WITHDRAWN: Modulation of the kinetics of 3ß-hydroxy-5-oxo-5,6-secocholestan-6-al/phosphatidylethanolamine Schiff base formation by cholesterol and cholesterol crystallization.

The Publisher regrets that this article is an accidental duplication of an article that has already been published, http://dx.doi.org/10.1016/j.chemphyslip.2015.01.001. The duplicate article has therefore been withdrawn. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.

Modulation of the kinetics of 3ß-hydroxy-5-oxo-5,6-secocholestan-6-al/phosphatidylethanolamine Schiff base formation by cholesterol and cholesterol crystallization.

We have previously shown that the oxidized cholesterol 3ß-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.

Room temperature ordering of dipalmitoyl phosphatidylserine bilayers induced by short chain alcohols.

Using differential scanning calorimetry and small and wide angle X-ray diffraction, we show that, following extended incubation at room temperature, methanol, propanol, and three of the isomers of butanol can induce ordering in dipalmitoyl phosphatidylserine (DPPS) gel phase bilayers. The organization of the bilayers in the presence of ethanol, described previously, is now observed to be a general effect of short chain alcohols. Evidence is presented for tilting of the acyl chains with respect to the bilayer normal in the presence of ethanol or propanol. However, the different chain lengths of the alcohols, and isomeric form, influence the thermal stability of the ordered gel to different extents. This behavior is unlike that of the gel state phosphatidylcholine analog which, in the presence of short chain alcohols, undergoes hydrocarbon chain interdigitation. Dipalmitoyl phosphatidylcholine added to DPPS in the presence of 20 vol% ethanol, acts to suppress the ordered gel phase.

The oxysterol 3ß-hydroxy-5-oxo-5,6-secocholestan-6-al changes the phase behavior and structure of phosphatidylethanolamine-phosphatidylcholine mixtures.

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 oxidized form of cholesterol 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al induces structural and thermotropic changes in phospholipid membranes.

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.

Kinetics of Schiff base formation between the cholesterol ozonolysis product 3-beta-hydroxy-5-oxo-5,6-secocholestan-6-al and phosphatidylethanolamine.

The kinetics of Schiff base formation between the cholesterol ozonolysis product 3beta-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.

Interaction of dipalmitoyl phosphatidylserine with ethanol: induction of an ordered gel phase at room temperature.

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.

Effect of gold adsorption on the conductive properties of cyclo-octasulfur microcrystals.

The formation of gold crystallites on the surface of S8 promotes diffusion of electrons and determines the conductive properties of the shell-core nanosystems. Conducting probe atomic microscopy and four-probe resistance measurements confirmed that Au/S8 shell-core systems exhibit electrical conductivity on the micro- as well as on the nanoscale in contrast to non-covered S8 crystals, which are insulating. The conductivity of Au/S8 systems on the microscale was measured to be 10+/-1 S cm(-1). In XPS measurements, a single peak at 163.6 eV was observed for bulk S8 whereas an additional peak corresponding to a binding energy of 161.4 eV appeared for S8 adsorbed on a Au substrate. This is interpreted to mean that a chemical reaction has taken place. A process which results in adsorption of uniform gold nanolayers on needle shaped or fibrous S8 crystallites is under investigation.

On the origin of the lattice constant anomaly in nanocrystalline ceria.

The lattice parameter of nanocrystalline ceria films prepared by sputtering was monitored as a function of annealing temperature. Within the temperature range of 150-420 degrees C, an equilibrium with atmospheric oxygen is established within a few hours, whereas grain growth does not occur. On the basis of the experimental results and analysis of literature data, we present a model that posits the formation of a non-uniform grain structure with stoichiometric interiors and oxygen deficient boundaries. This model, based on defect thermodynamics, correctly describes the dependence of the lattice parameter of nanocrystalline ceria on annealing temperature and grain size and can be extended to other materials as well.

Thermal denaturation of Bungarus fasciatus acetylcholinesterase: Is aggregation a driving force in protein unfolding?

A monomeric form of acetylcholinesterase from the venom of Bungarus fasciatus is converted to a partially unfolded molten globule species by thermal inactivation, and subsequently aggregates rapidly. To separate the kinetics of unfolding from those of aggregation, single molecules of the monomeric enzyme were encapsulated in reverse micelles of Brij 30 in 2,2,4-trimethylpentane, or in large unilamellar vesicles of egg lecithin/cholesterol at various protein/micelle (vesicle) ratios. The first-order rate constant for thermal inactivation at 45 degrees C, of single molecules entrapped within the reverse micelles (0.031 min(-1)), was higher than in aqueous solution (0.007 min(-1)) or in the presence of normal micelles (0.020 min(-1)). This clearly shows that aggregation does not provide the driving force for thermal inactivation of BfAChE. Within the large unilamellar vesicles, at average protein/vesicle ratios of 1:1 and 10:1, the first-order rate constants for thermal inactivation of the encapsulated monomeric acetylcholinesterase, at 53 degrees C, were 0.317 and 0.342 min(-1), respectively. A crosslinking technique, utilizing the photosensitive probe, hypericin, showed that thermal denaturation produces a distribution of species ranging from dimers through to large aggregates. Consequently, at a protein/vesicle ratio of 10:1, aggregation can occur upon thermal denaturation. Thus, these experiments also demonstrate that aggregation does not drive the thermal unfolding of Bungarus fasciatus acetylcholinesterase. Our experimental approach also permitted monitoring of recovery of enzymic activity after thermal denaturation in the absence of a competing aggregation process. Whereas no detectable recovery of enzymic activity could be observed in aqueous solution, up to 23% activity could be obtained for enzyme sequestered in the reverse micelles.

Phase separation of cholesterol and the interaction of ethanol with phosphatidylserine-cholesterol bilayer membranes.

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).

A new high-temperature transition of crystalline cholesterol in mixtures with phosphatidylserine.

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.

Regulated phase transitions of bacterial chromatin: a non-enzymatic pathway for generic DNA protection.

The enhanced stress resistance exhibited by starved bacteria represents a central facet of virulence, since nutrient depletion is regularly encountered by pathogens in their natural in vivo and ex vivo environments. Here we explore the notion that the regular stress responses, which are mediated by enzymatically catalyzed chemical transactions and promote endurance during the logarithmic growth phase, can no longer be effectively induced during starvation. We show that survival of bacteria in nutrient-depleted habitats is promoted by a novel strategy: finely tuned and fully reversible intracellular phase transitions. These non-enzymatic transactions, detected and studied in bacteria as well as in defined in vitro systems, result in DNA sequestration and generic protection within tightly packed and highly ordered assemblies. Since this physical mode of defense is uniquely independent of enzymatic activity or de novo protein synthesis, and consequently does not require energy consumption, it promotes virulence by enabling long-term bacterial endurance and enhancing antibiotic resistance in adverse habitats.

Water Solubilization in Nonionic Microemulsions Stabilized by Grafted Siliconic Emulsifiers.

Microemulsions containing octanol, decanol, or dodecanol as the oil phase and oligomeric, grafted nonionic amphiphiles based on ethoxylated polymethylsiloxanes (Silwets) have been studied. It was demonstrated that significant amounts of water can be solubilized only when the hydrophobic siliconic backbone is very short (trimers). The water solubilization was evaluated using SAXS, DSC, and conductivity measurements. It was found that up to 40 wt% of water can be solubilized in dodecanol and Silwet L-7607 (MW 1000 and 75 wt% ethylene oxide (EO)). Surprisingly, no free water was detected in the aggregate core. All the solubilized water was confined in the vicinity of the interphasal region and froze at -10 degrees C and below. Up to three molecules of water can be associated with each EO headgroup. Based on SAXS measurements, the structural units of the microemulsions were interpreted to be lamellar-like, a form previously found for the related monomeric microemulsions. Copyright 2001 Academic Press.

The language of becoming: helping children change how they think about themselves.

In this article, I describe the language of becoming, which is a way of speaking to children that enables them to see themselves as continually evolving and changing. By noticing and reflecting back the specific ways the child is becoming, the parent gives a meta-message that one's personality is not set in stone but, rather, evolves and changes over time. In order for parents to use the language of becoming, the child must actually act differently. I will describe a three-pronged approach to helping children try new behaviors. The language of becoming helps parents and children see the child's personality, not just his or her behavior, as fluid rather then static. This helps parents and children break the vicious circles and self-fulfilling prophesies of rigid definitions of self. An understanding of stages of cognitive development in the construction of beliefs about the self and the implications of these stages for using the language of becoming is also discussed.

The inter-rater reliability of estimating the size of burns from various burn area chart drawings.

The accuracy and variability of burn size calculations using four Lund and Browder charts currently in clinical use and two Rule of Nine's diagrams were evaluated. The study showed that variability in estimation increased with burn size initially, plateaued in large burns and then decreased slightly in extensive burns. The Rule of Nine's technique often overestimates the burn size and is more variable, but can be performed somewhat faster than the Lund and Browder method. More burn experience leads to less variability in burn area chart drawing estimates. Irregularly shaped burns and burns on the trunk and thighs had greater variability than less irregularly shaped burns or burns on more defined anatomical parts of the body.

Cholesterol crystalline polymorphism and the solubility of cholesterol in phosphatidylserine.

There is a marked hysteresis between the heating and cooling polymorphic phase transition of anhydrous cholesterol. At a scan rate of 0.05 degrees C/min the difference in transition temperatures between heating and cooling scans is approximately 10 degrees C. This phenomenon also occurs with mixtures of cholesterol with phosphatidylserine and can result in an underestimation of the amount of crystalline cholesterol in a sample that has not been cooled sufficiently. With 1-palmitoyl-2-oleoyl phosphatidylserine and 1-stearoyl-2-oleoyl phosphatidylserine the cholesterol crystallites form while the lipid remains in the L(alpha) phase. Sonication of dimyristoyl phosphatidylserine with a 0.4 mol fraction cholesterol results in the loss of cholesterol crystallite diffraction, but only a partial loss of the polymorphic transition detected by calorimetry. We therefore conclude that the thermal history of the sample can have profound effects on the appearance of the polymorphic phase transition of cholesterol by differential scanning calorimetry. Depending on the morphology of the vesicles, diffraction methods may underevaluate the amount of cholesterol crystallites present.

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.

Mechanical behavior of human trabecular bone after overloading.

With the etiology of osteoporotic fractures as motivation, the goal of this study was to characterize the mechanical behavior of human trabecular bone after overloading. Specifically, we quantified the reductions in modulus and strength and the development of residual deformations and determined the dependence of these parameters on the applied strain and apparent density. Forty cylindrical specimens of human L1 vertebral trabecular bone were destructively loaded in compression at 0.5% strain per second to strains of up to 3.0% and then immediately unloaded to zero stress and reloaded. (An ancillary experiment on more readily available bovine bone had been performed previously to develop this testing protocol.) In general, the reloading stress-strain curve had a short initial nonlinear region with a tangent modulus similar to Young's modulus. This was followed by an approximately linear region spanning to 0.7% strain, with a reduced residual modulus. The reloading curve always approached the extrapolated envelope of the original loading curve. Percent modulus reduction (between Young's and residual), a quantitative measure of mechanical damage, ranged from 5.2 to 91.0% across the specimens. It increased with increasing plastic strain (r2 = 0.97) but was not related to modulus or apparent density. Percent strength reduction, in the range of 3.6-63.8%, increased with increasing plastic strain (r2 = 0.61) and decreasing apparent density (r2 = 0.23). The residual strains of up to 1.05% depended strongly on applied strain (r2 = 0.96). Statistical comparisons with previous data for bovine tibial bone lend substantial generality to these trends and provide an envelope of expected behavior for other sites. In addition to providing a basis for biomechanical analysis of the effects of damage in trabecular bone at the organ level, these findings support the concept that occasional overloads may increase the risk of fracture by substantially degrading the mechanical properties of the underlying trabecular bone.

Application of the Tsai-Wu quadratic multiaxial failure criterion to bovine trabecular bone.

As a first step toward development of a multiaxial failure criterion for human trabecular bone, the Tsai-Wu quadratic failure criterion was modified as a function of apparent density and applied to bovine tibial trabecular bone. Previous data from uniaxial compressive, tensile, and torsion tests (n = 139 total) were combined with those from new triaxial tests (n = 17) to calibrate and then verify the criterion. Combinations of axial compression and radial pressure were used to produce the triaxial compressive stress states. All tests were performed with minimal end artifacts in the principal material coordinate system of the trabecular network. Results indicated that the stress interaction term F12 exhibited a strong nonlinear dependence on apparent density (r2 > 0.99), ranging from -0.126 MPa-2 at low densities (0.29 g/cm3) to 0.005 MPa-2 at high densities (0.63 g/cm3). After calibration and when used to predict behavior of new-specimens without any curve-fitting, the Tsai-Wu criterion had a mean (+/- SD) error of -32.6 +/- 10.6 percent. Except for the highest density triaxial specimens, most (15/17 specimens) failed at axial stresses close to their predicted uniaxial values, and some reinforcement for transverse loading was observed. We conclude that the Tsai-Wu quadratic criterion, as formulated here, is at best only a reasonable predictor of the multiaxial failure behavior of trabecular bone, and further work is required before it can be confidently applied to human bone.