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PubMed1970

Phase separation in solutions of noradrenaline and adenosine triphosphate: influence of bivalent cations and drugs.

1. From aqueous solutions of biogenic amines, such as noradrenaline plus adenosine triphosphate (ATP), a second liquid phase spontaneously separates in the presence of small amounts of bivalent cations such as calcium. This separation is reversible and temperature-dependent; the concentration of amine and ATP in the bottom phase is several times higher than in the supernatant.2. Analytical ultracentrifugation provides evidence that the second phase consists of high molecular weight aggregates of the amine and ATP.3. The separated second phase of the noradrenaline-ATP system dissolves isothermally on addition of tyramine and amphetamine which in vivo are known to liberate biogenic monoamines and which have a low tendency to aggregate with ATP. The apparent molecular weights of noradrenaline-ATP aggregates are decreased by tyramine and amphetamine. Dopamine does not diminish the second phase and it can also form aggregates of high molecular weight with ATP.4. Bivalent cations in high concentrations diminish or abolish the separation of a second phase.5. Small amounts of reserpine affect phase separation.6. It is concluded that the physico-chemical properties of aggregates of biogenic amines with ATP may be of importance for understanding the storage and release of the amines in vivo.

Adenosine Triphosphate↗
PubMed2000

Preparation of macroporous poly(2-hydroxyethyl methacrylate) hydrogels by enhanced phase separation.

Macroporous poly(2-hydroxyethyl methacrylate) (p(HEMA)) hydrogels were prepared in the presence of a 0.3-0.7 M NaCl solution. The pore morphology of the p(HEMA) hydrogels was dependent on the concentration of NaCl for a constant monomer solution to aqueous solution ratio. Swelling studies showed an increase in equilibrium water content and hydrogel porosity as the NaCl concentration in the polymerization medium increased from 0 to 0.7 M. The equilibrium water content, however, decreased as the NaCl concentration in the swelling medium increased. The frozen water content increased and non-frozen water decreased with an increase in the NaCl concentration in the polymerization medium. Mechanical testing indicated that the elastic modulus of the hydrogels was not affected by the increased porosity until the pores became interconnected. These data suggest that the addition of NaCl to the polymerization medium results in a multi-phase separation during fabrication that produces macroporous hydrogels of controlled morphology.

Calorimetry, Differential Scanning↗
PubMed2006

Phase separation in charge-stabilized colloidal suspensions: influence of nonlinear screening.

The phase behavior of charge-stabilized colloidal suspensions is modeled by a combination of response theory for electrostatic interparticle interactions and variational theory for free energies. Integrating out degrees of freedom of the microions (counterions, salt ions), the macroion-microion mixture is mapped onto a one-component system governed by effective macroion interactions. Linear response of microions to the electrostatic potential of the macroions results in a screened-Coulomb (Yukawa) effective pair potential and a one-body volume energy, while nonlinear response modifies the effective interactions [A. R. Denton, Phys. Rev. E 70, 031404 (2004)]. The volume energy and effective pair potential are taken as input to a variational free energy, based on thermodynamic perturbation theory. For both linear and first-order nonlinear effective interactions, a coexistence analysis applied to aqueous suspensions of highly charged macroions and monovalent microions yields bulk separation of macroion-rich and macroion-poor phases below a critical salt concentration, in qualitative agreement with predictions of related linearized theories [R. van Roij, M. Dijkstra, and J.-P. Hansen, Phys. Rev. E 59, 2010 (1999); P. B. Warren, J. Chem. Phys. 112, 4683 (2000)]. It is concluded that nonlinear screening can modify phase behavior but does not necessarily suppress bulk phase separation of de-ionized suspensions.

Journal Article↗
PubMed2006

Structure of silica xerogels synthesized with organoalkoxysilane co-reactants hints at multiple phase separation.

The microstructure of hybrid silica xerogels synthesized by the base-catalyzed polymerization of tetraethoxysilane (TEOS) in ethanol in the presence of 3-aminopropyltriethoxysilane (AES) and of 3-(2-aminoethylamino)propyltrimethoxysilane (EDAS) as co-reactants, and dried in subcritical conditions, is analyzed. A thorough structural characterization of the samples is performed combining nitrogen adsorption, small-angle X-ray scattering (SAXS), and transmission electron microscopy coupled with digital image analysis. The use of these methods shows that, for both co-reactants, the xerogels are made of macropores supported by filaments, with each filament being formed of smaller structures. The quantitative impact of the additive on each structural level is assessed. The data are compared with a previous time-resolved SAXS study conducted during the formation of the gels (J. Phys. Chem. B 2004, 108, 8983-8991). The results are analyzed in the framework of a double phase separation model.

Journal Article↗
PubMed1982

Phase separation induced by melittin in negatively-charged phospholipid bilayers as detected by fluorescence polarization and differential scanning calorimetry.

Interactions between melittin and a variety of negatively-charged lipid bilayers have been investigated by intrinsic fluorescence, fluorescence polarization of 1,6-diphenylhexatriene and differential scanning calorimetry. (1) Intrinsic fluorescence of the single tryptophan residue of melittin shows that binding of this peptide to negatively-charged phospholipids is directly related to the surface charge density, but is unaffected by the physical rate of lipids, fluid or gel, single-shell vesicles or unsonicated dispersions. (2) Changes in the thermotropic properties of negatively-charged lipids upon melittin binding allow to differentiate two groups of lipids: (i) A progressive disappearance of the transition, without any shift in temperature, is observed with monoacid C14 lipids such as dimyristoylphosphatidylglycerol and -serine (group 1). (ii) With a second group of lipids (group 2), a transition occurs even at melittin saturation, and two transitions are detected at intermediate melittin content, one corresponding to remaining unperturbed lipids, the other shifted downward by 10-20 degrees C. This second group of lipids is constituted by monoacid C16 lipids, dipalmitoylphosphatidylglycerol and -serine. Phosphatidic acids also enter this classification, but it is the net charge of the phosphate group which allows to discriminate: singly charged phosphatidic acids belong to group 2, whereas totally ionized ones behave like group 1 lipids, whatever the chain length. (3) It is concluded that melittin induces phase separations between unperturbed lipid regions which give a transition at the same temperature as pure lipid, and peptide rich domains in which the stoichiometry is 1 toxin per 8 phospholipids. The properties of such domains depend on the bilayer stability: in the case of C16 aliphatic chains and singly charged polar heads, the lipid-peptide domains have a transition at a lower temperature than the pure lipid. With shorter C14 chains or with two net charges by polar group, the bilayer structure is probably totally disrupted, and the new resulting phase can no longer lead to a cooperative transition.

Anions↗
PubMed2002

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

Thermotropic and structural effects of ethanol on phosphatidylserine (PS) membranes containing up to 0.4 mol fraction cholesterol were investigated by differential scanning calorimetry, X-ray diffraction and fluorescence spectroscopy. It was found that in the presence of cholesterol, 10% (v/v) added ethanol depresses the melting temperature of the phospholipid by approximately 2 degrees C, similar to what was observed in the absence of cholesterol. Below the melting temperature the progressive disordering effect of added cholesterol is weakly enhanced by the presence of ethanol. In the liquid crystalline state, the marked decrease in the thickness of the bilayer which ethanol causes in the absence of cholesterol (Chem. Phys. Lipids 92 (1998) 127), is also observed in its presence. We conclude that, in contrast to what has been observed for zwitterionic phospholipids, high concentrations of cholesterol do not diminish the interaction of ethanol with PS membranes. With addition of 10% (v/v) ethanol, crystalline cholesterol diffraction, an indication of phase separation of the sterol, appears at mol fraction cholesterol 0.34, as compared to 0.3 in the absence of ethanol (Chem. Phys. Lipids 92 (1998) 71).

Animals↗
PubMed1997

Vacuolar cytoplasmic phase separation in cultured mammalian cells involves the microfilament network and reduces motional properties of intracellular water.

Hep-2, human epithelial carcinoma cells, and human foreskin fibroblasts (FF9 and FF13) were exposed to either an ultrafiltrate (< 50 kD) of human sera or the weak base, procaine hydrochloride, to induce reversible cytoplasmic vacuolization. The formation of vacuoles was shown not to be due to imbibition of medium. Ultrastructural details obtained from various stages of vacuole formation were compared. In both cases of induction vacuoles were irregular and often appeared membraneless, with little in the way of electron-dense content. They started to form in the perinuclear cytoplasm and progressed towards the periphery. Osmotic stress was not involved since mitochondria remained normal throughout a vacuolization episode. Vacuoles were often seen in close contact with filamentous structures, and this association remained detectable at late stages of the phenomenon. Fluorescent visualization of F-actin confirmed that the vacuoles were frequently bordered by microfilaments. No major metabolic impairment was apparent in vacuolized cells as judged by protein synthesis measurements, but nuclear fluorescence (DNA content) and forward light scatter (nuclear volume) by flow cytometric analysis suggested late S phase and G2 retardation. 1H-nmr relaxation measurements indicated intracellular water restricted in motional characteristics in vacuolized cells. The possibility of a restricted cytoplasmic phase separation as part of a transient adaptation response is raised, and a hypothesis to explain the findings is discussed.

Actin Cytoskeleton↗
PubMed2002

A novel porous cells scaffold made of polylactide-dextran blend by combining phase-separation and particle-leaching techniques.

In this study, a kind of biodegradable material was developed by blending polylactide (PLA) with natural biodegradable dextran, and a novel sponge-like scaffold made of it was fabricated thereof using solvent-casting and particle-leaching technique. To obtain a uniform blend of PLA and dextran by simple solvent-casting method, hydroxyls of dextran should be protected via trimethylsilyl (TMS) groups to make dextran soluble in organic solvents. Benzene was found among the few solvents that could dissolve this TMS-protected dextran (TMSD) well, however, it was not a good solvent for PLA. Therefore, a homogeneous mixed solution of PLA and TMSD could be obtained when a mixture of dichloroform (DCM) and benzene (v/v = 6/4) was used. By this technique, PLA-dextran blend films and even PLA films were observed a microporous structure (pore size around 5-10 microm) formation throughout the films under scanning electron microscope (SEM). Scaffolds that were prepared by dissolving PLA and TMSD in mixed solvent of DCM and benzene and using salt as porogen, were observed the formation of micropores (pore size around 5-10 microm) in the cellular walls of macropores (pore size around 100-200 microm). This microporous structure was closely related to the phase separation occurring during films or foams formation, which was mainly due to the different solubility of PLA and TMSD in benzene, as well as the different evaporation rates of DCM and benzene. In comparison with PLA, the surface and bulk hydrophilicity of PLA-dextran blend films or foams were significantly improved after the TMS groups were removed in methanol, and the results of cell culture on these polymeric substrates exhibited an enhancement on cell attachment and proliferation.

3T3 Cells↗
PubMed1987

Correlation of mammalian cell killing by heat shock to intramembranous particle aggregation and lateral phase separation using fluorescence-activated cell sorting.

Heat shock induces a dose-dependent increase in the fraction of Chinese hamster ovary cells that stain the fluorescent membrane probe N-epsilon-dansyl-L-lysine (DL). Dansyl lysine has previously been shown to select for cholesterol-free membrane domains in phospholipid liposomes. We found that the fraction of cells excluding DL could be closely correlated to cell survival as assayed by 37 degrees C incubation following heat treatment. Fluorescence-activated cell sorting indicated that essentially all of the DL-staining cells were nonviable. Freeze fracture electron microscopy of sorted cells showed that all the cells that stained with DL also had highly suggested intramembranous particle (IMP) aggregation while DL-excluding cells did not. Furthermore, IMP aggregation was shown to occur immediately after heat shock and to precede DL staining. Treatment with other membrane-active agents such as ethanol, amphotericin B, filipin, procaine, and lidocaine (i) induced DL staining that was closely correlated to survival, (ii) induced dramatic cytotoxic sensitization when combined with heat, and (iii) induced aggregated IMPs at relevant cytotoxic concentrations. Several nonmembrane-active agents were examined; none induced DL staining, dramatic cytotoxic sensitization, or IMP aggregation. These results raise the possibility that heat shock inactivates mammalian cells primarily via nonspecific aggregation and denaturation of membrane proteins resulting in a lateral phase separation of membrane components, including the generation of phospholipid domains.

Animals↗
PubMed1988

Isolation of flagella from the archaebacterium Methanococcus voltae by phase separation with Triton X-114.

The flagella of Methanococcus voltae were isolated by using three procedures. Initially, cells were sheared to release the filaments, which were purified by differential centrifugation and banding in KBr gradients. Flagella were also prepared by solubilization of cells with 1% (vol/vol) Triton X-100 and purified as described above. Both of these techniques resulted in variable recovery and poor yield of flagellar filaments. Purification of intact flagella (filament, hook, and basal body) was achieved by using phase transition separation with Triton X-114. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified flagella revealed two major proteins, with molecular weights of 33,000 and 31,000. This result indicates the likely presence of two flagellins. The filament had a diameter of 13 nm. The basal structure consisted of a small knob, while a slight thickening of the filament immediately adjacent to this area was the only evidence of a hook region. Flagella from three other Methanococcus species were isolated by this technique and found to have the same ultrastructure as flagella from M. voltae. Isolation of flagella from three eubacteria and another methanogen (Methanospirillum hungatei [M. hungatii]) by the phase separation technique indicated that the detergent treatment did not affect the structure of basal bodies. Intact ring structures and well-differentiated hook regions were apparent in each of these flagellar preparations.

Archaea↗
PubMed2006

Preparation of glycerol dimethacrylate-based polymer monolith with unusual porous properties achieved via viscoelastic phase separation induced by monodisperse ultra high molecular weight poly(styrene) as a porogen.

The preparation of polymer-based monolith capillary was examined by the use of glycerol dimethacrylate (GDMA) as monomer and monodisperse standard polystyrene (PS) solution in chlorobenzene as porogen. Poly-GDMA monoliths were prepared in situ in test tubes with standard PS having the variety of molecular weight (defined as Mw hereafter) from 50,000 to 3,840,000, and their morphology was compared to that of poly-GDMA monolith prepared in situ with a poor porogenic solvent of GDMA. According to scanning electron micrograph (SEM) observation, the structure of poly-GDMA monolith prepared in situ with toluene as a poor porogenic solvent showed a typical agglomerated globular structure, whereas the morphology of poly-GDMA monolith prepared in situ with the polymer (PS) porogenic solution was transformed from the aggregated globule form to three dimensionally (3D) continuous skeletal structure with the increase of Mw of standard PS utilized. Along with this morphological transformation or change, in the case of poly-GDMA monolith prepared in situ with ultra high Mw standard PS porogenic solution, the pore size distribution showed a sharp bimodal distribution, with one peak being located around 4 nm in the mesopore range (2-50 nm) and the other peak located around 1-2 microm in the macropore range (>50 nm), respectively. The poly-GDMA capillaries were prepared in situ with toluene, low Mw (50,000, 600,000) PS solution in chlorobenzene and the above mentioned ultra high Mw PS solution in chlorobenzene as a porogen, respectively, and measured by mu-HPLC with benzene and n-alkyl phenyl ketone as solutes for the evaluation in aqueous methanol (MeOH/H(2)O = 50/50-80/20, v/v). The permeability of capillaries prepared in situ with ultra high Mw standard PS polymer porogenic solution was much larger, compared to those of the capillaries prepared in situ with low Mw standard PS polymer porogenic solution or with toluene as porogen. On the other hand, the column efficiency was better in the case of the capillary prepared in situ with the ultra high Mw PS solution than in the latter capillaries. Those observations indicated that the ultra high Mw standard PS polymer porogenic solution should delay dynamically the phase separation of polymerizing mixture because of its visco-elasticity and should contribute to the creation of three dimensionally continuous skeletal monolith structure better to afford high separation efficiency.

Acetophenones↗
PubMed2005

Possible mechanisms for early and intermediate stages of sperm chromatin condensation patterning involving phase separation dynamics.

During spermiogenesis in some internally fertilizing molluscs and insects, the post-meiotic spermatid nucleus develops via a sequence of complex patterns of the nuclear contents (chromatin and nucleoplasm) on the way to final chromatin condensation. We have examined the TEM data on these sequences for three species: Philaenus spumarius(a homopteran insect), Murex brandaris (a gastropod mollusc), and Eledone cirrhosa(a cephalopod mollusc). For each of these, spatially quantitative study reveals a constant spacing between pattern repeats through changes from granular to fibrillar to lamellar pattern, followed finally by a shrinkage of the spacing. Therefore we distinguish a "patterning" stage followed by a "condensation" stage. The former appears to demand a dynamic explanation, because there is no sign of structural connections to establish the part of the spacing that crosses the nucleoplasm. We consider types of dynamic mechanism, and show that for "nanostructural" dimensions (tens of nanometers as pattern spacing) reaction-diffusion dynamics are quite inappropriate, but that separation of two fluid phases by a mechanism similar to what is known as "spinodal decomposition" is a very attractive possibility.

Animals↗
PubMed1994

Phase separation of biomolecules in polyoxyethylene glycol nonionic detergents.

The advantage of aqueous two-phase systems based on polyoxyethylene detergents over other liquid-liquid two-phase systems lies in their capacity to fractionate membrane proteins simply by heating the solution over a biocompatible range of temperatures (20 to 37 degrees C). This permits the peripheral membrane proteins to be effectively separated from the integral membrane proteins, which remain in the detergent-rich phase due to the interaction of their hydrophobic domains with detergent micelles. Since the first reports of this special characteristic of polyoxyethylene glycol detergents in 1981, numerous reports have consolidated this procedure as a fundamental technique in membrane biochemistry and molecular biology. As examples of their use in these two fields, this review summarizes the studies carried out on the topology, diversity, and anomalous behavior of transmembrane proteins on the distribution of glycosyl-phosphatidylinositol-anchored membrane proteins, and on a mechanism to describe the pH-induced translocation of viruses, bacterial endotoxins, and soluble cytoplasmic proteins related to membrane fusion. In addition, the phase separation capacity of these polyoxyethylene glycol detergents has been used to develop quick fractionation methods with high recoveries, on both a micro- and macroscale, and to speed up or increase the efficiency of bioanalytical assays.

Animals↗
PubMed1994

Diffusion and chemical reactions in phase-separated membranes.

The biological membrane may be viewed as a two-dimensional solvent system, the lipid bilayer, in which the membrane components are either dissolved (intrinsic) or to the surface of which they are adsorbed (extrinsic). The solvent bilayer is made up of a large number of lipid chemical species derived from a few lipid classes. Experience with model systems has shown that in mixed lipid bilayers immiscibility of components is the rule rather than the exception. This suggests that the bilayer in a biological membrane is not a homogenous two-dimensional fluid but rather a heterogenous system consisting of a mosaic of co-existing phase domains in which the phases differ both chemically and physically from each other. A consequence of this is the physical separation of membrane components, including proteins, based on their phase solubility. The percolation in such a phase-separated system then determines the range over which free lateral diffusion is possible and bimolecular reactions can occur. Phase percolation and long-range translational diffusion have been studied in model systems using the fluorescence recovery after photobleaching (FRAP) technique, and theoretical work shows that bimolecular reaction yields can be seriously reduced in phase-separated membranes. Transitions between percolating and non-percolating states in biomembranes is proposed as a potential trigger mechanism in the control of membrane physiology.

Cell Membrane↗
PubMed1985

A lipid-phase separation model of low-temperature damage to biological membranes.

An hypothesis is proposed to explain the damage caused to biological membranes exposed to low temperatures. The thesis rests on the general observation that the lipid components of most membranes are heterogeneous and undergo phase transitions from gel-phase lamellae to liquid-crystalline lamellae and some to a non-lamellar, hexagonal-II phase over a wide range of temperatures. As a consequence of these phase transitions the lateral distribution of the lipids characteristic of the growth temperature is disturbed and redistribution takes place on the basis of the temperature at which phase transitions occur. When membranes are cooled, first the non-lamellar forming lipids pass through a transition to a fluid lamellar phase and are miscible with bilayer-forming lipids into which they diffuse. On further cooling the high-melting-point lipids begin to crystallize and separate into a lamellar gel phase, in the process excluding the low-melting point lipids and intrinsic proteins. The lipids in these remaining regions form a gel phase at the lowest temperature. It is suggested that, because the non-lamellar lipids tend to undergo a liquid-crystalline to gel-phase transition at higher temperatures than lamellar-forming lipids, these will tend to phase separate into a gel phase domain rich in these lipids. Damage results when the membrane is reheated, whereupon the hexagonal-II-forming lipids give rise to non-lamellar structures. These probably take the form of inverted micelles sandwiched within the lipid bilayer and they completely destroy the permeability barrier properties of the membrane. The model is consistent with the phase behavior of membrane lipids and the action of cryoprotective agents in modifying lipid phase properties.

Cold Temperature↗
PubMed2004

Development and assessment of a miniaturised centrifugal chromatograph for reversed-phase separations in micro-channels.

This paper describes the micro-fabrication and preliminary assessment of a miniature polydimethylsiloxane (PDMS) device for performing rapid, parallel liquid phase chromatographic separations driven by centrifugal force in microchannels. Device components include a main separating channel, into which a high performance liquid chromatography (HPLC) particulate stationary phase was packed under pressure by application of centrifugal force, in addition to solvent and sample reservoirs. Also described are methods for sealing such devices based upon partial polymerisation of PDMS. The mobile phase flow rate through a typical device was measured and several important chromatographic parameters determined from a test separation. An expression describing mobile phase flow through packed channels was also developed, based upon work on liquid flow in open micro-channels. Good agreement between predicted and measured flow rates were observed. Some predictions for potential uses of such devices and possibilities for further miniaturisation are discussed.

Journal Article↗