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Cholesterol suppresses the hemolysis and the detachment of cytoskeletal proteins from bilayer in the human erythrocyte membrane under stress conditions. However, there is little information on how cholesterol functions. So, examining the role of a short side chain of cholesterol, we used the plant sterols such as ß-sitosterol and stigmasterol. Incorporation of sterols into the membrane using a sterol/methyl-ß-cyclodextrin complex was confirmed by the mass spectrometry. Hemolysis of human erythrocytes under high hydrostatic pressure (200 MPa) or hypotonic conditions was suppressed by cholesterol, but not by ß-sitosterol and stigmasterol. Moreover, the bilayer-cytoskeleton interaction was also strengthened by cholesterol, but not by ß-sitosterol and stigmasterol. Taken together, we suggest that the short side chain of cholesterol plays an important role in the membrane stability of human erythrocytes.

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Of group 12 metals, zinc is an essential element to maintain our life, but other metals such as cadmium and mercury are toxic in cellular activities. Interactions of these metals with biomembranes are important to understand their effects on our living cells. Here, we describe the membrane perturbations induced by these metals in human erythrocytes. Of these metals, Zn2+ ions only induced the erythrocyte agglutination. Histidine residues in extracellular domains of band 3 participated in Zn2+-induced agglutination. Interestingly, it was found that band 3-cytoskeleton interactions play an important role in Zn2+-induced agglutination. In contrast with Hg2+ and Cd2+ ions, Zn2+ ions greatly suppressed pressure-induced hemolysis by cell agglutination. Such a suppression was removed upon dissociation of agglutinated erythrocytes by washing, indicating the reversible interactions of Zn2+ ions with erythrocyte membranes. Excimer fluorescence of pyrene indicated that spectrin is denatured by a pressure of 200 MPa irrespective of hemolysis suppression. Taken together, these results suggest that the agglutination of erythrocytes due to the interactions of Zn2+ ions with band 3 is stable under pressure, but spectrin, cytoskeletal protein, is denatured by pressure.

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Human erythrocytes are agglutinated by lectins such as concanavalin A (Con A). The behaviors of agglutinated erythrocytes under pressure are less well understood. Here, we report the effects of erythrocyte agglutination on pressure-induced membrane damages. Small clumps of intact erythrocytes by Con A were dissociated by a pressure of 200 MPa. Further, the observation by scanning electron microscopy demonstrated the generation of vesicles, fragmented particles, and membrane hole. On the other hand, large clumps of trypsin-digested erythrocytes by Con A seemed to be stable against 200 MPa. However, the erythrocytes dissociated from such pressure-treated clumps by methyl α-mannopyranoside also showed the existence of vesicles and fragmented particles except for the membrane hole. Pressure-induced hemolysis was greatly suppressed in such large clumps. Similar suppressive effects were observed in erythrocytes packed by centrifugation. However, the hemolysis occurred when the erythrocytes dissociated from 200 MPa-treated large clumps by methyl α-mannopyranoside were incubated at 0°C and atmospheric pressure. Pyrene excimer fluorescence due to spectrin denaturation was observed in Con A-agglutinated ghosts that were exposed to a pressure of 200 MPa. These results suggest that upon pressure treatment of tightly agglutinated erythrocytes, the hemolysis is greatly suppressed, but membrane damages occur such as spectrin denaturation and vesiculation.

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Self-aggregation of the zwitterionic surfactant 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) in water and isopropanol-water media, and interaction of the amphiphile with the biopolymer inulin in these media were investigated. The micellar properties of the zwitterionic surfactant and its associated interfacial and bulk properties along with the related energetic, and aggregation number were determined. The different stages of interaction of the CHAPS-inulin combines were identified and assessed. The complexes were formed and aggregated in solution at different stages of their molecular compositions. The aggregated sizes were determined by dynamic light scattering study and the morphology in the solvent removed states were examined using scanning electron microscope and transmission electron microscope techniques. The results witnessed formation of ensembles of varied and striking patterns.

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Recent studies on mixed surfactant systems were systematically overviewed, paying special attention to synergism observed in micellization as well as adsorbed film formation upon mixing of a few nonionic surfactants with a variety of surfactants (such as anionics including bile salts and a hybrid type surfactant, cationics including a Gemini type surfactant, different types of nonionics and a zwitterionic surfactant used as a membrane solubilizer) in addition to various combinations of anionics. Through the text, it was shown for each given binary mixed system composed of surfactants 1 and 2 how to estimate not only the composition of mixed micelles (Y(2)) equilibrated with singly dispersed surfactant species in bulk solution phase, where the mole fraction of 2 in the surfactant mixture is denoted as X(2), but also the composition of adsorbed film phase (Z(2)). Almost all combinations were discussed in terms of the respective interaction parameters, omega(R) and omega(A), in mixed micelles (3-D phase) and in mixed adsorbed film (2-D phase), respectively, surface excess concentration (Gamma), partial molecular area (PMA), minimum surface Gibbs energy (G(s)min), and such defined measures as pC(20), CMC/C(20) etc. for evaluation of synergism.

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For a mixed system of a typical membrane protein solubilizer CHAPS (a derivative of a bile acid cholic acid) combined with a bile salt (sodium salt of glycocholic acid, NaGC), which is also a candidate as a membrane protein solubilizer, micellization and adsorbed film formation in a phosphate buffer solution of pH 7.4 at 303 K were studied paying special attention to the synergistic effect upon mixing. The collection of sufficient data based on plots of surface tension (gamma) versus logarithmic concentration (C(t) or m(t)) in total molality at discrete mole fractions (X(2)) in the mixture of surfactants 1 and 2 (where 1 and 2 correspond to CHAPS and NaGC, respectively) allowed us to accurately determine critical micelle concentration (CMC), minimum surface tension at CMC (gamma(CMC)), and the slope (dgamma/dlnC(t)) from the gamma-lnC(t) curves in the concentration range just below CMC. These data enabled us to estimate surface excess (Gamma(t)), and mean molecular area (A(m)) in addition to such parameters as the minimum surface Gibbs energy (G(min)((S))), pC(20) and CMC/C(20) related to synergism accompanied by blending. Applying the regular solution theory (RST), the relation of compositions of the singly dispersed phase (X(2)) and the micellar phase (Y(2)) as well as the interaction parameter (omega(R)) (by using the Rubingh's equations) were estimated. The relation between the composition in the adsorbed film (Z(2)) and X(2) together with the interaction parameter (omega(A)) in the adsorbed film was also estimated. The partial molecular area (PMA), gamma(CMC), and G(min)((S)) were examined as functions of X(2) and/or Z(2.) The resultant CMC-X(2) and CMC-Y(2) curves and omega(R) and omega(A) values have demonstrated that mixed micelles and adsorbed film formation are attained accompanying to some extent enhanced intermolecular interaction (with negative omega(R) and omega(A) values). Comparing with previous results for mixed systems of CHAPS with n-acyl (octanoly, nonanoyl, and decanoyl)-N-methylglucamides [MEGA-n's (n=8, 9, and 10)] and of sodium chenodeoxycholate (NaCDC) with sodium ursodeoxycholate (NaUDC), the synergism observed for the mixed system of CHAPS with NaGC lies between both combinations. However the expected properties as a membrane protein solubilizer are judged to be sufficient.

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Different physicochemical properties of Langmuir films (monolayers) composed of 10 mixed systems of a bile acid, deoxycholic acid (DC) with various plant sterols, such as stigmasterol (Stig), beta-sitosterol (Sito) and campesterol (Camp) and a stanol, cholestanol (Chsta) in addition to an animal sterol, cholesterol (Ch) [these sterols and Chsta are abbreviated as St] and DC with 1:1 St mixtures; (Ch+Chsta), (Ch+Stig), (Stig+Chsta), (Ch+Sito) and (Ch+Camp) on the substrate of 5M aqueous NaCl solution (pH 1.2) at 25 degrees C, were investigated in terms of mean surface area per molecule (A(m)), the partial molecular area (PMA), surface excess Gibbs energy (DeltaG((ex))), interaction parameter (I(p)) as well as activity coefficients (f(1) and f(2)) in 2-D phase of each binary (or ternary) component system and elasticity (Cs(-1)) of formed films; these were analyzed on the basis of the respective surface pressure (pi) versus A(m) isotherms as a function of mole fraction of Sts (X(st)) in the DC/St(s) mixtures at discrete surface pressures. Notable findings are: (i) all the binary component systems did form patched film type monolayers consisting of (a) DC-dominant film solubilizing a trace amount of St molecules and (b) St dominant film dissolving a small amount of DC molecules, (ii) DC in 2-D phase exhibited a transition from LE film to LC film at a constant pressure (pi(C)(1)) accompanied by compression and (iii) DeltaG((ex)) as well as I(p) was found to be greatly dependent on (a) the combinations of DC with different St species and (b) to be markedly varied by a difference in mixing ratio of DC to Sts. Compressibility (or elasticity) analyses and fluorescence microscopy images could support the above findings as well as interpretation.

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Surface pressure-area (pi-A), surface potential-area (DeltaV-A), and dipole moment-area (mu( perpendicular)-A) isotherms were obtained for the Langmuir monolayer of two fluorinated-hydrogenated hybrid amphiphiles (sodium phenyl 1-[(4-perfluorohexyl)-phenyl]-1-hexylphosphate (F6PH5PPhNa) and (sodium phenyl 1-[(4-perfluorooctyl)-phenyl]-1-hexylphosphate (F8PH5PPhNa)), DPPC and their two-component systems at the air/water interface. Monolayers spread on 0.02 M Tris buffer solution (pH 7.4) with 0.13M NaCl at 298.2K were investigated by the Wilhelmy method, ionizing electrode method and fluorescence microscopy. Moreover, the miscibility of two components was examined by plotting the variation of the molecular area and the surface potential as a function of the molar fraction for the fluorinated-hydrogenated hybrid amphiphiles on the basis of the additivity rule. The miscibility of the monolayers was also examined by construction of two-dimensional phase diagrams. Furthermore, assuming the regular surface mixture, the Joos equation for analysis of the collapse pressure of two-component monolayers allowed calculation of the interaction parameter (xi) and the interaction energy (-Deltaepsilon) between the fluorinated-hydrogenated hybrid amphiphiles and DPPC. The observations by a fluorescence microscopy also supported our interpretation as for the miscibility in the monolayer state. Comparing the monolayer behavior between the two binary systems, no remarkable difference was found among various aspects. Among the two combinations, the mole fraction dependence in monolayer properties was commonly classified into two ranges: 0

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By means of surface tension measurement (Wilhelmy method), micellization and adsorbed film formation were investigated for three combinations of mixed surfactant systems, all of which are used for solubilizing membrane proteins: a typical zwitterionic surfactant, CHAPS (a derivative of cholic acid) with n-alkyl (octyl, nonyl and decyl)-N-glucamides, MEGA-n (n=8, 9, 10). The data based on plotting of surface tension (gamma) versus logarithmic total molarity (or molality) (Ct or Mt) as a function of mole fraction of surfactant 2 (2 corresponds to MEGA-n's) enabled us to determine critical micellization concentration (CMC), minimum surface tension at CMC (gammaCMC), surface excess (Gamma(t)), mean molecular surface area (Am), the minimum Gibbs energy (Gmin(S)) of adsorbed film of both single and mixed surfactant systems and partial molecular area (PMA) in addition to parameters such as pC20 and CMC/C20 being related to synergism accompanied by blending (mixing) in regard to surface activity as well as micelle forming ability. On the basis of the regular solution theory, the relations of compositions of singly dispersed phase (X2), micellar phase (Y2) and adsorbed film (Z2) were estimated, and then the interaction parameters in micelles (omegaR) and in the adsorbed film phase (omegaA) were also calculated. From both the CMC-X2 and CMC-Y2 curves, it was found for all the combinations to show synergistically enhanced ability of mixed micelle formation as well as surface tension reduction. The resultant synergism coming from blending CHAPS with MEGA-n's was discussed in comparison with different combinations of various types of surfactants including membrane proteins solubilizers.

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