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Results for “Phase Separation”

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PubMed1985

Effect of the vesicular stomatitis virus matrix protein on the lateral organization of lipid bilayers containing phosphatidylglycerol: use of fluorescent phospholipid analogues.

In order to investigate the mode of interaction of peripheral membrane proteins with the lipid bilayer, the basic (pI approximately 9.1) matrix (M) protein of vesicular stomatitis virus was reconstituted with small unilamellar vesicles (SUV) containing phospholipids with acidic head groups. The lateral organization of lipids in such reconstituted membranes was probed by fluorescent phospholipid analogues labeled with pyrene fatty acids. The excimer/monomer (E/M) fluorescence intensity ratios of the intrinsic pyrene phospholipid probes were measured at various temperatures in M protein reconstituted SUV composed of 50 mol % each of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG). The M protein showed relatively small effects on the E/M ratio either in the gel or in the liquid-crystalline phase. However, during the gel to liquid-crystalline phase transition, the M protein induced a large increase in the E/M ratio due to phase separation of lipids into a neutral DPPC-rich phase and DPPG domains presumably bound to M protein. Similar phase separation of bilayer lipids was also observed in the M protein reconstituted with mixed lipid vesicles containing one low-melting lipid component (1-palmitoyl-2-oleoylphosphatidylcholine or 1-palmitoyl-2-oleoylphosphatidylglycerol) or a low mole percent of cholesterol. The self-quenching of 4-nitro-2,1,3-benzoxadiazole (NBD) fluorescence, as a measure of lipid clustering in the bilayer, was also studied in M protein reconstituted DPPC-DPPG vesicles containing 5 mol % NBD-phosphatidylethanolamine (NBD-PE). The quenching of NBD-PE was enhanced at least 2-fold in M protein reconstituted vesicles at temperatures within or below the phase transition.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipid Bilayers↗

PubMed2001

Skin barrier structure and function: the single gel phase model.

A new model for the structure and function of the mammalian skin barrier is postulated. It is proposed that the skin barrier, i.e., the intercellular lipid within the stratum corneum, exists as a single and coherent lamellar gel phase. This membrane structure is stabilized by the very particular lipid composition and lipid chain length distributions of the stratum corneum intercellular space and has virtually no phase boundaries. The intact, i.e., unperturbed, single and coherent lamellar gel phase is proposed to be mainly located at the lower half of stratum corneum. Further up, crystalline segregation and phase separation may occur as a result of the desquamation process. The single gel phase model differs significantly from earlier models in that it predicts that no phase separation, neither between liquid crystalline and gel phases nor between different crystalline phases with hexagonal and orthorhombic chain packing, respectively, is present in the unperturbed barrier structure. The new skin barrier model may explain: (i) the measured water permeability of stratum corneum; (ii) the particular lipid composition of the stratum corneum intercellular space; (iii) the absence of swelling of the stratum corneum intercellular lipid matrix upon hydration; and (iv) the simultaneous presence of hexagonal and orthorhombic hydrocarbon chain packing of the stratum corneum intercellular lipid matrix at physiologic temperatures. Further, the new model is consistent with skin barrier formation according to the membrane folding model of Norlén (2001). This new theoretical model could fully account for the extraordinary barrier capacity of mammalian skin and is hereafter referred to as the single gel phase model.

Cholesterol↗

PubMed1997

Polyelectrolyte Complex Formation in Lamellar Liquid Crystalline Systems

The influence of polyelectrolytes on structure formation in liquid crystalline Na-dodecylsulfate/decanol/water systems was investigated by means of small angle X-ray diffraction, rheology, NMR spectroscopy, and microscopy. By adding Na-polyacrylate (PAA) into the mesophase, the one-phase region is left and phenomena of phase separation into a solvent-rich and a polymer/surfactant-rich phase occurs. By incorporating an anionic and cationic polyelectrolyte step by step the tendency of phase separation is increased drastically. The self-organization process can be regulated directly by varying the water content of the system. However, at a water content of 30% the properties of the resulting liquid crystal were changed drastically. X-ray diffraction shows a multitude of Bragg peaks, NMR shows a peak-splitting, and rheology shows a change from non-Newtonian to Newtonian-flow behavior. On the basis of the experimental results an ordered multilayer associate structure can be assumed.

Journal Article↗

PubMed2006

Structural and physical aspects of bacterial chromosome segregation.

Microscopic observations on the bacterial nucleoid suggest that the chromosome occurs in the cell as a compact nucleoid phase separate from the cytoplasm. Physical theory likewise predicts a phase separation, taking into consideration DNA supercoiling, nucleoid-binding proteins, and excluded-volume interactions between DNA and cytoplasmic proteins. Specific DNA loci, visualized as oriC-GFP spots in the densely packed nucleoid, exhibit a very low diffusion coefficient indicating that they are virtually immobile and may primarily be moved by overall length growth. Such gradual movement could be effectuated by replication, transertion (combined transcription, translation, and insertion of proteins), and actin- (MreB) directed surface synthesis. Differences in the movement and positioning of gene loci between Escherichia coli and Caulobacter crescentus are discussed. We propose that a low diffusion coefficient could explain the linear positioning of genes in the nucleoid and that differential transcriptional activity could induce different mobilities between either replichores (E. coli) or daughter strands (C. crescentus). The transertion process, possibly in combination with MreB cytoskeletal tracks, could overcome the compaction forces and move specific chromosomal regions and the nucleoid as a whole without invoking a dedicated mechanism.

Chromosome Segregation↗

PubMed2006

Processing and characterization of porous structures from chitosan and starch for tissue engineering scaffolds.

Natural biodegradable polymers were processed by different techniques for the production of porous structures for tissue engineering scaffolds. Potato, corn, and sweet potato starches and chitosan, as well as blends of these, were characterized and used in the experiments. The techniques used to produce the porous structures included a novel solvent-exchange phase separation technique and the well-established thermally induced phase separation method. Characterization of the open pore structures was performed by measuring pore size distribution, density, and porosity of the samples. A wide range of pore structures ranging from 1 to 400 microm were obtained. The mechanisms of pore formation are discussed for starch and chitosan scaffolds. Pore morphology in starch scaffolds seemed to be determined by the initial freezing temperature/freezing rate, whereas in chitosan scaffolds the shape and size of pores may have been determined by the processing route used. The mechanical properties of the scaffolds were assessed by indentation tests, showing that the indentation collapse strength depends on the pore geometry and the material type. Bioactivity and degradation of the potential scaffolds were assessed by immersion in simulated body fluid.

Amylopectin↗

PubMed2005

Thermodynamic behavior of poly(3-alkyl thiophene) blends: Equilibrium cocrystal formation and phase segregation.

The equilibrium cocrystal formation of poly(3-alkyl thiophene) (P3AT) blends has been studied by isothermal cocrystallization in a differential scanning calorimeter (DSC-7). The equilibrium melting points (T(m)0) of the cocrystals are measured using the Hoffman-Weeks extrapolation procedure. The equilibrium phase diagrams are of three different types: (a) concave upward, (b) linear, and (c) linear with phase separation at higher content of lower melting component. The phase diagram nature depends on the regioregularity difference and also on the difference in the number of carbon atoms in the pendent alkyl group of the components. The origin of biphasic nature of type "c" phase diagram has been explored from the glass transition temperature (Tg) measurement using a dynamic mechanical analyzer. The biphasic compositions show two glass transition temperatures (Tg) as well as two beta transition temperatures (T beta). The T(g)s of phase-separated regions correspond to almost the component values but the T(beta)s correspond to that of a lower (T beta) component value, and the other is higher than that of the higher (T beta) component value. Possible reasons are discussed from the interchain lamella thickness in the P3AT blends and molecular modeling using molecular mechanics program.

Journal Article↗

PubMed1999

Nematic ordering of rigid rods in a gravitational field.

The isotropic-to-nematic transition in an athermal solution of long rigid rods subject to a gravitational (or centrifugal) field is theoretically considered in the Onsager approximation. The new feature emerging in the presence of gravity is a concentration gradient that coupled with the nematic ordering. For rodlike molecules this effect becomes noticeable at centrifugal acceleration g approximately 10(3)-10(4) m/s(2), while for biological rodlike objects, such as tobacco mosaic virus, the effect is important even for normal gravitational acceleration conditions. Rods are concentrated near the bottom of the vessel, which sometimes leads to gravity induced nematic ordering. The concentration range corresponding to phase separation increases with increasing g. In the region of phase separation the local rod concentration, as well as the order parameter, follow a step function with height.

Journal Article↗

PubMed2001

Self-propulsion of cellular structures in chemically reacting mixtures.

An alternative model of phase-separating reactive mixtures is proposed. In this model both phase separation and chemical reactions simultaneously take place and a traveling coherent structure can be formed through a Hopf bifurcation at a finite wave number. Numerical simulations show that, depending on the parameters, either lamellar or hexagonal structures travel at constant speeds in two-dimensional systems.

Journal Article↗

PubMed1984

Sequence of centromere separation another mechanism for the origin of nondisjunction.

The most commonly accepted view about the origin of aneuploidy is that it is due to errors in meiotic division. However, its rare occurrence makes it difficult to explain recurrent births of trisomic children to some parents. This problem causes more serious concern when one accepts that an abnormal (n + 1 or n - 1) sperm would enter fertilization by overriding thousands, or even millions, of normal haploid sperms. Also, the failure of aneuploidy to be induced in the offspring of mammals treated with mutagens raises questions about the effectiveness of the accepted mode of origin of errors. Current concepts also do not explain why one observes more errors of meiotic I, than of meiotic II, origin. It is known that most chromosomes separating at meta-anaphase junction in mitosis follow a nonrandom, genetically controlled sequence of separation. The present proposal makes use of out-of-phase separation of a rare chromosome, like premature separation in mitosis of the X in elderly humans or of an 18 in parents of trisomy 18 children. The suggestion is made that such out-of-phase separation results in aneuploid cell lines by total failure of the centromere to separate or by it separating too early, before the spindle is formed. The prematurely separating centromeres, it appears, do not attach to spindle fibers and hence cause nondisjunction. Such nondisjunction in embryonic stages will produce apparently normal individuals with mosaicism in somatic and/or gametic tissue. An individual carrying mosaicism in gonadal tissue will produce a large number of abnormal gametes, one of which may have a reasonable chance of entering fertilization. This mode of origin of aneuploidy takes care of all questions raised above and finds support in the data available in the literature. Several of the suggestions made in the hypothesis are easily testable.

Adult↗

PubMed2026

Mediator at the Helm: Coordinating transcription and biomolecular condensates in hematopoiesis.

Hematopoiesis relies on precisely coordinated transcriptional programs that balance stem cell self-renewal, lineage commitment, and terminal differentiation. Central to this regulation is the Mediator complex, a large multi-subunit transcriptional co-regulator that integrates signals from transcription factors and chromatin regulators to control RNA polymerase Ⅱ (Pol Ⅱ) activity. The dynamic and modular composition of Mediator enables context-dependent transcriptional outputs, while individual subunits can exert specialized regulatory functions during hematopoietic lineage specification, thereby contributing to cell-fate-specific transcriptional outputs. Recent advances further reveal that transcriptional regulation is shaped by the spatial organization of regulatory machinery with biomolecular condensates formed through liquid-liquid phase separation (LLPS), particularly at super-enhancers. In this emerging framework, Mediator functions not only as a transcriptional integrator but also as a key coordinator of transcriptional machinery within condensates at cell-fate-related gene loci. In this chapter, we summarize how distinct Mediator subunits confer specific modes of transcriptional regulation and discuss how the interplay between Mediator and phase-separated condensates shapes transcriptional control during hematopoiesis. We highlight how specific subunits, including MED1 and MED26, participate in distinct regulatory modes in erythropoiesis, spanning super-enhancer-driven transcriptional activation, progenitor expansion, and condensate-associated mechanisms that influence Pol Ⅱ pausing and global transcription repression during terminal differentiation. Together, these findings support a model in which Mediator integrates transcriptional regulation with nuclear organization through condensate-mediated mechanisms, providing a conceptual framework for understanding hematopoietic cell fate decisions and transcriptional dysregulation in hematological diseases.

Hematopoiesis↗

PubMed2026

Biomolecular Condensates Integrate Transcriptional and Epigenetic Responses to Hypoxia.

Hypoxia is a defining feature of physiological stress and the core of solid tumors, where aberrant vascularization limits oxygen delivery; cells respond through mechanisms that extend beyond the canonical stabilization of hypoxia-inducible factors (HIFs). Recent studies suggest that hypoxia can promote the formation of specific biomolecular conden-sates, membraneless compartments generated through liquid-liquid phase separation in which regulatory proteins and RNAs become locally enriched at genomic regions, while chromatin mainly serves as an organizational scaffold. Transcription factors, the coacti-vators p300/CBP, Mediator, and BRD4, chromatin-modifying enzymes, and architectural RNAs such as NEAT1 and MALAT1 partition into these compartments, and their con-densation can help reorganize local chromatin structure and enhancer-promoter interac-tions. Because molecular oxygen is a shared co-substrate for the Jumonji-C histone demethylases and the ten-eleven translocation (TET) DNA dioxygenases, hypoxia reshapes histone methylation and DNA methylation in parallel, and readers that bridge these marks, including UHRF1, may participate in condensate-associated chromatin regulation. Hypoxia-driven condensation of ZHX2 rewires enhancer-promoter contacts and higher-order genome architecture, influencing cell identity, stemness, and metastatic potential, and Polycomb condensates represent another candidate epigenetic compartment that may be influenced by hypoxic signaling. These processes may be particularly important in cancer, where chronic hypoxia provides a sustained stimulus for condensate formation and epigenetic remodeling. Together, these findings support a model in which phase separation and epigenetic reprogramming are not separate layers but one integrated response to low oxygen, offering opportunities to target maladaptive condensates in disease.

Epigenesis, Genetic↗

PubMedDate not supplied

Reversed-phase HPLC separation of human serum employing a novel saw-tooth gradient: toward multidimensional proteome analysis.

A novel reversed-phase (RP-) HPLC gradient profile applicable to multidimensional separations of complex protein mixtures is reported. This gradient profile elutes small numbers of proteins from the RP-HPLC column in discrete intervals while minimizing the amount of band broadening between elution intervals and maintaining constant flow through the HPLC column. Eluting the proteins in discrete intervals eases the instrumental requirements necessary for performing multidimensional separations and can be used to aid in the collection of well-defined fractions. The saw-tooth gradient was applied to the successful isolation of albumin from less abundant proteins in whole human serum and provides adequate separation of proteins in a low-molecular weight (LMW) fraction of human serum with resolution comparable to that achieved using a typical linear gradient profile.

Blood Proteins↗

PubMed1975

The antagonistic effect of an inhalation anesthetic and high pressure on the phase diagram of mixed dipalmitoyl-dimyristoylphosphatidylcholine bilayers.

Several workers have shown that phase transition-related changes in membrane lipids have a profound effect on membrane-solvated protein function. This phase transition temperature dependence has been explained as resulting from the formation of lateral phase separations within the membrane bilayer. The present study demonstrates that a clinical concentration of an inhalation anesthetic produces changes in both the phase transition temperature of pure lipid bilayers and the lateral phase separation temperature of mixed dipalmitoyl- and dimyristoylphosphatidylcholine bilayers of a magnitude sufficient to influence protein function. It is further shown that pressure is able to antagonize the effect of the anesthetic on these transition temperatures. It is proposed that anesthetic action within nerve membranes may be the result of changes in the lateral phase separation-controlled environment of the membrane-solvated proteins essential to nerve function.

Anesthesia, Inhalation↗

PubMed2005

Interaction of fluoxetine with phosphatidylcholine liposomes.

Fluoxetine (Prozac) is one of the latest of a new generation of antidepressants, approved by FDA in 2002. The interactions of fluoxetine with multilamellar liposomes of pure phosphatidylcholine (PC) or containing cholesterol 10% molar were studied as a function of the lipid chain lengths, using differential scanning calorimetry and spin labelling EPR techniques. The DSC profiles of the gel-to-fluid state transition of liposomes of DMPC (C14:0) are broadened and shifted towards lower temperatures at increasing dopant concentrations and, with less than 10% fluoxetine, any detectable transition is destroyed. The broadened profiles and the lowered transition temperatures demonstrate that both the size and the packing of the cooperative units undergoing the transition are modified by fluoxetine, leading to a looser and more flexible bilayer. No phase separation was observed. The effects of fluoxetine on the thermotropic phase behaviour of DPPC (C16:0) and, even more, of DSPC (C18:0) are different from that of DMPC. In fact, in the former cases, two peaks appeared at increasing dopant concentrations, suggesting the occurrence of a phase separation phenomenon, which is a sign of a binding of fluoxetine in the phosphate region. In cholesterol containing membranes, fluoxetine, even at low concentrations, leads to a general corruption of the membrane, both in terms of packing and cooperativity, and the formation of any new phase is no longer observable. EPR spectra reflect the disordered motion of acyl chains in the bilayer. It was found that fluoxetine lowers the order of the lipid chains mainly in correspondence of the fifth carbon position of SASL, indicating a possible accumulation near the interfacial region.

Binding Sites↗

PubMed2006

Isotropic-nematic phase transition of nonaqueous suspensions of natural clay rods.

A novel model system for studying the behavior of hard colloidal rods is presented, consisting of sterically stabilized particles of natural sepiolite clay. Electron microscopy and scattering results confirmed that the organophilic clay particles were individual, rigid rods when dispersed in organic solvents. With a length-to-diameter ratio of approximately 27, the particles showed nematic ordering for volume fractions phi > 0.06. Polarizing microscopy revealed that the phase separation process involved nucleation, growth, and coalescence of nematic domains. The phase volumes and particle concentrations in the coexisting phases were determined. The dependence of these quantities on the total concentration of the suspension agrees well with Onsager's [Ann. N. Y. Acad. Sci. 51, 627 (1949)] isotropic-nematic phase transition theory extended to bidisperse and polydisperse rod systems, and with previous experimental results for rigid rodlike particles. Particle size distributions were obtained by analyzing transmission electron microscopy images. A significant fractionation with respect to rod length (but not diameter) was observed in the coexisting isotropic and nematic phases. The relative polydispersity of both daughter phases was distinctly smaller than that of the parent suspension. The phase behavior of these daughter fractions agrees well with the predictions for hard spherocylinders of corresponding aspect ratios. An isotropic-nematic-nematic phase equilibrium was seen to develop in phase separated samples after 1 month standing and is ascribed to the effect of polydispersity and possibly gravity. The second nematic phase appearing is dominated by very long rods.

Journal Article↗

PubMed2005

Oligomerization and phase transitions in aqueous solutions of native and truncated human beta B1-crystallin.

Human betaB1-crystallin is a major eye-lens protein that undergoes in vivo truncation at the N-terminus with aging. By studying native betaB1 and truncated betaB1DeltaN41, which mimics an age-related in vivo truncation, we have determined quantitatively the effect of truncation on the oligomerization and phase transition properties of betaB1 aqueous solutions. The oligomerization studies show that the energy of attraction between the betaB1DeltaN41 proteins is about 10% greater than that of the betaB1 proteins. We have found that betaB1DeltaN41 aqueous solutions undergo two distinct types of phase transitions. The first phase transition involves an initial formation of thin rodlike assemblies, which then evolve to form crystals. The induction time for the formation of rodlike assemblies is sensitive to oligomerization. The second phase transition can be described as liquid-liquid phase separation (LLPS) accompanied by gelation within the protein-rich phase. We refer to this process as heterogeneous gelation. These two phase transitions are not observed in the case of betaB1 aqueous solutions. However, upon the addition of poly(ethylene glycol) (PEG), we observe heterogeneous gelation also for betaB1. Our PEG experiments allow us to estimate the difference in phase separation temperatures between betaB1 and betaB1DeltaN41. This difference is consistent with the increase in energy of attraction found in our oligomerization studies. Our work suggests that truncation is a cataractogenic modification since it favors protein condensation and the consequent formation of light scattering elements, and highlights the importance of the N-terminus of betaB1 in maintaining lens transparency.

Circular Dichroism↗

PubMed2000

Novel use of a dual-zone restricted access sorbent: normal-phase solid-phase extraction separation of methyl oleate from polynuclear aromatic hydrocarbons stemming from semi-permeable membrane devices.

A normal-phase solid-phase extraction (SPE) method was developed utilizing a dual-zone restricted-access sorbent to separate methyl oleate from the 16 US Environmental Protection Agency priority-pollutant polynuclear aromatic hydrocarbons in a hexane matrix. This technique represents a new development in SPE methodology expanding the limited number of available normal-phase SPE sorbents. While based on a specific application (removal of methyl oleate from semipermeable membrane device extracts), this cleanup method could easily be adapted for other uses requiring the removal/isolation of methyl oleate and potentially related compounds.

Chromatography, Gas↗

PubMed2006

Novel chiral selectors anchored on polydimethylsiloxane as stationary phases for separation of derivatized amino acid enantiomers by capillary gas chromatography.

Six chiral selectors of S-(-)-t-Leu-cyclopropylamide, S-(-)-t-Leu-cyclopentylamide, S-(-)-t-Leu-cyclohexylamide, S-(-)-t-Leu-cycloheptylamide, S-(-)-t-Leu-cyclooctylamide, S-(-)-t-Leu-cyclododecylamide have been prepared and anchored individually through amide bonding to a polydimethylsiloxane functionalized with 2,2,2-trifluoroethyl ester groups by way of nucleophilic displacement reaction. The resulting chiral polysiloxanes have been provided as stationary phases for the separation of amino acid enantiomers by capillary GC. Amino acids were derivatized into N(O)-trifluoroacetyl isopropyl esters. Especially, polydimethylsiloxane anchored with S-(-)-t-Leu-cyclooctylamide was found to be efficient for the separation of aspartic acid (Asp) enantiomers. The method was applied to the estimation of ages from the extent of Asp racemization in human dentines.

Adult↗