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Ibuprofen, a putative anti-cataract drug, protects the lens against cyanate and galactose.

Cataract, the major cause of blindness world-wide, may be caused partly by modification of lens proteins by carbamylation and non-enzymic glycosylation (glycation) in some patients. Aspirin has been found to protect against these modifications and to prevent cyanate-induced opacification occurring in whole rate lenses. Ibuprofen is an aspirin-like anti-inflammatory drug which appeared as a protective factor against cataract in an Oxford case-control study. The binding of cyanate, galactose and glucose 6-phosphate to lens proteins, and the effect of ibuprofen on this reaction was investigated, as was cyanate-induced opacification in whole rat lenses. Labelled metabolite was incubated with bovine lens homogenate in the presence and absence of ibuprofen, and the incorporation of label into the lens homogenate was followed. Simultaneous and preincubation experiments were performed. Intact rat lenses were incubated in culture medium with and without cyanate and ibuprofen. The phase separation temperature was noted as the temperature at which opacity first appeared on cooling. Cyanate, galactose and glucose 6-phosphate bind progressively to lens proteins. Simultaneous incubation with ibuprofen reduces cyanate and galactose binding but not glucose 6-phosphate. Ibuprofen protects against opacities due to cyanate-induced phase separation. Ibuprofen has protected against cataract in the models of cataractogenesis in this study. It appears to have a different mechanism of action from that of aspirin. These studies provide some support for the idea, based on epidemiological findings, that ibuprofen might be a useful anti-cataract drug.

Animals↗

Development of protamine-bonded phase for separation of saccharides in liquid chromatography.

A protamine-bonded polymer gel was synthesized by passing this protein through an activated carbamate polyamine polymer gel layer. This new packing material was suitable for versatile separation of monosaccharides, oligosaccharides, sugar alcohols and uronic acids in liquid chromatography. The column packed with this gel could be operated at room temperature and gave excellent recovery for the reducing monosaccharides such as 2-deoxyribose, 2-deoxyglucose, ribose and mannose for which the conventional alkylamine-bonded phase column gave poor recovery. This column was used for the analysis of saccharides in urine using a post-column reaction detection system.

Amino Acid Sequence↗

Solid-fluid and solid-solid phase equilibrium in a model of n-alkane mixtures.

Solid-fluid and solid-solid phase equilibrium for binary mixtures of hard sphere chains modeling n-hexane, n-heptane, and n-octane has been calculated using Monte Carlo computer simulations. Thermodynamic integration was used to calculate the Gibbs free energy and chemical potentials in the solid and fluid phases from pure component reference values. A multiple stage free energy perturbation method was used to calculate the composition derivative of the Gibbs free energy. Equation of state and free energy data for the fluid phase indicate ideal solution behavior. Nonideality is much more significant in the solid phase with only partial solubility of shorter chains in the longer chains and essentially no solubility at the other end of the composition range. The miscibility decreases with increasing chain length difference between the components. For the model of n-hexane/n-octane mixtures solid--solid phase separation has been observed directly in some of the simulations, with the components segregating between the layers of the solid structure. The behavior is similar to that seen in some binary n-alkane mixtures with longer chain lengths but comparable chain length ratios between the components. Such phase separation, although indicated thermodynamically, is not seen directly in the simulations of the n-heptane/n-octane mixture due to the difference in the pure component crystal structures.

Journal Article↗

The preparation and drug-release behaviour of CTA/EC and PMS/EC composite microcapsules.

A cellulose triacetate (CTA) and three different molecular weights of poly(alpha-methyl styrene) (PMS) were used as co-wall materials to prepare composite microcapsules with ethylcellulose (EC). A non-solvent-addition phase-separation method was used. The core material was theophylline (TH) and the solvent-non-solvent pair was dichloromethane-n-hexane, and the drug-release rates of the microcapsules prepared from these two types of co-wall materials were compared. The effects of their phase-separation range on the properties of the microcapsules, such as particle size, release rate and the morphology of the microcapsules are also discussed. The release rate of microcapsules was also affected by the compatibility of the co-wall materials and the EC. The dissolution studies indicated that the drug-release time of CTA/EC and PMS/EC composite microcapsules was sustained to 10 and 3.5 times, respectively, in comparison with that for pure EC microcapsules.

Capsules↗

Synthesis, characterization, and platelet adhesion studies of novel aliphatic polyurethaneurea anionomers based on polydimethylsiloxane-polytetramethylene oxide soft segments.

Two novel aliphatic polyurethaneurea anionomers were synthesized based on polydimethylsiloxane (PDMS)-polytetramethylene oxide (PTMO) soft segments. The hard segments consisted of either 4,4'-methylene dicyclohexyl diisocyanate (H12MDI), sulfonic acid-containing diol and 1,4-butandiol (BD) or H12MDI, carboxylic acid-containing diol and BD. The nonionic counterpart chain extended with BD was prepared. In addition, the base nonionic polyurethaneurea containing a pure PDMS soft segment, which is denote H-D-BD, was also studied for comparison. The effects of soft segment type and ion incorporation on the physical properties, surface properties, and plateled adhesion are discussed. The ionic polyurethaneureas exhibited poor phase separation, a smaller fraction of PTMO present at the surface, and a smaller contact angle. On the other hand, it also showed a larger fraction of PDMS present at the surface and a higher water absorption value than its nonionic counterpart. H-D-BD had more phase-separated structure, a larger fraction of PDMS present at the surface, and larger contact angle but lower water absorption value than the PTMO-containing polyurethaneureas. The in vitro platelet adhesion experiments indicated that the ionic groups, especially for carboxylate, and surface enrichment PDMS soft segment could effectively inhibit platelet adhesion.

Anions↗

Magmatic events can produce rapid changes in hydrothermal vent chemistry.

The Endeavour segment of the Juan de Fuca ridge is host to one of the most vigorous hydrothermal areas found on the global mid-ocean-ridge system, with five separate vent fields located within 15 km along the top of the ridge segment. Over the past decade, the largest of these vent fields, the 'Main Endeavour Field', has exhibited a constant spatial gradient in temperature and chloride concentration in its vent fluids, apparently driven by differences in the nature and extent of subsurface phase separation. This stable situation was disturbed on 8 June 1999 by an earthquake swarm. Owing to the nature of the seismic signals and the lack of new lava flows observed in the area during subsequent dives of the Alvin and Jason submersibles (August-September 1999), the event was interpreted to be tectonic in nature. Here we show that chemical data from hydrothermal fluid samples collected in September 1999 and June 2000 strongly suggest that the event was instead volcanic in origin. Volatile data from this event and an earlier one at 9 degrees N on the East Pacific Rise show that such magmatic events can have profound and rapid effects on fluid-mineral equilibria, phase separation, 3He/heat ratios and fluxes of volatiles from submarine hydrothermal systems.

Journal Article↗

Antibodies to brain integral membrane proteins in systemic lupus erythematosus.

Wheat germ lectin affinity chromatography and temperature-induced phase separation with Triton X-114 were evaluated for the isolation of surface neuronal antigens from rat and human brain and from human neuroblastoma cell lines IMR-6 and SK-N-SH. Both techniques yielded surface proteins which were free of contamination by intracellular proteins but temperature-induced phase separation was technically less demanding and less expensive, required a shorter assay time and resulted in a superior quantity and quality of isolated proteins. Rat brain surface proteins were used for characterization of antineuronal antibody reactivity in sera from patients with systemic lupus erythematosus (SLE). Western blotting identified reactivity in 15 of 75 (20%) SLE sera compared to five of 95 (5%) normal controls (P 0.006). In rat brain the molecular weight of the individual proteins identified ranged from 59 kDa to 22 kDa. Six of these were also present in human brain and two were present in neuroblastoma cell lines. Absorption studies indicated that some of the antigenic proteins were either restricted to brain tissue or shared with other non-neuronal tissues. These techniques should facilitate the characterization of antineuronal antibody reactivities and lead to a clearer understanding of their role in the pathogenesis of autoimmune neurologic disease.

Adult↗

Effects of lecithin addition in oil or water phase on the stability of emulsions made with whey proteins.

The effects of lecithin addition in oil or water phase on the stability of oil-in-water emulsions made with 0.1 wt% whey protein and 10 wt% n-tetradecane at neutral and acidic pH were studied by monitoring the gravitational creaming and phase separation. The effects of lecithin addition on the interfacial behavior of beta-lactoglobulin were also studied to compare with the results of emulsion stability. At neutral pH, crude phosphatidylcholine (PC) from egg yolk or soybean increased the stability of the emulsion made with protein and lowered the interfacial tension of protein films more effectively than pure egg PC. A more remarkable effect on both the emulsion stability and the interfacial tension was found when crude PC was added in the oil phase rather than in the water phase. The purity of lecithins and the way to add them are suggested to be very important to make a stable emulsion with protein. On acidic pH (4.5 or 3.0), the increased creaming or phase separation in a whey protein-stabilized emulsion, but the lowered interfacial tension of beta-lactoglobulin films, were found upon the addition of pure or crude PC in oil or water phase. These results suggest that in acidic pH, densely packed films may be formed on a planar oil-water interface, but not on adsorbed layers around oil droplets in an emulsion.

Animals↗

Efficient bulk heterojunction photovoltaic cells using small-molecular-weight organic thin films.

The power conversion efficiency of small-molecular-weight and polymer organic photovoltaic cells has increased steadily over the past decade. This progress is chiefly attributable to the introduction of the donor-acceptor heterojunction that functions as a dissociation site for the strongly bound photogenerated excitons. Further progress was realized in polymer devices through use of blends of the donor and acceptor materials: phase separation during spin-coating leads to a bulk heterojunction that removes the exciton diffusion bottleneck by creating an interpenetrating network of the donor and acceptor materials. The realization of bulk heterojunctions using mixtures of vacuum-deposited small-molecular-weight materials has, on the other hand, posed elusive: phase separation induced by elevating the substrate temperature inevitably leads to a significant roughening of the film surface and to short-circuited devices. Here, we demonstrate that the use of a metal cap to confine the organic materials during annealing prevents the formation of a rough surface morphology while allowing for the formation of an interpenetrating donor-acceptor network. This method results in a power conversion efficiency 50 per cent higher than the best values reported for comparable bilayer devices, suggesting that this strained annealing process could allow for the formation of low-cost and high-efficiency thin film organic solar cells based on vacuum-deposited small-molecular-weight organic materials.

Journal Article↗

Glucagon-stimulated adenylate cyclase detects a selective perturbation of the inner half of the liver plasma-membrane bilayer achieved by the local anaesthetic prilocaine.

Prilocaine can increase the fluidity of rat liver plasma membranes, as indicated by a fatty acid spin-probe. This led to the activation of the membrane-bound fluoride-stimulated adenylate cyclase activity, but not the Lubrol-solubilized activity, suggesting that increased lipid fluidity can activate the enzyme. With increasing prilocaine concentrations above 10 mM, the membrane-bound fluoride-stimulated activity was progressively inhibited, even though bilayer fluidity continued to increase and the activity of the solubilized enzyme remained unaffected. Glucagon-stimulated adenylate cyclase was progressively inhibited by increasing prilocaine concentrations. Prilocaine (10 mM) had no effect on the lipid phase separation occurring at 28 degrees C and attributed to those lipids in the external half of the bilayer, as indicated by Arrhenius plots of both glucagon-stimulated adenylate cyclase activity and the order parameter of a fatty acid spin-probe. However, 10 mM-prilocaine induced a lipid phase separation at around 11 degrees C that was attributed to the lipids of the internal (cytosol-facing) half of the bilayer. It is suggested that prilocaine (10 mM) can selectively perturb the inner half of the bilayer of rat liver plasma membranes owing to its preferential interaction with the acidic phospholipids residing there.

Adenylyl Cyclase Inhibitors↗

Poly-DL-lactic acid: polyethylene glycol block copolymers. The influence of polyethylene glycol on the degradation of poly-DL-lactic acid.

ABA block copolymers of polyethylene glycol and poly-DL-lactic acid were prepared by ring-opening polymerization of DL-dilactide with alpha,omega-dihydroxy polyethylene glycol, Mn 1000 or 2000. The morphology of the resulting copolymers, with PEG:PLA ratios(mol/mol) of 1:2, 1:3 and 1:4, was characterized by DSC and ESR spectroscopy. The rate of water uptake was biphasic, reflecting the contribution of two processes: rapid diffusion of water into the initially miscible PEG and PLA blocks; then a slower rate of hydration possibly due to phase separation and hydrolytic cleavage of the PLA blocks. The rate of hydrolytic degradation of the block copolymers in DI water at 37 degrees C was measured by two methods: weight loss and colorimetric analysis of the carboxy end group concentration resulting from chain scission of PLA blocks. As a result of phase separation, the rate of scission of PLA blocks in the copolymers was similar to that of the PLA homopolymer. The more rapid onset of weight loss of the copolymers, relative to PLA, is attributed to the greater water solubility of PEG-PLA oligomers and their greater diffusivity in the more highly hydrated copolymers.

Biocompatible Materials↗

Heterogeneous molecular distribution in supported multicomponent lipid bilayers.

Membrane domains contribute important structural and functional attributes to biological membranes. We describe the heterogeneous nanoscale distribution of lipid molecules within microscale membrane domains in multicomponent lipid bilayers composed of dipalmitoylphosphatidylcholine (DPPC), dilauroylphosphatidylcholine (DLPC), and cholesterol (chol). The lipids were labeled with the fluorescent lipid analogues Bodipy-PC and DiI-C20:0 to identify the distribution of individual membrane components. We used a near-field scanning optical microscope (NSOM) at room temperature to identify the nanoscale structures in the membrane. Simultaneous multicolor NSOM imaging at the emission maxima of the fluorescent analogues revealed a patchy distribution of Bodipy-PC and DiI-C20:0 indicative of phase separations in the bilayer. In a cholesterol-free system (DPPC/DLPC = 1:1), NSOM images proved that the two phosphatidylcholine molecules can coexist in domains at the micrometer level but form nanoscopic patches within the domains; DPPC occurs at the edge of the domains, whereas DLPC is present throughout the domains. In the presence of cholesterol (DPPC/DLPC = 7:3, chol = 18.9%), the two lipid molecules were more miscible but incomplete phase separations also occurred. The average domain sizes were 140-200 nm, well below the resolution capabilities of diffraction-limited light microscopy techniques; the domains were unresolvable by confocal microscopy. Our high-resolution NSOM studies of membrane domain behavior provide a better understanding of complex membrane phase phenomena in multicomponent biological membranes.

1,2-Dipalmitoylphosphatidylcholine↗

Efficient purification of recombinant proteins using hydrophobins as tags in surfactant-based two-phase systems.

In this work we describe the new concept of using fungal hydrophobins as efficient tags for purification of recombinant fusion proteins by aqueous two-phase separation. Hydrophobins are a group of small surface-active proteins produced by filamentous fungi. Some characteristics of hydrophobins are that they are relatively small (approximately 100 amino acids), they contain eight disulfide-forming Cys residues in a conserved pattern, and they self-assemble on interfaces. The aqueous two-phase systems studied were based on nonionic surfactants that phase-separate at certain temperatures. We show that the use of hydrophobins as tags has many advantages such as high selectivity and good yield and is technically very simple to perform. Fusion proteins with target proteins of different molecular size were compared to the corresponding free proteins using a set of different surfactants. This gave an understanding on which factors influence the separation and what rationale should be used for optimization. This unusually strong and specific interaction between polymeric surfactants and a soluble protein shows promise for new developments in interfacing proteins and nonbiological materials for other applications as well.

Biological Assay↗

Dynamics of spinodal decomposition in finite-lifetime systems: Nonlinear statistical theory based on a coarse-grained lattice-gas model.

We study theoretically dynamics of the spinodal decomposition in finite-lifetime systems to clarify effects of the interparticle interactions beyond the Ginzburg-Landau-Wilson phenomenology. Our theory is based on the coarse-grained Hamiltonian derived from the interacting lattice-gas model with a finite lifetime. The information of a system is reduced to closed-form coupled integrodifferential equations for the single-point distribution function and the dynamical structure factor. These equations involve explicitly the interparticle interactions. The finite lifetime prevents the phase separation and the order formation in the cw creation case; domains cannot grow to be larger than an asymptotic characteristic size [k(max)(t --> infinity)](-1). Power-law dependence of k(max)(t --> infinity) on the interparticle interaction and the particle lifetime is also found numerically. The finite lifetime prevents the phase separation, i.e., the lower critical wave number k((1))(c) appears and domains of size larger than [k((1))(c)](-1) cannot grow.

Journal Article↗

The organization and dynamics of viral factories.

Viral factories (VFs) are dynamic, virus-induced microcompartments that serve as centralized hubs in the host cell for viral genome replication, transcription, and virion assembly. These structures employ unique viral mechanisms for remodeling cellular architecture to create specialized replication organelles and improve the efficiency of viral propagation. VFs exhibit striking structural and functional diversity among RNA and DNA viruses, from reoviruses and poxviruses to the Nucleocytoviricota phylum. Some are enclosed by host-derived membranes, while others exist as biomolecular condensates from liquid-liquid phase separation. VFs recruit host lipids, cytoskeletal elements, and metabolic enzymes, effectively reprogramming the intracellular environment to favor viral replication. This review provides a comprehensive examination of the molecular composition, ultrastructure, and biogenesis of viral factories across a wide range of viral lineages and host systems. We describe membrane-bound and phase-separated VFs and the mechanisms by which they hijack host machinery to create these replication organelles and explore viral strategies to shield replication intermediates from host immune responses. Additional emphasis is placed on the complex VFs formed by giant viruses in the Nucleocytoviricota, whose ability to spatially compartmentalize replication and transcription, exclude ribosomes, and recruit host mitochondria and membranes blurs the line between viral and cellular organization. By integrating findings from cell biology and evolutionary virology, this review proposes that viral factories offer a conceptual framework for understanding virus-host coevolution and provides new insights into how their organization may have shaped the emergence of eukaryotic complexity.

Nucleocytoviricota↗

The lipid environment of the glucagon receptor regulates adenylate cyclase activity.

1. The lipids composition of rat liver plasma membranes was substantially altered by introducing synthetic phosphatidylcholines into the membrane by the techniques of lipid substitution or lipid fusion. 40-60% of the total lipid pool in the modified membranes consisted of a synthetic phosphatidylcholine. 2. Lipid substitution, using cholate to equilibrate the lipid pools, resulted in the irreversible loss of a major part of the adenylate cyclase activity stimulated by F-, GMP-P(NH)P or glucagon. However, fusion with presonicated vesicles of the synethic phosphatidylcholines causes only small losses in adenylate cyclase activity stimulated by the same ligands. 3. The linear form of the Arrhenius plots of adenylate cyclase activity stimulated by F- or GMP-(NH)P was unaltered in all of the membrane preparations modified by substitution or fusion, with very similar activation energies to those observed with the native membrane. The activity of the enzyme therefore appears to be very insensitive to its lipid environment when stimulated by F- or gmp-p(nh)p. 4. in contrast, the break at 28.5 degrees C in the Arrhenius plot of adenylate cyclase activity stimulated by glucagon in the native membrane, was shifted upwards by dipalmitoyl phosphatidylcholine, downwards by dimyristoyl phosphatidylcholine, and was abolished by dioleoyl phosphatidylcholine. Very similar shifts in the break point were observed for stimulation by glucagon or des-His-glucagon in combination with F- or GMP-P(NH)P. The break temperatures and activation energies for adenylate cyclase activity were the same in complexes prepared with a phosphatidylcholine by fusion or substitution. 5. The breaks in the Arrhenius plots of adenylate cyclase activity are attributed to lipid phase separations which are shifted in the modified membranes according to the transition temperature of the synthetic phosphatidylcholine. Coupling the receptor to the enzyme by glucagon or des-His-glucagon renders the enzyme sensitive to the lipid environment of the receptor. Spin-label experiments support this interpretation and suggest that the lipid phase separation at 28.5 degrees C in the native membrane may only occur in one half of the bilayer.

Adenylyl Cyclases↗

Pb2+ promotes lipid oxidation and alterations in membrane physical properties.

Experimental evidence suggests that cellular damage mediated by oxidants could be involved in the pathology associated with lead (Pb) toxicity. We investigated the effect of Pb2+ on lipid oxidation in liposomes using different initiators. In the presence of Fe2+, Pb2+ (12.5-200 microM) stimulated lipid oxidation in phosphatidylcholine:phosphatidylserine-containing liposomes, measured as 2-thiobarbituric acid-reactive substances (TBARS) and conjugated dienes. This stimulatory effect depended on the presence of membrane negative charges and on bilayer integrity. Pb2+ did not stimulate TBARS formation in the presence of 25 mM 2,2'-azo-bis (2,4 dimethylvaleronitrile (AMVN) and 2,2' azobis (2-amidinopropane) (AAPH). Pb2+ significantly stimulated TBARS production and NADH oxidation in the presence of photoactivated rose Bengal. The use of specific inhibitors indicated that several reactive oxygen species were involved in the pro-oxidant action of Pb2+. Pb2+ (12.5-200 microM) caused membrane lateral phase separation and this effect was positively correlated with its capacity to stimulate Fe2+ and rose Bengal-initiated TBARS production. Pb2+ could bind to the membrane and act to stimulate lipid oxidation by causing changes in membrane physical properties. Through this mechanism Pb2+ would favor the propagation of lipid oxidation. By causing lateral phase separation and/or by increasing lipid oxidation rates, Pb2+ could be cytotoxic by altering membrane-related processes.

Amidines↗

Microencapsulation of ascorbic acid: effect of process variables on product characteristics.

This study deals with a comparative investigation of the characteristics of ascorbic acid microcapsules prepared by different methods, such as thermal phase separation, melt dispersion, solvent evaporation and spray drying. Scanning electron microscopy (SEM), release tests and size distribution were used for the evaluation of product characteristics. The results show that microencapsulated ascorbic acid could prevent the ascorbic acid colour change, retard its core release rate, and generally mask its acid taste. In the thermal phase separation, molecular weight (Mw) of ethyl cellulose (EC) and the addition of polyisobutylene (PIB) significantly influenced the aggregation and release rate of microcapsules. In the melt dispersion method, spherical particles were prepared by using carnauba. The ascorbic acid release rate was found to be slower in the case of carnauba-encapsulated ascorbic acid than that made by EC using other methods. In the solvent evaporation method, a higher Mw of EC and the addition of plastizer were also found to be important for good encapsulation. In the spray drying method, loss of ascorbic acid was found to be minimum during microencapsulation. Starch and beta-cyclodextrin encapsulated ascorbic acid delayed the degradation of ascorbic acid during storage at 38 degrees C and relative humidity 84.0%.

Antioxidants↗