Rigid Gel Phase Research Articles

Sphingolipid-enriched domains in fungi.

Plasma membrane carries out multiple physiological functions that require its dynamic and tightly regulated organization into specialized domains of different size, stability, and lipid/protein composition. Sphingolipids are a group of lipids in which the plasma membrane is particularly enriched, thus being crucial for its structure and function. A specific type of sphingolipid-enriched plasma membrane domains, where ergosterol is depleted and lipids are tightly packed in a rigid gel phase, has recently been found in several fungal species, including yeasts and moulds. After presenting the main biophysical features of gel domains and the experimental method for their detection in the fungal plasma membrane, we review these sphingolipid-enriched gel domains and illustrate their importance to both unicellular and multicellular fungi. First, the biophysical properties of the fungal sphingolipid-enriched domains will be analysed taking into consideration the plasma membrane sphingolipidome. Next, their possible biological roles will be summarized, including their relations with plasma membrane compartments and involvement in stress responses. Moreover, since the plasma membrane is a target for several antifungal compounds, a biophysical connection between sphingolipid-enriched domains and antifungal action will be explored.

NMR relaxation and self-diffusion in aqueous micellar gels of pluronic F-127

Abstract We studied the transverse NMR relaxation of protons of different chemical groups and diffusion of molecules in an aqueous pluronic F-127 system. The system was studied at concentrations of 15, 21 and 28 wt% and in the temperature range of 293–333 K. The dynamic peculiarities in different phases were analyzed based on the NMR relaxation and diffusion data. Our study demonstrated a correlation between phase states, T2 relaxation times of “solid-like” and “liquid” protons of selected chemical groups of polypropylene oxide (PPO) and polyethylene oxide (PEO) blocks, and diffusion coefficients. Transverse NMR relaxation established that the presence of a “solid-like” component of CH3 protons of PEO blocks is observed only at temperatures and concentrations corresponding to the rigid-gel phase, due to entanglements between micellar coronas. At all temperatures and concentrations, some CH3 protons of PPO blocks and some CH2 protons of PEO blocks show “liquid-like” transverse NMR relaxation. Under the conditions corresponding to formation of the rigid-gel phase of the pluronic, relaxation of the “liquid-like” protons additionally accelerates due to entanglements of micellar coronas. Transition of the system to the rigid-gel phase is accompanied by decrease of diffusion coefficients by a factor of 10–104 relative to the diffusivity of free-moving pluronic micelles in the sol phase. Diffusion measurements show that there are pre-transition phenomena, which are characteristic for temperatures and concentrations near sol-gel phase boundaries.

Cell Cycle Position Determines Critical Temperatures in Plasma Membrane Vesicles

Giant plasma membrane vesicles (GPMVs) isolated from RBL-2H3 cells appear uniform at physiological temperatures, contain coexisting liquid-ordered and liquid-disordered phases at low temperatures, and experience micron-sized critical fluctuations close to their critical temperature. Individual vesicles have well-defined critical temperatures yet there is significant vesicle to vesicle variation even when GPMVs are isolated from a plate of seemingly identical cells. In this study, we explore if heterogeneity in critical temperatures arise, at least in part, from cells being at different stages of the cell cycle. Populations of cells were synchronized at S, G2, M, and G1 stages using a double Thymidine block that arrests cells at the border between G1 and S phases. Critical temperatures were elevated in GPMVs isolated from cells in cell cycle phases that precede cell division (G2 and M) compared to other stages (G1 and S). In unsynchronized cells, critical temperatures were found to be inversely proportional to cell density, suggesting that contact inhibition and associated arrest of cell growth results in lower plasma membrane critical temperatures. Lower critical temperatures were also observed when growth was arrested through overnight serum starvation, and elevated critical temperatures were restored 24h after the addition of serum containing medium. Transition temperatures are also lowered in GPMVs prepared from cells undergoing apoptosis through the application of TRAIL, and vesicles contain a more rigid liquid-ordered or gel phase at low temperature. These results are in agreement with past studies that have indicated that plasma membrane composition varies within the cell cycle. Since GPMV critical temperatures are hypothesized to reflect on the magnitude of lipid-mediated heterogeneity in intact cells, these results suggest that membrane heterogeneity is greatest in cells undergoing rapid growth and cell division and is suppressed in cells under low growth conditions.

Physical properties of aqueous mixtures of the ionic 1-ethl-3-methyl imidazolium octyl sulfate: A new ionic rigid gel

We report the existence of hydrophobically driven lyotropic rigid gel phase in aqueous mixtures of the ionic liquid (IL) 1-ethyl-3-methyl imidazolium octyl sulfate (EMIM-OS), and we characterize the physical properties of the gel phase by means of density, electrical conductivity and viscosity measurements. Also we include polarized microscopy images, showing the existence of a crystalline-like gel phase with the cations and anions (mainly the alkyl chain of this last ones) ordered in a fashion similar to a liquid crystal, which is induced by the presence of the water molecules. As was pointed out briefly in a previous paper, some of the mixtures of that system under goes a (liquid+gel) phase transition only in a quite narrow concentration interval: for ionic liquid molar fraction, xIL, from 0.09 to 0.5. Below xIL=0.09 and above xIL=0.5, the mixtures crystallize at low temperature becoming a hard solid crystal. Here we report the complete phase diagram of this binary system, as well as measurements of the temperature behavior of several physical properties around the (liquid+gel) and (liquid+crystal) transitions. Curiously enough, we did not detect any change in the temperature dependence of the measured magnitudes at the (liquid+gel) phase transition, but for viscosity, even in the case of the electrical conductivity. Viscosity increases enormously at the transition going from less than 0.3Pa·s three degrees above the liquid to gel transition temperature to more than 20Pa·s three degrees below it, indicating a deep change in the mass transport in the system as molecular diffusion is frozen out. No similar change is registered in the charge transport mechanism nor in density from liquid to gel phases in agreement with previous theoretical predictions of pseudolattice structure of ILs in their liquid phase.

Fluorescence studies on potential antitumor 6-(hetero)arylthieno[3,2-b]pyridine derivatives in solution and in nanoliposomes

Abstract The photophysical properties (absorption and fluorescence) of four 6-(hetero)arylthieno[3,2- b ]pyridine derivatives, the methyl 3-amino-6-(thien-3-yl)thieno[3,2- b ]pyridine-2-carboxylate 1 , the methyl 3-amino-6-(2,2′-bithienyl-5-yl)thieno[3,2- b ]pyridine-2-carboxylate 2 , the methyl 3-amino-6-(thien-2-yl)thieno[3,2- b ]pyridine-2-carboxylate 3 and the methyl 3-amino-6-(fur-3-yl)thieno[3,2- b ]pyridine-2-carboxylate 4 , evaluated previously as potential antitumor compounds, were studied in solvents of different polarity. All compounds have reasonable fluorescence quantum yields and exhibit a solvatochromic behaviour. The thienopyridine derivatives were incorporated in lipid membranes of neat egg-yolk phosphatidylcholine (egg-PC), dipalmitoyl phosphatidylcholine (DPPC), dipalmitoyl phosphatidylglycerol (DPPG) and dioctadecyldimethylammonium bromide (DODAB). Fluorescence measurements indicate that all compounds are mainly located in the lipid bilayer, feeling the transition from the rigid gel phase to the liquid-crystalline phase. The most promising antitumor compounds, the thien-3-yl and the 2,2′-bithienyl-5-yl thienopyridine derivatives 1 and 2 , were encapsulated in different nanoliposome formulations, considering future drug delivery applications using liposomes as carriers. Almost all the liposomes with incorporated compounds have diameters lower than 165 nm and generally low polydispersity. The formulation DPPC:DMPG:DSPE-PEG (1:1:0.1) exhibits a small diameter (below 100 nm), low polydispersity and reasonable negative zeta-potential values for both thienopyridines 1 and 2 .

New 1,3-diarylureas linked by CC Suzuki coupling to the methyl 3-aminothieno[3,2-b]pyridine-2-carboxylate moiety: Synthesis and fluorescence studies in solution and in lipid membranes

► Six new 1,3-diarylurea derivatives in the thieno[3,2- b ]pyridine series were synthesized. ► The fluorescence properties of the new compounds were studied in solution and in lipid membranes. ► The compounds were incorporated in lipid membranes of DPPC, DPPG, egg lecithin and DODAB. ► These compounds are useful to monitor changes in fluidity of lipid membranes. New six fluorescent 1,3-diarylureas linked by C C Suzuki coupling to the 6-position of the methyl 3-aminothieno[3,2- b ]pyridine-2-carboxylate moiety were prepared by reaction of the amino groups on the ortho or meta positions relative to the C C bond of the Suzuki coupling products, with different para -substituted arylisocyanates (H, OMe, CN), in high to excellent yields. The fluorescence properties of the 1,3-diarylureas in solution and in lipid membranes of egg yolk phosphatidylcholine (Egg-PC), dipalmitoyl phosphatidylcholine (DPPC), dipalmitoyl phosphatidylglycerol (DPPG) or dioctadecyldimethylammonium bromide (DODAB), with or without cholesterol (Ch), were studied. The six 1,3-diarylureas have reasonable fluorescence quantum yields in several solvents (0.02 ≤ Φ F ≤ 0.69) and present a moderately solvent sensitive emission, but are not fluorescent in alcohols and water. The compounds bearing the arylurea moiety in the meta position relative to the C C bond, especially with the OMe and CN substituents, present the better solvatochromic properties. Incorporation of the six compounds in lipid membranes indicates that all the compounds are deeply located in the hydrophobic region of the lipid bilayers, feeling the transition between the rigid gel phase and fluid phases.

The Effect of the Lipid Environment on Drug Binding and Transport by the P‐Glycoprotein Multidrug Transporter

The P‐glycoprotein multidrug transporter (Pgp, ABCB1) is an ABC protein that effluxes hundreds of structurally unrelated compounds from cells, and is implicated in multidrug resistance in many human cancers. The membrane environment is critically important for Pgp function since lipophilic substrates partition into, and are pumped out of, the membrane. Using purified Pgp reconstituted into proteoliposomes of defined lipids, the effect of the bilayer melting transition on the lipid partition coefficient, binding affinity, and initial rate of transport was measured for Hoechst 33342, LDS‐751 and MK‐571. All drugs partitioned best into liquid crystalline lipid, and bound to Pgp with millimolar affinities within the membrane, suggesting that they interact with the protein weakly. Pgp drug binding affinity was modulated by both the lipid phase state and acyl chain length, and was higher for all drugs in the rigid gel phase. The kcat values for Pgp‐mediated drug transport were also sensitive to lipid melting, however, they were higher in the liquid crystalline phase. Transition state analysis showed the existence of entropy‐enthalpy compensation and an isokinetic relationship between ΔH‡ and ΔS‡. The transport rate is proposed to be controlled by the drug binding affinity and a large conformational change, rather than the rate of ATP hydrolysis.This research is funded by the Canadian Cancer Society (grant #700248).

Effects of Curcumin Concentration and Temperature on the Spectroscopic Properties of Liposomal Curcumin

The spectroscopic properties of liposomal curcumin in pH 7.0 sodium phosphate buffer were studied at various curcumin concentrations and temperatures. At 25 °C, liposomal curcumin exhibited much higher values than free curcumin in absorption maximum, fluorescence maximum, and fluorescence anisotropy. When curcumin concentration was increased from 2 to 20 μM, the values of fluorescence anisotropy of liposomal curcumin decreased gradually, consistent with the reduction of phase transition temperature of liposome. This observation revealed that liposomal curcumin can disrupt the packing of phospholipid bilayer and give a loose and disordered structure. On the other hand, as the temperature was increased from 25 to 80 °C, the relative intensity of maximum absorption of liposomal curcumin showed a more pronounced decrease above the phase transition temperature than lower temperatures, suggesting a weaker curcumin protection from the liquid crystalline phase of phospholipid bilayer than the rigid gel phase. However, it was observed that the fluorescence anisotropy of liposomal curcumin had higher values as the temperature increased. This phenomenon was explained as the result of location change of curcumin toward the core of phospholipid bilayer, although the structure of the phospholipid bilayer tended to be looser at higher temperatures.

Partitioning of ethanol into lipid membranes and its effect on fluidity and permeability as seen by X-ray and neutron scattering

We present a combined neutron and X-ray scattering investigation to study the effect of ethanol on the molecular structure and dynamics of lipid membranes. 1,2-Dimyristoyl-sn-glycero-3-phoshatidylcholine (DMPC) powder hydrated with a 5 wt% ethanol solution (corresponding to 2 mol% of ethanol) was used in this study. From high-resolution X-ray experiments the position and partitioning of the ethanol molecules in the phospholipid bilayers was determined in their gel and fluid phases. We find that the ethanol molecules reside in the head group region of the bilayers, with 1.6 ethanol molecules per lipid molecule in the gel phase and 1.2 ethanol molecules per lipid molecule in the fluid phase. We find evidence for enhanced permeability in both fluid and gel phases of the phospholipid bilayers in the presence of ethanol molecules. Elastic and quasi-elastic neutron scattering data, obtained using a neutron backscattering spectrometer, was used to study slow, nanosecond molecular dynamics on length scales corresponding to lipid diffusion, acyl chain dynamics and solvent dynamics. While the presence of ethanol molecules had no observable effect on these types of dynamics in the fluid (Lα) phase, the membranes appeared to have a higher degree of order in gel (Lβ) and ripple (Pβ′) phases. In particular, lipid diffusion was found to be slower by a factor of two in the more rigid gel phase when ethanol was present.

Fluorescence properties of a potential antitumoral benzothieno[3,2-b]pyrrole in solution and lipid membranes

Fluorescence properties of the antitumoral methyl 3-(benzo[b]thien-2-yl)-benzothieno[3,2-b]pyrrole-2-carboxylate (BTP) were studied in solution and in lipid bilayers of dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylethanolamine (DOPE) and egg yolk phosphatidylcholine (Egg-PC). BTP presents good fluorescence quantum yields in all solvents studied (0.20≤ΦF≤0.32) and a bathochromic shift in polar solvents. The results indicate an ICT character of the excited state, with an estimated dipole moment of μe=7.38D.Fluorescence (steady-state) anisotropy measurements of BTP incorporated in lipid membranes of DPPC, DOPE and Egg-PC indicate that this compound is deeply located in the lipid bilayer, feeling the difference between the rigid gel phase and fluid phases.BTP inhibits the growth of three human tumour cell lines, MCF-7 (breast adenocarcinoma), SF-268 (glioma) and NCI-H460 (non-small cell lung cancer), being significantly more potent against the NCI-H460 tumour cells.

Fluorescence Studies on Potential Antitumoral Heteroaryl and Heteroannulated Indoles in Solution and in Lipid Membranes

Fluorescence properties of three potential antitumoral compounds, a 3-(dibenzothien-4-yl)indole 1, a phenylbenzothienoindole 2 and a 3-(dibenzofur-4-yl)indole 3, were studied in solution and in lipid aggregates of dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylethanolamine (DOPE) and egg yolk phosphatidylcholine (Egg-PC). The 3-(dibenzofur-4-yl)indole 3 exhibits the higher fluorescence quantum yields in all solvents studied (0.32 <or= Phi(F) <or= 0.51). All the compounds present a solvent sensitive emission, with significant red shifts in alcohols. The results point to an ICT character of the excited state, more pronounced for compound 1. Fluorescence (steady-state) anisotropy measurements of the compounds incorporated in lipid aggregates of DPPC, DOPE and Egg-PC indicate that the three compounds are deeply located in the lipid bilayer, feeling the difference between the rigid gel phase and fluid phases.

Excitation Wavelength Dependence of Solvation Dynamics in a Gel. (PEO)20−(PPO)70−(PEO)20 Triblock Copolymer

In a 30 wt % aqueous solution, the triblock copolymer, (polyethylene oxide, PEO)20−(polypropylene oxide, PPO)70−(PEO)20 (Pluronic P123) forms a cubic gel phase. In the gel, the micelles associate to form an interconnected network with intermicellar distance (140 A) less than the sum (160 A) of the radii of the micelles. Fluorescence anisotropy decay and solvation dynamics of coumarin 480 (C480) in P123 gel have been investigated as a function of excitation wavelength (λex) using femtosecond and picosecond fluorescence spectroscopy. The anisotropy decay of C480 in the macroscopically rigid (“solid”) gel phase is quite fast and only ∼1.6 times slower than that in the sol (micellar) phase. Solvation dynamics in the P123 gel displays three components. First, even in the “solid” gel, there is a bulk water-like ultrafast component (2 ps and <0.3 ps), which represents response of the “free” water molecules in the pores of the gel. Second, there is an ultraslow 4500 ps component, which is ascribed to the hydropho...

Drug transport by reconstituted P‐glycoprotein in proteoliposomes

The P‐glycoprotein multidrug transporter (Pgp) is an active efflux pump for chemotherapeutic drugs, natural products and hydrophobic peptides. Pgp is envisaged as a ‘hydrophobic vacuum cleaner’, and drugs are believed to gain access to the substrate binding sites from within the membrane, rather than from the aqueous phase. The intimate association of both Pgp and its substrates with the membrane suggests that its function may be regulated by the biophysical properties of the lipid bilayer. Using the high affinity fluorescent substrate tetramethylrosamine (TMR), we have monitored, in real time, transport in proteoliposomes containing reconstituted Pgp. The TMR concentration gradient generated by Pgp was collapsed by the addition of either the ATPase inhibitor, vanadate, or Pgp modulators. TMR transport by Pgp obeyed Michaelis–Menten kinetics with respect to both of its substrates. The Km for ATP was 0.48 mm, close to the Km for ATP hydrolysis, and the Km for TMR was 0.3 µm. TMR transport was inhibited in a concentration‐dependent fashion by verapamil and cyclosporin A, and activated (probably by a positive allosteric effect) by the transport substrate colchicine. TMR transport by Pgp reconstituted into proteoliposomes composed of two synthetic phosphatidylcholines showed a highly unusual biphasic temperature dependence. The rate of TMR transport was relatively high in the rigid gel phase, reached a maximum at the melting temperature of the bilayer, and then decreased in the fluid liquid crystalline phase. This pattern of temperature dependence suggests that the rate of drug transport by Pgp may be dominated by partitioning of drug into the bilayer.

Drug transport by reconstituted P-glycoprotein in proteoliposomes Effect of substrates and modulators, and dependence on bilayer phase state

The P-glycoprotein multidrug transporter (Pgp) is an active efflux pump for chemotherapeutic drugs, natural products and hydrophobic peptides. Pgp is envisaged as a 'hydrophobic vacuum cleaner', and drugs are believed to gain access to the substrate binding sites from within the membrane, rather than from the aqueous phase. The intimate association of both Pgp and its substrates with the membrane suggests that its function may be regulated by the biophysical properties of the lipid bilayer. Using the high affinity fluorescent substrate tetramethylrosamine (TMR), we have monitored, in real time, transport in proteoliposomes containing reconstituted Pgp. The TMR concentration gradient generated by Pgp was collapsed by the addition of either the ATPase inhibitor, vanadate, or Pgp modulators. TMR transport by Pgp obeyed Michaelis--Menten kinetics with respect to both of its substrates. The Km for ATP was 0.48 mM, close to the K(m) for ATP hydrolysis, and the K(m) for TMR was 0.3 microM. TMR transport was inhibited in a concentration-dependent fashion by verapamil and cyclosporin A, and activated (probably by a positive allosteric effect) by the transport substrate colchicine. TMR transport by Pgp reconstituted into proteoliposomes composed of two synthetic phosphatidylcholines showed a highly unusual biphasic temperature dependence. The rate of TMR transport was relatively high in the rigid gel phase, reached a maximum at the melting temperature of the bilayer, and then decreased in the fluid liquid crystalline phase. This pattern of temperature dependence suggests that the rate of drug transport by Pgp may be dominated by partitioning of drug into the bilayer.

In vitro/in vivo characterisation of polyhedral niosomes

Non-ionic surfactant vesicles (niosomes) formed by a hexadecyl diglycerol ether (C 16G 2) and a series of polyoxyethylene alkyl ethers exhibit a variety of shapes dependant on their membrane composition. These surfactants form with an equimolar amount of cholesterol a mixture of largely spherical and tubular niosomes. In the absence of cholesterol, they form faceted polyhedral structures. The physicochemical and biological differences between polyhedral and spherical/tubular niosomes were studied. Polyhedral niosomes undergo a reversible shape transformation into spherical structures on heating above their phase transition temperature ( T m). The viscosity of polyhedral niosomes at room temperature is higher than their spherical counterparts due to their faceted and relatively rigid shape, and is more dependant on temperature due to shape transformation. At room temperature, polyhedral niosomes possess more rigid gel phase membranes and are less osmotically sensitive; however, they are more permeable because of a lack of or low levels of cholesterol in their membranes. Polyhedral niosomes loaded with luteinising hormone releasing hormone (LHRH), nonetheless, slow the release of drug compared to solution, albeit to a small extent.

Selective Adsorption of Lipopolysaccharide in Protein Solution by Polyion-Complexed Lipid Membrane. Influence of the Membrane Rigidity on the Adsorption Selectivity

A polyion-complexed lipid membrane was prepared from dioctadecyldimethylammonium bromide (2C 18 N2C 1 ) and sodium dextran sulfate. Controlled-pore glass (CPG) was coated with the obtained lipid membrane and applied to the adsorption of fluorescein-lipopolysaccharide (FITC-LPS) and bovine serum albumin (BSA). At temperatures not higher than the lipid phase transition (T c = 43 °C, DSC), FITC-LPS was selectively adsorbed to CPG coated with the lipid membrane from BSA/FITC-LPS mixed solution (pH 6.8). On the other hand, at the phase transition temperature or higher both FITC-LPS and BSA were adsorbed. When polyion-complexed lipid membrane of didodecyldimethylammonium bromide (2C 12 N2C 1 ) or octadecyltrimethylammonium bromide (C 18 N3C 1 ) was used, phase transition was not observed and both FITC-LPS and BSA were adsorbed. FITC-LPS is probably adsorbed by inserting its fatty acid alkyl chains into the lipid membrane on CPG. Because of the structural similarity, FITC-LPS could interact with not only the lipid membrane in fluid liquid crystalline phase but that in rigid gel phase. On the other hand, the lipid membrane probably has to be in relatively fluid state to interact with the hydrophobic region of BSA. Therefore the selective adsorption of FITC-LPS from BSA/FITC-LPS solution was achieved by only 2C 18 N 2 C 1 polyion-complexed membrane in the rigid gel phase.

A comparison of physical behaviour amongst four glycosphingolipid families

In the general search for information regarding glycosphingolipid behaviour and arrangement in membranes there has arisen the question of whether the answers may be very different for different glycosphingolipid families. We show here that in fact considerable basic similarity exists at least amongst a certain number of families as sensed by a spin label probe covalently attached to the glycosphingolipid fatty acid. This potential for similarity may be as important to take account of as is the clearly demonstrated potential for differences based on headgroup structure (Maggio, B., Albert, J. and Yu, R.K. (1988) Biochim. Biophys. Acta 945, 145–160). The ganglioside, G M1, was examined, as were the neutral species, galactosyl ceramide, lactosyl ceramide, and globoside, in phospholipid bilayers at 2 mol% and 10 mol%. In each case EPR spectra of a spin probe at carbon 16 of the single glycolipid fatty acid chain were compared. This was done for both 18-carbon and 24-carbon species in order to test the phenomenon over the range of common glycosphingolipid fatty acid chain length. The most telling result was that spectra of these four families were identical within experimental error in both rigid gel phase and semi-fluid non-cooperative bilayer matrices as sensed by a nitroxide spin probe near the centre of the membrane hydrophobic interior. No glycolipid family showed a detectable difference in organization either laterally or vertically within the membranes, nor in membrane dynamics. In addition, the membrane phase transition sensed by each family fell within the same narrow range. Freeze-etch visualization of glycolipid lateral distribution was attempted using a variety of native lectins and antibodies as markers. Success was severely limited by low affinity of marker attachment to neutral species; however, where testable, no differences were observed amongst families.