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A biomembrane-related fibrillogenesis of Amyloid β from Alzheimer' disease (Aβ) is closely related to its accumulation behavior. A binding property of Aβ 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 Aβ, Δ f max , was determined from the measurement of the maximal frequency reduction using QCM. The plots of Δ f max to Aβ concentration gave the slope and saturated value of Δ f max , (Δ f max)sat that are the parameters for binding property of Aβ to lipid membranes. Therefore, the Aβ-binding property on lipid membranes was classified by the slope and (Δ f max)sat. The plural lipid system was described as X + Y where X = L 1 , L 1 /L 2 , and L 1 /L 2 /L 3. The slope and (Δ f max)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 Aβ-specific binding site, generated at the boundary between l d and l o phases. The LP violation observed only in the slope resulted from glycolipid-rich domain acting as Aβ-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 Aβ binding property on lipid membranes appeared to be related to the fibrillogenesis with a certain morphology. • The lipid lanar membrane system made of 41 kinds of lipids was constructed. • Aβ-specific binding and glucoside cluster effect resulted in the strong accumulation of Aβ. • Aβ accumulation behavior on lipid planar membrane likely corresponds to the fibrillogenesis of Aβ. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Product temperature (T b) 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 T b 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 (C r) was estimated via the water sublimation test. The use of C r value made it possible to obtain the time course of T b 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. [ABSTRACT FROM AUTHOR]

The objective of this review is to survey the development of the optimization of lyophilization. The optimization study of the lyophilizer has been roughly developing by the order of (i) trial-and-error approach, (ii) process modeling using mathematical models, (iii) scalability, and (iv) quality-by-design. From the conventional lyophilization studies based on the trial-and-error, the key parameters to optimize the operation of lyophilization were found out, i.e., critical material attributes (CMAs), critical process parameters (CPPs), and critical quality attributes (CQAs). The mathematical models using the key parameters mentioned above have been constructed from the viewpoints of the heat and mass transfer natures. In many cases, it is revealed that the control of the primary drying stage determines the outcome of the lyophilization of products, as compared with the freezing stage and the secondary drying stage. Thus, the understanding of the lyophilization process has proceeded. For the further improvement of the time and economical cost, the design space is a promising method to give the possible operation range for optimizing the lyophilization operation. This method is to search the optimized condition by reducing the number of key parameters of CMAs, CPPs, and CQAs. Alternatively, the transfer of lyophilization recipe among the lab-, pilot-, and production-scale lyophilizers (scale-up) has been examined. Notably, the scale-up of lyophilization requires the preservation of lyophilization dynamics between the two scales, i.e., the operation of lab- or pilot-scale lyophilizer under HEPA-filtrated airflow condition. The design space determined by focusing on the primary drying stage is large and involves the undesired variations in the quality of final products due to the heterogeneous size distribution of ice crystals. Accordingly, the control of the formation of the ice crystal with large size gave impact on the product quality and the productivity although the large water content in the final product should be improved. Therefore, the lyophilization should take into account the quality by design (QbD). The monitoring method of the quality of the product in lyophilization process is termed the "process analytical technology (PAT)." Recent PAT tools can reveal the lyophilization dynamics to some extent. A combination of PAT tools with a model/scale-up theory is expected to result in the QbD, i.e., a quality/risk management and an in situ optimization of lyophilization operation. [ABSTRACT FROM AUTHOR]

The hydrolysis of disaccharides was conducted using a microcapillary system under hydrothermal conditions (up to 190°C at 10 MPa and pH 4–11). The hydrolysis reaction showed a sigmoidal progression with time, especially under alkaline conditions. Analysis using a kinetic model yielded the reaction induction period. The specific pH value (pHamb) at the induction time, which is the pH value corresponding to the progression of disaccharide hydrolysis, was peculiar to each disaccharide. Finally, the calculation of the electron density around the oxygen atom of the glycosidic bond between saccharides was found to roughly predict the pHamb value required for the progression of hydrolysis. [ABSTRACT FROM AUTHOR]

Senile plaques that is characterized as an amyloid deposition found in Alzheimer's disease are composed primarily of fibrils of an aggregated peptide, amyloid β (Aβ). 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 Aβ fibrils in the presence of liposomes incorporating β-cholesteryl-D-glucose (β-CG) was examined using total internal reflection fluorescence microscopy, transmittance electron microscopy, and other spectroscopic methods. We found that β-CG on the liposome membrane induced the spontaneous formation of spherulitic Aβ fibrillar aggregates. The β-CG cluster formed on liposome membranes appeared to induce the accumulation of Aβ, followed by the growth of the spherulitic Aβ 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 Aβ fibrils and liposomes. This agglomeration required the spontaneous formation of spherulitic Aβ 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. • β-CG on liposomes induced a spontaneous formation of spherulitic fibrillary aggregatie of Aβ. • β-CG enhanced the accumulation of Aβ on liposome membranes to accelerate its nucleation. • Spherulite formation of Aβ resulted from a synergetic effect of glucoside group and cholesterol in β-CG. [ABSTRACT FROM AUTHOR]

Woody thin boards were prepared from lignin, cellulose, and water by compression molding at 180 °C and 25 MPa for 10 min. Boards with higher contact angles gave lower values of relative permittivity on their surface. Attenuated-total reflection Fourier transfer infrared spectroscopy suggested that more lignin existed on the surface of the boards with the high contact angle, which was also supported by scanning electron microscopy and atomic force microscopy. Our findings thus revealed that the orientation of lignin at the surface resulted in increased hydrophobicity of the surface and contributed to the enhancement of water repellency. [ABSTRACT FROM AUTHOR]

Wet torrefaction is an effective process to improve the fuel properties of a biomass. However, different biomasses have different component weight ratios and it is possible that not all biomasses are suitable for wet torrefaction treatment. Here we conducted wet torrefaction using three types of biomass with different component weight ratios: Humulus lupulus (HL, the common hop), Plumeria alba (PA, an evergreen shrub) and Calophyllum inophyllum L . (CIL, an evergreen tree). The fuel properties of the obtained solid fuels were characterized. We found that lignin made the main contribution to the solid fuel yield. The reactivity of cellulose and hemicellulose in each biomass was affected by the biomass species and the component weight ratio of the biomass. The wet torrefaction was observed to efficiently elevate the fuel properties of carbon content, atomic H/C and O/C ratios, higher heating value (HHV) and hydrophobicity of all three types of biomass. The HHVs of the solid fuels prepared at 260 °C are comparable to those of commercial coals such as Northumerland No. 81/2 Sem. Anth. Coal, Jhanjra Bonbahal Seam Coal-R-VII, and German Braunkohole lignite. These solid fuels could be co-combusted with German Braunkohole lignite without a significant change in the combustion properties of German Braunkohole lignite because of their similar atomic H/C and O/C ratios as well as HHVs. At the wet torrefaction temperature of 260 °C, the solid fuel delivered from CIL had lower HHV compared to those from HL and PA although CIL contained the highest lignin content, which has a higher HHV than those of cellulose and hemicellulose. Scanning electron microscopy images of the solid fuels revealed that wet torrefaction was able to completely destroy the biomass surface and create numerous pores and cracks on the solid fuels surface, indicating that the solid fuels have the potential to be used as a source of carbon materials such as activated carbon in addition to their use as bio-fuels. [ABSTRACT FROM AUTHOR]

The adsorption behaviour of amyloid beta (Aβ), thought to be a key peptide for understanding Alzheimer's disease, was investigated by means of time-of-flight secondary ion mass spectrometry (ToF-SIMS). Aβ aggregates depending on the lipid membrane condition though it has not been fully understood yet. In this study, Aβ samples on different lipid membranes, 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC) and 1,2-dioleoyl- sn-glycero-3-phosphocholine (DOPC), were observed with ToF-SIMS and the complex ToF-SIMS data of the Aβ samples was interpreted using data analysis techniques such as principal component analysis (PCA), gentle-SIMS (G-SIMS) and g-ogram. DOPC and DMPC are liquid crystal at room temperature, while DPPC is gel at room temperature. As primary ion beams, Bi and Ar cluster ion beams were used and the effect of an Ar cluster ion for evaluating biomolecules was also studied. The secondary ion images of the peptide fragment ions indicated by G-SIMS and g-ogram were consistent with the PCA results. It is suggested that Aβ is adsorbed homogeneously on the liquid-crystalline-phase lipid membranes, while it aggregates along the lipid on the gel-phase lipid membrane. Moreover, in the results using the Ar cluster, the influence of contamination was reduced. [ABSTRACT FROM AUTHOR]

Abstract: Proteins could affect the headgroup mobility of phospholipid within liposome membranes through the protein–liposome interaction. The variation of headgroup mobility of phospholipid was then investigated by using the dielectric dispersion analysis. The eight proteins (Mw = 4.2–28.7 kDa) were used to investigate the protein–liposome interaction. It has been revealed that the strength of the protein–liposome interaction at 25 °C was linearly correlated with the stability of intramolecular hydrogen bondings of proteins, better than with their hydrophobicity and the surface charge density. Overall, liposomes composed of binary lipid system, appeared to strongly interact with proteins, in contrast to liposomes composed of single, ternary, and quaternary lipid systems. This is probably because liposomes composed of binary lipid system favored to form the microscopic environment where proteins could interact. The present result suggested the heterogeneous phase state of lipid membranes was one of dominant factors for the interaction between proteins and lipid membranes. [Copyright &y& Elsevier]

Abstract: The growth of giant vesicles (GVs) can be considered as a consecutive process of swelling/detachment/deformation, which is a response of lipid membranes on solid surfaces to the solvent and environmental factors such as temperature and ionic strength. The electroformation method allows to visualize the responses to such factors. The additive effect of the protein on the growth of GVs, composed of zwitterionic phospholipids, was herein investigated using the electroformation method. Proteins denatured by a pH-shift (to be in the Molten Globule state) perturbed the lipid membranes, resulting in the acceleration of GV growth. The GVs detached from the electrode showed deformation close to a stomatocyte. It was revealed that common factor for the response of lipid membranes was the variation of the apparent area elastic modulus associated with the interaction between proteins and lipid membranes. The present finding affords better understanding about the response of lipid membranes on solid surfaces under a variety of environment factors. [Copyright &y& Elsevier]

Abstract: Phospholipase C (PLC) is considered to be one of key enzymes for the design of drug delivery system using the endocytosis route, because PLC can catalyze the membrane fusion between cell membranes and phospholipid vehicles (liposomes). Membrane fusion by PLC was then studied under various pHs to model the endosomal environment. The used liposomes were composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and cholesterol (Ch). The membrane fusion was dominated by the enzymatic reaction at pH 6–7.5. In contrast, the membrane perturbation effect due to the conformational change of PLC could induce the membrane fusion at around pH 4. The maximal value of membrane fusion was observed at around pH 5 for three liposomes in the order of DOPC

Abstract: The inhibition of fibril formation of amyloid β (Aβ) and the disaggregation of Aβ fibrils are the promising approaches for a medical treatment of Alzheimer''s disease (AD) therapy. In this study, we investigated the effects of liposomes on dopamine-induced disaggregation of Aβ fibrils by using the variety of liposomes. The used liposomes were normal liposomes, raft-forming liposomes, charged liposomes and oxidized liposomes. Those liposome could accelerate the disaggregation rate of fibrils. From the comparison of normal and charged liposomes, a certain contribution of dopamine via an electrostatic interaction to the disaggregation was confirmed. From raft-forming and oxidized liposomes, we revealed a significant contribution of bound water to liposomes, which could assist the formation of the quinine-form of dopamine by a removal of its proton. It is, therefore, concluded that the membrane surface of liposomes is considered to be an adequate environment for the dopamine-induced disaggregation of fibrils. [Copyright &y& Elsevier]

Abstract: The protofibrils of amyloid fibrils have recently drawn an attention due to their cytotoxicity although the physicochemical properties of the protofibrils as an intermediate of fibrillation process have still remained unclear. We investigated the growth behavior of the protofibrils of amyloid β protein with 40 amino acid residues in the presence of various liposomes and the membrane perturbation (calcein leakage). The growth behavior of protofibrils was apparently correlated with the protofibrils-induced calcein leakage. From the binding experiment using a hydrophobic fluorescence probe and the quartz crystal microbalance method combined with the planar lipid membrane, it is suggested that the apparent correlation between the growth behavior of protofibrils and their membrane perturbation resulted from the physicochemical properties of protofibrils such as hydrophobicity and the stability of hydrogen bonds. The protofibrils were found to show the highest hydrophobicity and the highest instability of hydrogen bonds of the proteins used in this study (monomer, protofibrils, matured fibrils, and other three typical proteins). It is anticipated that such properties of protofibrils were advantageous both for the binding of protofibrils with monomer (growth behavior) and for the binding of lipid bilayer membranes (membrane perturbation). [Copyright &y& Elsevier]

Abstract: Alzheimer’s disease is the most common form of senile dementia. This neurodegenerative disorder is characterized by an amyloid deposition in senile plaques, composed primarily of fibrils of an aggregated peptide, amyloid β (Aβ). The modeling of a senile plaque formation on a model neuronal membrane under the physiological condition is an attractive issue. In this study, we used anionic liposomes to model the senile plaque formation by Aβ. The growth behavior of amyloid Aβ fibrils was directly observed, revealing that the induction of the spherulitic Aβ aggregates could result from the growth of seeds in the presence of anionic liposomes. The seeds of Aβ fibrils strongly interacted with negatively charged liposome and the subsequent association of the seeds were induced to form the seed cluster with many growth ends, which is advantageous for the formation of spherulitic Aβ aggregates. Therefore, anionic liposomes mediated not only fibril growth but also the aggregation process. These results imply that anionic liposome membranes would affect the aggregate form of Aβ fibrils. The modeling of senile plaque reported here is considered to have great potential for study on the amyloidosis. [Copyright &y& Elsevier]

ABSTRACT: Amyloid β protein (Aβ) from Alzheimer''s disease formed fibrillar aggregates and their morphology depended on oxidized and negatively charged liposomes. The morphology of fibrillar aggregates was affected by Cu2+, together with their growth kinetics. This is because Cu2+ inhibited the nucleation step in the formation of amyloid Aβ fibrillar aggregates by forming Aβ/Cu complex inactive to the growth of fibrillar aggregates. In addition, this is probably because Cu2+ affected the fibrillar aggregate formed on the surface of liposomes. These findings would give a better understanding of the formation mechanism of amyloid fibrils on biomembranes. [Copyright &y& Elsevier]

Abstract: In this study, we investigated the dynamics of a membrane interface of liposomes prepared by eight zwitterionic phosphatidylcholines in terms of their headgroup mobility, with spectroscopic methods such as dielectric dispersion analysis (DDA), fluorescence spectroscopy. The DDA measurement is based on the response of the permanent dipole moment to a driving electric field and could give the information on the axial rotational Brownian motion of a headgroup with the permanent dipole moment. This motion depended on kinds of phospholipids, the diameter of the liposomes, and the temperature. The activation energy required to overcome the intermolecular force between headgroups of phospholipids depended on the strength of the interaction between headgroups such as hydrogen bonds and/or dipole–dipole interaction. Hydration at the phosphorous group of phospholipid and the molecular order of lipid membrane impaired the interaction between headgroups. Furthermore, the hydrophobicity of membrane surface increased parallel to the increase in headgroup mobility. It is, therefore, concluded that hydration of headgroup promoted its mobility to make the membrane surface hydrophobic. The lipid membrane in liquid crystalline phase or the lipid membrane with the larger curvature was more hydrophobic. [Copyright &y& Elsevier]

Abstract: The surface properties of Span 80 vesicles at various cholesterol contents, together with those of various liposomes, were characterized by using fluorescence probes. The membrane fluidity of the Span 80 vesicles was measured by 1,6-diphenyl-1.3.5-hexatriene (DPH) and trimethlyammonium-DPH (TMA-DPH), and the results suggested that the surface of the Span 80 vesicles was fluid due to the lateral diffusion of Span 80 molecules. The depolarization measured by TMA-DPH and the headgroup mobility measured by dielectric dispersion analysis indicated the high mobility of the head group of Span 80 vesicles. This suggested that the surface of Span 80 vesicles was flexible due to the head group structure of Span 80, sorbitol. In addition, spectrophotometric analysis with 6-dodecanoyl-N, N-dimethyl-2-naphthylamine and 8-anilino-1-naphthalenesulfonic acid indicated that the water molecules could easily invade into the interior of the Span 80 vesicle membrane, suggesting that the membrane surface was more wet than the liposome surface. These surface properties indicated that the protein could interact with the interior of vesicle membranes, which was similar to the case of cholesterol. Thus the present results confirmed that the Span 80 vesicle surfaces showed the unique characteristics of fluidity, flexibility, and “wetness”, whereas the liposome surfaces did not. [Copyright &y& Elsevier]

Abstract: The inhibition of fibril formation of amyloid β proteins (Aβ) would be attractive therapeutic targets for the treatment of Alzheimer''s disease (AD). Dopamine (DA) and other catechol derivatives were used as inhibitory factors for Aβ fibril formation. The fibril formation of Aβ was monitored by Thioflavin T fluorescence, a transmission electron microscopy (TEM) and a total internal reflection fluorescence microscopy (TIRFM). Catechol and its derivatives showed the dose-dependent inhibitory effects on the spontaneous Aβ fibril formation. The inhibitory activity depended on the chemical structure of catechol derivatives both in the presence and absence of the liposome a model of biomembrane. Formation of catechol quinone-conjugated-Aβ adduct by a Schiff-base is a key step for the inhibition effect of Aβ fibril formation. [Copyright &y& Elsevier]

Abstract: The transfer of 22-NBD-cholesterol (22-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3-ol) between two liposome membranes was quantitatively analyzed by using the fluorescence resonance energy transfer (FRET) method. Liposomes labeled with both 22-NBD-cholesterol and a rhodamine-labeled phosphatidylethanolamine (Rh-DHPE) were used as donor liposomes, and the 22-NBD-cholesterol transfer from these donor liposomes to acceptor liposomes prepared from same type of phosphatidylcholine was monitored. The transfer kinetics was found to be composed of a fast and a slow phase, and all kinetic measurements could be fitted with a bi-exponential model. The results obtained indicate that the 22-NBD-cholesterol transfer kinetics between liposome membranes depends on the fluidity of the liposome used and that the curvature may affect the kinetics. Furthermore, the behavior of 22-NBD-cholesterol in lipid membrane is similar to that of the oxysterol 25-hydroxycholesterol rather than cholesterol. It is proposed that 22-NBD-cholesterol can be a useful fluorescent probe to mimic the intermembrane transfer of oxidized cholesterols like 25-hydroxycholesterol, rather than that of cholesterol itself. [Copyright &y& Elsevier]

Abstract: The amyloid β protein with 42 amino acid residues (Aβ), which is a causative protein of Alzheimer''s disease (AD), forms the complex with copper (II) to induce the cholesterol oxidase-like activity by the proton transfer from the cholesterol. In this study, the oxidation of cholesterol by Aβ/Cu complex was investigated on the surface of the zwitterionic phospholipid liposome including the bound water advantageous for the enhancement of the proton transfer. The bound water was pooled by the formation of cholesterol-rich domain within liposomes. The resulting reactivity was enhanced by the proton transfer mediated by the bound water. [Copyright &y& Elsevier]

Abstract: NBD-cholesterol (NBD-Ch)-modified liposome was immobilized on indium tin oxide (ITO) electrode via the covalent binding method. The transfer of NBD-Ch between the immobilized liposomes and the target liposomes was observed by using a fluorescent microscope. The addition of liposome suspension co-incubated with α-chymotrypsin or stimuli-responsive polymer to the surface of the above ITO electrode, enhanced the liposome–liposome interaction, resulting in the promotion of NBD-Ch transfer. The apparent transfer rate constant of NBD-Ch was found to be correlated with the index for the liposome–liposome interaction evaluated by an immobilized liposome chromatography. This suggests that the present method using the liposome-immobilized ITO electrode was effective to evaluate the liposome–liposome interaction induced by the protein or the stimuli-responsive polymer under stress conditions. [Copyright &y& Elsevier]

The possible use of an electrode modified with electroactive conductive poly(3-methylthiophene) (PMeT)/Nafion as a chemical sensor was investigated for the voltammetric analysis of Dopamine (DA). The electrochemical behavior of dopamine was examined by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) techniques. By using a PMeT-modified glassy carbon (GC/PMeT) electrode, DA and Ascorbic Acid (AA) signals could be separated but the AA at high concentrations still caused significant interference by overlapping the DA peak. In comparison to the GC/PMeT electrode, the glassy carbon (GC/Nafion/PMeT) electrode modified with hybrid film Nafion/PMeT was found to permit a superior separation by shifting the oxidation of AA peak toward the less positive potential. The DPV curves for a mixture of DA and AA at an GC/Nafion/PMeT electrode in a 0.1 M H2SO4 solution showed peaks of DA and AA, at 0.45 and 0.21 V, respectively, indicating that the difference in the oxidation potential was 240 mV. In the 0.1 M H2SO4 solution, the oxidation peak current on the differential pulse voltammograms for the GC/PMeT electrode increased linearly with the concentration of DA in the range 1 × 10−6 to 1 × 10−3 M, and the oxidation peak current on the differential pulse voltammograms for the GC/Nafion/PMeT electrode in the range 5 × 10−7 to 2 × 10−4 M. The DA detection sensitivity of the GC/Nafion/PMeT electrode (26.7 μA μM−1 cm−2) was 22 times higher than that of the GC/PMeT electrode (1.21 μA μM−1 cm−2). [ABSTRACT FROM AUTHOR]

Abstract: The permeation of calcein across the phospholipid bilayer membrane is a key phenomenon in the detection system using liposomes as a sensor unit. The behavior of the calcein release from the liposome was analyzed by a first-order kinetic to obtain the permeability coefficient, P s [cm/s]. The P s value for the neutral liposome, prepared by zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), was found to depend on both the diameter of liposome and the temperature. The membrane fluidity of the POPC liposome, evaluated by the hydrophobic probe, 1-(4-trimethyl-aminophenyl)-6-diphenyl-1,3,5-hexatriene, was also dependent on the liposome diameter and the temperature. The P s values for various neutral liposomes under gel phase or liquid-crystalline phase were correlated with their membrane fluidity, although some data were a little scattered, possibly due to the lamellarity. It is therefore considered that the membrane fluidity dominates the permeability of calcein across the neutral phospholipid membrane. Based on the above results, the P s value for liposomes immobilized on the solid surface is discussed. [Copyright &y& Elsevier]

Abstract: Immobilization of model cell membranes (liposomes) on sensing devices is useful for the detection, evaluation, and analysis of interaction between cell membranes and proteins or other biological materials. The method to immobilize the liposomes on the solid surface was investigated by using the quartz crystal microbalance (QCM) method, focusing on the density of immobilized liposomes and their stability. The different liposomes were applied onto the planar surface of the QCM electrode by varying the immobilization method. The results showed that the immobilized liposomes depended not only on the immobilization method but also on the properties of the liposomes. The liposome with low membrane fluidity likely showed intact immobilization on a solid surface. An electrostatic interaction also affected the amount of immobilized liposomes on the functionalized quartz crystal. By using the amino-coupling method, intact liposomes could be immobilized on a solid surface and immobilized liposomes could stabilize for 10h with frequency changes less than 5%. [Copyright &y& Elsevier]

[Display omitted] • Hydrothermal treatment in microcapillary made it possible to form W/O/W emulsion. • W/O/W emulsion yielded vesicles of less than 100 nm in diameter by the solvent diffusion. • Span20/Tween20 vesicles indicated the dynamic interface. 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. [ABSTRACT FROM AUTHOR]

Abstract: Based on a number of experiments it is concluded that the fluorescein labeled β-heptapeptide fluoresceinyl-NH-CS-(S)-β 3hAla-(S)-β 3hArg-(R)-β 3hLeu-(S)-β 3hPhe-(S)-β 3hAla-(S)-β 3hAla-(S)-β 3hLys-OH translocates across lipid vesicle bilayers formed from DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine). The conclusion is based on the following observations: (i) addition of the peptide to the vicinity of micrometer-sized giant vesicles leads to an accumulation of the peptide inside the vesicles; (ii) if the peptide is injected inside individual giant vesicles, it is released from the vesicles in a time dependent manner; (iii) if the peptide is encapsulated within sub-micrometer-sized large unilamellar vesicles, it is released from the vesicles as a function of time; (iv) if the peptide is submitted to immobilized liposome chromatography, the peptide is retained by the immobilized DOPC vesicles. Furthermore, the addition of the peptide to calcein-containing DOPC vesicles does not lead to significant calcein leakage and vesicle fusion is not observed. The finding that derivatives of the β-heptapeptide (S)-β 3hAla-(S)-β 3hArg-(R)-β 3hLeu-(S)-β 3hPhe-(S)-β 3hAla-(S)-β 3hAla-(S)-β 3hLys-OH can translocate across phospholipid bilayers is supported by independent measurements using Tb3+-containing large unilamellar vesicles prepared from egg phosphatidylcholine and wheat germ phosphatidylinositol (molar ratio of 9:1) and a corresponding peptide that is labeled with dipicolinic acid instead of fluorescein. The experiments show that this dipicolinic acid labeled β-heptapeptide derivative also permeates across phospholipid bilayers. The possible mechanism of the translocation of the particular β-heptapeptide derivatives across the membrane of phospholipid vesicles is discussed within the frame of the current understanding of the permeation of certain oligopeptides across simple phospholipid bilayers. [Copyright &y& Elsevier]

Abstract: Conformational transitions of bovine β-lactoglobulin A (β-LG) induced by carboxylic acid were systematically studied by steady-state tryptophan (Trp) fluorescence. The behavior of β-LG denaturation depends upon the species and concentration of carboxylic acid, as well as on the pH of solutions. The order of the effectiveness of the respective carboxylic acids was described as follows: MeCOOH

The dielectric response of lipid bilayer membrane vesicles (liposomes) prepared using either phosphatidylcholine from egg (EPC) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was analyzed at a frequency range of 0.1 to 100 MHz. A marked dielectric dispersion for EPC and POPC liposome suspensions was observed above 1 MHz. An appropriate analysis of the dielectric dispersion curve was performed using the Cole-Cole equation and the Debye equation and was found to provide a method for the determination of dielectric parameters. Among the dielectric parameters, the characteristic frequency of a second dispersion around 50 MHz varied corresponding with changes in the test conditions. Of particular note is that an anomalous change in the characteristic frequency in the presence of protein corresponded to the degree of hydrophobic interaction between proteins and liposomes. The value of the frequency around 50 MHz, as well as the decrease in permittivity over the frequency range tested, are indicators of the interaction between proteins and liposomes. [Copyright &y& Elsevier]

The N-terminal fragment of apolipoprotein A-I (apoA-I), comprising residues 1–83, contains three segments prone to aggregation: residues 14–22, 53–58, and 67–72. We previously demonstrated that residues 14–22 are critical in apoA-I fibril formation while residues 53–58 entropically drove the nucleation process. Here, we investigated the impact of amyloidogenic mutations (Δ60–71/VT, Δ70–72, and F71Y) located around residues 67–72 on fibril formation by the apoA-I 1–83 fragment. Thioflavin T fluorescence assay demonstrated that the Δ60–71/VT mutation significantly enhances both nucleation and fibril elongation rates, whereas the Δ70–72 and F71Y mutations had minimal effects. Circular dichroism measurements and microscopic observations revealed that all variant fragments formed straight fibrils, transitioning from random coils to β-sheet structures. Kinetic analysis demonstrated that primary nucleation is the dominant step in fibril formation, with fibril elongation reaching saturation at high protein concentrations. Thermodynamically, both nucleation and fibril elongation were enthalpically and entropically unfavorable in all apoA-I 1–83 variants, in which the entropic barrier of nucleation was almost eliminated for the Δ60–71/VT variant. Taken together, our results suggest the presence of new aggregation-prone segment in the Δ60–71/VT variant that promotes nucleation through entropic effects. [ABSTRACT FROM AUTHOR]

Regulation of α-synuclein (αS) fibril formation is a potent therapeutic strategy for αS-related neurodegenerative disorders. αS, an intrinsically disordered 140-residue intraneural protein, comprises positively charged N-terminal, hydrophobic non-amyloid β component (NAC), and negatively charged C-terminal regions. Although mouse and human αS share 95% sequence identity, mouse αS forms amyloid fibrils faster than human αS. To evaluate the kinetic regulation of αS fibrillation, we examined the effects of mismatched residues in human and mouse αS on fibril formation and intramolecular interactions. Thioflavin T fluorescence assay using domain-swapped or C-terminal-truncated αS variants revealed that mouse αS exhibited higher nucleation and fibril elongation than human αS. In mouse αS, S87N substitution in the NAC region rather than A53T substitution is dominant for enhanced fibril formation. Fӧrester resonance energy transfer analysis demonstrated that the intramolecular interaction of the C-terminal region with the N-terminal and NAC regions observed in human αS is perturbed in mouse αS. In mouse αS, S87N substitution is responsible for the perturbed interaction. These results indicate that the interaction of the C-terminal region with the N-terminal and NAC regions suppresses αS fibril formation and that the human-to-mouse S87N substitution in the NAC region accelerates αS fibril formation by perturbing intramolecular interaction. [ABSTRACT FROM AUTHOR]

A new fluorescent arrayed biosensor has been developed to discriminate species and concentrations of target proteins by using plural different phospholipid liposome species encapsulating fluorescent molecules, utilizing differences in permeation of the fluorescent molecules through the membrane to modulate liposome-target protein interactions. This approach proposes a basically new label-free fluorescent sensor, compared with the common technique of developed fluorescent array sensors with labeling. We have confirmed a high output intensity of fluorescence emission related to characteristics of the fluorescent molecules dependent on their concentrations when they leak from inside the liposomes through the perturbed lipid membrane. After taking an array image of the fluorescence emission from the sensor using a CMOS imager, the output intensities of the fluorescence were analyzed by a principal component analysis (PCA) statistical method. It is found from PCA plots that different protein species with several concentrations were successfully discriminated by using the different lipid membranes with high cumulative contribution ratio. We also confirmed that the accuracy of the discrimination by the array sensor with a single shot is higher than that of a single sensor with multiple shots. [ABSTRACT FROM AUTHOR]

Amyloid beta (Aβ) adsorption onto lipid membranes depending on the condition of a lipid was investigated by means of time-of-flight secondary ion mass spectrometry (ToF-SIMS). Aβ aggregation depending on the different hardness of lipid membrane has not been clarified yet although it is important to understand Alzheimer's disease. Aβ (1-40) on three different lipid membranes having different transition temperatures has been evaluated using ToF-SIMS in the previous study, and in this study, the differences between Aβ on liquid crystalline-phase lipid membranes and that on gel-phase lipid membranes were investigated in order to clarify the mechanisms of aggregation and peptide folding change. ToF-SIMS secondary ion images clearly showed Aβ distribution on lipid membranes. Because ToF-SIMS data is extremely complicated although it contains rich information, it was analysed by principal component analysis (PCA). Score images indicated by PCA are consistent with the images of secondary ions related to Aβ and are clearer than the secondary ion images. Moreover, PCA results suggest the difference between Aβ on different lipid membranes in terms of amino acid fragment ions, and the orientation of Aβ on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine was indicated. Copyright © 2016 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

The microphase separation of lipid molecules on a vesicle membrane can be induced, depending on the difference in the geometric structures of their headgroups. Through cryo-transmission-electron-microscopy analysis, a lens-like vesicle was prepared by mixing 50 wt% Span 40 (sorbitan monopalmitate) and 50 wt% Tween 40 [polyoxyethylene (20) sorbitan monopalmitate]. Considering the molecular structures of Span 40 and Tween 40, the high-curvature region was mainly formed by Tween 40. As determined by Fourier-transform infrared spectroscopy, dielectric-dispersion analysis, and differential scanning calorimetry, a hydration layer was likely formed because polyoxyethylene conjugates with the headgroups of Tween 40. These investigations of the obtained self-assembled aggregates of nonionic surfactants with heterogeneous surfaces could contribute to the development of new types of biomaterials. [ABSTRACT FROM AUTHOR]

Aggregation of α-synuclein (α-syn) into amyloid fibrils is closely associated with Parkinson's disease (PD). Familial mutations or posttranslational truncations in α-syn are known as risk factor for PD. Here, we examined the effects of the PD-related A30P or A53T point mutation and C-terminal 123–140 or 104–140 truncation on the aggregating property of α-syn based on the kinetic and thermodynamic analyses. Thioflavin T fluorescence measurements indicated that A53T, Δ123‒140, and Δ104–140 variants aggregated faster than WT α-syn, in which the A53T mutation markedly increases nucleation rate whereas the Δ123‒140 or Δ104‒140 truncation significantly increases both nucleation and fibril elongation rates. Ultracentrifugation and western blotting analyses demonstrated that these mutations or truncations promote the conversion of monomer to aggregated forms of α-syn. Analysis of the dependence of aggregation reaction of α-syn variants on the monomer concentration suggested that the A53T mutation enhances conversion of monomers to amyloid nuclei whereas the C-terminal truncations, especially the Δ104–140, enhance autocatalytic aggregation on existing fibrils. In addition, thermodynamic analysis of the kinetics of nucleation and fibril elongation of α-syn variants indicated that both nucleation and fibril elongation of WT α-syn are enthalpically and entropically unfavorable. Interestingly, the unfavorable activation enthalpy of nucleation greatly decreases for the A53T and becomes reversed in sign for the C-terminally truncated variants. Taken together, our results indicate that the A53T mutation and the C-terminal truncation enhance α-syn aggregation by reducing unfavorable activation enthalpy of nucleation, and the C-terminal truncation further triggers the autocatalytic fibril elongation on the fibril surfaces. [ABSTRACT FROM AUTHOR]

Abstract: We have focused on the interaction between the Span 80 vesicle and phospholipid vesicle (liposome). The Span 80 vesicle was mixed with a phospholipid vesicle (liposome), used as a simplified model of plasma membrane. From calcein leakage experiments, it was revealed that the interaction between the Span 80 vesicles and the liposomes could perturb the liposome membrane. In the experiments based on fluorescence resonance energy transfer, the lipid mixing between Span 80 vesicle and liposome was observed. The above phenomena were also supported by the fluorescence spectroscopic analysis using laurdan, resulting in the variation in the membrane properties of liposome after its mixing with Span 80 vesicle. These results suggest that the Span 80 vesicles can easily interact and hemifuse with the liposome membrane, which depend on the membrane properties of the Span 80 vesicle, “flexible” and “molecular structure” of the components. [Copyright &y& Elsevier]

Abstract: The strategy to prepare a novel biocatalyst by the immobilization of chitosanase onto liposome (ICL) was carried out based on the direct interaction of liposomes with cell membrane of Streptomyces griseus cell. The ICL was characterized in relation to the molecular weight of protein, the chitosanase activity, the effect of the surface hydration of various liposomes on hydrolysis activity of immobilized chitosanase and the stability of ICL under various extreme conditions. The SDS-PAGE analysis of the purified ICL sample shows the existence of a protein with approximately 39kDa that corresponded to the sum of weight of the mature chitosanase and its signal peptide (38.8kDa). The above protein of ICL also expresses the chitosanase activity that is significantly higher than that of the conventional chitosanase. Furthermore, the surface hydration of liposomes used to prepare ICL that affected the activity of immobilized chitosanase verified the importance of liposome surfaces. Indeed, the stability of ICL assayed by measuring the chitosanase activity is significantly higher than that of conventional chitosanase under various temperatures and pH conditions. These characteristics of ICL show the possible preparation of the biocatalysts that can be prepared by immobilizing enzymes onto liposome vesicles properly. [Copyright &y& Elsevier]

Abstract: The interaction between the neutral 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) liposomes and cell membrane of Streptomyces griseus induced by the heat treatment at specific temperature was investigated, focusing on the internalization of the neutral POPC liposomes with S. griseus cells. In an attempt to clarify the modes of liposome internalization, various kinds of inhibitors of endocytotic pathways were used to treat S. griseus cells. The efficiency of the heat treatment on liposome–cell membrane interactions was finally characterized based on the hydrophobic, electrostatic interactions and hydration effect. In fact, the internalization of the neutral liposomes into these cells was found to show higher rate and greater amount at higher temperatures. The kinetic study showed that the maximum amount of the internalized liposomes was, respectively, 469×105 and 643×105 liposomes/cell at 37 and 41°C. The internalization of the neutral liposomes induced by the heat treatment was characterized, implying that the endocytosis occurred. The interactions involving the internalization, adsorption, and fusion of these liposomes with S. griseus cells were mainly contributed by the hydrophobic interaction and the unstable hydrogen bonds caused by the loss of water of surface hydration of cell membrane rather than the electrostatic interaction under the specific heat condition. [Copyright &y& Elsevier]

Abstract: A mechanism for liposome-recruited activity of oxidized and fragmented superoxide dismutase (Fr.-SOD) [Tuan LQ, Umakoshi H, Shimanouchi T, Kuboi R. Liposome-recruited activity of oxidized and fragmented superoxide dismutase. Langmuir 2008;24:350–4] was further investigated, focusing on the secondary structure of Fr.-SOD. Liposome membrane was found to assist the conformational change of Fr.-SOD and reactivate the enzymatic activity, like molecular and metal chaperones. The loss of SOD activity and its secondary structure was observed during 6h oxidation in 2mM hydrogen peroxide. The contents of the α-helix and β-sheet structures in the oxidized and fragmented SOD (2μM) were increased only in the presence of 10μM Cu2+ and Zn2+ together, or in the presence of 2mM POPC liposomes. The mixture of all of these elements (fragmented SOD and POPC liposomes with Cu2+ and Zn2+) gave not only the increase of the α-helix and β-sheet contents but also the mediation of the high SOD-like activity. [Copyright &y& Elsevier]

A direct interaction of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes with membrane of Streptomyces griseus cell under the heat stress at 41°C increased the chitosanase production and release to 2.2 times higher than that at 37°C without the POPC liposomes. Amount of chitosanase released across the lipid mimicking cell membrane (LMCM) liposome under a heat at 41°C in the presence of POPC liposomes was 17% of initially-entrapped chitosanase while it was only 1% in the absence of POPC liposomes, even under a heat stress at 41°C, clearly showing the importance of the direct interaction between membrane and membrane. [Copyright &y& Elsevier]

The effects of heat stress and liposome treatment on the growth of Streptomyces griseus cells and chitosanase production were investigated on the basis of using the designed strategy of a stress-mediated bioprocess. The effective conditions for increasing the interaction between chitosanase and the 1-palmitoyl-2-oleoyl-3-phosphocholine (POPC) liposome under heat stress condition were determined on the basis of the results of circular dichroism (CD) and dielectric dispersion analysis (DDA). Under these effective conditions, S. griseus cells were cultivated for the effective production of chitosanase. The results obtained from both CD spectra and DDA showed that heat stress enhances the interaction of the POPC liposomes and chitosanase. The strongest interaction between them could be obtained in the specific temperature range of 40–45°C. The enhancement of the target chitosanase production was conducted under heat stress at 41°C in the presence and absence of the POPC liposomes. The growth rates of S. griseus cells in the cases of heat (41°C) and heat (41°C)/POPC treatments were respectively 1.2 and 1.4 times higher than that obtained under the control condition. In the heat (41°C) and heat (41°C)/POPC treatments, chitosanase activity increased to 1.8 and 2 times, respectively, higher than that obtained under the control condition. Heat stress and the addition of the POPC liposomes could therefore be utilized to induce the potential functions of bacterial cells for the enhancement of the final target production. [Copyright &y& Elsevier]

A catalytic reaction of H2O2 production by an amyloid β-peptide (Aβ)–Cu complex with cholesterol incorporated in a liposome was kinetically analyzed. The Michaelis–Menten model was applied to the H2O2 production reaction using cholesterol as the substrate catalyzed by the Aβ–Cu complex. The K m value for the Aβ–Cu complex catalytic reaction with cholesterol-containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes (K m=0.436 μM for Aβ(1–40); K m=0.641 μM for Aβ(1–42)) was found to be smaller than that with cholesterol-containing 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes (K m=0.585 μM for Aβ(1–40), K m=0.890 μM for Aβ(1–42)). The results imply that membrane properties could play an important role in the interactions of the Aβ–Cu complex with cholesterol in these liposomes. Considering the physical states of the cholesterol/POPC (liquid disordered phase) and cholesterol/DPPC (liquid ordered phase) liposomes in the present reaction conditions, the data obtained suggests that the H2O2-generating activity of the Aβ–Cu complex, accompanied by oxidation of membrane-incorporated cholesterol, could be effected by the phase of the liposome membranes. [Copyright &y& Elsevier]

The process of fusion of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes in the presence of stimuli responsive copolymers (poly(N-isopropylacrylamide-co-methacrylic acid) and poly(N-isopropylacrylamide-co-methacrylic acid-co-octadecylacrylamide)) or proteins (α-chymotrypsin (α-CT), bovine carbonic anhydrase (CAB), and β-galactosidase (β-gal)) was quantitatively characterized under the stimuli by varying pH and temperature. After the liposomes were exposed to the specific pH and heat stimuli in the presence of the stimuli responsive polymers or proteins, the percentage of the fusion was determined by using two kinds of liposomes entrapping cobalt-calcein and EDTA (cobalt-calcein method). In the presence of stimuli responsive copolymers, the percentage of fusion was increased to 20% above the phase transition condition of the copolymer (above 37 °C and below pH 5.7). The percentage of fusion in the presence of proteins was varied under the stimuli, depending on the type of proteins. In the case of the α-CT, the maximal percentage of fusion was only 8%. The addition of the CAB and β-gal improved the percentage of fusion to 15 and 20%, respectively, by selecting the optimal stimuli conditions. Although some disagreements were observed in the region of strong acidic conditions, it was found that the increase of the percentage of fusion was well corresponding with the increase of the membrane fluidity of the POPC liposome, followed by the increase of the local hydrophobicity of proteins or copolymers under the stimuli. Based on the above results, a possible model for the stimuli-induced fusion of POPC liposome membranes was finally presented. [Copyright &y& Elsevier]

Novel gas sensor devices have been developed using polythiophene (pTh) film and poly(3-n-dodecylthiophene) (pDpTh) film coated over pTh film. These polymer films were electrochemically deposited and doped by cyclic voltammetry on thin-film electrodes where the isolation gap was formed by a micromachining process. We examined the response characteristics of the conducting polymer films against various sample gases over a range of temperatures of the sensitive layer. The resistance changes of both sensitive layers of pTh and pDpTh were highly dependent on the kind of layer. In particular, pTh film responded to ammonia gas and pDpTh films clearly responded to hydrophobic gases, such as chloroform, methane and ethanol. The response of these films to several gases was analyzed with a pattern recognition (PARC) algorithm. It was found that our simple gas sensor device could discriminate between the gases that were used here. [Copyright &y& Elsevier]

Membrane lipid composition is known to influence aggregation and fibril formation of many amyloidogenic proteins. Here, we found that phosphatidylethanolamine (PE) accelerates aggregation of the N‐terminal 1‒83 fragment of an amyloidogenic G26R variant of apoA‐I on lipid membranes. Circular dichroism and isothermal titration calorimetry measurements demonstrated that PE does not affect the α‐helical structure and lipid binding property of apoA‐I 1‐83/G26R. Rather, fluorescence measurements indicated that PE induces more ordered lipid packing at the interfacial and acyl chain regions, providing more hydrophobic environments especially around the highly amyloidogenic regions in apoA‐I on the membrane surface. These results suggest that PE promotes aggregation of the amyloidogenic N‐terminal fragment of apoA‐I on lipid membranes by inducing hydrophobic membrane environments. [ABSTRACT FROM AUTHOR]