formation.

Surfactant-free O/W emulsion formation of oleic acid and its esters with ultrasonic dispersion

Abstract Dispersibility and stabilizing factors for surfactant-free O/W emulsion were investigated with oleic acid (OA) and its esters, focusing on the effects of their weak polarity, molecular length and branched chain structure, in comparison to normal hydrocarbons. The droplet size distributions obtained by the dynamic light scattering method appeared to be discrete but almost singly peaked except for OA. For OA monoesters, the droplet growth was found to be continuous and retarded as the ester chain length increased, in contrast to the discrete, fast growth in OA dispersion. In the case of glycerol trioleate (GTO), a branched ester, aqueous dispersions of extremely fine droplets could be prepared and the number distribution of droplet diameters showed a single peak in the nanometer range. This high dispersibility remained unchanged for about a year after preparation to give the dispersions a good stability. Changes of the observed ξ potential, Fourier transform-infra red (FT-IR) spectrum, fluorescence spectrum of probes indicated that a particular carboxyl acid group network is formed in the droplet sphere to make it more stable than expected while the interior of oil droplets is hydrophobic. The ξ potential change, in particular, was found to be highly correlated with these of the carboxyl CO stretching frequency and the reciprocal droplet diameter. The droplet stability evaluated by increase in the diameter revealed a biphasic growth consisting of fast and slow modes. The fast growth at early stages (in hours) observed for OA and methyl oleate (MO) was found to proceed by the Ostwald ripening mechanism through rate analysis. On the other hand, the slow growth at later stages (in days) found for the other esters showed a semi-logarithmic dependence on the oil viscosity. This seems to be caused by an Arrhenius-type activation factor in the stepwise flocculation/coalescence rate.

Freeze-fracture electron microscopic and calorimetric studies on microscopic states of surface-modified liposomes with poly(ethylene glycol) chains

Abstract The differential scanning calorimetry (DSC) and the freeze-fracture electron microscopy of dipalmitoyl phosphatidylcholine (DPPC) liposomes containing distearoyl-N-monomethoxy poly(ethylene glycol)-succinyl-phosphatidylethanolamines (PEG-DSPE) were carried out. The DSC peak of DPPC liposomes containing PEG-DSPE had a shoulder. The main phase transition temperature of DPPC bilayer membranes containing PEG-DSPE whose molecular weight of PEG is less than 3000 was slightly shifted to a higher temperature, while that containing PEG-DSPE whose molecular weight of PEG is more than 5000 was slightly shifted to a lower temperature. The electron micrographs of freeze-fracture replicas of DPPC liposomes containing PEG-DSPE quenched from 37±2°C exhibited banded and planar textures, suggesting the lateral phase separation in the bilayer membranes.

Solution properties of amino acid-type new surfactant

The solution properties of an amino acid-type new surfactant, N-[3-lauryloxy-2-hydroxypropyl]-L-arginine L-glutamate (C12HEA-Glu), were examined by means of pK(a), surface tension, light scattering and fluorescence measurements. The dissociation state of C12HEA-Glu was changed by varying pH of the aqueous solution. The critical micelle concentration (cmc) of aqueous solution of C12HEA-Glu was lowest at pH 5.6 and the cmc increased with decreasing pH. The aggregation number of C12HEA-Glu micelles was largest at pH 5.6 and decreased with decreasing pH. The surface tension-concentration curve for C12HEA-Glu in water (non-buffered solution) showed a minimum. This is considered to be due to the coexistence of a trace species and a main species of C12HEA-Glu with various dissociation states since the pH of aqueous solution of C12HEA-Glu changes with the concentration of C12HEA-Glu. The micropolarity in the micelles of C12HEA-Glu was almost independent of pH, while the microfluidity in the micelles increased with increasing pH.

Atomic force microscopic study on the surface properties of phospholipid monolayers containing Ceramide 3

Abstract The interactions between l -α-dipalmitoylphosphatidylcholine (DPPC) and 2 S ,3 S ,4 R -2-stearoylamide-1,3,4-octadecanetriol (Ceramide 3) in the mixed monolayers were investigated by using a surface pressure measurement and an atomic force microscopy (AFM). Mixed DPPC/Ceramide 3 monolayers were deposited on mica using the Langmuir–Blodgett (LB) technique for AFM. In the case of DPPC alone system, the phase transition from the liquid-expanded film to the liquid-condensed film was observed at 17 mN m −1 of surface pressure. Whereas in the case of Ceramide 3 alone system, no phase transition and only the liquid-condensed film were observed. In the mixed DPPC/Ceramide 3 systems, the average area per molecule in the liquid-condensed film was almost independent of the mole fraction of Ceramide 3, while that in the liquid-expanded film decreased with increasing the mole fraction of Ceramide 3. The observed negative deviation of the average area per molecule from the ideality relation indicated an attractive interaction between Ceramide 3 and DPPC in the liquid-expanded films, suggesting the higher packing density in the mixed DPPC/Ceramide 3 monolayers as compared with the pure components monolayers. The AFM images for the mixed DPPC/Ceramide 3 monolayers deposited at 10 mN m −1 of surface pressure on mica indicated elliptical domains, while those deposited at 30 mN m −1 of surface pressure indicated no domains. Ceramide 3 is likely to interact with DPPC in the liquid-expanded phase and consequently bring about phase separation.

Inhibition effects of gangliosides GM1, GD1a and GT1b on base-catalyzed isomerization of prostaglandin A2

Micellar inhibition effect of gangliosides on a degradation of drug was investigated, where ganglioside G(M1) (GM1), G(D1a) (GD1a) and G(T1b) (GTlb) whose sialic acid residue is one, two and three, respectively, were used. The base-catalyzed isomerization of prostaglandin A(2) (PGA(2)) to prostaglandin B(2) (PGB(2)) was chosen as a model experiment. The rate for the isomerization of PGA(2) was determined by measuring the concentration of PGA(2) (and PGB(2)) with a high-performance liquid chromatography. Gangliosides micelles inhibited the isomerization of PGA(2). The inhibition effect of GT1b micelles was larger than that of GD1a micelles. This result would be due to the larger absolute value of surface potential of GT1b micelles, which brings about a larger electrostatic repulsion between micellar surface and OH(-). The terminal sialic acid residue of ganglioside was effective to inhibit the isomerization of PGA(2). GM1 micelles without terminal sialic acid residue but with large aggregation number exhibited a superior steric shielding effect rather than an electrostatically repulsive effect. The inhibition effect of GM1 micelles was enhanced by the mixed micellization with the other ganglioside with a terminal sialic acid residue. GM1-GD1a or GM1-GT1b mixed micelles remarkably inhibited the isomerization of PGA(2). The physiological activity of PGs in the biological membranes containing gangliosides was also discussed.