Phase diagram of 1,2-dioleoylphosphatidylethanolamine (DOPE):water system at subzero temperatures and at low water contents.

The phase behavior of partially hydrated 1, 2-dioleoylphosphatidylethanolamine (DOPE) has been studied using differential scanning calorimetry and X-ray diffraction methods together with water sorption isotherms. DOPE liposomes were dehydrated in the H(II) phase at 29 degrees C and in the L(alpha) phase at 0 degrees C by vapor phase equilibration over saturated salt solutions. Other samples were prepared by hydration of dried DOPE by vapor phase equilibration at 29 degrees C and 0 degrees C. Five lipid phases (lamellar liquid crystalline, L(alpha); lamellar gel, L(beta); inverted hexagonal, H(II); inverted ribbon, P(delta); and lamellar crystalline, L(c)) and the ice phase were observed depending on the water content and temperature. The ice phase did not form in DOPE suspensions containing <9 wt% water. The L(c) phase was observed in samples with a water content of 2-6 wt% that were annealed at 0 degrees C for 2 or more days. The L(c) phase melted at 5-20 degrees C producing the H(II) phase. The P(delta) phase was observed at water contents of <0.5 wt%. The phase diagram, which includes five lipid phases and two water phases (ice and liquid water), has been constructed. The freeze-induced dehydration of DOPE has been described with the aid of the phase diagram.     

Citrate increases glass transition temperature of vitrified sucrose preparations

The aim of this study was to investigate the effect of sodium citrate on the properties of dried amorphous sucrose glasses. Addition of sodium citrate to a sucrose solution followed by freeze-drying or convective drying resulted in a glass transition temperature (Tg) that was higher than the well-studied sucrose Tg. This result was obtained either at reduced water content of the analysed sample or by removal of water during Modulated DSC analysis. After removal of the remaining water ( < 3.5% w/w), a Tg of approximately 105 degrees C was obtained at a mass ratio of sodium citrate to sucrose of 0.3. FTIR analysis showed a similar increase in Tg as was found with Modulated DSC analysis. The Tg values were derived from breaks in the vibrational frequency vs. temperature plots in the OH stretching and bending regions. Elevated average strength of hydrogen bonding in the sucrose/citrate glass was concluded from the downshift of the OH stretching band of 25 cm(-1) and from the reduced wavenumber temperature coefficient (WTC). The antisymmetric carboxylate stretch of citrate sensed the glass transition of the mixture, from which we conclude that citrate interacts with the sucrose OH via its carboxylate groups.     

The nonequilibrium phase and glass transition behavior of beta-lactoglobulin

Concentrated solutions of bovine beta-lactoglobulin were studied using osmotic stress and rheological techniques. At pH 6.0 and 8.0, the osmotic pressure was largely independent of NaCl concentration and could be described by a hard sphere equation of state. At pH 5.1, close to the isoelectric point, the osmotic pressure was lower at the lower NaCl concentrations (0 mM, 100 mM) and was fitted by an adhesive hard sphere model. Liquid-liquid phase separation was observed at pH 5.1 at ionic strengths of 13 mM and below. Comparison of the liquid-liquid and literature solid-liquid coexistence curves showed these solutions to be supersaturated and the phase separation to be nonequilibrium in nature. In steady shear, the zero shear viscosity of concentrated solutions at pH 5.1 was observed at shear rates above 50 s(-1). With increasing concentration, the solution viscosity showed a progressive increase, a behavior interpreted as the approach to a colloidlike glass transition at approximately 60% w/w. In oscillatory shear experiments, the storage modulus crossed the loss modulus at concentrations of 54% w/w, an indication of the approaching glass transition. Comparison of the viscous behavior with predictions from the Krieger-Dougherty equation indicates the hydrodynamic size of the protein decreases with increasing concentration, resulting in a slower approach to the glass transition than a hard sphere system.     

Staphylococcal growth and enterotoxins (A—D) and thermonuclease synthesis in the presence of dehydrated garlic

E. GONZÁLEZ-FANDOS, M.L. GARCÍA-LÓPEZ, M.L. SIERRA AND A. OTERO. 1994. The inhibition of Staphylococcus aureus growth and enterotoxin and thermonuclease production by various concentrations of garlic ( Allium sativum ) was studied in BHI broth. The growth of Staph. aureus was inhibited by dehydrated garlic at levels of 1.5% (w/v) and over. Enterotoxins A, B and C₁ were only detectable in broth containing < 1% of garlic while enterotoxin D was produced at a level of 2%. Garlic also inhibited thermonuclease (TNAse) production, complete inhibition being observed at levels ≥ 1.5%. TNAse was not always detected when enterotoxin was present.     

Volume recovery, surface properties and membrane integrity of Lactobacillus delbrueckii subsp. bulgaricus dehydrated in the presence of trehalose or sucrose

AIMS: Although the practical importance of adding sugars before drying is well known, the mechanism of protection of bacteria by sugars is not clear. The response of the dehydrated micro-organisms to rehydration is analysed in terms of structural and functional changes, and correlated with their potentiality to grow in rich media. These aspects are related with the membrane integrity and the metabolic state of the rehydrated bacteria, measured by means of surface properties and permeability. To attain this objective, Lactobacillus delbrueckii subsp. bulgaricus was dehydrated in the presence and in the absence of sucrose and trehalose. The bacterial response upon rehydration was investigated by determining: (i) the lag time of the bacterial growing in rich media, (ii) the restoration of the surface properties and the cellular volume and (iii) the membrane integrity. METHODS AND RESULTS: Lactobacillus delbrueckii subsp. bulgaricus was grown in MRS at 37 degrees C overnight [De Man et al. (1960)J Appl Bacteriol 23, 130] and then dehydrated for 10, 20 and 30 min at 70 degrees C in a vacuum centrifuge. The lag time of micro-organisms was determined by optical density changes after rehydration. The surface properties were determined by measuring the zeta potential of the bacteria suspended in aqueous solution. The cellular volume recovery was measured, after stabilization in saline solution, by light scattering and by the haematocrit method [Alemohammad and Knowles (1974)J Gen Microbiol 82, 125]. Finally, the membrane integrity has been determined by using specific fluorescent probes [SYTO 9 and propidium iodide, (PI)] that bind differentially depending on the integrity of the bacterial membrane. The lag time of Lact. delbrueckii subsp bulgaricus, dehydrated by heat in the presence of sucrose or trehalose and after that rehydrated, was significantly shortened, when compared with that obtained for bacteria dried in the absence of sugars. In these conditions, trehalose and sucrose maintained the zeta potential and the cell volume close to the control (nondried) cells. However, the membrane integrity, measured with fluorescent probes, was maintained only when cells were dehydrated for 10 min in the presence of sugars. For larger times of dehydration, the membrane integrity was not preserved, even in the presence of sugars. CONCLUSIONS: When the micro-organisms are dehydrated in the absence of protectants, the membrane damage occurs with a decrease in the absolute value of the zeta potential and a decrease in the cellular volume recovered after rehydration. In contrast, when the zeta potential and the cellular volume are restored after rehydration to that corresponding to nondried cells, the micro-organisms are able to recover and grow with a reduced lag time. This can only be achieved when the dehydration is carried out in the presence of sugars. At short dehydration times, the response is associated with the preservation of the membrane integrity. However, for longer times of dehydration the zeta potential and volume recovery occurs in the presence of sugars in spite of a severe damage at membrane level. In this condition, cells are also recovered. In conclusion, to predict the ability of growing after dehydration, other bacterial structural parameters besides membrane integrity, such as zeta potential and cellular volume, should be taken into account. SIGNIFICANCE AND IMPACT OF THE STUDY: The correlation of the lag time with the surface and permeability properties is of practical importance because the correlation of these two parameters with cell viability, allow to determine the potential bacterial capacity to grow in a rich medium after the preservation procedure, without necessity of performing a kinetic curve of growth, which is certainly time-consuming.     

The glass transition behavior of the globular protein bovine serum albumin

The glass-like transition behavior of concentrated aqueous solutions of bovine serum albumin was examined using rheological techniques. At mass fractions >0.4, there was a marked concentration dependence of viscosity with a glass-like kinetic arrest observed at mass fractions in the region of 0.55. At mass fractions >0.6 the material behaved as a solid with a Young's modulus rising from approximately 20 MPa at a mass fraction of 0.62-1.1 GPa at 0.86. The solid was viscoelastic and exhibited stress relaxation with relaxation times increasing from 33 to 610 s over the same concentration range. The concentration dependence of the osmotic pressure was measured, at intermediate concentrations, using an osmotic stress technique and could be described using a hard sphere model, indicating that the intermolecular interactions were predominantly repulsive. In summary, a major structural relaxation results from the collective motion of the globules at the supra-globule length scale and, at 20 degrees C, this is arrested at water contents of 40% w/w. This appears to be analogous to the glass transition in colloidal hard spheres.     

Ion activity decrease in the presence of sucrose

The conductivity of sucrose solutions containing different salts was measured. It was shown that conductivity, and hence ion activity, decreased with the increase of sucrose concentration of the solutions.     

Phase transition properties of 1,2- and 1,3-diacylphosphatidylethanolamines with modified head groups

The phase transition properties of dilute aqueous suspensions of "nonhydrated" (i.e., lipid suspensions which had not been heated above room temperature or above the main phase transition temperature of the fully hydrated lipid, whichever was lower) and hydrated 1,2(alpha)- and 1,3(beta)-dipalmitoylphosphatidylethanolamines with modified head groups have been determined by high-sensitivity differential scanning calorimetry at a scan rate of 0.1 K min-1. In both the 1,2 and 1,3 series, the head-group modifications of the phosphoethanolamine moiety included N-methyl, N,N-dimethyl, and N,N,N-trimethyl (phosphocholine). In the 1,2 series, additional modifications were dinitrophenyl, trinitrophenyl, N-(dinitrophenyl)aminocaproyl, N-(trinitrophenyl)aminocaproyl, and N-4-nitro-2,1,3-benzoxadiazole. Also included in this study were 1,2-dihexadecylphosphatidylethanolamine and the corresponding N-methyl-substituted lipid. In general, increasing bulkiness of the head-group substituent caused increasing lowering of the transition temperature, the most extreme cases among the hydrated lipids being the 45 degrees C lowering produced by the N-(dinitrophenyl)aminocaproyl substitution and its trinitrophenyl analogue in the 1,2 series. No simple trend is evident in the changes produced in the calorimetric enthalpy of transitions.     

Thermostability of alcohol dehydrogenase in the presence of sucrose

Summary The influence of sucrose on the thermostability of pure alcohol dehydrogenase is investigated for various temperatures (50–70°C) in the presence and absence of sucrose (0, 80 wt.%). The thermal inactivation clearly exhibits nonlinear biphasic behavior. The thermal inactivation rate constants and the magnitude of the heat-stable and heat-labile fractions of the enzyme are quantified.     

Phase transition behavior, protein adsorption, and cell adhesion resistance of poly(ethylene glycol) cross-linked microgel particles

Thermoresponsive poly(N-isopropylacrylamide) (pNIPAm) microgel particles cross-linked with various concentrations of PEG diacrylates of 3 different PEG chain lengths were synthesized via free-radical precipitation polymerization in order to investigate the phase transition and protein adsorption behavior as the hydrophilicity of the network is increased. Photon correlation spectroscopy (PCS) reveals that, as the concentration of PEG cross-linker incorporated into the particles is increased, an increase in the temperature and breadth of the phase transition occurs. Qualitative differences in particle density using isopycnic centrifugation confirm that higher PEG concentrations result in denser networks. The efficient incorporation of PEG cross-linker was confirmed with (1)H NMR, and variable temperature NMR studies suggest that, in the deswollen state, the longer PEG cross-links protrude from the dense globular network. This behavior apparently manifests itself as a decrease in nonspecific protein adsorption with increasing PEG length and content. Furthermore, when electrostatically attached to a glass surface, the particles containing the longer chain lengths exhibited enhanced nonfouling behavior and were resistant to cell adhesion in serum-containing media. The excellent performance of these particulate films and the simplicity with which they are assembled suggests that they may be applicable in a wide range of applications where nonfouling coatings are required.     

Physicochemical characteristics associated with transfection of cationic cholesterol-based gene delivery vectors in the presence of DOPE

The physicochemical properties of a novel series of cholesterol-based cationic lipids in the presence of DOPE were studied by various techniques in an effort to correlate cationic lipid structure with transfection efficacy. It was found that while DOPE improves the β-gal activity of the active AC and MC derivatives, the overall zeta potential of the particles, pDNA complexation and condensation is not improved. This is in stark contrast with the tertiary amine derivative DC whose dispersion properties were improved and its monolayer surface potential is restored at high molecular surface density in the presence of DOPE. Overall the transfection activity mediated by DC and the quaternary ammonium TC derivative was greatly improved in the presence of DOPE and is attributed to decreased cytotoxicity, improved fusogenicity and cellular association.     

The phase transition behavior of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) model membrane influenced by 2,4-dichlorophenol--an FT-Raman spectroscopy study

The effect of 2,4-dichlorophenol (DCP) on the structures and phase transitions of fully hydrated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes was studied using FT-Raman spectroscopy. Whereas the Raman frequency shifts of the most frequently investigated bands of C-C and C-H stretching regions only indicate the main phase transition (P(beta')-L(alpha)) of the pure DPPC/water system, the Raman shift of C-H scissoring vibration at 1440 cm(-1) was found to be able to reveal the pretransition (L(beta')-P(beta')) as well. Analyzing the spectral parameters of the trans band at 1128 cm(-1), which does not overlap with DCP vibrational modes, a continuous decrease of trans conformations was found with increasing DCP concentration at 26 degrees C accompanying the phase transitions L(beta')-P(beta') and P(beta')-L(alpha). The intensity ratio of the symmetrical and asymmetrical methylene stretching bands (at 2850 cm(-1) and 2880 cm(-1)), defined as the disorder parameter by Levin [Levin, I.W., 1985. Two types of hydrocarbon chain interdigitation in sphingomielin bilayers. Biochemistry 24, 6282-6286], indicated that in the interdigitated phase (L(I)) the order is markedly high and comparable with that of L(beta). Both the phase transition P(beta')-L(alpha) in the DCP/DPPC molar ratio range of 10/100-50/100 and the phase transition L(I)-L(alpha) led to a significant increase of disordered chains and the presence of DCP molecules induced a more disordered chain region than that observed in the L(alpha) phase of DPPC. Nevertheless, it was found that the L(alpha) phase with DCP contains approximately the same amount of trans conformers than that without DCP.     

Kauzmann's paradox and the glass transition

Kauzmann showed that the entropy of a liquid decreases rapidly on cooling towards the kinetic glass transition temperature and extrapolates to unreasonable values at lower temperature. The temperature where the extrapolated liquid entropy meets the crystal entropy is now called the Kauzmann temperature. Thermodynamics, with Planck's statement of the third law, shows that the entropy of a liquid cannot be less than the entropy of a glass with the same enthalpy. This is the thermodynamic condition violated by the Kauzmann extrapolation and it suggests a thermodynamic glass transition. Simulations show that, for the simple models studied and regardless of how the liquid entropy is extrapolated, the Kauzmann temperature cannot be reached because the entropy of glasses with the same enthalpy as the liquid is greater than that of the crystal.     

B-Z transition in poly(dG-dC).poly(dG-dC) in the presence of formaldehyde amino derivatives

It was shown by circular dichroism that the B-Z transition of poly(dG-dC).poly(dG-dC) in high NaCl concentrations occurred more rapidly in the presence of formaldehyde and Tris. The product of formaldehyde and glycine interaction induces changes in the poly(dG-dC).poly(dG-dC) CD spectral characteristics of a 'B-like' conformation. It is supposed that the B-Z transition occurs without large-scale hydrogen bond breakage.     

Viscometric and potentiometric study of high-methoxyl pectins in the presence of sucrose

The polyelectrolytic behaviour of high-methoxyl pectins (degree of esterification = 72·9) was studied in the presence of sucrose. Potentiometric measurements were carried out on the acidic form by titration with NaOH. Variations of the apparent dissociation constant pK_a as a function of the degree of dissociation α were studied in salt-free solutions on a range of sucrose contents (0–60% w/v). Experimental plots could be described by Lifson-Katchalsky's (LK) treatment up to a 20% sucrose content. The intrinsic apparent dissociation constant pK₀ value was 3·1 ± 0·1 in agreement with previous data on pectinic acid. Above the 20% sucrose content, the LK theory was not appropriate owing to aggregation phenomena of macromolecular chains. Viscometric measurement were performed on the sodium form (α = 1) and for three sucrose contents (0%, 40% and 60%). By carrying out isoionic dilutions, the effect of ionic strength (I_t) on the intrinsic viscosity [η] was studied. The same infinite limit value [η]_∞ was obtained for each sucrose content and linear variations of [η] as a function of $\text{I}{}_{\mathrm{<mtext>t</mtext>}}{}^{\mathrm{<mtext>?1</mtext><mtext>2</mtext>}}$ allowed an estimate of the relative stiffness of the macromolecular chain. This stiffness was slightly increased by sucrose addition.From these data, it could be concluded that a high sucrose content did not significantly change the macromolecular size (for α = 1), as shown by viscometry. However, when α decreased, potentiometric investigations with sucrose showed a higher value of pK_a and a down-curvature of pK_a(α) which could be interpreted by assuming aggregation. These occurred at about 40% sucrose and above which was a much lower concentration than the gelling conditions (about 60%). Therefore aggregation phenomena could be present for relatively low sucrose contents if α < 0·5. It was postulated that interchain associations could occur progressively (from 40 to 60%) and involve aggregate formation with a critical size for gelation.     

Hygienic behavior of the honey bee (Apis mellifera) is independent of sucrose responsiveness and foraging ontogeny

Hygienic behavior in honey bees is a behavioral mechanism of disease resistance. Bees bred for hygienic behavior exhibit an increased olfactory sensitivity to odors of diseased brood, which is most likely differentially enhanced in the hygienic line by the modulatory effects of octopamine (OA), a noradrenaline-like neuromodulator. Here, we addressed whether the hygienic behavioral state is linked to other behavioral activities known to be modulated by OA. We specifically asked if, during learning trials, bees from hygienic colonies discriminate better between odors of diseased and healthy brood because of differences in sucrose (reward) response thresholds. This determination had to be tested because sucrose response thresholds are susceptible to OA modulation and may have influenced the honey bee's association of the conditioned stimulus (odor) with the unconditioned stimulus (i.e., the sucrose reward). Because the onset of first foraging is also modulated by OA, we also examined whether bees from hygienic colonies differentially forage at an earlier age compared to bees from non-hygienic colonies. Our study revealed that 1-day- and 15- to 20-day-old bees from the hygienic line do not have lower sucrose response thresholds compared to bees from the non-hygienic lines. In addition, hygienic bees did not forage at an earlier age or forage preferentially for pollen as compared to non-hygienic bees. These results support the idea that OA does not function in honey bees simply to enhance the detection of all chemical cues non-selectively or control related behaviors regardless of their environmental milieu. Our results indicate that the behavioral profile of the hygienic bee is sculpted by multiple factors including genetic, neural, social and environmental systems.     

Determination of L(alpha)-H(II) phase transition temperature for 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine

The thermodynamic properties of fully-hydrated lipids provide important information about the stability of membranes and the energetic interactions of lipid bilayers with membrane proteins (Nagle and Scott, Physics Today, 2:39, 1978). The lamellar/inverse hexagonal (L(alpha)-H(II)) phase transition of 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) water mixtures is a first-order transition and, therefore, at constant pressure, must have a thermodynamically well-defined equilibrium transition temperature. The observed transition temperature is known to be dependent upon the rate at which the temperature is changed, which accounts for the many different values in the literature. X-ray diffraction was used to study the phase transition of fully-hydrated DOPE to determine the rate-independent transition temperature, T(LH). Samples were heated or cooled for a range of rates, 0.212 < r < 225 degrees C/hr, and the rate-dependent apparent phase transition temperatures, T(A)(r) were determined from the x-ray data. By use of a model-free extrapolation method, the transition temperature was found to be T(LH) = 3.33 +/- 0.16 degrees C. The hysteresis, /T(A)(r) - T(LH)/, was identical for heating and cooling rates, +/-r, and varied as /r/beta for beta approximately 1/4. This unexpected power-law relationship is consistent with a previous study (Tate et al., Biochemistry, 31:1081-1092, 1992) but differs markedly from the exponential behavior of activation barrier kinetics. The methods used in this study are general and provide a simple way to determine the true mesomorphic phase transition temperatures of other lipid and lyotropic systems.