The partition of cholesterol between ordered and fluid bilayers of phosphatidylcholine: A synchrotron X-ray diffraction study

The structure and composition of coexisting bilayer phases separated in binary mixtures of dipalmitoylphosphatidylcholine and cholesterol and ternary mixtures of equimolar proportions of dipalmitoyl- and dioleoylphosphatidycholines containing different proportions of cholesterol have been characterized by synchrotron X-ray diffraction methods. The liquid-ordered phase is distinguished from gel and fluid phases by a disordering of the hydrocarbon chains intermediate between the two phases as judged from the wide-angle X-ray scattering profiles. Electron density distribution calculated in coexisting bilayer phases shows that liquid-ordered phase is enriched in dipalmitoylphosphatidylcholine and cholesterol and a higher electron density in the methylene chain region of the bilayer ascribed to the location of the sterol ring of cholesterol. The ratio of the two constituents in the liquid-ordered phase is not constant because the stoichiometry is temperature-dependent as seen by respective changes in bilayer thickness over the range 20° to 36 °C where coexisting phases are observed. Three coexisting phases were deconvolved in the ternary mixture at 20 °C. From an analysis of the ternary mixtures containing mole fractions of cholesterol from 0.09 to 0.15 it was found that the liquid-crystal and gel phases each contained about 10% of the cholesterol molecules and the liquid-ordered phase was comprised of 30% cholesterol molecules.     

Effect of dehydroepiandrosterone on phosphatidylserine or phosphatidylcholine bilayers: DSC and X-ray diffraction study

The effect of dehydroepiandrosterone (DHEA) on the thermotropic and structural properties of phosphatidylserine or phosphatidylcholine membranes was investigated by differential scanning calorimetry and X-ray diffraction. At molar fractions of sterol, X (sterol), less than approximately 0.2, DHEA interacts with both types of model membranes, depressing the melting temperature and reducing the enthalpy of melting. At higher concentrations, phase separation of DHEA occurs with appearance of crystallites of the S2 monohydrate form.     

Influence of local anesthetics on the phosphatidylcholine model membrane: small-angle synchrotron X-ray diffraction and neutron scattering study

The phase preferences of egg yolk phosphatidylcholine (EYPC) have been examined in the presence of tertiary amine anesthetics [2-(propyloxy)phenyl]-2-(1-piperidinyl)ethyl ester of carbamic acid (C3A) and [2-(heptyloxy)phenyl]-2-(1-piperidinyl)ethyl ester of carbamic acid (C7A, heptacaine). Using the synchrotron small-angle X-ray diffraction (SAXD), it is shown that the C3A anesthetic induces the cubic and hexagonal (H(I)) phases at 2 > or = C3A:EYPC > 0.5 and H2O:EYPC < or = 40 molar ratios. In contrast, longer alkyloxy chain homolog C7A has no effect on the bilayer arrangement of EYPC at C7A:EYPC < = 1 molar ratios as observed by SAXD in C7A + EYPC mixtures hydrated at H2O:EYPC < = 40 molar ratios, as well as in sonicated C7A + EYPC mixtures hydrated in excess water as proved by the small-angle neutron scattering (SANS). The bilayer thickness d(L) decreases and the bilayer C7A surface area SC7A increases with the increase of C7A:EYPC molar ratio. It is suggested that the ability of tertiary amine local anesthetics to influence the dL and SC7A values and EYPC polymorphism is caused by their effective molecular shape and by charge. The possibility that anesthetic molecules may exert some of their biological effects by virtue of these properties is discussed.     

The structure of DNA-DLPC-cationic gemini surfactant aggregates: a small angle synchrotron X-ray diffraction study

The structure of aggregates formed by interaction of DNA with unilamellar dilauroylphosphatidylcholine (DLPC) vesicles (DNA:DLPC=1:1 base/mol) in the presence of gemini surfactant butane-1,4-diyl-bis(dodecyldimethylammonium bromide) (C12GS) was investigated using synchrotron small angle X-ray diffraction. In the concentration range C12GS+:DLPC< or =1 mol/mol, a condensed lamellar Lalphac phase was found with a repeat period of lipid bilayer stacking in the range d approximately 5.70-6.53 nm and the DNA interhelical distance d(DNA) approximately 3.52-3.99 nm, depending on the concentration of C12GS. At molar ratio C12GS+:DLPC> or =0.35:1, the diffractograms have shown the presence of a second lamellar phase with the repeat period d approximately 5.31 nm which slightly decreases with increasing concentration of C12GS+. The increasing fraction of this phase in the aggregates with increasing concentration of C12GS supports the association of this phase with microscopic domains enriched by surfactant molecules. The temperature behaviour of aggregates was investigated in the range 25-60 degrees C and the transversal thermal expansivities of the observed phases were determined.     

An X-ray diffraction and differential scanning calorimetric study on the effect of sucrose on the properties of phosphatidylcholine bilayers

The influence of sucrose, between 0 and 70% in the aqueous phase, upon multilamellar liposomes of dimyristoylphosphatidylcholine was examined by differential scanning calorimetry and X-ray diffraction analysis. Increasing concentrations of sucrose increase the temperatures of both the main transition and the pretransition of the lipid. The effect is greater on the pretransition than on the main transition. At 35 degrees C the interlamellar spacing in the multilamellar liposomes is reduced by increasing sucrose concentration in the aqueous phase and no significant effects are seen in the chain lattice of the bilayers. This result is interpreted as a dehydrating effect of sucrose upon the bilayer-water system at 35 degrees C. At 5 degrees C the interlamellar spacing is increased and this increase is, at high (70%) sucrose concentrations, attributable to an untilting of the lipid acyl chains with no change in the thickness of the aqueous layers in the multilamellae.     

The effect of cholesterol on the structure of phosphatidylcholine bilayers

The effect of cholesterol on the structure of phosphatidylcholine bilayers was investigated by X-ray diffraction methods. Electron density profiles at 5 Å resolution along with chain tilt and chain packing parameters were obtained and compared for phosphatidylcholine/cholesterol bilayers and for pure phosphatidylcholine bilayers in both the gel and liquid crystalline states. The cholesterol in the bilayer was localized by noting the position of discrete elevations in the electron density profiles. Cholesterol can either increase or decrease the width of the bilayer depending on the physical state and chain length of the lipid before the introduction of cholesterol. For saturated phosphatidylcholines containing 12–16 carbons per chain, cholesterol increases the width of the bilayer as it removes the chain tilt from gel state lipids or increases the trans conformations of the chains for liquid crystalline lipids. However, cholesterol reduces the width of 18 carbon chain bilayers below the phase transition temperature as the long phospholipid chains must deform or kink to accomodate the significantly shorter cholesterol molecule. Although cholesterol has a marked effect on hydrocarbon chain organization, it was found that, within the resolution limits of the data, the phosphatidylcholine head group conformation is unchanged by the addition of cholesterol to the bilayer. The head group is oriented parallel to the plane of the bilayer for phosphatidylcholine in the gel and liquid crystalline states and this orientation is not changed by the addition of cholesterol.     

The diversity of the liquid ordered (Lo) phase of phosphatidylcholine/cholesterol membranes: a variable temperature multinuclear solid-state NMR and x-ray diffraction study

To investigate the properties of a pure liquid ordered (Lo) phase in a model membrane system, a series of saturated phosphatidylcholines combined with cholesterol were examined by variable temperature multinuclear (1H, 2H, 13C, 31P) solid-state NMR spectroscopy and x-ray scattering. Compositions with cholesterol concentrations>or=40 mol %, well within the Lo phase region, are shown to exhibit changes in properties as a function of temperature and cholesterol content. The 2H-NMR data of both cholesterol and phospholipids were used to more accurately map the Lo phase boundary. It has been established that the gel-Lo phase coexistence extends to 60 mol % cholesterol and a modified phase diagram is presented. Combined 1H-, 2H-, 13C-NMR, and x-ray scattering data indicate that there are large changes within the Lo phase region, in particular, 1H-magic angle spinning NMR and wide-angle x-ray scattering were used to examine the in-plane intermolecular spacing, which approaches that of a fluid Lalpha phase at high temperature and high cholesterol concentrations. Although it is well known for cholesterol to broaden the gel-to-fluid transition temperature, we have observed, from the 13C magic angle spinning NMR data, that the glycerol region can still undergo a "melting", though this is broadened with increasing cholesterol content and changes with phospholipid chain length. Also from 2H-NMR order parameter data it was observed that the effect of temperature on chain length became smaller with increasing cholesterol content. Finally, from the cholesterol order parameter, it has been previously suggested that it is possible to determine the degree to which cholesterol associates with different phospholipids. However, we have found that by taking into account the relative temperature above the phase boundary this relationship may not be correct.     

Short-range interactions between lipid bilayers measured by X-ray diffraction

Interactions between lipid bilayers are critical in many biological processes in which membrane surfaces come close together. Recent X-ray diffraction analyses of bilayers subjected to known osmotic pressures have provided critical information on the magnitude of both the repulsive and the attractive forces that exist between phospholipid and glycolipid membranes.     

Interaction of long-chain n-alcohols with fluid DOPC bilayers: a neutron diffraction study

Lamellar phases composed of fluid dioleoylphosphatidylcholine (DOPC) bilayers containing alkan-1-ols (CnOH, n = 8, 10, 14, 16, 18 is the number of carbon atoms) at CnOH : DOPC = 0.3 molar ratio and hydrated with heavy water at 20.2 ≥ D2O : DOPC ≥ 14.4 molar ratio were studied by neutron diffraction. The bilayer thickness d(L) and the bilayer surface area A(L) per DOPC at the bilayer-water interface were obtained from the lamellar repeat period d using molecular volumes of DOPC, CnOH and D2O, and the Luzatti's method. Both the d(L) and A(L) increase with the CnOH chain length n at CnOH : DOPC = 0.3 molar ratio: d(L) = (3.888 ± 0.066) + (0.016 ± 0.005)·n (in nm), A(L) = (0.6711 ± 0.0107) + (0.0012 ± 0.0008)·n (in nm2).     

The influence of cholesterol on the interaction between N-dodecyl-N,N-dimethyl-N-benzylammonium halides and phosphatidylcholine bilayers

Effects of N-dodecyl-N,N-dimethyl-N-benzylammonium halides (DBeAX) on thermotropic phase behavior of phosphatidylcholine/cholesterol bilayers as well as on 1H NMR spectra were studied. The surfactants were added either to the water phase or directly to the lipid phase (a mixed film was formed). The benzyl group, opposite to liposomes without cholesterol, is not incorporated into the bilayer in the gel state but only in the liquid state. All the halides DBeAX (particularly the chloride DBeAC) showed greater ability to destabilize the membrane structure in the presence than in the absence of cholesterol. The interaction of DBeAX with DPPC/cholesterol bilayers and subsequent changes in the phospholipid bilayer organization depended on the kind of counterion. The strongest effects were observed for chloride (most electronegative ion) and for iodide (largest ion). The effects of chloride and bromide on phase transition and 1H NMR spectra in the presence and absence of cholesterol were opposite. This is discussed in terms of the influence of counterions on the pair cholesterol-DPPC interactions.     

Maximum solubility of cholesterol in phosphatidylcholine and phosphatidylethanolamine bilayers

In any lipid bilayer membrane, there is an upper limit on the cholesterol concentration that can be accommodated within the bilayer structure; excess cholesterol will precipitate as crystals of pure cholesterol monohydrate. This cholesterol solubility limit is a well-defined quantity. It is a first-order phase boundary in the phospholipid/cholesterol phase diagram. There are many different solubility limits in the literature, but no clear picture has emerged that can unify the disparate results. We have studied the effects that different sample preparation methods can have on the apparent experimental solubility limit. We find that artifactual demixing of cholesterol can occur during conventional sample preparation and that this demixed cholesterol may produce artifactual cholesterol crystals. Therefore, phospholipid/cholesterol suspensions which are prepared by conventional methods may manifest variable, falsely low cholesterol solubility limits. We have developed two novel preparative methods which are specifically designed to prevent demixing during sample preparation. For detection of the cholesterol crystals, X-ray diffraction has proven to be quantitative and highly sensitive. Experiments based on these methods yield reproducible and precise cholesterol solubility limits: 66 mol% for phosphatidylcholine (PC) bilayers and 51 mol% for phosphatidylethanolamine (PE) bilayers. We present evidence that these are true, equilibrium values. In contrast to the dramatic headgroup effect (PC vs. PE), acyl chain variations had no effect on the cholesterol solubility limit in four different PC/cholesterol mixtures.     

Characterization of the sub-transition of hydrated dipalmitoylphosphatidylcholine bilayers: X-ray diffraction study

The structural changes accompanying the recently described sub-transition of hydrated dipalmitoylphosphatidylcholine (Chen, S.C., Sturtevant, J.M. and Gaffney, B.J. (1980) Proc. Natl. Acad. Sci. USA 77, 5060–5063) have been defined using X-ray diffraction methods. Following prolonged storage at ?4°C the usual L_β′ gel form of hydrated dipalmitoylphosphatidylcholine (DPPC) is converted into a more ordered stable ‘crystal’ form. The bilayer periodicity is 59.1 Å and the most striking feature is the presence of a number of X-ray reflections in the wide angle region. The most prominent of these are a sharp reflection at $\text{1}\text{4.4}\text{A}\text{?}{}^{\mathrm{?1}}$ and a broader reflection at $\text{1}\text{3.9}\text{A}\text{?}{}^{\mathrm{?1}}$. This diffraction pattern is indicative of more ordered molecular and hydrocarbon chain packing modes in this low temperature ‘crystal’ bilayer form. At the sub-transition (T_rmsub = 15–20°C) an increase in the bilayer periodicity occurs ($\text{d=63.6}\text{A}\text{?}$) and a strong reflection at approx. $\text{1}\text{4.2}\text{A}\text{?}{}^{\mathrm{?1}}$ with a shoulder at approx. $\text{1}\text{4.1}\text{A}\text{?}{}^{\mathrm{?1}}$ is observed. This diffraction pattern is identical to that of the bilayer gel (L_β′) form of hydrated DPPC. Thus, the sub-transition corresponds to a bilayer ‘crystal’ → bilayer L_β′ gel structural rearrangement accompanied by a decrease in the lateral hydrocarbon chain interactions. Differential scanning calorimetry and X-ray diffraction show that on further heating the usual structural changes L_β′ → P_β′ and P_β′ → L_α occur at the pre- and main transitions, at approx. 35°C and 41°C, respectively.     

Lateral distribution of a pyrene-labeled phosphatidylcholine in phosphatidylcholine bilayers: fluorescence phase and modulation study

The lateral distribution of 1-palmitoyl-2-[10-(1-pyrenyl)decanoyl]phosphatidylcholine (PyrPC) was studied in small unilamellar vesicles of 1,2-dipalmitoyl-, 1,2-dimyristoyl-, and 1-palmitoyl-2-oleoyl-phosphatidylcholine (DPPC, DMPC, and POPC, respectively) under anaerobic conditions. The DPPC and DMPC experiments were carried out over temperature ranges above and below the matrix phospholipid phase transition temperature (Tm). The excimer to monomer fluorescence intensity ratio (E/M) was determined as a function of temperature for the three PyrPC/lipid mixtures. Phase and modulation data were used to determine the temperature dependence of pyrene fluorescence rate parameters in gel and in liquid-crystalline bilayers. These parameters were then used to provide information about excited-state fluorescence in phospholipid bilayers, calculate the concentration of the probe within liquid-crystalline and gel domains in the phase transition region of PyrPC in DPPC, and simulate E/M vs. temperature curves for three systems whose phase diagrams are different. From the simulated curves we could determine the relationship between the shape of the three simulated E/M vs. temperature curves and the lateral distribution of the probe. This information was then used to interpret the three experimentally derived E/M vs. temperature curves. Our results indicate that PyrPC is randomly distributed in pure gel and fluid phosphatidylcholine bilayers.(ABSTRACT TRUNCATED AT 250 WORDS)     

X-ray diffraction studies of lecithin bilayers.

Counting the individuals in a population before and after an annual period of environmental stress allows the proportion s of the initial population surviving the period to be computed. A series of such observations over n annual periods gives a sequence s₁, s₂, …, s_n. A statistical model is formulated from axioms describing the survival process, and it is concluded that these observed values may usefully be regarded as realizations of a random variable that arises from the normal generated distribution (n.g.d.). Equations for estimating the n.g.d. parameters ζ and τ² from observed survival proportions by the method of moments and maximum likelihood are given. The distributions of parameter estimates ζ and τ² are obtained and discussed in the context of testing hypotheses comparing survival among different populations. Finally, the dependence of the n.g.d. upon parameters ζ and τ² is examined in terms of altering survival, either by population self-regulation mechanisms or man-induced controls. The intent is to provide insight into the relationship between the n.g.d. and its supporting axioms and, more generally, basic knowledge of population processes.     

The structure of DNA-DOPC aggregates formed in presence of calcium and magnesium ions: a small-angle synchrotron X-ray diffraction study

The structure of aggregates formed due to DNA interaction with dioleoylphosphatidylcholine (DOPC) vesicles in presence of Ca(2+) and Mg(2+) cations was investigated using synchrotron small-angle X-ray diffraction. For DOPC/DNA=1:1 mol/base and in the range of concentration of the cation(2+) 0-76.5 mM, the diffractograms show the coexistence of two lamellar phases: L(x) phase with repeat distance d(Lx) approximately 8.26-7.39 nm identified as a phase where the DNA strands are intercalated in water layers between adjacent lipid bilayers, and L(DOPC) phase with repeat distance d(DOPC) approximately 6.45-5.65 nm identified as a phase of partially dehydrated DOPC bilayers without any divalent cations and DNA strands. The coexistence of these phases was investigated as a function of DOPC/DNA molar ratio, length of DNA fragments and temperature. If the amount of lipid increases, the fraction of partially dehydrated L(DOPC) phase is limited, depends on the portion of DNA in the sample and also on the length of DNA fragments. Thermal behaviour of DOPC+DNA+Ca(2+) aggregates was investigated in the range 20-80 degrees C. The transversal thermal expansivities of both phases were evaluated.     

The structure of lipid bilayers and the effects of general anaesthetics: An X-ray and neutron diffraction study

We have looked for the effects of three clinically used inhalational anaesthetics (nitrous oxide, halothane and cyclopropane) on the structure of lecithin/ cholesterol bilayers. The anaesthetics were delivered to the membranes in the gaseous phase, so that effects at clinical concentrations could be determined.High-resolution X-ray diffraction patterns were recorded out to 4 Å and analyzed using swelling experiments. Parallel neutron diffraction experiments were performed and analyzed using H₂O-²H₂O exchange. Methods were developed which enabled us to obtain confidence limits for the X-ray and neutron structure factors.The resultant X-ray and neutron scattering density profiles clearly define the positions of the principal molecular groups in the unperturbed bilayer. In particular, the high-resolution electron density profiles reveal features directly attributable to the cholesterol molecule. A comparison with the neutron scattering density profiles shows that cholesterol is anchored with its hydroxyl group at the water/hydrocarbon interface, aligned with the fatty acid ester groups of the lecithin molecule. We suggest that this positioning of the cholesterol molecule allows it to act as a thickness buffer for plasma membranes.In the presence of very high concentrations of general anaesthetics, the bilayers show increased disorder while maintaining constant membrane thickness. At surgical concentrations, however, there are no significant changes in bilayer structure at 95% confidence levels. We briefly review the literature previously used to support lipid bilayer hypotheses of general anaesthesia. We conclude that the lipid bilayer per se is not the primary site of action of general anaesthetics.     

Synergistic perturbation of phosphatidylcholine/sphingomyelin bilayers by diacylglycerol and cholesterol

An angiogenic factor, platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP), stimulates the chemotaxis of endothelial cells and confers resistance to apoptosis induced by hypoxia. 2-deoxy-D-ribose, a degradation product of thymidine generated by TP enzymatic activity partially prevented hypoxia-induced apoptosis. 2-Deoxy-D-ribose inhibits a number of components of the caspase-mediated hypoxia-induced apoptotic pathway. It inhibits hypoxia-induced caspase 3 activation, mitochondrial cytochrome c release, downregulation of Bcl-2 and Bcl-x(L), upregulation of hypoxia-inducible factor (HIF)-1 alpha, and loss of mitochondrial transmembrane potential in human leukemia HL-60 cell line. These findings suggest a molecular mechanism by which 2-deoxy-d-ribose confers the resistance to apoptosis. Thus 2-deoxy-D-ribose-modulated suppression of HIF-1 alpha expression could prevent the hypoxia-induced decrease of the anti-apoptotic Bcl-2 and Bcl-x(L) on the mitochondria. 2-Deoxy-L-ribose and its analogs may enhance apoptosis and suppress the growth of tumors by competitively inhibiting the activities of 2-deoxy-d-ribose and thus these analogs show promise for anti-tumor therapy.