Classification of binding property of amyloid ß to lipid membranes: Membranomic research using quartz crystal microbalance combined with the immobilization of lipid planar membranes.

A biomembrane-related fibrillogenesis of Amyloid ß from Alzheimer' disease (Aß) is closely related to its accumulation behavior. A binding property of Aß peptides from Alzheimer' disease to lipid membranes was then classified by a quartz crystal microbalance (QCM) method combined with an immobilization technique using thiol self-assembled membrane. The accumulated amounts of Aß, Δfmax, was determined from the measurement of the maximal frequency reduction using QCM. The plots of Δfmax to Aß concentration gave the slope and saturated value of Δfmax, (Δfmax)sat that are the parameters for binding property of Aß to lipid membranes. Therefore, the Aß-binding property on lipid membranes was classified by the slope and (Δfmax)sat. The plural lipid system was described as X + Y where X = L1, L1/L2, and L1/L2/L3. The slope and (Δfmax)sat values plotted as a function of mixing ratio of Y to X was classified on a basis of the lever principle (LP). The LP violation observed in both parameters resulted from the formation of the crevice or pothole, as Aß-specific binding site, generated at the boundary between ld and lo phases. The LP violation observed only in the slope resulted from glycolipid-rich domain acting as Aß-specific binding site. Furthermore, lipid planar membranes indicating strong LP violation favored strong fibrillogenesis. Especially, lipid planar membranes indicating the LP violation only in the slope induced lateral aggregated and spherulitic fibrillar aggregates. Thus, the classification of Aß binding property on lipid membranes appeared to be related to the fibrillogenesis with a certain morphology.

Amyloid-ß aggregates induced by ß-cholesteryl glucose-embedded liposomes.

Senile plaques that is characterized as an amyloid deposition found in Alzheimer's disease are composed primarily of fibrils of an aggregated peptide, amyloid ß (Aß). The ability to monitor senile plaque formation on a neuronal membrane under physiological conditions provides an attractive model. In this study, the growth behavior of amyloid Aß fibrils in the presence of liposomes incorporating ß-cholesteryl-D-glucose (ß-CG) was examined using total internal reflection fluorescence microscopy, transmittance electron microscopy, and other spectroscopic methods. We found that ß-CG on the liposome membrane induced the spontaneous formation of spherulitic Aß fibrillar aggregates. The ß-CG cluster formed on liposome membranes appeared to induce the accumulation of Aß, followed by the growth of the spherulitic Aß aggregates. In contrast, DMPC and DMPC incorporated cholesterol-induced fibrils that are laterally associated with each other. A comparison study using three types of liposomes implied that the induction of glucose contributed to the agglomeration of Aß fibrils and liposomes. This agglomeration required the spontaneous formation of spherulitic Aß fibrillary aggregates. This action can be regarded as a counterbalance to the growth of fibrils and their toxicity, which has great potential in the study of amyloidopathies.

Microfluidic and hydrothermal preparation of vesicles using sorbitan monolaurate/polyoxyethylene (20) sorbitan monolaurate (Span 20/Tween 20).

Here, we present a method for preparing vesicles by combining hydrothermal emulsification with solvent diffusion (SD). The sorbitan monolaurate/polyoxyethylene (20) sorbitan monolaurate (Span 20/Tween 20) system was used as the target lipid because these lipids are cheap and advantageous for the production scale. The water-in-oil (W/O) emulsion stabilized with lipids was formed under hydrothermal conditions (240 °C under 10 MPa), followed by mixing with water that included lipids to obtain a W/O-in-water (W/O/W) emulsion. The SD for the W/O/W emulsion as a subsequent process yielded vesicles. The optimal preparation conditions were 50:50 wt% Span 20/Tween 20 as a mixing ratio (final lipid concentration 12 mM), octanoic acid as an organic solvent, 240 °C for 4 min during the hydrothermal treatment, and 4 °C for 24 h in the SD process. The diameter of the vesicles obtained was at most 100 nm, which was comparable to that of the W/O/W emulsion before SD. This suggested that the W/O/W emulsion acted as a template for vesicle formation. The number density, diameter, and membrane properties of vesicles depend on the mixing ratio of the water/oil/lipid system. Specifically, the number density of vesicles was low relative to that of vesicles prepared by the conventional method.

Application of liposome membrane as the reaction field: A case study using the Horner-Wadsworth-Emmons reaction.

The properties of the liposome membrane as a reaction field were investigated by focusing on the Horner-Wadsworth-Emmons reaction as a case study. Use of the liposomes existing in the gel phase resulted in the enhanced activity of the substrates and furnished the products with same E/Z stereoselectivity as in the liposome-free system. The membrane environment in the gel phase most likely assisted the formation of adducts that induced selective generation of the E-isomer. The possible role of liposomes is to assist the proton removal from the reactant, rather than providing the basic interfacial environment.

Temperature Measurement by Sublimation Rate as a Process Analytical Technology Tool in Lyophilization.

Product temperature (Tb) and drying time constitute critical material attributes and process parameters in the lyophilization process and especially during the primary drying stage. In the study, we performed a temperature measurement by the sublimation rate (TMbySR) to monitor the Tb value and determine the end point of primary drying. First, the water vapor transfer resistance coefficient through the main pipe from the chamber to the condenser (Cr) was estimated via the water sublimation test. The use of Cr value made it possible to obtain the time course of Tb from the measurement of pressure at the drying chamber and at the condenser. Second, a Flomoxef sodium bulk solution was lyophilized by using the TMbySR system. The outcome was satisfactory when compared with that obtained via conventional sensors. The same was applicable for the determination of the end point of primary drying. A laboratory-scale application of the TMbySR system was evidenced via the experiment using 220-, 440-, and 660-vial scales of lyophilization. The outcome was not dependent on the loading amount. Thus, the results confirmed that the TMbySR system is a promising tool in laboratory scale.

Effect of Controlled Nucleation of Ice Crystals on the Primary Drying Stage during Lyophilization.

The freezing stage cannot be directly controlled, which leads to variation in product quality and low productivity during the lyophilization process. Our objective was to establish a robust design space for the primary drying stage using ice nucleation control based on the pressurization and depressurization technique. We evaluated the specific surface area (SSA), water content, scanning electron microscopy (SEM) images, and water vapor transfer resistance of the dried layer (Rp) of the products. The ice nucleation control resulted in a reduction of the SSA value and in an increase in water content. SEM observation suggested that the ice nucleation control enabled formation of large ice crystals, which was consistent with the reduction in the Rp value. As a result, the generation of collapsed cakes was inhibited, whereas 18% of the collapsed cakes were observed without ice nucleation control. Finally, this technique succeeded in determining a robust design space for the primary drying stage to produce uniform products of higher productivity. It was considered, from the present findings, that controlling the formation of large ice crystals impacted the product quality and productivity.

Scale-Up Procedure for Primary Drying Process in Lyophilizer by Using the Vial Heat Transfer and the Drying Resistance.

The objective of this study is to design primary drying conditions in a production lyophilizer based on a pilot lyophilizer. Although the shelf temperature and the chamber pressure need to be designed to maintain the sublimation interface temperature of the formulation below the collapse temperature, it is difficult to utilize a production lyophilizer to optimize cycle parameters for manufacturing. In this report, we assumed that the water vapor transfer resistance (Rp) in the pilot lyophilizer can be used in the commercial lyophilizer without any correction, under the condition where both lyophilizers were operated in the high efficiency particulate air (HEPA)-filtrated airflow condition. The shelf temperature and the drying time for the commercial manufacturing were designed based on the maximum Rp value calculated from the pilot lyophilizer (1008 vials) under HEPA-filtrated airflow condition and from the vial heat transfer coefficient of the production lyophilizer (6000 vials). And, the cycle parameters were verified using the production lyophilizer of 60000 vials. It was therefore concluded that the operation of lab- or pilot-scale lyophilizer under HEPA-filtrated airflow condition was one of important factors for the scale-up.

Microfluidic fabrication of monodisperse polylactide microcapsules with tunable structures through rapid precipitation.

We describe a versatile and facile route to the continuous production of monodisperse polylactide (PLA) microcapsules with controllable structures. With the combination of microfluidic emulsification, solvent diffusion, and internal phase separation, uniform PLA microcapsules with a perfluorooctyl bromide (PFOB) core were successfully obtained by simply diluting monodisperse ethyl acetate (EA)-in-water emulsion with pure water. Rapid extraction of EA from the droplets into the aqueous phase enabled the solidification of the polymer droplets in a nonequilibrium state during internal phase separation between a concentrated PLA/EA phase and a PFOB phase. Higher-molecular-weight PLA generated structural complexity of the microcapsules, yielding core-shell microcapsules with covered with small PFOB droplets. Removal of the PFOB via freeze drying gave hollow microcapsules with dimpled surfaces. The core-shell ratios and the diameter of these microcapsules could be finely tuned by just adjusting the concentration of PFOB and flow rates on emulsification, respectively. These biocompatible microcapsules with controllable size and structures are potentially applicable in biomedical fields such as drug delivery carriers of many functional molecules.

Absorption of the indigestible disaccharide, ß-1,4-mannobiose, from coconut by the rat portal vein.

The intestinal absorption of ß-1,4-mannobiose by rats was investigated. Mannobiose was detected in the portal vein plasma by matrix-assisted laser desorption ionization/time of flight mass spectrometry after its administration to rats. The presence of mannobiose in the rat plasma was confirmed by an experiment using ß-mannosidase. These results indicate that mannobiose was directly absorbed through the intestines even without being hydrolyzed.

Synthesis of glyceryl ferulate by immobilized ferulic acid esterase.

Glyceryl ferulate was synthesized by the condensation of ferulic acid with glycerol using Pectinase PL "Amano" from Aspergillus niger, which contained ferulic acid esterase, to improve the water-solubility of ferulic acid. The optimum reaction medium was glycerol/0.1 M acetate buffer, pH 4.0, (98:2 v/v). The enzyme immobilized onto Chitopearl BCW3003 exhibited the highest activity among the those immobilized onto various kinds of Chitopearl BCW resins. The optimum temperature for the immobilized enzyme was 50 degrees C, and it could be reused at least five times without a significant loss in activity for the synthesis of glyceryl ferulate in batch reaction. Storage of the reaction mixture at 25 degrees C improved the molar fraction of glyceryl ferulate relative to the dissolved ferulic residues.

Continuous synthesis of glyceryl ferulate using immobilized Candida antarctica lipase.

Glyceryl ferulate was synthesized through condensation of ferulic acid and glycerol at 50 degrees C in glycerols with different water contents using an immobilized lipase from Candida antarctica in a batch reactor, and condensation in the glycerol with a 7.5% (w/w) water content was shown to be the favorite. The solubility of ferulic acid was higher at higher temperature in glycerol with a lower water content. The viscosity was lower at higher temperature for the glycerol with a higher water content. The condensation was carried out using a batch reactor at a temperature from 50 degrees C to 90 degrees C. These observations indicated that the condensation at 80 degrees C in the glycerol with a 7.5% (w/w) water content was the most adequate for continuously synthesizing glyceryl ferulate. A reactor system was constructed for the continuous synthesis and was steadily operated to realize a productivity of 430 kg/(m(3)-reactor(day) without any decrease in the conversion for at least 6 days.

Production and characterization of functional substances from a by-product of rice bran oil and protein production by a compressed hot water treatment.

A by-product of rice bran oil and protein production was treated with water and compressed hot water at 20 degrees C to 260 degrees C for 5 min, and at 200 degrees C and 260 degrees C for 5 to 120 min. Each extract was evaluated for its yield, radical scavenging activity, carbohydrate, protein, total phenolic and furfural contents, molecular-mass distribution and antioxidative activity. The maximum yield was obtained at 200 degrees C. The radical scavenging activity and the protein, total phenolic and furfural contents of the extract increased with increasing temperature. However, the carbohydrate content abruptly decreased when treated at above 200 degrees C. The extract treated at 260 degrees C for 5 min exhibited suppressive activity toward the autoxidation of linoleic acid. Each extract obtained at temperatures lower than or equal to 200 degrees C exhibited emulsifying ability.

Properties of extracts from defatted rice bran by its subcritical water treatment.

Defatted rice bran was extracted with water and subcritical water at 50-250 degrees C for 5 min. The highest extract yield was achieved at 200 degrees C, at which the maximum amounts of protein and carbohydrate were also obtained. The total phenolic and furfural contents, radical scavenging activity, and antioxidative activity for the autoxidation of linoleic acid increased with increasing treatment temperature. The bran extracts exhibited emulsifying activity except for the extract prepared at 250 degrees C, which was concomitant with the disappearance of its high-molecular-mass substances. The extract prepared at 200 degrees C also had the highest emulsion-stabilizing activity.

Hydrolysis kinetics of trisaccharides consisting of glucose, galactose, and fructose residues in subcritical water.

The hydrolysis kinetics of trisaccharides consisting of glucose, galactose, and fructose residues with different glycosidic bonds, 1-kestose, d-melezitose, d-raffinose, and lactosucrose, in subcritical water were conducted over the temperature range of 150-230 degrees C and at a constant pressure of 10 MPa. The hydrolysis of trisaccharides in subcritical water proceeded consecutively, i.e., one cleavage of the two bonds antedated the other. The preceding cleavage was not expressed by the first-order kinetics, but by the kinetics considering the concentration of the acidic compounds, which were produced by the degradation of the constituent monosaccharides. The hydrolysis of the constituent disaccharides, except sucrose composed of the alpha-Glc-(1-->2)-beta-Fru bond, obeyed first-order kinetics. All of the rate constants of the hydrolytic kinetics were determined, and the values were found to depend on the type of bond.

Continuous synthesis of alkyl ferulate by immobilized Candida antarctica lipase at high temperature.

1-Pentyl, 1-hexyl and 1-heptyl ferulates were continuously synthesized at 60-90 degrees C using a reactor system in which a column packed with ferulic acid powders and another column packed with immobilized Candida antarctica lipase particles were connected in series. Conversions greater than 0.9 were achieved for the synthesis of the 1-hexyl and 1-heptyl ferulates at 90 degrees C. The system could be stably operated for the 1-heptyl ferulate synthesis at 90 degrees C for at least two weeks.

Thermal inactivation of immobilized lipase in 1-alcohols.

The thermal inactivation of immobilized lipase from Candida antarctica in 1-alcohols with four to eight carbons at various temperatures could be well expressed using a model in which heterogeneity in the susceptibility of the enzyme to the inactivation was assumed. It was found that the enthalpy-entropy compensation held for inactivation in 1-alcohols.

Kinetics of maltooligosaccharide hydrolysis in subcritical water.

The kinetics of the hydrolysis of maltooligosaccharides with a degree of polymerization (DP) of 3-6 in subcritical water was studied using a tubular reactor at temperatures between 200 and 260 degrees C and at a constant pressure of 10 MPa. The maltooligosaccharide disappearance and product formation at residence times shorter than 50 s could be expressed by first-order kinetics. The rate constants for the hydrolysis of each maltooligosaccharide were evaluated. There was a tendency that the exosite glucosidic bond was hydrolyzed faster than the endo-site one irrespective of the DP of the maltooligosaccharide. The hydrolysis of the maltooligosaccharides was consecutively preceded, and the time dependence of the hydrolysis for maltooligosaccharides with different DPs could be calculated by simultaneously solving the mass balance equations for all the possible saccharides.

Synthesis of linoleoyl disaccharides through lipase-catalyzed condensation and their surface activities.

Monolinoleoyl trehalose, maltose and cellobiose were synthesized by Candida antarctica lipase-catalyzed condensation in an organic solvent with a low water content. The use of a mixture of pyridine and tert-butanol as the reaction medium resulted in a high product concentration on the order of mmol/l for the synthesis of linoleoyl trehalose and maltose. The highest product concentration was achieved with the 0.4 volumetric fraction of pyridine. Linoleoyl cellobiose was also synthesized although its concentration was approximately one tenth the concentrations of linoleoyl trehalose and maltose. The surfactant properties of linoleoyl trehalose, maltose and cellobiose were measured. Among the esters, linoleoyl trehalose showed the strongest surface activity.

Condensation reaction between angiotensin II and dicarboxylic acid in water at high temperature without any catalytic agent additive.

The condensation reaction of angiotensin II and tartaric acid in water at 100-140 degrees C proceeded without any added catalytic agent. One of the products, N-CO-tartarylangiotensin II, was confirmed by LC-MS, positive-ion MALDI-MS, and the fluorescamine method as a condensation product. The initial concentration of the substrates, temperatures, and initial pH affected the yield of the product. The higher concentrations and temperatures increased the concentration of the condensation product. The optimal pH for the condensation reaction was 3.0. The other C4 saturated dicarboxylic acids, malic and succinic acids, were also condensed by angiotensin II.

Preparation of finely dispersed O/W emulsion from fatty acid solubilized in subcritical water.

A novel method for preparing a finely dispersed oil-in-water emulsion is proposed. Octanoic acid dissolved in water at a high temperature of 220 or 230 degrees C at 15 MPa was combined with an aqueous solution of a surfactant and then the mixture was cooled. When a nonionic surfactant, decaglycerol monolaurate (ML-750) or polyoxyethylene sorbitan monolaurate (Tween 20), was used, fine emulsions with a median oil droplet diameter of 100 nm or less were successfully prepared at ML-750 and Tween 20 concentrations of 0.083% (w/v) and 0.042%, respectively, or higher. The diameters were much smaller than those of oil droplets prepared by the conventional homogenization method using a rotor/stator homogenizer. However, an anionic surfactant, sodium dodecyl sulfate, was not adequate for the preparation of such fine emulsions by the proposed method. Although the interfacial tensions between octanoic acid and the surfactant solutions were measured at different temperatures, they were not an indication for selecting a surfactant for the successful preparation of the fine emulsion by the proposed method.