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Results for “Phase Separation”

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At least 1,531 records · Page 85Linked to original sources

PubMed2003

Evaluation of different organic phases for water-in-oil xanthan fermentation.

Water-in-oil (W/O) fermentation technology has the potential for overcoming the problems related with high broth viscosity in xanthan fermentations. By dispersing the aqueous broth in a continuous organic phase, the broth-thickening mechanisms are confined within the aqueous droplets without significantly increasing the overall viscosity. In this study, xanthan fermentations were made with perfluorocarbon (PFC) or vegetable oil as the organic phase. The results were compared with those obtained previously using n-hexadecane as the organic phase, to evaluate the effects of various properties. PFC provided easy phase separation at the end of fermentation but required higher power input for agitation, a direct result of its high density. The aqueous droplets formed were large (400-450 microm), limiting the cell concentration employable due to the occurrence of oxygen starvation in the inner core. One main advantage of using vegetable oil was its low cost. In addition, vegetable oil provided much finer droplets (<120 microm) and produced high xanthan concentrations (>100 g l(-1)). However, complete phase separation for product recovery was difficult to achieve. Fermentations in both organic phases were terminated by the occurrence of phase inversion to highly viscous O/W dispersions at aqueous-phase volume fractions of 0.53-0.56. The initial fraction was 0.3 but changed due to base addition for pH adjustment and nutrient addition for prolonged production.

Alkanes↗

PubMed2001

Transparent nematic phase in a liquid-crystal-based microemulsion.

Complex fluids are usually produced by mixing together several distinct components, the interactions between which can give rise to unusual optical and rheological properties of the system as a whole. For example, the properties of microemulsions (composed of water, oil and surfactants) are determined by the microscopic structural organization of the fluid that occurs owing to phase separation of the component elements. Here we investigate the effect of introducing an additional organizing factor into such a fluid system, by replacing the oil component of a conventional water-in-oil microemulsion with an intrinsically anisotropic fluid--a nematic liquid crystal. As with the conventional case, the fluid phase-separates into an emulsion of water microdroplets (stabilized by the surfactant as inverse micelles) dispersed in the 'oil' phase. But the properties are further influenced by a significant directional coupling between the liquid-crystal molecules and the surfactant tails that emerge (essentially radially) from the micelles. The result is a modified bulk-liquid crystal that is an ordered nematic at the mesoscopic level, but which does not exhibit the strong light scattering generally associated with bulk nematic order: the bulk material here is essentially isotropic and thus transparent.

Journal Article↗

PubMed1982

X-ray diffraction analysis of wet isolated bovine rod outer segment disks. A dehydration study.

Sequences of X-ray diffraction patterns were obtained from dehydrating, artificially oriented multilayers of isolated, bovine rod outer segment disks. A direct-phase analysis was applied to highly hydrated specimens to determine sequences of low resolution (approx. 30 A) electron density profiles of the disks as dehydration proceeded. The profiles were found to evolve smoothly as the multilayer lattice simultaneously shrank and became increasingly ordered. The bilayer profiles were largely invariant under dehydration and the evolution of the diffraction consistent with simple decreases in fluid spacings. The specimens were observed to phase separate into characteristic primary and a secondary lattices when the multi-layer became too dehydrated. The small unit cell size of the secondary lattice was suggestive of a lipid phase. Large changes in the diffraction patterns from phase separated specimens were observed upon bleaching of the specimen. The changes were consistent with a reversible disordering of the primary lattice.

Animals↗

PubMed2001

[Study on chitosan-gelatin/hydroxyapatite composite scaffolds--preparation and morphology].

OBJECTIVE: To prepare chitosan-gelatin/hydroxyapatite (CS-Gel/HA) composite scaffolds, and to investigate the influence of components and preparing conditions to their micromorphology. METHODS: The CS-Gel/HA composite scaffolds were prepared by phase-separation method. Micromorphology and porosity were detected by using scanning electron microscope and liquid displacement method respectively. RESULTS: Porous CS-Gel/HA composite scaffolds could be prepared by phase-separation method, and their density and porosity could be controlled by adjusting components and quenching temperature. CONCLUSION: The study suggests the feasibility of using CS-Gel/HA composite scaffolds for the transplantation of autogenous osteoblasts to regenerate bone tissue.

Biocompatible Materials↗

PubMed2001

Microphase separation in bioerodible copolymers for drug delivery.

This research examines the microstructure of bioerodible polyanhydrides with an eye towards precise design of drug delivery devices. Our main hypothesis is that the bioerodible copolymer poly(1,6-bis-p-carboxyphenoxyhexane-co-sebacic anhydride) (CPH : SA) undergoes micro-phase separation at certain copolymer compositions due to differences in relative hydrophobicity of the co-monomers, resulting in thermodynamic partitioning of drugs incorporated into these copolymers. We investigate the thermal properties, degree of crystallinity, and surface microstructure of several compositions of CPH : SA using differential scanning calorimetry (DSC), wide-angle X-ray diffraction (WAXD), and atomic force microscopy (AFM). We observe that the degree of crystallinity decreases, while the crystal lamellar thickness increases with CPH content. Phase-imaging using AFM indicates the presence of micro-domains in 20 : 80 and 80 : 20 CPH : SA, while poly(SA) and 50 : 50 CPH : SA show no micro-phase separation. Finally, drug-polymer interactions are studied by loading the polymers with different amounts of brilliant blue (hydrophilic) and p-nitroaniline (hydrophobic). DSC and WAXD analysis shows that loading hydrophobic drugs into relatively hydrophobic polymers (poly(SA)) lowers melting point that becomes more pronounced with increased drug loading.

Biodegradation, Environmental↗

PubMed1986

The migration of osteoblasts over substrata of discrete surface charge.

Osteoblasts, from the endocranial surface of neonate rat parietal bones, migrated over ion-exchange resin beads and phase-separation beads in vitro. The morphology of migrated osteoblasts was examined using a scanning electron microscope. The ion-exchange resin beads were either positively or negatively surface charged. The phase-separation model system described, using poly(methyl methacrylate) (PMMA) and lead poly-acrylate (LPA), presented morphologically discrete regions of positive and negative surface charge to single migrating osteoblasts. Osteoblasts migrated over both positively and negatively charged substrata. Close approximation of cells was noted on positively charged surfaces while a 'stand-off' morphology was characteristic of cells on negatively charged substrata. Dorsal cell-surface membrane activity was influenced by the surface charge of the colonized substratum.

Animals↗

PubMed1995

Filipin fluorescence quenching by spin-labeled probes: studies in aqueous solution and in a membrane model system.

A detailed photophysical study of the fluorescence quenching (transient and steady state) of the macrolide antibiotic filipin by nitroxide-substituted fatty acids and a cholesterol derivative was carried out, aimed at determining its transverse position in a model system of membranes (multilamellar vesicles of dipalmitoylphosphatidylcholine). Filipin partitions efficiently into membranes (Kp = (5.0 +/- 1.0).10(3), 20 degrees C) and it was concluded that the antibiotic is buried in the membrane, away from the lipid-water interface. In addition, information on the organization of the quenchers was also obtained. The 5-nitroxide derivative of the fatty acid is essentially randomly distributed, while the 16-nitroxide is aggregated at concentrations higher than approximately 5% molar. For the cholesterol compound the results point to a phase separation at concentrations higher than 3% molar (below this limit concentration filipin associates with the derivatized sterol with KA = 20 M-1, assuming a 1:1 interaction). We propose that this phase separation and the aggregation state of filipin in the aqueous solution may be key processes in the antibiotic mode of action. A systematic and general approach to fluorescence quenching data analysis in complex (e.g., biochemical) systems is also presented.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2000

Sustained drug release characteristics of biodegradable composite poly(d,l)lactic acid-poly(l)lactic acid microcapsules containing ciprofloxacin.

Ciprofloxacin polylactic microcapsules were prepared by the phase separation process. Two types of polylactic acid, poly(d,l)lactic acid and poly(l)lactic acid were combined as membrane materials to prevent the aggregation which happened frequently in the phase separation process. The polymer compositions of the microcapsules can influence the release rate of Ciprofloxacin. The optimal release rate of the drug can be obtained by modifying microcapsule compositions. Poly(d,l)lactic acid is superior in slowing the rate of drug release than poly(l)lactic acid. However, poly(l)lactic acid is necessary in the preparation of the microcapsules to prevent aggregation.

Anti-Infective Agents↗

PubMed2001

Enhanced crystallization of the Cys18 to Ser mutant of bovine gammaB crystallin.

The cysteine residues of the gamma crystallins, a family of ocular lens proteins, are involved in the aggregation and phase separation of these proteins. Both these phenomena are implicated in cataract formation. We have used bovine gammaB crystallin as a model system to study the role of the individual cysteine residues in the aggregation and phase separation of the gamma crystallins. Here, we compare the thermodynamic and kinetic behavior of the recombinant wild-type protein (WT) and the Cys18 to Ser (C18S) mutant. We find that the solubilities of the two proteins are similar. The kinetics of crystallization, however, are different. The WT crystallizes slowly enough for the metastable liquid-liquid coexistence to be easily observed. C18S, on the other hand, crystallizes rapidly; the metastable coexisting liquid phases of the pure mutant do not form. Nevertheless, the coexistence curve of C18S can be determined provided that crystallization is kinetically suppressed. In this way we found that the coexistence curve coincides with that of the WT. Despite the difference in the kinetics of crystallization, the two proteins were found to have the same crystal forms and almost identical X-ray structures. Our results demonstrate that even conservative point mutations can bring about dramatic changes in the kinetics of crystallization. The implications of our findings for cataract formation and protein crystallization are discussed.

Amino Acid Substitution↗

PubMed2006

Probing perturbation of bovine lung surfactant extracts by albumin using DSC and 2H-NMR.

Lung surfactant (LS), a lipid-protein mixture, forms films at the lung air-water interface and prevents alveolar collapse at end expiration. In lung disease and injury, the surface activity of LS is inhibited by leakage of serum proteins such as albumin into the alveolar hypophase. Multilamellar vesicular dispersions of a clinically used replacement, bovine lipid extract surfactant (BLES), to which (2% by weight) chain-perdeuterated dipalmitoylphosphatidycholine (DPPG mixtures-d(62)) had been added, were studied using deuterium-NMR spectroscopy ((2)H-NMR) and differential scanning calorimetry (DSC). DSC scans of BLES showed a broad gel to liquid-crystalline phase transition between 10-35 degrees C, with a temperature of maximum heat flow (T(max)) around 27 degrees C. Incorporation of the DPPC-d(62) into BLES-reconstituted vesicles did not alter the T(max) or the transition range as observed by DSC or the hydrocarbon stretching modes of the lipids observed using infrared spectroscopy. Transition enthalpy change and (2)H-NMR order parameter profiles were not significantly altered by addition of calcium and cholesterol to BLES. (2)H-NMR spectra of the DPPC-d(62) probes in these samples were characteristic of a single average lipid environment at all temperatures. This suggested either continuous ordering of the bilayer through the transition during cooling or averaging of the DPPC-d(62) environment by rapid diffusion between small domains on a short timescale relative to that characteristic of the (2)H-NMR experiment. Addition of 10% by weight of soluble bovine serum albumin (1:0.1, BLES/albumin, dry wt/wt) broadened the transition slightly and resulted in the superposition of (2)H-NMR spectral features characteristic of coexisting fluid and ordered phases. This suggests the persistence of phase-separated domains throughout the transition regime (5-35 degrees C) of BLES with albumin. The study suggests albumin can cause segregation of protein bound-lipid domains in surfactant at NMR timescales (10(-5) s). Persistent phase separation at physiological temperature may provide for a basis for loss of surface activity of surfactant in dysfunction and disease.

Animals↗

PubMed1980

Permeability of mitochondria to sucrose induced by hydrostatic pressure.

When subjected to increasing pressure at 0 degree C, rat liver mitochondria become permeable to sucrose, causing them to swell and their outer membrane to rupture. Afterwards they are lysed and their matrix content is released into the medium. This permeation to sucrose may be prevented to some extent by increasing the temperature at which compression is carried out. 0.75 mM imipramine protects mitochondria against lysis caused by hydrostatic pressure, but does not oppose their permeation to sucrose nor the swelling resulting from the compression. At this concentration, the drug does not exhibit a significant effect on the lateral phase separations which take place in the inner mitochondrial membrane under pressure. The mitochondria of rat fetal liver (21 days), kidney and Morris hepatoma 16 become permeable to sucrose when they are subjected to compression; under these conditions, lateral phase separations occur in their inner membrane. Contrary to liver mitochondria, the former do not undergo lysis. Taking into account both present and previous results, events leading to mitochondrial membrane deterioration by hydrostatic pressure may be summarized in the following way. Pressure first leads to a phase transition of the membrane lipids, thus causing a permeation to sucrose; as a result the mitochondria swell because they have absorbed osmotic water. The membrane lipids freeze increasingly as the pressure increases; the inner membrane becomes fragile and finally, in the case of the adult liver organelles, can no longer resist the swelling. All these events can be avoided by increasing the temperature; imipramine only prevents inner membrane lysis.

Animals↗

PubMed1994

Non-proteolytic release of carcinoembryonic antigen from normal human colonic epithelial cells cultured in collagen gel.

Recent studies have shown that, even with a minimal content of carcinoembryonic antigen (CEA), normal human colonic epithelial cells express substantial amounts of CEA mRNA and colonic mucosal fragments cultured in vitro produce CEA quite actively, indicating that CEA should no longer be considered to be of an oncofetal nature. To understand the basis of the usefulness of CEA as a tumor marker, we analyzed the release of CEA, a glycosyl-phosphatidylinositol (GPI)-anchored protein, from colonic epithelial cells, by culturing isolated colonic crypts in collagen gel. The crypts appeared to preserve their morphological and biochemical integrity in the gel for at least 16 hr, and released CEA spontaneously. Three forms of CEA--spontaneously released CEA, CEA liberated with phosphatidylinositol-specific phospholipase C (PI-PLC) and CEA in cell lysates--were indistinguishable on SDS-PAGE. This is in contrast to recombinant CEA spontaneously released from CHO transfectants, which showed a smaller molecular mass than that of PI-PLC-cleaved recombinant CEA. By phase separation using Triton X-114, CEA in the cell lysates of crypts was separated mostly into the detergent phase, while the spontaneously released and the PI-PLC-cleaved CEA were separated into the aqueous phase. When the cells were metabolically labeled with the precursors of the GPI-anchor, 3H-ethanolamine but not 3H-palmitic acid was found in the spontaneously released CEA. These findings suggest that, in contrast to the proteolysis-like release of the recombinant CEA from CHO cells, CEA in normal colonic epithelial cells is released by a non-proteolytic cleavage, which probably occurs through the action of some endogenous phospholipase.

Animals↗

PubMed2002

Evidence for a physiological role for membrane rafts in human platelets.

We have investigated raft formation in human platelets in response to cell activation. Lipid phase separation and domain formation were detected using the fluorescent dye 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (diI-C(18)) that preferentially partitions into gel-like lipid domains. We showed that when human platelets are activated by cold and physiological agonists, rafts coalesce into visible aggregates. These events were disrupted by depletion of membrane cholesterol. Using Fourier transform infrared spectroscopy (FTIR), we measured a thermal phase transition at around 30 degrees C in intact platelets, which we have assigned as the liquid-ordered to the liquid-disordered phase transition of rafts. Phase separation of the phospholipid and the sphingomyelin-enriched rafts could be observed as two phase transitions at around 15 and 30 degrees C, respectively. The higher transition, assigned to the rafts, was greatly enhanced with removal of membrane cholesterol. Detergent-resistant membranes (DRMs) were enriched in cholesterol (50%) and sphingomyelin (20%). The multi-functional platelet receptor CD36 selectively partitioned into DRMs, whereas the GPI-linked protein CD55 and the major platelet integrin alpha(IIb)beta(3a) did not, which suggests that the clustering of proteins within rafts is a regulated process dependent on specific lipid protein interactions. We suggest that raft aggregation is a dynamic, reversible physiological event triggered by cell activation.

Blood Platelets↗

PubMed1984

Freeze-etch study of an unmodified lectin interacting with its receptors in model membranes.

Experiments are described in which liposomes bearing ganglioside or glycophorin as receptor were exposed to the native (unmodified) lectin, wheat germ agglutinin, and subsequently examined by freeze-etch electron microscopy. Visualized in this way in the absence of lectin, phosphatidylcholine bilayer membranes show no features attributable to the presence of small amounts of glycolipid. Similarly bilayers bearing glycophorin show no obvious etch face (outer surface) features attributable to that species, although they do have intramembranous particles associated with the hydrophobic interior. However, the otherwise smooth and featureless model membrane outer surface permitted ready visualization of bound lectin molecules, and thus the indirect localization of receptors. The lipid membrane itself in the vicinity of receptors was not visibly affected by lectin binding to glycolipid or glycoprotein. The only identifiable lectin-induced change was lateral redistribution of receptor glycoproteins. Ganglioside molecules showed some evidence of existing in small clusters; but their distribution was apparently unaffected by lectin binding. Clumps of lectin bound to glycophorin were found associated predominantly with fluid bilayer regions of phase separated membranes; and lectin distribution on the etch face correlated with intramembranous particle distribution in the fracture face. In contrast, ganglioside lateral distribution was not appreciably influenced by the host lipid matrix phase separation.

Cholesterol↗

PubMed2004

Localization of bovine serum albumin in double-walled microspheres.

Phase separation of binary blends of various combinations of poly (L-lactide) (PLA), and poly (D,L-lactide-co-glycolide) (PLGA), was investigated using differential scanning calorimetry (DSC). Based on this phase separation phenomenon, double-walled microspheres were fabricated. A model agent, bovine serum albumin (BSA) labeled with fluorescein isothiocyanate (FITC-BSA) was localized in each layer. Scanning electron microscopy (SEM) and fluorescence microscopy (FM) were used to assess the formation of double-walled microspheres and the localization of the drug, respectively. When a 1:1 polymer ratio was used, the FITC-BSA was localized in the outer layer. When the relative ratio of PLGA to PLA was increased to 3:1 using the same overall polymer concentration, the FITC-BSA was localized in the inner core. Release studies were carried out to evaluate the advantage of double-walled microspheres compared to single walled microspheres. Microspheres made with FITC-BSA localized in the inner core exhibited a significantly lower initial release rate compared to microspheres where the drug was located in the outer layer, or compared to microspheres made from PLA only. Hence microspheres with a double-walled morphology have the potential for therapeutic use where a high burst might be detrimental.

Animals↗

PubMed1994

Interaction of bombolitin III with phospholipid monolayers and liposomes and effect on the activity of phospholipase A2.

This study is focused on the characterization of the interaction of the amphiphilic peptide bombolitin III (from the bumblebee Megabombus pennsylvanicus) with phospholipid monolayers and vesicles. It is shown that due to the amphiphilic character of its alpha-helical conformation this water-soluble peptide is able to interact in an ordered fashion with phospholipid organized structures. Depending on the temperature, the subphase, and the particular phosphatidylcholine used, the mixed peptide-phospholipid monolayers can be homogeneous or display phase separation. This behavior was observed by means of the Langmuir film balance technique, coupled with an epifluorescence microscope. In well-defined conditions it is possible to visualize the formation of phase-separated peptide domains at the air-water interface and to study the effect of their presence on the organization of the lipid. The action of phospholipase A2 at the lipid-peptide interface was also followed by means of fluorescence microscopy: some evidence that the enzyme preferentially hydrolyzes the phospholipid that is in contact with the peptide is presented. Furthermore, the presence of bombolitin III in L-alpha-DLPC monolayers causes an increase in the initial speed of degradation with phospholipase A2. These results are in agreement with previous findings that show that the bombolitins are activators in vitro of phospholipase A2. Experiments were also performed with peptide fragments corresponding to the alpha-helical sequences of the protein uteroglobin: despite some amphiphilic character, these peptides do not interact strongly with phospholipid monolayers. Only one of these peptides (corresponding to the helix 4-14 in uteroglobin) is adsorbed in the monolayer in a similar fashion to bombolitin III but does not cause an increase in the activity of phospholipase A2.

Amino Acid Sequence↗

PubMed1995

Endopeptidase-24.11 is the integral membrane peptidase initiating degradation of somatostatin in the hippocampus.

The membrane metalloenzyme endopeptidase-24.11 has been localized by immunocytochemistry in the porcine hippocampus in the stratum oriens and stratum radiatum. Endopeptidase-24.11 was found to be approximately 10-fold more abundant in a striatal than a hippocampal membrane preparation. Both somatostatin-28 and somatostatin-14 were metabolized by endopeptidase-24.11, but the kinetics of hydrolysis markedly favoured the smaller form of the neuropeptide. After phase separation with Triton X-114 of striatal and hippocampal membrane preparations, and by using selective inhibitors, the major (> 80%) somatostatin-metabolizing activity was found to partition into the detergent-rich phase and was attributable predominantly to endopeptidase-24.11. The residual activity observed in the presence of the selective endopeptidase-24.11 inhibitor phosphoramidon was blocked by Pro-Ile or N-[1-(RS)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate, inhibitors of endopeptidase-24.16 and endopeptidase-24.15, respectively. However, Pro-Ile, at comparable concentrations, was shown to inhibit endopeptidase-24.11, challenging the validity of its use as a selective inhibitor of endopeptidase-24.16. The immunocytochemical and Triton X-114 phase-separation data implicate endopeptidase-24.11, rather than endopeptidase-24.16 or endopeptidase-24.15, as the major physiological somatostatin-degrading neuropeptidase in the striatum and hippocampus.

Animals↗

PubMed1992

Solid-liquid phase boundaries of lens protein solutions.

We report measurement of the solid-liquid phase boundary, or liquidus line, for aqueous solutions of three pure calf gamma-crystallin proteins: gamma II, gamma IIIa, and gamma IIIb. We also studied the liquidus line for solutions of native gamma IV-crystallin calf lens protein, which consists of 85% gamma IVa/15% gamma IVb. In all four proteins the liquidus phase boundaries lie higher in temperature than the previously determined liquid-liquid coexistence curves. Thus, over the range of concentration and temperature for which liquid-liquid phase separation occurs, the coexistence of a protein crystal phase with a protein liquid solution phase is thermodynamically stable relative to the metastable separated liquid phases. The location of the liquidus lines clearly divides these four crystallin proteins into two groups: those in which liquidus lines flatten at temperatures greater than 70 degrees C: gamma IIIa and gamma IV, and those in which liquidus lines flatten at temperatures less than 50 degrees C: gamma II and gamma IIIb. We have analyzed the form of the liquidus lines by using specific choices for the structures of the Gibbs free energy in solution and solid phases. By applying the thermodynamic conditions for equilibrium between the two phases to the resulting chemical potentials, we can estimate the temperature-dependent free energy change upon binding of protein and water into the solid phase.

Animals↗