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PubMed2004

Structural diversity of sphingomyelin microdomains.

In cells plasma membrane, sphingomyelin (SM) plays a key role in the formation of a category of lipid microdomains enriched in cholesterol (Chl) often referred to as rafts. Atomic force microscopy (AFM) was used to analyze the mesoscopic topography of enriched SM microdomains in supported bilayers made of SM/dioleoylphosphatidylcholine (SM/DOPC) and SM/palmitoyl-oleoyl-phosphatidylcholine (SM/POPC) equimolar mixtures, in buffer, at room temperature. Gel-fluid phase separation occurs in both SM/DOPC and SM/POPC bilayers. The gel phase SM-enriched microdomains adopt a variety of size, shape and mesoscopic structure, from homogeneous flat domains of a few hundreds of nanometer in diameter to domains of several micrometers made of closely packed globular structures. Gel-gel phase separation in SM domains is also observed which gives rise to different structures for the diunsaturated and the mixed-saturated PC species. These differences could also extend to the interactions with Chl. This suggests that studies on rafts formation commonly performed using SM/DOPC mixture as a model should also include the physiologically more relevant POPC species.

Lipid Bilayers↗

PubMed1979

Amphotericin B induced structural changes of the erythrocyte membrane.

Incubation of human erythrocytes in isotonic NaCl-sucrose medium with a relative high concentration of amphotericin B results in the occurrence of distinct structures on the membrane faces as visible by freeze-fracturing. Such kind of structures is known to be formed by phase separation of the lipid components (domain formation). The induction of phase separation is considered to be an additional effect of amphotericin B.

Amphotericin B↗

PubMed1996

Evidence for the formation of amphotericin B-phospholipid complexes in Langmuir monolayers.

PURPOSE: To study the interaction of the polyene antifungal amphotericin B with phospholipid Langmuir monolayers and to correlate with stability of phospholipid-stabilized drug emulsions. METHODS: Pressure-area isotherms of mixed monolayers of amphotericin B (0-20 mol%) and different phospholipid types were recorded using conventional Langmuir trough methods. Emulsion stability of amphotericin B-containing lipid emulsions was measured using dynamic light scattering. RESULTS: Incorporation of amphotericin B into monolayers composed of saturated phospholipids (Lipoid E80-3) had a profound effect on the shape of the isotherm. This effect was directly related to the concentration of amphotericin B in the monolayer. At high drug concentrations, the shape of the isotherms became progressively similar to that of pure DPPC, thus exhibiting regions attributable to phospholipid in different phase states. This effect on isotherm shape was not observed following incorporation of the drug into monolayers composed of the equivalent unsaturated lecithin (Lipoid E80). CONCLUSIONS: These results are interpreted as indicating the formation of an amphotericin B-phospholipid complex, resulting in phase separation within the monolayer. The extent and nature of this phase separation was dependent on both the concentration of drug in the system, and the saturation state of the phospholipid component. The relevance of these observations to the stability of amphotericin B drug emulsions stabilised by saturated and unsaturated phospholipid emulsifiers is discussed. These observations may also be relevant to the toxicity of these, and other novel amphotericin B formulations.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2003

Phase stability of concentrated dairy products.

The interactions between the two most important colloids in milk, fat globules and casein micelles, were investigated. Mixtures of oil droplets (as a model for fat globules) and casein micelles were prepared, and their phase behavior was studied. It was found that the oil droplets and the casein micelles phase separate as a result of depletion interaction. The experimentally determined phase boundary is consistent with a prediction by theory that involves no adjustable parameters. Furthermore, it is shown that the kinetics of phase separation can be explained by the differences in viscosity of the samples. The results are of relevance to the behavior of concentrated dairy products such as whipping cream and evaporated milk.

Animals↗

PubMedDate not supplied

Polysaccharide-protein interaction: a rheological study of the gel-sol transition of a gelatin-methylcellulose-water system.

Viscoelastic parameters for mixtures of gelatin and methylcellulose were measured as a function of temperature, in order to study the gel-sol transition of the system in which biopolymers forming thermo-setting and thermo-melting gels coexist. At higher temperatures than 45 degrees C, the gel network is mainly formed by methylcellulose while at lower temperatures around 5 degrees C, it is mainly formed by gelatin. At higher temperatures than 45 degrees C, gelatin inhibits the gelation of methylcellulose. A small amount of methylcellulose helps gelatin to form a network at lower temperatures. However, excessive amounts of methylcellulose inhibit the growth of network structure. Therefore, this mixture forms a phase separated gel at higher temperatures while it is not completely phase separated at lower temperatures, probably by some interaction between non-substituted hydroxyl groups in methylcellulose with carboxylic groups in gelatin.

Animals↗

PubMed1998

Separation of proteins and viruses using two-phase aqueous micellar systems.

We discuss the utilization of a novel two-phase aqueous nonionic micellar system for the purification and concentration of biomolecules, such as proteins and viruses, by liquid-liquid extraction. The nonionic surfactant n-decyl tetra(ethylene oxide), C10E4, phase separates in water into two coexisting aqueous micellar phases by increasing temperature. The mild interactions of the C10E4 nonionic surfactant with biomolecules, combined with the high water content of the two coexisting micellar phases, suggest the potential utility of two-phase aqueous C10E4 micellar systems for the purification and concentration of biomolecules. In this paper, we review our recent experimental and theoretical studies involving the partitioning of several water-soluble proteins, including cytochrome c. soybean trypsin inhibitor, ovalbumin, bovine serum albumin, and catalase, in the two-phase aqueous C10E4 micellar system. In addition, we present results of our preliminary experimental investigation on the partitioning of bacteriophages, including phiX174, P22, and T4.

Bacteriophages↗

PubMed1991

Hydrophilic shielding of hydrophobic, cation- and anion-exchange phases for separation of small analytes: direct injection of biological fluids onto high-performance liquid chromatographic columns.

Shielded hydrophobic phases (SHPs) have an external hydrophilic network that prevents larger protein molecules from interacting with hydrophobic zones. Smaller analytes are not sterically hindered from interacting with the hydrophobic zones and are retained. This mechanism allows the separation of the proteins from the analytes of interest. SHPs have been shown to be useful for the direct-injection analysis of drugs in biological fluids. In this paper, two kinds of shielded phases are discussed: bonded micellar phases and embedded polymeric phases. For some compounds of interest, the hydrophobic retention and selectivity is insufficient to obtain the desired resolution. To increase selectivity without affecting protein exclusion, shielded phases were prepared with ion-exchange groups added to the hydrophobic zones. Such modified phases with cation-exchange and anion-exchange capabilities were examined for additional selectivities. Usage of these additional selectivities will be demonstrated to achieve better analysis and resolution for basic, acidic, and neutral compounds.

Animals↗

PubMed2002

High-resolution computer simulation of the dynamics of isoelectric focusing using carrier ampholytes: the post-separation stabilizing phase revisited.

A dynamic electrophoresis simulator that accepts 150 components and voltage gradients employed in the laboratory was used to provide a detailed description of the stabilizing phase in isoelectric focusing under conditions that were hitherto inaccessible. High-resolution focusing data are presented for pH gradients spanning 7 units (pH 3-10 and pH 4-11 with 20 carrier ampholytes/pH unit) and 3.5 units (pH 7-10.5 and pH 5-8.5 with 40 carrier ampholytes/pH unit). Stabilizing phase behavior for configurations (i) with the focusing column ends only permeable to OH(-) and H(+) at cathode and anode, respectively, and (ii) with the focusing column being sandwiched between NaOH (catholyte) and phosphoric acid (anolyte) are described. Simulation data reveal the stabilizing phase to be diffusion-controlled and characterized by changes that progress from the column ends towards neutrality (i.e., towards the center in case of pH gradients bracketing neutrality). Transient states are characterized by moving concentration valleys of carrier ampholytes that significantly alter the distributions of pH and conductivity. Nonlinear pH gradients are produced. The magnitude of the changes occuring is dependent on the span of the pH gradient. Gradients that encompass greater extremes of pH show more pronounced stabilizing phases. For all systems subjected to a constant 300 V/cm, the initial separation and subsequent stabilization require less than 10 min and more than 7000 min, respectively. The presence of electrolytes at the column ends disrupts the stabilizing phase, with the degree of disruption dependent on the concentrations of the acid and base employed as electrode solutions. The data not only indicate that a true steady state is never attained in the average laboratory experiment, they also suggest that a true steady state in absence of immobilized pH gradients cannot be achieved experimentally at all.

Buffers↗

PubMed2006

Phase coexistence and connectivity in the apical membrane of polarized epithelial cells.

Although it is well described in model membranes, little is known about phase separation in biological membranes. Here, we provide evidence for a coexistence of at least two different lipid bilayer phases in the apical plasma membrane of epithelial cells. Phase connectivity was assessed by measuring long-range diffusion of several membrane proteins by fluorescence recovery after photobleaching in two polarized epithelial cell lines and one fibroblast cell line. In contrast to the fibroblast plasma membrane, in which all of the proteins diffused with similar characteristics, in the apical membrane of epithelial cells the proteins could be divided into two groups according to their diffusion characteristics. At room temperature ( approximately 25 degrees C), one group showed fast diffusion and complete recovery. The other diffused three to four times slower and, more importantly, displayed only partial recovery. Only the first group comprises proteins that are believed to be associated with lipid rafts. The partial recovery is not caused by topological constraints (microvilli, etc.), cytoskeletal constraints, or protein-protein interactions, because all proteins show 100% recovery in fluorescence recovery after photobleaching experiments at 37 degrees C. In addition, the raft-associated proteins cannot be coclustered by antibodies on the apical membrane at 12 degrees C. The interpretation that best fits these data is that the apical membrane of epithelial cells is a phase-separated system with a continuous (percolating) raft phase <25 degrees C in which isolated domains of the nonraft phase are dispersed, whereas at 37 degrees C the nonraft phase becomes the continuous phase with isolated domains of the raft phase dispersed in it.

Animals↗

PubMed1986

Acidic phospholipid species inhibit adenylate cyclase activity in rat liver plasma membranes.

Incubation of rat liver plasma membranes with liposomes of dioleoyl phosphatidic acid (dioleoyl-PA) led to an inhibition of adenylate cyclase activity which was more pronounced when fluoride-stimulated activity was followed than when glucagon-stimulated activity was followed. If Mn2+ (5 mM) replaced low (5 mM) [Mg2+] in adenylate cyclase assays, or if high (20 mM) [Mg2+] were employed, then the perceived inhibitory effect of phosphatidic acid was markedly reduced when the fluoride-stimulated activity was followed but was enhanced for the glucagon-stimulated activity. The inhibition of adenylate cyclase activity observed correlated with the association of dioleoyl-PA with the plasma membranes. Adenylate cyclase activity in dioleoyl-PA-treated membranes, however, responded differently to changes in [Mg2+] than did the enzyme in native liver plasma membranes. Benzyl alcohol, which increases membrane fluidity, had similar stimulatory effects on the fluoride- and glucagon-stimulated adenylate cyclase activities in both native and dioleoyl-PA-treated membranes. Incubation of the plasma membranes with phosphatidylserine also led to similar inhibitory effects on adenylate cyclase and responses to Mg2+. Arrhenius plots of both glucagon- and fluoride-stimulated adenylate cyclase activity were different in dioleoyl-PA-treated plasma membranes, compared with native membranes, with a new 'break' occurring at around 16 degrees C, indicating that dioleoyl-PA had become incorporated into the bilayer. E.s.r. analysis of dioleoyl-PA-treated plasma membranes with a nitroxide-labelled fatty acid spin probe identified a new lipid phase separation occurring at around 16 degrees C with also a lipid phase separation occurring at around 28 degrees C as in native liver plasma membranes. It is suggested that acidic phospholipids inhibit adenylate cyclase by virtue of a direct headgroup specific interaction and that this perturbation may be centred at the level of regulation of this enzyme by the stimulatory guanine nucleotide regulatory protein NS.

Adenylyl Cyclase Inhibitors↗

PubMed2002

Percolative Superconductivity in Mg1-xB2.

Our results from various transport experiments on Mg1-xB2 indicate a surprising effect associated with the presence of a Mg deficiency in MgB2: the phase separation between Mg-vacancy rich and Mg-vacancy poor phases. The Mg-vacancy poor phase is superconducting, but the insulating nature of the Mg-vacancy rich phase probably originates from the Anderson (disorder-induced) localization of itinerant carriers. Furthermore, electron diffraction measurements indicate that within vacancy-rich regions these defects tend to order with intriguing patterns. This electronic phase separation in Mg1-xB2 shows similar, but also distinct characteristics compared with that observed in La(2)CuO(4+delta).

Journal Article↗

PubMed2000

The leptospiral major outer membrane protein LipL32 is a lipoprotein expressed during mammalian infection.

We report the cloning of the gene encoding the 32-kDa lipoprotein, designated LipL32, the most prominent protein in the leptospiral protein profile. We obtained the N-terminal amino acid sequence of a staphylococcal V8 proteolytic-digest fragment to design an oligonucleotide probe. A Lambda-Zap II library containing EcoRI fragments of Leptospira kirschneri DNA was screened, and a 5.0-kb DNA fragment which contained the entire structural lipL32 gene was identified. Several lines of evidence indicate that LipL32 is lipid modified in a manner similar to that of other procaryotic lipoproteins. The deduced amino acid sequence of LipL32 would encode a 272-amino-acid polypeptide with a 19-amino-acid signal peptide, followed by a lipoprotein signal peptidase cleavage site. LipL32 is intrinsically labeled during incubation of L. kirschneri in media containing [(3)H]palmitate. The linkage of palmitate and the amino-terminal cysteine of LipL32 is acid labile. LipL32 is completely solubilized by Triton X-114 extraction of L. kirschneri; phase separation results in partitioning of LipL32 exclusively into the hydrophobic, detergent phase, indicating that it is a component of the leptospiral outer membrane. CaCl(2) (20 mM) must be present during phase separation for recovery of LipL32. LipL32 is expressed not only during cultivation but also during mammalian infection. Immunohistochemistry demonstrated intense LipL32 reactivity with L. kirschneri infecting proximal tubules of hamster kidneys. LipL32 is also a prominent immunogen during human leptospirosis. The sequence and expression of LipL32 is highly conserved among pathogenic Leptospira species. These findings indicate that LipL32 may be important in the pathogenesis, diagnosis, and prevention of leptospirosis.

Acylation↗

PubMed1985

Reverse-phase h.p.l.c. separation, quantification and preparation of bilirubin and its conjugates from native bile. Quantitative analysis of the intact tetrapyrroles based on h.p.l.c. of their ethyl anthranilate azo derivatives.

We describe a facile and sensitive reverse-phase h.p.l.c. method for analytical separation of biliary bile pigments and direct quantification of unconjugated bilirubin (UCB) and its monoglucuronide (BMG) and diglucuronide (BDG) conjugates in bile. The method can be 'scaled up' for preparative isolation of pure BDG and BMG from pigment-enriched biles. We employed an Altex ultrasphere ODS column in the preparative steps and a Waters mu-Bondapak C18 column in the separatory and analytical procedures. Bile pigments were eluted with ammonium acetate buffer, pH 4.5, and a 20 min linear gradient of 60-100% (v/v) methanol at a flow rate of 2.0 ml/min for the preparative separations and 1.0 ml/min for the analytical separations. Bile pigments were eluted in order of decreasing polarity (glucuronide greater than glucose greater than xylose conjugates greater than UCB) and were chemically identified by t.l.c. of their respective ethyl anthranilate azo derivatives. Quantification of UCB was carried out by using a standard curve relating a range of h.p.l.c. integrated peak areas to concentrations of pure crystalline UCB. A pure crystalline ethyl anthranilate azo derivative of UCB (AZO . UCB) was employed as a single h.p.l.c. reference standard for quantification of BMG and BDG. We demonstrate that: separation and quantification of biliary bile pigments are rapid (approximately 25 min); bile pigment concentrations ranging from 1-500 microM can be determined 'on line' by using 5 microliters of bile without sample pretreatment; bilirubin conjugates can be obtained preparatively in milligram quantities without degradation or contamination by other components of bile. H.p.l.c. analyses of a series of mammalian biles show that biliary UCB concentrations generally range from 1 to 17 microM. These values are considerably lower than those estimated previously by t.l.c. BMG is the predominant, if not exclusive, bilirubin conjugate in the biles of a number of rodents (guinea pig, hamster, mouse, prairie dog) that are experimental models of both pigment and cholesterol gallstone formation. Conjugated bilirubins in the biles of other animals (human, monkey, pony, cat, rat and dog) are chemically more diverse and include mono-, di- and mixed disconjugates of glucuronic acid, xylose and glucose in proportions that give distinct patterns for each species.

Animals↗

PubMed1990

Interaction of gramicidin S analogs with lipid bilayer membrane.

One of the side chains of Orn residues in gramicidin S (GS) was connected with alanine (AGS), sarcosine (SGS), or histidine (HGS) residue, aiming at developing membrane-active artificial enzymes by virtue of the membrane-associating property of GS. The conformation of the GS analogs was similar to that of GS. However, the affinity of GS and its analogs for dipalmitoylphosphatidylcholine (DPPC) vesicles decreased in the order of GS greater than SGS greater than HGS congruent to AGS. The addition of GS analogs at 10 microM to DPPC vesicles decreased the membrane fluidity, indicating that GS analogs did not disrupt the vesicular structure of DPPC vesicles. On the other hand, GS analogs enhanced carboxyfluorescein-leakage from DPPC vesicles. It was therefore considered that the GS analogs induced the phase-separation of the lipid bilayer membrane. Hydrolytic reactions of HGS in the presence of DPPC vesicles were studied using N-methylindoxyl alkanoate as substrate. HGS reacted only with N-methylindoxyl hexanoate below the phase-transition temperature of the membrane. The substrate specificity of HGS was ascribed to the condensation of HGS in the neighbourhood of the substrate in the lipid bilayer membrane due to the phase-separation below the phase-transition temperature of the membrane.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1987

Effect of myelin basic protein on the thermotropic behavior of aqueous dispersions of neutral and anionic glycosphingolipids and their mixtures with dipalmitoylphosphatidylcholine.

The thermotropic behavior of the natural glycosphingolipids galactosylceramide, asialo-Gal beta 1-3GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-4Glc beta 1-Cer (GM1), sulfatide, GM1, NeuAc alpha 2-3Gal beta 1-3GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-4Glc beta 1-1Cer (GD1a), and NeuAc alpha 2-3Gal beta 1-3GalNAc beta 1-4Gal(3-2 alpha NeuAc8-2 alpha NeuAc)beta 1-4Glc beta 1-1 Cer (GT1b), and their mixtures with dipalmitoylphosphatidylcholine (DPPC) in the presence of myelin basic protein (MBP) was studied by high sensitivity differential scanning calorimetry. The transition temperature of DPPC, galactosylceramide, and asialo-GM1 is affected little by MBP while their transition enthalpy is decreased in proportion to the amount of protein in the mixture. The thermotropic behavior of anionic glycosphingolipids is considerably perturbed by MBP. The transition temperature of gangliosides increases in the presence of MBP, whereas that of sulfatide decreases. The enthalpy of the transition of anionic glycosphingolipids increases markedly in the presence of MBP. The excess heat capacity function of these systems can be resolved into two independent phase transitions. Phase separation of enriched lipid/protein domains occurs in a magnitude that depends on the amount of MBP; the rest of the lipid phase exhibits some altered thermodynamic properties. In mixtures of glycosphingolipids with DPPC, phase separation is also present but no phase transition with the characteristic of pure DPPC is found. MBP is changing the properties of the lipid mixture as a whole and does not interact exclusively with the glycosphingolipids. The proportion of MBP required to produce the maximal changes is greater the greater the complexity of the glycosphingolipids polar head group. Relatively small variations of the amount of MBP induce large shifts in the proportion of the different phases present.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1999

High-performance liquid chromatographic methods for monitoring of isomers of 17-hydroxy-16-hydroxymethyl-3-methoxyestra-1,3,5(10)-triene.

Claisen condensation and consecutive reduction of 3-methoxyestra-1,3,5(10)-trien-17-one theoretically leads to four diastereomers of 17-hydroxy-16-hydroxymethyl-3-methoxyestra-1,3,5(10)-triene and their further transformations give different compounds with different biological activities. High-performance liquid chromatographic methods were developed for separation of the four isomers of 17-hydroxy-16-hydroxymethyl-3-methoxyestra-1,3,5(10)-triene: reversed-phase separation on a Nucleosil ODS C18 column with water-methanol as mobile phase; and normal-phase separation on an APEX Silica column with hexane-dichloromethane-2-propanol as mobile phase. The effects of eluent composition and flow-rate on the separation were investigated. This is the first chromatographic evidence for the formation of the 16alpha,17alpha isomer in the reduction of 16-hydroxymethylene-3-methoxyestra-1,3,5(10)-trien-17-one.

Chromatography, High Pressure Liquid↗

PubMed2001

Reversed-phase liquid chromatographic separation of enantiomers on polysaccharide type chiral stationary phases.

The direct chiral separation by chiral stationary phases (CSPs) is one of the most important techniques to analyze enantiomeric purity as well as to get enantiomerically pure material quickly. Among various types of CSPs, polysaccharide type CSPs are well known by their versatility and durability. They are not only effective under normal-phase conditions, but also under reversed-phase conditions. In order to get a good separation under the reversed-phase conditions, it is the key to choose an appropriate mobile phase. For example, a simple mixture of water/acetonitrile or water/methanol are sufficient for a neutral analyte, while it is necessary to use an acidic solution instead of water for an acidic analyte and a solution of a chaotropic salt (or a basic solution) for a basic analyte, respectively. The paper also presents lists of more than 350 separation examples that include 22 validated methods for drug analyses from serum, plasma, or urine samples on polysaccharide type CSPs under reversed-phase conditions.

Chromatography, Liquid↗

PubMed1998

Preparation of Poly(methylmethacrylate) Microcapsules with Liquid Cores.

Particles with liquid cores and solid shells have been prepared by the controlled phase separation of poly(methylmethacrylate) (PMMA) within the droplets of an oil-in-water emulsion. The oil phase of the emulsion contained poly(methylmethacrylate), a good solvent for the polymer (CH2Cl2), a poor solvent (hexadecane, decane, octanol, or tetrachloromethane), and in some cases acetone (a water soluble co-solvent) to aid emulsification. Emulsions were prepared using a Silverson high-speed stirrer, and the droplet size distributions were determined using a Coulter particle counter. Size distributions were found to be dependent on the nature of the emulsifier, the concentration of acetone in the oil phase, and the concentration of polymer in the oil phase. The good solvent was then removed under reduced pressure, causing the poly(methylmethacrylate) to phase separate within the emulsion droplets. The resultant two-phase particles were characterized by optical microscopy and scanning electron microscopy. Particle morphologies depended strongly on the nature of the non-solvent and also the emulsifier employed. Spreading coefficients were calculated from interfacial tension and contact angle measurements, and were used to account for the morphologies observed. Core/shell microcapsules were formed when hexadecane or decane was used as non-solvent, and only when polymeric emulsifiers were employed. All other combinations yielded "acorn"-shaped particles. The thickness of microcapsule walls was found to be a constant fraction of the overall capsule diameter for all microcapsule sizes and depended, as expected, on the concentration of polymer in the oil phase. Copyright 1998 Academic Press.

Journal Article↗