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Small-angle neutron scattering and coarse-grained molecular dynamics simulations have been used to determine the structural parameters (bilayer thickness D, polar region thickness D(H), interfacial lateral area of the unit cell A(UC) and alcohol partial interfacial area A(CnOH)) of fluid dioleoylphosphatidylcholine:dioleoylphosphatidylserine (PCPS, DOPC:DOPS=24.7mol:mol) bilayers in extruded unilamellar vesicles with incorporated aliphatic alcohols (CnOH, n=8-18 is the even number of carbons in alkyl chain). External ((2))H(2)O/H(2)O contrast variation experiments revealed that D(H) decreases as a function of alkyl chain length and CnOH:PCPS molar ratio. Using measurements at single 100% ((2))H(2)O contrast we found that (i) D decreases with CnOH:PCPS molar ratio and increases with CnOH chain length (at 0.4 molar ratio); (ii) A(UC) significantly increases already in the presence of shortest CnOH studied (at 0.4 molar ratio), further increase is observed with longer CnOHs and at higher molar ratios; (iii) A(CnOH) of alcohol molecules in PCPS bilayer increases linearly with the alkyl chain length, A(CnOH) obtained for CnOHs with n≤10 corresponds to A(CnOH)≤20Å(2) - a value specific for the crystalline or solid rotator phase of alkanes. All these structural modifications induced by studied CnOHs were reproduced in MD simulations. The computational results give an accurate description of the alcohol effects at the molecular level, explaining the experimental data. The anomaly in A(CnOH) is discussed via the "umbrella" effect described for cholesterol.

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The influence of cholesterol on the structural parameters of phosphatidylcholine bilayers is studied by small-angle neutron scattering on unilamellar liposomes. Monounsaturated diacylphosphatidylcholines diCn:1PC with the length of acyl chains n=14, 18 and 22 carbons are used. We confirm that the bilayer thickness increases with increasing concentration of cholesterol for all studied diCn:1PCs. However, partial areas per diCn:1PC and cholesterol molecule on lipid-water interface are found not to depend of cholesterol concentration. The partial area per cholesterol molecule is 0.24nm². In addition, the partial area per diC18:1PC is larger than that for diC14:1PC and diC22:1PC.

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The influence of a mammalian sterol cholesterol and a plant sterol beta-sitosterol on the structural parameters and hydration of bilayers in unilamellar vesicles made of monounsaturated diacylphosphatidylcholines (diCn:1PC, n=14-22 is the even number of acyl chain carbons) was studied at 30 degrees C using small-angle neutron scattering (SANS). Recently published advanced model of lipid bilayer as a three-strip structure was used with a triangular shape of polar head group probability distribution (Kucerka et al., Models to analyze small-angle neutron scattering from unilamellar lipid vesicles, Physical Review E 69 (2004) Art. No. 051903). It was found that 33 mol% of both sterols increased the thickness of diCn:1PC bilayers with n=18-22 similarly. beta-sitosterol increased the thickness of diC14:1PC and diC16:1PC bilayers a little more than cholesterol. Both sterols increased the surface area per unit cell by cca 12 A(2) and the number of water molecules located in the head group region by cca 4 molecules, irrespective to the acyl chain length of diCn:1PC. The structural difference in the side chain between cholesterol and beta-sitosterol plays a negligible role in influencing the structural parameters of bilayers studied.

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Sarcoplasmic reticulum Ca-transporting ATPase (EC 3.6.1.38) was isolated from rabbit white muscle, purified and reconstituted into vesicles of synthetic diacylphosphatidylcholines with monounsaturated acyl chains using the cholate dilution method. In fluid bilayers at 37 degrees C, the specific activity of ATPase displays a maximum (31.5+/-0.8 IU/mg) for dioleoylphosphatidylcholine (diC18:1PC) and decreases progressively for both shorter and longer acyl chain lengths. Besides the hydrophobic mismatch between protein and lipid bilayer, changes in the bilayer hydration and lateral interactions detected by small angle neutron scattering (SANS) can contribute to this acyl chain length dependence. When reconstituted into dierucoylphosphatidylcholine (diC22:1PC), the zwitterionic surfactant N-dodecyl-N,N-dimethylamine N-oxide (C12NO) stimulates the ATPase activity from 14.2+/-0.6 to 32.5+/-0.8 IU/mg in the range of molar ratios C12NO:diC22:1PC=0/1.2. In dilauroylphosphatidylcholines (diC12:0PC) and diC18:1PC, the effect of C12NO is twofold-the ATPase activity is stimulated at low and inhibited at high C12NO concentrations. In diC18:1PC, it is observed an increase of activity induced by C12NO in the range of molar ratios C12NO:diC18:1PC< or =1.3 in bilayers, where the bilayer thickness estimated by SANS decreases by 0.4+/-0.1 nm. In this range, the 31P-NMR chemical shift anisotropy increases indicating an effect of C12NO on the orientation of the phosphatidylcholine dipole N(+)-P- accompanied by a variation of the local membrane dipole potential. A decrease of the ATPase activity is observed in the range of molar ratios C12NO:diC18:1PC=1.3/2.5, where mixed tubular micelles are detected by SANS in C12NO+diC18:1PC mixtures. It is concluded that besides hydrophobic thickness changes, the changes in dipole potential and curvature frustration of the bilayer could contribute as well to C12NO effects on Ca(2+)-ATPase activity.

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Equoral oral solution is a novel drug delivery system for cyclosporine consisting mainly of non-ionic surfactants, polyglycerol esters and polyoxyethylated fatty acids aggregates, and gives microdispersions in the aqueous enviroment. To simulate dispergation, Equoral was mixed with varying amounts of water. Changes in the structure of the prepared aggregates were studied using synchrotron x-ray small- and wide-angle scattering. A lamellar phase is the most probable structure, arising spontaneously after dispergation of Equoral in the region of 30-70 wt% H2O.

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Small-angle neutron scattering on extruded unilamellar vesicles in water was used to study bilayer thickness when cholesterol (CHOL) was added to dilauroylphosphatidylcholine (DLPC) and dioleoylphosphatidylcholine (DOPC) bilayers in molar fraction 0.44. Using the H2O/2H2O contrast variation and the small-angle form of Kratky-Porod approximation, the bilayer gyration radius at infinite contrast R(g,infinity) and the bilayer thickness parameter d(g,infinity) = 12(0.5)R(g,infinity) were obtained at 25 degrees C. Addition of CHOL to DLPC increased the d(g,infinity) from 4.058 +/- 0.028 nm to 4.62 +/- 0.114 nm, while in case of DOPC the d(g,infinity) values were the same in the absence (4.618 +/- 0.148 nm) and in the presence (4.577 +/- 0.144 nm) of CHOL within experimental errors. The role of CHOL-induced changes of bilayer thickness in the protein insertion, orientation and function in membranes is discussed.

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The effect of cholesterol (CHOL) on the rate of autoperoxidation of chromatographically pure phosphatidylcholine from hen egg yolks (EYPC) in aerated multilamellar liposomes was examined at eight CHOL:EYPC molar ratios from 0.1 up to 1.0 at 45 degrees C. Autoperoxidation was measured as conjugated diene (CD) formation by UV-VIS spectrophotometry. In the presence of cholesterol, the initiation and propagation phases of autoperoxidation were prolonged, the autoperoxidation rate in the propagation phase was decreased, and the CD concentration in the termination phase was lowered. The inhibition of EYPC autoperoxidation by cholesterol can originate a) from the increased lateral separation of polyunsaturated EYPC acyl chains caused by intercalation of cholesterol between EYPC molecules, b) from the increased molecular packing of both the bilayer polar and hydrophobic regions due to the reduced bilayer hydration, c) from the decreased concentration of the molecular oxygen in the bilayer interior, and d) from the cholesterol epoxidation and hydroperoxidation induced by the oxidation products of EYPC polyunsaturated acyl chains.

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The lipid bilayer thickness d(L), the transbilayer distance of lipid phosphate groups d(pp/inf> and the lipid surface area A(L) of fluid hydrated bilayers of lamellar phases of egg phosphatidylcholine or dipalmitoylphosphatidylcholine containing N-alkyl-N,N-dimethylamine N-oxides (CnNO), 1,4-butanedi-ammonium-N,N'-dialkyl-N,N,N',N'-tetramethyl dibromides (GSn) or mono-hydrochlorides of [2-(alkyloxy)phenyl]-2-(1-piperidinyl)ethylesters of carbamic acid (CnA) were obtained by X-ray diffraction, and the bilayer thickness in extruded unilamellar dioleoylphosphatidylcholine vesicles containing C12NO was obtained by the neutron scattering. The values of d(L), d(pp/inf> and A(L) change linearly up to the 1:1 amphiphile:lipid molar ratio. The slopes of these dependencies increase for d(L) and d(pp/inf> and decrease for AL) with an increasing number of carbons n in the amphiphile long hydrocarbon substituent (18> or =n> or =8 for CnNO, 16> or =n> or =9 for GSn, 12> or =n> or =5 for CnA), while the opposite trends are observed for the short substituent (8> or =n>/=6 for CnNO, 9> or =n> or =7 for GSn, 5> or =n> or =3 for CnA). In case of long substituents, the effects on dL), dpp/inf> and AL) are caused by the decrease in the difference between the lipid and amphiphile hydrocarbon chain lengths and by the increase in their van der Waals attraction. The short substituent amphiphiles are mobile and exchange between multiple binding sites in the bilayer, minimizing the bilayer surface area.

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Mixtures of N-dodecyl-N,N-dimethylamine N-oxide (DDAO) and 1,2-dioleoylphosphatidyl choline (DOPC) in chloroform/methanol were evaporated, dried and hydrated in excess 2H2O. Aqueous dispersions thus prepared were extruded through polycarbonate filter with pores of diameter 500A. These samples were studied using small-angle neutron scattering. DDAO destabilizes the bilayer in unilamellar liposomes and solubilizes it into mixed micelles whose shape changes with the DDAO : DOPC molar ratio. Bilayers or/and bilayer fragments have been observed up to DDAO : DOPC = 1.5, rod-like particles (tubular, cylindric micelles) at 2.5 < DDAO : DOPC < 3.5, and transition to globular particles (spheroid micelles) at DDAO: DOPC > 4. In bilayers or/and bilayer fragments, DDAO modulates the thickness of the bilayer.

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Conjugated dienes (CD) and thiobarbituric acid reactive substances (TBARS) have been estimated during the autoperoxidation of chromatographically pure phosphatidylcholine from hen eggs (EYPC) in multilamellar liposomes by UV-VIS spectrophotometry. During the propagation phase of autoperoxidation reaction, n-decane (C10) and n-octadecane (C18) gradually inhibit EYPC peroxidation up to 0:2:1 and 1:1 alkane:EYPC molar ratios, respectively. At higher molar ratios, the yield of estimated autoperoxidation products increases. At the highest molar ratio studied (alkane:EYPC = 2:1), the CD and TBARS concentrations exceed their levels in control sample without alkane added. The changes in the lipid bilayer thickness estimated from the small-angle neutron scattering (SANS) curves of unilamellar dioleoylphosphatidylcholine (DOPC) liposomes have indicated that C10 is located in the bilayer hydrophobic region parallel to the DOPC acyl chains at low molar ratios (C10:DOPC < or = 0.4). At higher molar ratios (0.6 < or = C10:DOPC < or = 1.0), the alkane changes its location into the center of the bilayer between the apposing monolayers. The alkane location parallel to polyunsaturated lipid fatty acyl chains RH separates these chains resulting in a decreased frequency of their encounters, in decreased yields of ROO. + RH-->ROOH + R., 2 ROOH-->RO. + ROO. + H2O and RO. + RH-->R. + ROH free radical reactions, and consequently, in decreased autoperoxidation. Autoperoxidation returns to the control values due to alkane redistribution into the bilayer center. 1H decoupled 31P NMR spectra of EYPC + C10 aqueous dispersions have shown that the lipid bilayer transforms into nonbilayer phases at C10:EYPC > 1:1 molar ratios. The inverted hexagonal HII phase with C10 (or C18) preferentially located in the interstitial regions of the HII unit cell displays higher autoperoxidation than the control bilayer sample due to greater motional freedom of RH chains in the HII phase.

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Small-angle neutron scattering (SANS) experiments have been performed on large unilamellar liposomes prepared from 1,2-dilauroylphosphatidylcholine (DLPC), 1,2-dimyristoyl-phosphatidylcholine (DMPC) and 1,2-distearoylphosphatidylcholine (DSPC) in heavy water by extrusion through polycarbonate filters with 500 A pores. The neutron scattering intensity I(Q) in the region of scattering vectors Q corresponding to 0.0015 A(-2) < or = Q(2) < or = 0.0115 A(-2) was fitted using a step function model of bilayer neutron scattering length density and supposing that the liposomes are spherical and have a Gaussian distribution of radii. Using the lipid volumetric data, and supposing that the thickness of bilayer polar region equals to d(H) = 9+/-1 A and the water molecular volume intercalated in the bilayer polar region is the same as in the aqueous bulk aqueous phase, the steric bilayer thickness d(L), the lipid surface area A(L) and the number of water molecules per lipid molecule N intercalated in the bilayer polar region were obtained: d(L) = 41.58+/-1.93 A, A(L) = 57.18+/-1.00 A(2) and N = 6.53+/-1.93 in DLPC at 20 degrees C, d(L) = 44.26+/-1.42 A, A(L) = 60.01+/-0.75 A(2) and N = 7.37+/-1.94 in DMPC at 36 degrees C, and d(L) = 49.77+/-1.52 A, A(L) = 64.78+/-0.46 A(2) and N = 8.67+/-1.97 in DSPC at 60 degrees C. After correcting for area thermal expansivity alpha approximately 0.00417 K(-1), the lipid surface area shows a decrease with the lipid acyl chain length at 60 degrees C: A(L) = 67.56+/-1.18 A(2) in DLPC, A(L) = 66.33+/-0.83 A(2) in DMPC and A(L) = 64.78+/-0.46 A(2) in DSPC. It is also shown that a joint evaluation of SANS and small-angle X-ray scattering on unilamellar liposomes can be used to obtain the value of d(H) and the distance of the lipid phosphate group from the bilayer hydrocarbon region d(H1).

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Lipid peroxidation of aerated multilamellar liposomes composed of egg yolk phosphatidylcholine (EYPC) and cholesterol (CHOL) at molar ratios CHOL:EYPC = 0, 0.1, 0.2, 0.3, 0.4, 0.6 and 1.0 was studied during autooxidation and during Fe2+/H2O2-induced peroxidation by following the formation of conjugated diene and thiobarbituric acid reactive substances. The presence of cholesterol in the fluid lipid bilayers has an inhibiting effect on the EYPC peroxidation in the propagation phase of both the autooxidation and Fe(2+)-induced peroxidation free radical chain reactions. This inhibiting effect increases with the increase in CHOL:EYPC molar ratio. The inhibition of EYPC peroxidation by cholesterol probably originates a) from the increased lateral separation of polyunsaturated EYPC acyl chains caused by insertion of cholesterol between EYPC molecules, b) from the increased molecular packing of both the bilayer polar and hydrophobic regions due to the reduced bilayer hydration, and c) from the antioxidant properties of cholesterol.

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Dioleoylphosphatidylcholine (DOPC) and n-decane were mixed and hydrated afterwards in an excess of heavy water at 1 wt.% of DOPC. From this dispersion, unilamellar liposomes were prepared by extrusion through polycarbonate filter with 500-A pores. Small-angle neutron scattering (SANS) was conducted on these liposomes. From the Kratky-Porod plot ln[I(Q)Q2] vs. Q2 of SANS intensity I(Q) in the range of scattering vectors Q corresponding to the interval 0.001 A(-2) < or = Q2 < or = 0.006 A(-2), the liposome bilayer radius of gyration Rg and the bilayer thickness parameter d(g) = 12(0.5)Rg were obtained. The values of d(g) indicated that the bilayer thickness is within the experimental error constant up to n-decane/DOPC approximately 0.5 molar ratio, and then increases by 2.4 +/- 1.3 A up to n-decane/DOPC = 1.2 molar ratio.

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Local anesthetic monohydrochlorides of [2-(alkoxy)phenyl]-2-(1-piperidinyl)ethyl esters of carbamic acid (CnA, n = 2, 3, 4, 6, 8, 10, 12 is the number of carbon atoms in the alkyloxy substituent) increase the probability of formation of gauche isomers p(g) and decrease the effective energy difference between gauche and trans conformation E(g) in egg yolk phosphatidylcholine (EYPC) acyl chains, as determined by electron spin resonance spectroscopy using dipalmitoylphosphatidylcholines labeled with the paramagnetic dimethyloxazolidinyl group on the 12-th or 16-th carbon atoms of their sn-2 acyl chain, and oriented EYPC bilayers hydrated at 81% relative water vapour pressure. CnAs also increase the hydration of EYPC in non-oriented bilayers at the same relative water vapour pressure. At the molar ratio of CnA:EYPC = 0.4:1, the maximum effect on p(g), E(g) and hydration has been observed for intermediate alkyloxy chain lengths n approximately 4/6.

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The partition coefficient of the local anaesthetic heptacaine (monohydrochloride of [2-(heptaloxy)phenyl]-2-(1-piperidinyl)ethyl ester of carbamic acid) between unilamellar liposomes prepared from chromatographically pure egg yolk phosphatidylcholine and the aqueous phase (NaCl solution, ionic strength I = 0,1,pH 4,5, temperature t = 20 degrees C) was determined using UV-VIS spectrophotometry. The contribution to the anaesthetic spectra due to light scattering on liposomes was eliminated numerically using the scattering function. This procedure gave more precise results than the subtraction of liposomes spectra. The values of the partition coefficient defined with the use of molar concentrations, weight concentrations and molar fractions estimated at a wavelength of lambda = 292 nm were Kp = 1116 +/- 49, Kp(m) = 1098 +/- 48 and Kp(x) = 47575 +/- 2089, respectively.

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A theoretical model is proposed according to which the quasi-parabolic dependence of the biological activity of amphiphilic compounds on their hydrophobic substituent chain length (the "cut-off" effect) is caused by a combination of partition equilibria and geometrical parameters of these compounds interacting with the bilayer of phospholipid membranes. The most important parameter seems to be a change in the thickness of the phospholipid bilayer. The model is quantified for the case of normal alcohols.

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It is proposed a simple mathematical model explaining the quasi parabolic dependence of the local anesthetic activity on the length of hydrophobic substituent in the homologous series of tertiary amines (TA). It is suggested that the molecules of TA intercalate between the lipid molecules in bilayers. Due to the mismatch between the lengths of lipid and TA hydrocarbon chains the intercalation results in a decrease in the bilayer thickness. The quasi parabolic dependence is the result of combination of partition equilibria and of geometrical parameters of interacting molecules in the bilayer. The model predicts that the TA chain length at which the maximum activity is observed should be dependent on the lipid: aqueous phase volume ratio. The empirical parameters used in the model are obtained from the X-ray diffraction on multilamellar egg yolk phosphatidylcholine (EYPC) dispersions with the monohydrochloride of [2-(heptyloxy)phenyl]-2-(1-piperidinyl)ethyl ester of carbamic acid and from the partition equilibria of its alkyloxy homologs with unilamellar EYPC liposomes.

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The local anesthetic carbisocaine (1), the monohydrochloride of [2-(heptyloxy)phenyl]-2-(diethylamino)-1-methylethyl ester of carbamic acid, exerts a biphasic effect on the fluidity of egg yolk phosphatidylcholine model membranes as detected by the stearic acid spin probes with the paramagnetic doxyl group bound to C-5 or C-16. The fluidity initially increases with an increase in the concentration of 1, but at concentrations above 25 mmol/l a decrease of fluidity has been observed. 31P NMR spectroscopy indicates changes in the phosphocholine head-group conformation in the presence of 1. The decrease of fluidity is caused by the increased packing density in the bilayer hydrophobic region at high concentrations of 1, probably due to interdigitation of hydrocarbon chains from apposing monolayers in the hydrophobic center of the bilayer.

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The spin-lattice 13C-NMR relaxation time T1 of carbons in egg yolk phosphatidylcholine (EYPC) unilamellar liposomes was measured at 15 MHz, 25 MHz and 75 MHz in the presence of diamagnetic La3+ and paramagnetic Gd3+ ions. Supposing formation of ML2 complexes (where M is the metal ion and L the lipid) and using a simplified Solomon-Bloembergen-Morgan equation, a value of less than r-3IS greater than 2 = 0.1880 +/- 0.0005 nm-6 was obtained for C omega carbon of lipid chains, where rIS is the distance of Gd3+ unpaired electron and C omega nucleus, and angle brackets denote a weighted average. This value may serve as the basis for testing the application of statistical mechanics to lipid chain conformation and chain terminal group distribution in lipid bilayers.

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