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PubMed1974

Physical and physiological evidence for two phase transitions in cytoplasmic membranes of animal cells.

Electron spin resonance analysis of suspensions of animal cell plasma membranes consistently reveals four characteristic temperatures for lateral phase separations in the membrane lipids. Similar analysis of an aqueous dispersion of lipids extracted from these membranes reveals only two characteristic temperatures, indicating that some aspect of lipid organization in membranes is destroyed by the extraction procedure. The characteristic temperatures for surface membranes from two different species of homeothermic animals were nearly identical and were approximately 37 degrees , 31 degrees , 21 degrees , and 15 degrees . A treatment of the physical data revealed that these temperatures could identify independent phase transitions for two hydrocarbon compartments of approximately equal size with lower and upper characteristic temperatures of 21 degrees and 37 degrees , and of 15 degrees and 31 degrees . The analysis of the effects of temperature on a number of physiological parameters indicates that 21 degrees and 37 degrees are likely to define the boundaries for lateral phase separations in the inner monolayer and 15 degrees and 31 degrees the boundaries for lateral phase separations in the outer monolayer.

Adenosine Triphosphatases↗
PubMed2006

Random anisotropy nematic model: nematic-non-nematic mixture.

The influence of a random-anisotropy- (RA-) type disorder on the phase separation of the nematogen-non-nematogen mixture is studied. A combination of the phenomenological Landau-de Gennes and Flory-Huggins theories is used. We assume that the non-nematogen component (i.e., impurity) enforces the RA disorder to the enclosing thermotropic liquid-crystal (LC) phase. The Imry-Ma argument is used according to which the lower-temperature phase exhibits a domain-type pattern. The disorder strength is measured in terms of the dimensionless parameter Lambda. We consider the case in which the LC molecules and impurities mix in the isotropic phase for Lambda=0. The impurities enforce a finite degree of orientational ordering even in the high-temperature paranematic phase. In the low-temperature phase they give rise to a domain-type structure, resulting in the distorted nematic (speronematic) phase. We show that the onset of orientational ordering increases the phase separation tendency. The RA field, however, opposes this tendency. With increasing value of Lambda the difference between the paranematic and speronematic ordering decreases. Consequently the structure of the phase-separated pattern can be much more complex in comparison to the Lambda=0 case.

Journal Article↗
PubMed1974

Correlation between thermal death and membrane fluidity in Bacillus stearothermophilus.

Paramagnetic resonance spectra of spin labels partitioned into spheroplast membranes of Bacillus stearothermophilus indicate lateral lipid phase separations. Cells adjust their lipid composition in response to temperature changes so that the same change of state in membrane phospholipids is achieved at the respective growth temperature. A temperature-sensitive mutant that fails to change its lipid composition above a certain temperature can survive only up to the higher temperature boundary for lateral phase separation. These data are interpreted to indicate that the maximal and minimal growth temperatures of thermophiles are regulated by the onset and conclusion of phase separations of the particular lipid composition they synthesize. It is suggested that isolated lipid domains are required for functional membrane assembly.

Bacillus↗
PubMed2000

Hydrodynamic-flow-driven wetting in thin film polymer blends: growth kinetics and morphology

A thin film of deuterated poly(methyl methacrylate) (A) and poly(styrene-ran-acrylonitrile) at the critical composition is annealed in the two phase region to induce simultaneous phase separation and wetting of the A-rich phase at the surface. Using forward recoil spectrometry, the wetting layer thickness is found to grow linearly with time at 185 degrees C and 190 degrees C. After selective etching of A, atomic force microscopy reveals a depletion layer having a bicontinuous, phase separated morphology. The A-rich tubes in this layer provide a pathway for rapid transport of the wetting phase from the bulk to the surface via hydrodynamic flow. Taken together, fast wetting layer growth t(1) and connectivity between the wetting layer and bulk provide unambiguous support for hydrodynamic-flow-driven wetting in thin film polymer blends.

Journal Article↗
PubMed1984

Wide-pore silica-based ether-bonded phases for separation of proteins by high-performance hydrophobic-interaction and size-exclusion chromatography.

This paper examines the use of wide-pore silica-based hydrophilic ether-bonded phases for the chromatographic separation of proteins under mild elution conditions. In particular, ether phases of the following structure identical to Si-(CH2)3-O-(CH2-CH2-O)n-R, where n = 1, 2, 3 and R = methyl, ethyl or n-butyl, have been prepared. These phases can be employed either in high-performance hydrophobic-interaction or size-exclusion chromatography, depending on mobile phase conditions. In the hydrophobic-interaction mode, a gradient of decreasing salt concentration, e.g., from 3 M ammonium sulfate (pH 6.0, 25 degrees C), yields sharp peaks with high mass recovery of active proteins. In this mode, retention can be controlled by salt type and concentration, as well as by column temperature. In the size-exclusion mode, use of medium ionic strength, e.g., 0.5 M ammonium acetate (pH 6.0) yields linear calibration of log (MW[eta]) vs. retention volume. Even at 0.05 M salt concentration, no stationary phase charge effects on protein elution are observed. These bonded-phase columns exhibit good column-to-column reproducibility and constant retention for at least five months of continual use. Examples of the high-performance separation of proteins in both modes are illustrated.

Chromatography, Gel↗
PubMed1992

Cholesterol modulation of lipid intermixing in phospholipid and glycosphingolipid mixtures. Evaluation using fluorescent lipid probes and brominated lipid quenchers.

Carbazole- and indole-labeled phospholipids have been used to monitor the homo- or heterogeneity of lipid mixing in several types of lipid bilayers combining a brominated and a nonbrominated lipid with varying amounts of cholesterol. Experimental quenching curves (relating the normalized probe fluorescence intensity to the mole fraction of brominated lipid) show a characteristic smooth, monophasic form for homogeneous liquid-crystalline lipid mixtures. However, for mixtures exhibiting lipid lateral segregation, such curves show marked perturbations in form over the region of composition where segregation occurs. Using this approach, it is found that high mole fractions of cholesterol (40-50 mol %) promote the formation of apparently homogeneous solutions in mixtures of disaturated and monounsaturated phosphatidylcholines (PCs) that exhibit extensive thermotropic phase separations in the absence of sterol. At only slightly lower levels of cholesterol, however, these systems exhibit inhomogeneous lipid mixing over a wide range of relative proportions of the two PC components. Mixtures of cerebroside and monounsaturated PCs, even at high bilayer cholesterol contents, exhibit significant inhomogeneity in lipid mixing over a wide range of cerebroside/PC ratios. Phase-separating PC/PC and PC/cerebroside mixtures can readily form long-lived metastable solutions when the level of the higher-melting component in the liquid-crystalline phase exceeds its equilibrium solubility by as much as 20-30 mol %; this tendency is significantly increased by cholesterol. Cholesterol shows no significant ability to enhance lipid intermixing in a third type of phase-separating lipid system, combining a monounsaturated PC with a monounsaturated phosphatidic acid--calcium complex. Experiments using cleavable phospholipid conjugates, linking a fluorescent lipid to a brominated lipid, suggest that each fluorescent molecule probes a local lipid domain comprising approximately less than 40-50 nearby acyl chains.

Animals↗
PubMedDate not supplied

Recent developments in electrospray mass spectrometry including implementation on an ion trap.

Mass spectrometry (MS) may be the ultimate detection technique when combined with modern condensed phase separation sciences. The technique combines sensitivity with excellent specificity, so the pharmaceutical analyst can obtain definitive information regarding components separated in a mixture. Thus, mass spectrometric detection not only provides evidence of a chromatographic peak, but it also provides important information including molecular weight and structural information enabling identification of the components. The coupling of an atmospheric pressure ionization (API) mass spectrometer to most of the separation science techniques offers a simpler alternative from earlier non-routine, less sensitive systems where the vacuum systems struggled to handle the liquid effluent from these systems. Contemporary sensitive and analytically rugged API systems can be operated unattended for extended periods of time thus reducing the cost per sample to a reasonable value especially given the wealth of information provided. Although the mass spectrometer is more complicated than conventional spectroscopic detectors, present day API systems effectively decouple the liquid-phase separation inlet from the high-vacuum system where mass analysis occurs. The ability to form gas-phase ions at atmospheric pressure and sample primarily the analyte ions into the mass spectrometer promises a bright future for combining on-line condensed phase separation science techniques with mass spectrometry. The increasing ease of performing these experiments offers new analytical opportunities for pharmaceutical laboratories.

Carbohydrate Sequence↗
PubMed1994

Chilling Injury Induces Lipid Phase Changes in Membranes of Tomato Fruit.

Wide-angle x-ray diffraction has provided evidence for lipid phase separations in microsomal membranes from chill-injured tomato (Lycopersicon esculentum Mill. cv Caruso) fruit. Mature-green fruit stored for 20 d at 5[deg]C had not begun to ripen and were essentially free of chilling injury symptoms. Within 4 d of being returned to 25[deg]C, however, the fruit displayed characteristic symptoms of chilling injury, including translucent water-soaked patches, surface pitting, and irregular pigmentation. Membrane damage measured as electrolyte leakage from pericarp discs intensified after the fruit were returned to ambient temperature. Wide-angle x-ray diffraction patterns recorded at 25[deg]C for microsomal membranes isolated from untreated, mature-green fruit indicated that the membrane bilayers were exclusively liquid-crystalline. Diffraction patterns for microsomal membranes from fruit stored for 20 d at 5[deg]C showed only trace amounts of gel phase lipid, but within 4 d of subsequent exposure of the fruit to ambient temperature, there was evidence for a pronounced lateral phase separation of lipids within the membranes that would render them leaky. Inas-much as the phase separations were detectable at 25[deg]C and became pronounced only subsequent to the chilling episode, they appear to be an indirect rather than direct effect of exposure to low temperature. The diffraction data thus support the notion that the lipid phase changes observed here are not directly induced by low temperature but rather reflect subsequent biochemical changes in the bilayers that may contribute to the development of chilling symptoms.

Journal Article↗
PubMed1991

Comparative study of chromatography on thin layers impregnated with organic stationary phases. Chromatographic separation of nitrophenols.

The chromatographic behaviour of nitrophenols on thin layers of silica gel and cellulose was compared, both without impregnation and impregnated with non-aqueous polar stationary phases (formamide, dimethylformamide) and less polar stationary phases (liquid paraffin, octan-1-ol, 1-bromonaphthalene). Cellulose is preferred when using formamide or dimethylformamide if a pure partition process is required. For each particular analyte a certain amount of the stationary phase is always required to suppress the adsorption activity of silica gel. Separation by reversed-phase thin-layer chromatography is strongly affected by the type of stationary phase (the possibility of forming charge-transfer complexes with 1-bromonaphthalene) and its support (the acidic properties of silica gel and its adsorption activity), the mobile phase (content of organic modifier, pH, presence of salts) and the properties of the solutes (polarity, ionizability).

Cellulose↗
PubMed2004

Demixing in simple fluids induced by electric field gradients.

Phase separation in liquid mixtures is mainly controlled by temperature and pressure, but can also be influenced by gravitational, magnetic or electric fields. However, the weak coupling between such fields and concentration fluctuations limits this effect to extreme conditions. For example, mixing induced by uniform electric fields is detectable only at temperatures that are within a few hundredths of degree or less of the phase transition temperature of the system being studied. Here we predict and demonstrate that electric fields can control the phase separation behaviour of mixtures of simple liquids under more practical conditions, provided that the fields are non-uniform. By applying a voltage of 100 V across unevenly spaced electrodes about 50 micro m apart, we can reversibly induce the demixing of paraffin and silicone oil at 1 K above the phase transition temperature of the mixture; when the field gradients are turned off, the mixture becomes homogeneous again. This direct control over phase separation behaviour depends on field intensity, with the electrode geometry determining the length-scale of the effect. We expect that this phenomenon will find a number of nanotechnological applications, particularly as it benefits from field gradients near small conducting objects.

Journal Article↗
PubMed1999

Purification and immunocytochemical localization of neuraminidase from Tritrichomonas foetus.

Lysis of Tritrichomonas foetus with a solution of the non-ionic detergent Triton X-114 at 0 degree C, followed by low-speed centrifugation, resulted in a detergent-insoluble pellet and a detergent-soluble supernatant. The supernatant was further fractionated by phase separation at 30 degrees C into a detergent-rich phase and an aqueous phase. Neuraminidase activity was mostly located in the detergent-insoluble pellet. When the parasites were incubated with bacterial phosphatidylinositol phospholipase C (PI-PLC) prior to detergent solubilization and phase separation neuraminidase activity was predominantly recovered in aqueous phase, rather than in the pellet and detergent phase. The molecular mass determined by gel permeation in high performance liquid chromatography (HPLC) and SDS-PAGE was 80,000 Da. Indirect immunofluorescence microscopy using polyclonal antibodies raised in rabbits against the purified neuraminidase, indicated that the enzyme is exposed on the cell surface. Previous treatment of the cells with PI-PLC significantly reduced antibody binding. Incubation of cryo-sections with the antibodies followed by detection using gold-labelled anti-rabbit IgG confirmed the presence of neuraminidase in the plasma membrane enclosing the cell body and flagella and in the membrane of vesicles preferentially located at the peripheral region of the protozoan.

Animals↗
PubMed2006

Loose complexation of weakly charged microemulsion droplets and a polyelectrolyte.

The effect of polyelectrolyte addition on the properties of an oil-in-water (O/W) microemulsion of weakly charged spherical micelles is studied. The 81 A radius O/W droplets in this system can be charged by the partial substitution of the nonionic surfactant by a cationic surfactant. The effect of the addition of poly(acrylic acid) (PAA), which is a charged pH-dependent polyelectrolyte, on the interactions between charged or noncharged droplets has been investigated using SANS. We have characterized the phase behavior of this pH-smart system as a function of the microemulsion and the polyelectrolyte concentration and the number of charges per droplet at three pH values: pH = 2, 4.5, and 12. In particular, an associative phase separation due to the bridging of the droplets by the neutral PAA chains through H-bonds is observed with extremely low PAA addition at low pH. At the opposite, an addition of PAA at pH = 4.5 generates a strong repulsive contribution between neutral droplets. Electrostatic bonds between charged droplets and PAA, controlled by the number of charges per droplet, are responsible for a pH drift and then for an associative phase separation similar to that observed at low pH. Finally, at high pH, the creation of electrostatic bonds between fully charged PAA and charged droplets liberates sufficiently counterions in solution at high droplet charge density to screen the electrostatic interactions and to allow an associative phase separation.

Journal Article↗
PubMedDate not supplied

Effect of infusing fat emulsion into extracorporeal membrane oxygenation circuits.

Our objectives were to identify problems associated with the administration of fat emulsion by extracorporeal membrane oxygenation (ECMO) circuits, and gather information from other institutions on standards of practice and the complications associated with infusion of fat emulsion by ECMO to infants and children. In vitro analysis was performed using six circuits. Fat emulsion was infused into a prereservoir port at 3 ml/hour. Circuits and blood samples collected distal to the oxygenator were inspected visually for layering (separation of fat emulsion from blood), agglutination, and phase separation (formation of an oil layer) at 0.5, 1, 2, 4, 6, 12, and 24 hours. At 24 hours, samples were reevaluated and circuits dissected. All circuits showed layering and agglutination. Blood clots were present in five circuits during the simulation. There was no evidence of phase separation in the samples. Adhesion of emulsion to the equipment was present in all circuits. Five of the membrane oxygenators contained clots. One contained long strands of fat; separation of its mesh revealed an oily residue indicating disruption of the stability of the emulsion. Survey responses from 54 centers found that 78% used fat emulsion routinely in neonatal or pediatric patients receiving ECMO. Most used both ECMO and separate venous access for the infusion, depending on availability. Twenty-two (52%) of the 42 centers using fat emulsion had a policy in place regarding site selection. Of those, 73% preferred central venous access, another 18% used a prereservoir port of the ECMO circuit. The most frequently reported problems with administration through the circuit were cracking of stopcocks, clogging and malfunction of the membrane oxygenator, agglutination of the emulsion, and increase in blood clot formation. Our results suggest that fat emulsion should be infused through a separate intravenous site whenever possible. Based on these results and current practices of most ECMO centers, a clinical trial is currently being conducted to provide additional information.

Blood Coagulation↗
PubMed2006

Manufacture of multimicrotubule chitosan nerve conduits with novel molds and characterization in vitro.

Multimicrotubule chitosan conduits (M-conduits) were fabricated using novel molds and a thermal-induced phase-separation technique. Hollow chitosan conduits (H-conduits) with an inner diameter of 1-5 mm and a wall thickness of 0.2-1.0 mm were made, and then a novel mold composed of a styrofoam insulating pedestal with several holes and a stainless steel cover plate was used to make M-conduits. In brief, corresponding H-conduits were inserted upright into the holes of the styrofoam pedestal, and filled with chitosan solution, then rapidly covered with the precooled stainless steel cover plate, and then placed in a freezer. The styrofoam insulating pedestal enclosing the conduits could reduce the heat transfer through the side wall of the conduits. Gradual phase separation then occurred uniaxially in the presence of a unidirectional temperature gradient from the top end to the bottom end of the chitosan conduits. The phase-separated polymer/solvent systems were then dried in a freeze-dryer. The microtubule diameters were controlled by adjusting the polymer concentration and cooling temperature. In vitro characterization demonstrated that the mold-based multimicrotubule chitosan conduits possessed suitable mechanical strength, microtubule diameter distribution, porosity, swelling, biodegradability, and nerve cell affinity, and so they showed potential for application as nerve tissue engineering scaffolds.

Absorbable Implants↗
PubMed2005

Travelling lipid domains in a dynamic model for protein-induced pattern formation in biomembranes.

Cell membranes are composed of a mixture of lipids. Many biological processes require the formation of spatial domains in the lipid distribution of the plasma membrane. We have developed a mathematical model that describes the dynamic spatial distribution of acidic lipids in response to the presence of GMC proteins and regulating enzymes. The model encompasses diffusion of lipids and GMC proteins, electrostatic attraction between acidic lipids and GMC proteins as well as the kinetics of membrane attachment/detachment of GMC proteins. If the lipid-protein interaction is strong enough, phase separation occurs in the membrane as a result of free energy minimization and protein/lipid domains are formed. The picture is changed if a constant activity of enzymes is included into the model. We chose the myristoyl-electrostatic switch as a regulatory module. It consists of a protein kinase C that phosphorylates and removes the GMC proteins from the membrane and a phosphatase that dephosphorylates the proteins and enables them to rebind to the membrane. For sufficiently high enzymatic activity, the phase separation is replaced by travelling domains of acidic lipids and proteins. The latter active process is typical for nonequilibrium systems. It allows for a faster restructuring and polarization of the membrane since it acts on a larger length scale than the passive phase separation. The travelling domains can be pinned by spatial gradients in the activity; thus the membrane is able to detect spatial clues and can adapt its polarity dynamically to changes in the environment.

Biophysics↗
PubMed1982

The activity of 5'-nucleotidase in liver plasma membranes is affected by the increase in bilayer fluidity achieved by anionic drugs but not by cationic drugs.

The 5'-nucleotidase activity of rat liver plasma membranes could be selectively modulated by the anionic drugs phenobarbital and pentobarbital, whereas the corresponding activity of a Lubrol-solubilized preparations remained unaltered. The perturbation in the outer half of the bilayer induced by phenobarbital, which lead to a depression in the high temperature onset of the lipid phase separation occurring in this half of the bilayer, concomitantly lowered the break temperature in Arrhenius plots of 5'-nucleotidase activity from 28 degrees C to 16 degrees C. The stimulation of the membrane-bound activity achieved by low anionic drug concentrations was attributed to a preferential fluidization of the outer half of the bilayer. Contrarily, the cationic drugs prilocaine and carbocaine, when tested over agent concentrations that dramatically increase the fluidity of the inner half of the bilayer, achieved no selective effects on the membrane-bound enzyme. Prilocaine (10 mM) was previously found to induce a lipid phase separation at 11 degrees C that was attributed to the lipids of the internal (cytosol-facing) half of the bilayer, but had no effect on the onset of the lipid phase separation occurring at 28 degrees C. Since Arrhenius plots of 5'-nucleotidase activity in the presence of 10 mM prilocaine concentrations demonstrated only the single break at 28 degrees C, we suggest that prilocaine is unable to selectively perturb the enzyme because this cationic drug preferentially interacts with the acidic phospholipids residing in the inner half of the bilayer. The activity of the ectoenzyme 5'-nucleotidase in rat liver plasma membranes appears to be regulated by the external half of the bilayer only. These results support the view that independent modulation of he fluidity or chemical constituents of each half of the bilayer can distinctly affect the activity of proteins that are themselves asymmetrically orientated within the bilayer.

5'-Nucleotidase↗
PubMed1998

Properties of a peptide containing DL-lactide/glycolide copolymer nanospheres prepared by novel emulsion solvent diffusion methods.

Novel emulsion solvent diffusion methods in water or oil were developed to prepare the peptide (TRH and elcatonin)-loaded PLGA nanospheres, via the coprecipitation of drug and polymer in the emulsion droplets induced by the diffusion of solvent. The PLGA nanospheres prepared by the emulsion solvent diffusion method in water were unimodally dispersed particles with an average diameter of about 250 nm, whereas those prepared by the emulsion solvent diffusion method in oil and the phase separation method exhibited bimodal distribution with average diameter of about 700 and 800 nm, respectively. The content and recovery of the peptide and nanospheres prepared by the emulsion solvent diffusion method in oil were superior to those of nanospheres prepared using other methods. The drug release properties from nanospheres depended strongly on their preparation processes. The nanospheres prepared by the emulsion solvent diffusion method in oil, released the drug continuously over 14 days, the rate of which was determined by the diffusion of drug in the rigid matrix structure of the nanosphere. The drug release behavior of nanospheres prepared by the phase separation method exhibited a triphasic release pattern with an initial burst and an induction period followed by the diffusion of drug through the walls of the reservoir type nanospheres, which were produced via the phase separation of polymer deposited around the emulsion droplets of the drug.

Biocompatible Materials↗