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Background Clostridioides difficile is a major cause of infectious antibiotic resistant diarrhea. C. difficile spores are shed in patient stool, are hearty and difficult to kill. Bedpans are often used by patients with C. difficile infections and require proper handling and cleaning or disposal to prevent the transmission of C. difficile spores and other infectious microorganisms into the environment. Disposable bedpans are often used for convenience, which has consequences from an environmental sustainability perspective. Aim This study evaluates the ability for a washer-disinfector device (WD) to efficaciously clean and disinfect C. difficile spores and Escherichia coli from bedpans for sanitary reuse. Methods A commercially available WD device was evaluated for both efficacy and thermal disinfection against C. difficile spores and Escherichia coli using one disinfection cycle per test. Bedpans were not rinsed or dumped prior to placement in the WD. Bedpans were sampled using swabs. Microorganisms were eluted from the swabs and log-kill was calculated. Findings The average log-kill for C. difficile spores was 3.99 and >7.69 for E. coli. Thermal disinfection results showed an average log kill of 4.31 for C. difficile and >7.23 for E. coli. Conclusions The WD was efficacious against both C. difficile spores and E. coli when used according to manufacturer's instructions for use, suggesting a viable alternative to disposable bedpan waste management.

Cholesterol has limited solubility in phospholipid bilayers. The solubility limit is strongly dependent on the nature of the lipid with which the cholesterol is mixed while properties of the crystals formed can be modified by phospholipid-cholesterol interactions. In this review we summarize the various methods that have been developed to prepare hydrated mixtures of cholesterol and phospholipid. We point out some of the factors that determine the form adopted when cholesterol crystallizes in such mixtures, i.e. two- or three-dimensional, monohydrate or anhydrous. These differences can greatly affect the ability to experimentally detect the presence of these crystals in a membrane. Several methods for detecting cholesterol crystals are discussed and compared including DSC, X-ray and GIXRD diffraction methods, NMR and EPR spectroscopy. The importance of the history of the sample in determining the amount and nature of the cholesterol crystals formed is emphasized.

An oxidized form of cholesterol, atheronal, is a form found in vivo that has been associated with human pathologies. We have studied mixtures of this oxidized sterol with the phospholipids phosphatidylethanolamine and phosphatidylcholine. We used phospholipids either with palmitoyl and oleoyl acyl chains on the C1 and C2 carbon atoms of glycerol or with both acyl chains being palmitoleoyl. We also compared the effects of atheronal on the curvature properties of these lipids with the action of cholesterol. We studied the bilayer to hexagonal phase transition temperature of mixtures of these lipids using differential scanning calorimetry as well as the dimensions of the hexagonal phase cylinders using X-ray diffraction. Disordering of the lamellar phase was also qualitatively assessed by the loss of sharp diffraction peaks. Our results demonstrate that the modulation of membrane curvature in these systems depends not only on the nature of the sterol, but also on the acyl chain composition of the phospholipids used. In addition, some of the effects of atheronal could be ascribed to reaction of the aldehyde and ketone groups of this oxidized sterol with the amino group of phosphatidylethanolamine.

The effect of an oxidized form of cholesterol, 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al on the thermotropic and structural properties of phospholipid membranes was investigated by differential scanning calorimetry and X-ray diffraction and compared with that of cholesterol. The phospholipids studied included 1-palmitoyl-2-oleoylphosphatidylserine, dipalmitoleoylphosphatidylethanolamine, 1-palmitoyl-2-oleoylphosphatidylethanolamine, dipalmitoleoylphosphatidylcholine, 1-palmitoyl-2-oleoylphosphatidylcholine. Depending on the constituent phospholipids, the oxidized cholesterol is observed to shift phase transitions, disrupt stacking, modify interbilayer spacings and promote increased negative membrane curvature. We determined by absorption spectroscopy that the amino group of phosphatidylserine forms a Schiff base with the aldehyde group of the 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al as was previously found for the amino group of phosphatidylethanolamine. This result strengthens the biologically significant finding that not only amino groups of proteins but also amino groups of phospholipids are able to form a Schiff base with oxidized cholesterol. The marked triangular shape of the Schiff base complex with phosphatidylethanolamine may explain how 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al can promote increased negative curvature in the hexagonal phase, as compared to cholesterol, even though its increased polarity would favor a location closer to the interface with water.

The kinetics of Schiff base formation between the cholesterol ozonolysis product 3β-hydroxy-5-oxo-5,6-secocholestan-6-al and dimyristoyl phosphatidylethanolamine was investigated. The activation energy of Schiff base formation at temperatures above and below the phase transition of the phospholipid was calculated. Increase in the activation energy derived from perturbation of the surface structure by the process of Schiff base formation was demonstrated. The presence of the Schiff base with the aldolization product of the oxysterol was also observed and its significance is discussed.

7-Ketocholesterol is one of the major forms of oxidized cholesterol found in vivo. Several toxic effects of this sterol have been documented, and it is suggested to have a role in atherosclerosis. We have studied how this oxysterol modifies the physical properties of bilayers composed of the major lipid components of the cytoplasmic leaflet of the plasma membrane. 7-Ketocholesterol is much less effective in promoting the formation of the H ii phase in phosphatidylethanolamines than cholesterol. This is likely due to the fact that 7-ketocholesterol is more polar than cholesterol and hence would be located closer to the membrane interface. However, in ternary mixtures of dipalmitoleoylphosphatidylethanolamine with low concentrations of both sterols, the effect of 7-ketocholesterol on lowering T H is enhanced. Both cholesterol and 7-ketocholesterol are very soluble in bilayers of phosphatidylethanolamine, particularly with 1-palmitoyl-2-oleoylphosphatidylethanolamine. There is, however, a much greater solubility of 7-ketocholesterol in bilayers of 1-stearoyl-2-oleoylphosphatidylserine than is the case for cholesterol. In ternary mixtures of 1-stearoyl-2-oleoylphosphatidylserine with both sterols, it appears that the solubility of cholesterol is enhanced by the presence of 7-ketocholesterol. It is thus to be expected that several of the biophysical properties of a membrane would change as a result of the oxidation of cholesterol to 7-ketocholesterol.

Using differential scanning calorimetry and small and wide-angle X-ray diffraction, we show that, unlike the saturated phosphatidylcholines, for which ethanol induces chain interdigitation in the gel state, and unlike natural phosphatidylserine in which the gel state is almost unaffected by the addition of ethanol, dipalmitoyl phosphatidylserine (DPPS) assumes an ordered structure after incubation at room temperature in the presence of as little as 5% (v/v) ethanol. In the liquid crystalline state, a progressive decrease in the interbilayer spacing is observed as a function of ethanol concentration, similar to what is found for natural phosphatidylserine (PS) and 1-palmitoyl-2-oleoyl-phosphatidylserine (POPS). The 0.37 molar fraction of cholesterol in the DPPS dispersion in the presence of 10% (v/v) ethanol, does not prevent the formation of the ordered gel.

As authors of the “Comment on ‘Cholesterol solubility limit in lipid membranes probed by small angle neutron scattering and MD simulations’”, we wish to comment on both the form and content of the Reply cited above.

There is evidence that some products of the reaction of ozone with cholesterol contribute to atherosclerosis. One of these compounds is 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al. We have synthesized this compound and have demonstrated that it reacts with phosphatidylethanolamine to form a Schiff base. The 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al also affects the physical properties of phosphatidylethanolamines. We show by both DSC and X-ray diffraction that it increases the negative curvature of the membrane. In addition, 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al causes the lamellar phase to become disorganized, resulting in the loss of lamellar periodicity. The chemical and physical interactions of 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al with phosphatidylethanolamines may contribute to damaging effects of this lipid on cell membranes, resulting in pathology.

In the present work, we demonstrate that phosphatidylcholine with (16:1)9 acyl chains undergoes polymorphic rearrangements in mixtures with 0.6-0.8 mol fraction cholesterol. Studies were performed using differential scanning calorimetry, X-ray diffraction, cryo-electron microscopy, 31P NMR static powder patterns and 13C MAS/NMR. Mixtures of phosphatidylcholine with (16:1)9 acyl chains and 0.6 mol fraction cholesterol, after being heated to 100 degrees C, can form an ordered array with periodicity 14 nm which may be indicative of a cubic phase. Our results indicate that the formation of highly curved bilayer structures, such as those required for membrane fusion, can occur in mixtures of cholesterol with certain phosphatidylcholines that do not form non-lamellar structures in the absence of cholesterol. We also determine the polymorphic behavior of mixtures of symmetric phosphatidylcholines with cholesterol. Species of phosphatidylcholine with (20:1)11, (22:1)13 or (24:1)15 acyl chains in mixtures with 0.6-0.8 mol fraction cholesterol undergo a transition to the hexagonal phase at temperatures 70-80 degrees C. This is not the case for phosphatidylcholine with (18:1)6 acyl chains which remains in the lamellar phase up to 100 degrees C when mixed with as much as 0.8 mol fraction cholesterol. Thus, the polymorphic behavior of mixtures of phosphatidylcholine and cholesterol is not uncommon and is dependent on the intrinsic curvature of the phospholipid. Crystals of cholesterol can be detected in mixtures of all of these phosphatidylcholines at sufficiently high cholesterol mole fraction. What is unusual about the formation of these crystals in several cases is that cholesterol crystals are present in the monohydrate form in preference to the anhydrous form. Furthermore, after heating to 100 degrees C and recooling, the cholesterol crystals are again observed to be in the monohydrate form, although pure cholesterol crystals require many hours to rehydrate after being heated to 100 degrees C. Both the nature of the acyl chain as well as the mole fraction cholesterol determine whether cholesterol crystals in mixtures with the phospholipids will be in the monohydrate or in the anhydrous form.

We have studied the properties of mixtures of cholesterol with dioleoylphosphatidylcholine (DOPC), and with several other phospholipids, including 1-stearoyl-2-oleoylphosphatidylcholine (SOPC) and dioleoleoylphosphatidylserine (DOPS), as a function of cholesterol molar fraction and of temperature. Mixtures of DOPC with a cholesterol molar fraction of 0.4 or greater display polymorphic behavior. This polymorphism includes the formation of structures that give rise to isotropic peaks in 31P NMR at cholesterol molar fractions between 0.4 and 0.6, dependent on the thermal history of the sample. Cryo-electron microscopy studies demonstrate the formation of small globular aggregates that would contribute to a narrowing of the 31P NMR powder pattern. At molar fraction cholesterol 0.6 and higher and at temperatures above 70 degrees C, the mixtures with DOPC convert to the hexagonal phase. Lipid polymorphism is accompanied by the phase separation of cholesterol crystals in the anhydrous form and/or the monohydrate form. The crystals that are formed have substantially altered kinetics of hydration and dehydration, compared with both pure cholesterol monohydrate crystals and with crystals formed in the presence of the other phospholipids that do not form the hexagonal phase in the presence of cholesterol. This fact demonstrates that these cholesterol crystals are in intimate contact with the DOPC phospholipid and are not present as morphologically separate structures.

Experimental data that define conditions under which cholesterol crystallites form in cholesterol/phospholipid model membranes are reviewed. Structural features of the phospholipids that determine cholesterol crystallization include the length and degree of unsaturation of the acyl chains, the presence of charge on the headgroups and interheadgroup hydrogen bonds.

The stability and the ionic conductivity of biological membranes and of lipid bilayers depend on their hydration. A small number of water molecules adhere strongly to the different residues of the lipid headgroups and are oriented by them. An additional number of water molecules adhere more weakly, preserving their freedom of rotation, but are essential for bestowing the thermodynamic properties of hydrated bilayers and of biological membranes. Around six water molecules are attached so strongly to the headgroups of different phospholipids (PL) that they are rendered unfreezable, or their freezing is extended over such a wide range of temperatures that it cannot be detected by differential scanning calorimetry (DSC). If cholesterol is added to the PL above the concentration at which phase separation of the cholesterol phase occurs, the number of unfreezable water molecules per PL increases, indicating that the PL molecules on the border line between the two phases attach nearly twice as many water molecules as those in the middle of the phase. The orientation of about seven or eight water molecules attached to PL headgroups (seven to phosphatidyl serine (PS)) can be detected by polarized FTIR. The dichroic ratio of the successively adhering water molecules to the headgroup of PS fluctuates between 2.6 and 2.9, with the cumulative value of about 2.8 for the seven water molecules adhering to the headgroup of PS. In addition, in this case, the number of water molecules oriented by PL molecule residues on the border line of the two phases is much larger (f13 for PS). Interaction between two opposite negatively charged layers containing PS approaching each other may lead, after correlated electrostatic attraction, to change in the conformation of the headgroups with concomitant dehydration. This process is enhanced by Ca + and by Li + , but it may also occur with Na + and K + as counter-ions if the layers are mutually aligned. This process may be important in the fusion mechanism of biological membranes, and its molecular modeling has been carried out. D 2002 Published by Elsevier Science B.V.

The behavior of cholesterol is different in mixtures with phosphatidylcholine as compared with phosphatidylserine. In (13)C cross polarization/magic angle spinning nuclear magnetic resonance spectra, resonance peaks of the vinylic carbons of cholesterol are a doublet in samples containing 0.3 or 0.5 mol fraction cholesterol with 1-palmitoyl-2-oleoyl phosphatidylserine (POPS) or in cholesterol monohydrate crystals, but a singlet with mixtures of cholesterol and 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC). At these molar fractions of cholesterol with POPS, resonances of the C-18 of cholesterol appear at the same chemical shifts as in pure cholesterol monohydrate crystals. These resonances do not appear in samples of POPS with 0.2 mol fraction cholesterol or with POPC up to 0.5 mol fraction cholesterol. In addition, there is another resonance from the cholesterol C18 that appears in all of the mixtures of phospholipid and cholesterol but not in pure cholesterol monohydrate crystals. Using direct polarization, the fraction of cholesterol present as crystallites in POPS with 0.5 mol fraction cholesterol is found to be 80%, whereas with the same mol fraction of cholesterol and POPC none of the cholesterol is crystalline. After many hours of incubation, cholesterol monohydrate crystals in POPS undergo a change that results in an increase in the intensity of certain resonances of cholesterol monohydrate in (13)C cross polarization/magic angle spinning nuclear magnetic resonance, indicating a rigidification of the C and D rings of cholesterol but not other regions of the molecule.

Thermotropic and structural effects of ethanol on phosphatidylserine (PS) membranes containing up to 0.4 mol fraction cholesterol were investigated by differential scanning calorimetry, X-ray diffraction and fluorescence spectroscopy. It was found that in the presence of cholesterol, 10% (v/v) added ethanol depresses the melting temperature of the phospholipid by approximately 2 degrees C, similar to what was observed in the absence of cholesterol. Below the melting temperature the progressive disordering effect of added cholesterol is weakly enhanced by the presence of ethanol. In the liquid crystalline state, the marked decrease in the thickness of the bilayer which ethanol causes in the absence of cholesterol (Chem. Phys. Lipids 92 (1998) 127), is also observed in its presence. We conclude that, in contrast to what has been observed for zwitterionic phospholipids, high concentrations of cholesterol do not diminish the interaction of ethanol with PS membranes. With addition of 10% (v/v) ethanol, crystalline cholesterol diffraction, an indication of phase separation of the sterol, appears at mol fraction cholesterol 0.34, as compared to 0.3 in the absence of ethanol (Chem. Phys. Lipids 92 (1998) 71).

We have previously shown that the oxidized cholesterol 3β-hydroxy-5-oxo-5,6-secocholestan-6-al (atheronal A) reacts covalently with the free amino group of phosphatidylethanolamine (PE) or phosphatidylserine (PS) to produce a Schiff base. Accompanying this interaction, the biophysical properties of the phospholipid membranes are also changed. In the present report, we extend our earlier study of the rate of Schiff base formation in dimyristoyl PE/atheronal A binary mixtures to the more biologically relevant case in which varying amounts of cholesterol are also present. Using optical spectroscopy to monitor reaction kinetics, we demonstrate that the presence of cholesterol reduces the accessibility of the aldehyde moiety of the atheronal A to the free headgroup amine. We also find that the presence of atheronal A promotes the early onset of cholesterol crystallization in the ternary mixtures, perhaps with the Schiff base serving as a site for heterogeneous nucleation.

Phosphatidylserine and cholesterol are two major components of the cytoplasmic leaflet of the plasma membrane. The arrangement of cholesterol is markedly affected by the presence of phosphatidylserine in model membranes. At relatively low mol fractions of cholesterol in phosphatidylserine, compared with other phospholipids, cholesterol crystallites are formed that exhibit both thermotropic phase transitions as well as diffraction of x-rays. In the present study we have observed and characterized a novel thermotropic transition occurring in mixtures of phosphatidylserine and cholesterol. This new transition is observed at 96 degrees C by differential scanning calorimetry (DSC), using a heating scan rate of 2 degrees C/min. Observation of the transition requires that the hydrated lipid mixture be incubated for several days, depending on the temperature of incubation. The rate of formation of the material exhibiting a transition at 96 degrees C is more rapid at higher incubation temperatures. At 37 degrees C the half-time of conversion is approximately 7 days. Concomitant with the appearance of the 96 degrees C peak the previously known transitions of cholesterol, occurring at approximately 38 degrees C and 75 degrees C on heating scans of freshly prepared suspensions, disappear. These two transitions correspond to the polymorphic transition of anhydrous cholesterol and to the dehydration of cholesterol monohydrate, respectively. The loss of the 75 degrees C peak takes a longer time than that of the 38 degrees C peak, indicating that anhydrous cholesterol first gets hydrated to the monohydrate form exhibiting a transition at 75 degrees C and subsequently is converted by additional time of incubation to an altered form of the monohydrate, showing a phase transition at 96 degrees C. After several weeks of incubation at 37 degrees C, only the form with a phase transition at 96 degrees C remains. If such a sample undergoes several successive heating and cooling cycles, the 96 degrees C peak disappears and the 38 degrees C transition reappears on heating. For samples of 1-palmitoyl-2-oleoyl phosphatidylserine or of 1-stearoyl-2-oleoyl phosphatidylserine having mol fractions of cholesterol between 0.4 and 0.7, the 38 degrees C transition that reappears after the melting of the 96 degrees C component generally has the same enthalpy as do freshly prepared samples. This demonstrates that, at least for these samples, the amount of anhydrous cholesterol crystallites formed is indeed a property of the lipid mixture. We have also examined variations in the method of preparation of the sample and find similar behavior in all cases, although there are quantitative differences. The 96 degrees C transition is partially reversible on cooling and reheating. This transition is also scan rate dependent, indicating that it is, at least in part, kinetically determined. The enthalpy of the 96 degrees C transition, after incubation of the sample for 3 weeks at 37 degrees C is dependent on the ratio of cholesterol to 1-palmitoyl-2-oleoyl phosphatidylserine or to 1-stearoyl-2-oleoyl phosphatidylserine, with the enthalpy per mole cholesterol increasing between cholesterol mol fractions of 0.2 and 0.5. Dimyristoyl phosphatidylserine at a 1:1 molar ratio with cholesterol, after incubation at 37 degrees C, exhibits a transition at 95 degrees C that reverses on cooling at 44 degrees C, instead of 60 degrees C, as observed with either 1-palmitoyl-2-oleoyl phosphatidylserine or 1-stearoyl-2-oleoyl phosphatidylserine. These findings along with the essential absence of the 96 degrees C transition in pure cholesterol or in cholesterol/phosphatidylcholine mixtures, indicates that the phospholipid affects the characteristics of the transition, and therefore the cholesterol crystallites must be in direct contact with the phospholipid and are not simply in the form of pure crystals of cholesterol. These observations are particularly important in view of recent observations of the presence of cholesterol crystals in biological systems.

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.

Miscibility of cholesterol in dimyristoyl phosphatidylserine (DMPS) bilayers was investigated by differential scanning calorimetry and X-ray scattering. The onset of cholesterol phase separation as detected by X-ray diffraction is at the molar ratio DMPS:cholesterol of 2:1 and 1.7:1 in the gel and in the liquid crystalline states of the phospholipid, respectively.

Differential scanning calorimetry (DSC) was used for determining the number of unfreezable water molecules per molecule of phosphatidylserine from bovine spinal cord (PS) or dimyristoyl phosphatidylserine (DMPS) and dimyristoyl phosphatidylcholine (DMPC), alone or in mixtures with cholesterol. It was assumed that the unfreezable water molecules are tightly bound to the phospholipid. It was found that when the phospholipids are in the gel state and in the absence of cholesterol, PS binds 2.5 water molecules, DMPS 3.8 water molecules and DMPC 3.5 water molecules. In the presence of cholesterol the number of water molecules bound increases in the region where phase separation of cholesterol takes place [D. Bach, Chem. Phys. Lipids 35 (1984) 385–392; E.J. Wachtel, N. Borochov, D. Bach, Biochim. Biophys. Acta 1066 (1991) 63–69; D. Bach, N. Borochov, E. Wacktel, Chem. Phys. Lipids, submitted].

Miscibility of cholesterol and cholesteryl hemisuccinate was investigated by differential scanning calorimetry and small-angle X-ray scattering. In an excess of cholesterol, above 2:1 mol ratio, phase separation takes place into a mixed phase and an almost pure cholesterol phase.

The interaction of cholesterol with a series of saturated phosphatidylglycerols was investigated using differential scanning calorimetry and X-ray diffraction. We find that the miscibility of cholesterol in phosphatidylglycerol bilayers is lower than in the corresponding phosphatidylcholine bilayers and decreases with increasing acyl chain length of the phospholipid. The influence of the negative charge of the phosphatidylglycerol on cholesterol miscibility is discussed.

The thermotropic behavior of multilamellar vesicles composed of mixtures of dimyristoyl phosphatidylcholine-glucosyl ceramide and of egg phosphatidylcholine-glucosyl ceramide was investigated using differential scanning calorimetry. Macroscopic demixing of the lipid components occurred when multilamellar vesicles were prepared from mixtures of glucosyl ceramide and egg phosphatidylcholine by conventional methods. This problem was overcome by a technique based on spray drying of the lipid mixture. The results obtained for the two systems are compared with data available for dipalmitoyl phosphatidylcholine-glucosyl ceramide mixtures ( Biochemistry 22 (1983) 3497–3501). All three phosphatidylcholines perturb the complex thermotropic behavior of glucosyl ceramide. The data suggest that the interference with intermolecular interactions among glycosyl ceramide molecules by phospholipid molecules is related to the molecular miscibility of the two components. This is strongly dependent on the acyl chain composition of the phosphatidylcholine and the water activity of the ambient aqueous phase.

The oxidation of cholesterol with ozone produces 3β-hydroxy-5-oxo-5,6-secocholestan-6-al. This oxysterol has been implicated in a number of pathological conditions in vivo including atherosclerotic plaque formation and amyloidogenesis. We have shown previously that this oxysterol strongly modifies the physical properties of model membranes composed of different phosphatidylethanolamines or phosphatidylserine. In the present work we have extended our studies to ternary mixtures composed of phosphatidylethanolamine and phosphatidylcholine with sterols, either 3β-hydroxy-5-oxo-5,6- secocholestan-6-al or cholesterol. We use differential scanning calorimetry and small angle X-ray diffraction to characterize the phase behavior of mixtures of dipalmitoleoylphosphatidylethanolamine (diPoPE) and dipalmitoleoylphosphatidylcholine (diPoPC) or 1-palmitoyl-2-oleoyl-phosphatidylethanoloamine (POPE) and 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC). We compare the effect of the two sterols on the temperature of the transition of the ternary system from the liquid crystalline to the hexagonal phase (TH) and on the curvature of the resulting cylindrical micelles. Addition of low concentrations of diPoPC increases TH while adding cholesterol to this mixture significantly lowers TH. The effect of 3β-hydroxy-5-oxo-5,6-secocholestan-6-al is much weaker than that of cholesterol. With regard to the curvature of the cylindrical micelles, the addition of diPoPC and 3β-hydroxy-5-oxo-5,6-secocholestan-6-al have opposing effects, while cholesterol does not effect the curvature at all. Low concentrations of POPC in POPE cause an increase in TH and decrease the curvature of the cylindrical micelles, while cholesterol has the opposite effect.

The phase behavior of mixtures of cholesterol with 1-palmitoyl-2-oleoyl phosphatidylserine or with 1-stearoyl-2-oleoyl phosphatidylserine has been examined using differential scanning calorimetry and X-ray diffraction. We found that the miscibility of cholesterol in the bilayer is strongly affected by the small difference in length between the fully saturated fatty acid chains of the two phospholipids. Cholesterol crystallites are detected in the 1-palmitoyl-2-oleoyl-phosphatidylserine/cholesterol mixtures at a mol fraction of cholesterol of approximately 0.36, whereas for the mixtures with 1-stearoyl-2-oleoyl-phosphatidylserine they appear at a cholesterol mol fraction of approximately 0.2. A phase diagram, based on differential scanning calorimetry experiments without cryoprotectant, is presented for the 1-stearoyl-2-oleoyl phosphatidylserine/cholesterol system. This phase diagram indicates the marked immiscibility of cholesterol with either the gel or the liquid crystalline phase of this phospholipid.

The keto-aldehyde 3β-hydroxy-5-oxo-5,6- secocholestan-6-al is formed by the oxidation of cholesterol with ozone. This oxidized form of cholesterol is associated with a number of pathological conditions including atherosclerotic plaques, Alzheimer's and Parkinson's diseases. We have shown earlier that the compound can react covalently with the amino group of phosphatidylethanolamine to form a Schiff base . Here, using a spectroscopic technique, we determine the kinetics of the Schiff base formation between 3β-hydroxy-5-oxo-5,6- secocholestan-6-al and dimyristoylphosphatidylethanolamine in both the gel and liquid crystalline states of the phospholipid. The activation energies of this reaction in the two states are also calculated. In addition, we determine that a Schiff base can also be formed with the amino group of phosphatidylserine, albeit with slower kinetics. These findings are significant as they show that oxidized cholesterol can react covalently not only with the amino groups of proteins, but also with the amino groups of phospholipids, potentially influencing the structure of biological membranes.

Mixtures of cholesterol with dimyristoyl phosphatidylserine or deuterated dimyristoyl phosphatidylserine were investigated by polarized and non polarized attenuated total reflection (ATR) Fourier transform infrared (FTIR) Spectroscopy. From polarized spectra the dichroic ratios of various vibrations as a function of cholesterol were calculated. Dichroic ratios of methylene vibration (CH(2)) 2934 cm(-1) of cholesterol decreases with increase of cholesterol concentration leveling off in the region where cholesterol phase separation takes place. The orientation of deuterated methylene (CD(2)) symmetric and asymmetric bands of the deuterated dimyristoyl phosphatidylserine is influenced little by cholesterol. In the polar region of dimyristoyl phosphatidylserine no effect of cholesterol on the dichroic ratios of carbonyl (C==O) and asymmetric phosphate (PO(2)(-)) vibrations were detected. For nonpolarized spectra the broad bands in the polar region of the phospholipid were deconvoluted. The carbonyl band (C==O) in pure dimyristoyl phosphatidylserine is composed of five bands; in the presence of increasing concentrations of cholesterol conformational change of these vibrations takes place evolving into one predominant band. Similar conformational change takes place in the presence of 75 molecules water/molecule DMPS. For the asymmetric phosphate band very small shifts due to interaction with cholesterol were detected.

Thermotrophic and structural effects of ethanol on phosphatidylserine (PS) membranes were investigated by differential scanning calorimetry (DSC) and X-ray diffraction. It was found that up to 15% (v/v) added ethanol, there is little change in the melting temperature of the phospholipid and no change in the interbilayer (d) spacing in the gel phase, indicating that there is no interdigitation of the hydrocarbon chains. Above the melting temperature of the phospholipid, a large decrease of the d spacing, due primarily to a decrease in the thickness of the bilayer, was found. Ethanol molecules located in the headgroup region apparently expand the area available to the headgroups with concomitant coiling of the acyl chains, resulting in marked thinning of the lipid layer.

MinK (Isk) is a voltage-dependent K+ channel whose gene has been recently cloned and which consists of 130 amino acids [Takumi, T., Ohkubo, H., & Nakanishi, S. (1988) Science 242, 1042-1045]. The protein contains one putative transmembrane segment by hydropathy analysis. Whether this putative transmembrane segment is involved in the function of the protein was studied. A 32 amino acid peptide (residues 41-72) with the sequence SKLEALYILMVLGFFGFFTLGIMLSYIRSKKL, containing the hypothesized transmembrane domain, designed TM-minK, was synthesized and fluorescently labeled. The alpha-helical content of TM-minK, assessed in methanol using circular dichroism (CD), was 57%. The fluorescent emission spectrum of 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-labeled TM-minK displayed a blue shift upon binding to small unilamellar vesicles (SUV), reflecting a relocation of the fluorescent probe to an environment of increased apolarity, i.e., within the lipid bilayer. The increase in NBD's fluorescence upon mixing NBD-labeled TM-minK with small unilamellar vesicles (SUV) was used to generate a binding isotherm, from which was derived a surface partition coefficient of 5.5 x 10(4) M-1. Fluorescence energy transfer measurements between carboxyfluoresceine-labeled and rhodamine-labeled analogues suggest that TM-minK aggregates within membranes. In addition, single-channel experiments revealed that TM-minK can form single channels in planar lipid membranes only when a trans negative potential is applied. The findings herein experimentally support a role of the transmembrane segment of minK both in the assembly and as a constituent of the pore formed by the protein.

The influence of protons or calcium ions on the miscibility of cholesterol in phosphatidylserine has been examined using differential scanning calorimetry and X-ray diffraction. At pH 2.6, where the carboxyl group of the serine moiety is protonated, two endothermic transitions are observed in cholesterol-phosphatidylserine mixtures. The midpoint of the first is at 35 degrees C in the absence of cholesterol and decreases to approx. 15 degrees C for molar fraction of cholesterol 0.5. The second transition is centered at approx. 44 degrees C, almost independent of cholesterol content. The two lower temperature phases are lamellar and the high temperature phase has hexagonal symmetry. Cholesterol is more miscible in protonated phosphatidylserine than in the sodium form: cholesterol crystals are detected at a molar ratio of phosphatidylserine to cholesterol of about 1.7:1 as compared to about 2.3:1 at neutral pH. In the presence of calcium ions (1.3 Ca2+ per phosphatidylserine), a lamellar phase is observed with layer spacing 53 A which is independent of temperature (25 degrees C-65 degrees C) and of cholesterol content. Calcium ions cause reduced cholesterol solubility: crystallites are detected already at a molar ratio of 4:1.

In our world, influenced by microelectronics and science fiction, if we have to imagine the next generation of diagnostic biosensors, it is clear that removal of blood from the body will not be required to monitor the health of an individual. Rather, sensing devices will be inserted into the body, where, after detecting the desired chemical, they will relay by continuous telemetry, the levels of the chemical species being monitored.