Matrix regeneration agents improve wound healing in non-stressed human corneal epithelial cells.

PurposeMatrix regenerating agents (RGTAs) emerged as promising in vivo wound-healing agents. These agents could prove beneficial for the treatment of dry eye disease-associated corneal micro-erosions; therefore, we aimed to evaluate the wound healing efficacy of regenerative agents (RGTAs or serum) in an in vitro model of hyperosmolarity (HO) stressed and non-stressed human corneal epithelial cells.Patients and methodsThe migration and proliferation induced by the regenerative agents was evaluated using an in vitro scratch wound assay and brome-deoxy-uridine incorporation. The inflammatory profile and effects of osmoregulators were also investigated. The two-tailed paired t-test calculated the statistical significance, with P-value<0.05 considered significant.ResultsThe most efficient inducer of re-epithelization was 2% serum, followed closely by 2% RGTA with an average improvement in cell migration of 1.8- and 1.4-fold, respectively, when compared with the non-treated control. Hyperosmolar stress significantly reduced the restorative effects of both serum and RGTAs; these effects were, however, neutralized by the osmoregulator betaine.ConclusionThese findings suggest that RGTAs could provide efficient treatment for dry-eye associated corneal micro-lesions if ocular surface HO is neutralized.

Void volume variations in contact lens polymers.

AIM: In this study, void size and free volume properties in different contact lens materials have been investigated in their hydrated state using positron annihilation lifetime spectroscopy (PALS). METHODS: PALS is used to characterize the void size distributions inside the lens materials. Three different types of contact lenses were used (Balafilcon A, Hilafilcon B and Polymacon). RESULTS: Measurements on different contact lenses reveal significant differences between the materials, up to ∼ 100% difference in void volume was observed between Hilafilcon B and Balafilcon A, the latter having larger voids. As oxygen diffusion is strongly correlated with the void sizes, the results are in good agreement with the usage recommendations of the specific lens types (daily disposable lenses or 1 month continuous use lenses). The void sizes in monthly lenses (Balafilcon A) were found to decrease 25% under artificial aqueous tear (albumin-water solution) exposure in 4 weeks leading to a significant decrease in the oxygen permeation rate through the contact lens. Yet, the voids were still significantly larger than in disposable or semi-disposable lenses. CONCLUSIONS: We have showed that PALS is a viable method to probe the microstructure of biotechnologically relevant polymers and can be used to quantify the void properties in different types of contact lenses. Usage recommendations correlate well with measured void sizes and the median void size decreases during the incubation of albumin solution as a function of time. We anticipate the use of PALS for any polymer-based intracorneal/intraocular device in which diffusivity plays a crucial role.

Corneal recovery after lasik for high myopia: a 2-year prospective confocal microscopic study.

AIM: To quantify human corneal recovery after moderate to high myopic laser in situ keratomileusis (LASIK) in a 2-year prospective follow-up study. METHODS: Fifteen eyes of 15 patients (mean refraction -10.1 (SD 2.4) D) were examined preoperatively and postoperatively at day 1, 5 days, 2 weeks, 1, 3 and 6 months and 2 years. Biomicroscopy, visual acuity and refraction were examined prior to imaging studies. An in vivo tandem scanning confocal microscope was used to obtain images from the central cornea. Subbasal nerve density was measured as the total length of nerve trunks in confocal image per mm2. Keratocyte density was calculated manually from stromal sublayers. The thickness of the altered keratocyte zone was measured on both sides of the LASIK interface. RESULTS: At the end of the follow-up, all patients had a 20/20 BCVA, and nine of 15 patients were within +/-0.5 D of the intended correction. The total corneal thickness remained unaltered, but epithelial hyperplasia was seen at 2 years. Keratocyte density in the anterior stroma and posterior to the flap interface showed a slight decrease during the follow-up. Subbasal nerve density decreased 82% in 5 days after LASIK. A gradual increase was observed from 2 weeks postoperatively, but even 2 years after the operation the nerve density was only 64% from the preoperative values. CONCLUSIONS: Subbasal nerve fibre density shows a gradual recovery throughout the follow-up. However, only three subjects showed totally regenerated subbasal nerve fibres at 2 years. This may correlate with the observed decrease in the density of the most anterior keratocytes. Corneal remodelling seemed to continue for at least 2 years.

Elevated lysosomal pH in neuronal ceroid lipofuscinoses (NCLs).

We report here the intracellular (pHi) and lysosomal pH in fibroblasts of six forms of neuronal ceroid lipofuscinoses (NCLs). Acid extrusion rate and pH(i) values were measured by the membrane-permeant acetoxymethyl ester of the indicator dye, 2',7'-bis(carboxyethyl)-5-(and-6)-carboxy-fluorescein (BCECF) and lysosomal pH by a spectrofluorometric assay utilizing a novel acidotropic probe, Lysosensor yellow/blue. Intracellular pH was normal in all NCLs. Elevated lysosomal pH was detected in all NCL forms except CLN2 and CLN8. Elevated pH most probably disturbs the catalytic activity of lysosomes and is one important factor in explaining accumulation of ceroid and lipofuscin-like autofluorescent lipopigments characteristic of NCLs. Using the novel spectrofluorometric assay introduced in this study provides a fast and repeatable technique to measure intralysosomal pH from cell suspensions.

Comparison of the membrane association of two antimicrobial peptides, magainin 2 and indolicidin.

Interactions of two antimicrobial peptides, magainin 2 and indolicidin, with three different model biomembranes, namely, monolayers, large unilamellar vesicles (LUVs), and giant liposomes, were studied. Insertion of both peptides into lipid monolayers was progressively enhanced when the content of an acidic phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) in a film of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) was increased. Indolicidin and magainin 2 penetrated also into lipid monolayers containing cholesterol (mole fraction, X = 0.1). Membrane association of magainin 2 attenuated lipid lateral diffusion in POPG-containing LUVs as revealed by the decrease in the excimer/monomer fluorescence ratio I(e)/I(m) for the pyrene fatty-acid-containing phospholipid derivative 1-palmitoyl-2-[10-(pyren-1-yl) decanoyl]-sn-glycero-3-phospho-rac-glycerol (PPDPG). Likewise, an increase in steady-state fluorescence anisotropy of the membrane-incorporated diphenylhexatriene (DPH) was observed, revealing magainin 2 to increase acyl chain order and induce segregation of acidic phospholipids. Similar effects were observed for indolicidin. The topological effects of magainin 2 and indolicidin on phospholipid membranes were investigated using optical microscopy of giant vesicles. Magainin 2 had essentially no influence on either SOPC or SOPC:cholesterol (X = 0.1) giant liposomes. However, effective vesiculation was observed when acidic phospholipid (X(PG) = 0.1) was included in the giant vesicles. Indolicidin caused only a minor shrinkage of giant SOPC vesicles whereas the formation of endocytotic vesicles was observed when the giant liposome contained POPG (X(PG) = 0.1). Interestingly, for indolicidin, vesiculation was also observed for giant vesicles composed of SOPC/cholesterol (X(chol) = 0.1). Possible mechanisms of membrane transformation induced by these two peptides are discussed.

Characterization of mixed monolayers of phosphatidylcholine and a dicationic gemini surfactant SS-1 with a langmuir balance: effects of DNA.

Monolayers of a cationic gemini surfactant, 2,3-dimethoxy-1,4-bis(N-hexadecyl-N;N-dimethyl-ammonium)butane dibromide (abbreviated as SS-1) and its mixtures with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were studied using a Langmuir balance. More specifically, we measured the force-area (pi-A) curves and determined the elastic area compressibility modulus (C) as a function of lateral packing pressure and the mole fraction of the cationic lipid (X(SS-1)), with and without DNA in the subphase. Both SS-1 and POPC exhibited smooth compression isotherms, indicating their monolayers to be in the liquid expanded state. Even low contents (X(SS-1) < 0.05) of SS-1 in a POPC monolayer condensed the film dramatically, up to 20% at 30 mN/m. This effect is suggested to reflect reorientation of the P(-)-N(+) dipole of the POPC headgroup. Accordingly, the magnitude of the condensing effect diminishes with X(SS-1) and is not observed for mixed films of dioleoylglycerol and SS-1. Reorientation of the P(-)-N(+) dipole is further supported by the pronounced increase in monolayer dipole potential psi due to SS-1. The presence of DNA in the subphase affected the mixed POPC/SS-1 monolayers differently depending on the constituent lipid stoichiometry as well as on the DNA/SS-1 charge ratio. At a DNA concentration of 0.63 microM (in base pairs) condensation of neat POPC monolayers was evident, and this effect remained up to X(SS-1) < 0.5, corresponding to DNA/SS-1 charge ratio of 1.25. An expansion due to DNA, evident as an increase in DeltaA/molecule, was observed at X(SS-1) > 0.5. At a higher concentration of DNA (1.88 microM base pairs) in the subphase corresponding to DNA/SS-1 charge ratio of 3.75 at X(SS-1) = 0.5, condensation was observed at all values of X(SS-1).

Study on liposomes by capillary electrophoresis.

Liposomes made of mixtures of zwitterionic and anionic lipids were investigated by means of capillary electrophoresis and dynamic light scattering. The influence of the molar lipid ratio and of the buffers, used in the running electrolyte solution, on the physical characteristics of the liposomes were investigated. Data on effective electrophoretic mobilities, total charges as well as sizes of the liposomes are given. In addition, examples on the use of liposomes as carriers in electrokinetic capillary electrophoresis for the separation of benzene derivatives, steroids, and phenols are shown.

Interfacial interactions of ceramide with dimyristoylphosphatidylcholine: impact of the N-acyl chain.

The mixing behavior of dimyristoylphosphatidylcholine (DMPC) with either N-palmitoyl-sphingosine (C16:0-ceramide) or N-nervonoyl-sphingosine (C24:1-ceramide) was examined using monomolecular films. While DMPC forms highly elastic liquid-expanded monolayers, both neat C16:0-ceramide and C24:1-ceramide yield stable solid condensed monomolecular films with small areas and low interfacial elasticity. Compression isotherms of mixed C16:0-ceramide/DMPC films exhibit an apparent condensation upon increasing X(cer16:0) at all surface pressures. The average area isobars, coupled with the lack of a liquid-expanded to condensed phase transition as X(cer16:0) is increased, are indicative of immiscibility of the lipids at all surface pressures. In contrast, isobars for C24:1-ceramide/DMPC mixtures show surface pressure-dependent apparent condensation or expansion and surface pressure-area isotherms show a composition and surface pressure-dependent phase transition. This suggests miscibility, albeit non-ideal, of C24:1-ceramide and DMPC in both liquid and condensed surface phases. The above could be verified by fluorescence microscopy of the monolayers and measurements of surface potential, which revealed distinctly different domain morphologies and surface potential values for the DMPC/C16:0- and DMPC/C24:1-ceramide monolayers. Taken together, whereas C16:0-ceramide and DMPC form immiscible pseudo-compounds, C24:1-ceramide and DMPC are partially miscible in both the liquid-expanded and condensed phases, and a composition and lateral pressure-dependent two-phase region is evident between the liquid-expanded and condensed regimes. Our results provide novel understanding of the regulation of membrane properties by ceramides and raise the possibility that ceramides with different acyl groups could serve very different functions in cells, relating to their different physicochemical properties.

The assembly factor P17 from bacteriophage PRD1 interacts with positively charged lipid membranes.

The interactions of the assembly factor P17 of bacteriophage PRD1 with liposomes were investigated by static light scattering, fluorescence spectroscopy, and differential scanning calorimetry. Our data show that P17 binds to positively charged large unilamellar vesicles composed of the zwitterionic 1-palmitoyl-2-oleoyl-phosphatidylcholine and sphingosine, whereas only a weak interaction is evident for 1-palmitoyl-2-oleoyl-phosphatidylcholine vesicles. P17 does not bind to negatively charged membranes composed of 1-palmitoyl-2-oleoyl-phosphatidylglycerol and 1-palmitoyl-2-oleoyl-phosphatidylcholine. Our differential scanning calorimetry results reveal that P17 slightly perturbs the phase behaviour of neutral phosphatidylcholine and negatively charged multilamellar vesicles. In contrast, the phase transition temperature of positively charged dimyristoylphosphatidylcholine/sphingosine multilamellar vesicles (molar ratio 9 : 1, respectively) is increased by approximately 2.4 degrees C and the half width of the enthalpy peak broadened from 1.9 to 5.6 degrees C in the presence of P17 (protein : lipid molar ratio 1 : 47). Moreover, the enthalpy peak is asymmetrical, suggesting that lipid phase separation is induced by P17. Based on the far-UV CD spectra, the alpha-helicity of P17 increases upon binding to positively charged micelles composed of Triton X-100 and sphingosine. We propose that P17 can interact with positively charged lipid membranes and that this binding induces a structural change on P17 to a more tightly packed and ordered structure.

Liposomes as carriers in electrokinetic capillary chromatography.

Liposomes are small membrane-enclosed vesicles composed of either natural or synthetic lipids. Their size can be adjusted on a wide scale and they can be made with well-defined compositions. While liposomes have been extensively used as model biomembranes they have also gained a considerable degree of attention as carriers for drugs as well as for genetic material. The physical properties of liposomes are critically dependent on their chemical composition. In this study liposomes were applied as pseudostationary phases in electrokinetic capillary chromatography. Various negatively charged liposomes, consisting of mixtures of zwitterionic and anionic lipids, were investigated. Major emphasis was put on clarifying the effects of the total lipid concentration, the lipid molar ratio, the lipid head group, and the buffer on the capillary electrophoretic separation of neutral analytes. In addition, the influence of the physical state of the membrane, ie., gel vs. fluid, on the separation was investigated. Corticosteroids were applied as model analytes.

Characterisation of three novel cationic lipids as liposomal complexes with DNA.

Cationic lipids (CLs) are being increasingly exploited as transfection vectors for the delivery of DNA into eukaryotic cells. To obtain further insight to the complex formation and interactions between cationic liposomes and DNA, we characterised three novel cationic lipids, viz. bis[2-(11-phenoxyundecanoate)ethyl]-dimethylammonium bromide, N-hexadecyl-N-¿10-[O-(4-acetoxy)-phenylundecanoate]ethyl¿- dimethylammonium bromide, and bis[2-(11-butyloxyundecanoate)ethyl]dimethylammonium bromide. These lipids bear the same charged headgroup yet have different hydrophobic parts. Accordingly, we may anticipate their electrostatic interactions with DNA to be similar while differing in both thermal phase behaviour and physicochemical properties of their complexes with DNA. In keeping with the above all three lipids formed complexes with DNA as evidenced by light scattering, fluorescence spectroscopy and Langmuir film balance. Differential scanning calorimetry revealed very different phase behaviours for the binary mixtures of the three CLs with dimyristoylphosphatidylcholine and also provided evidence for DNA-induced lipid phase separation. These data were confirmed by compression isotherms and fluorescence microscopy of monolayers residing on an aqueous buffer, recorded both in the presence and absence of DNA. Importantly, binding to cationic liposomes appears to prevent thermal denaturation of DNA upon heating of the complexes. Likewise, renaturation of heat-treated DNA complexed with the cationic liposomes appears to be abolished as well.

Sphingomyelinase activity associated with human plasma low density lipoprotein.

Isolated human plasma low density lipoprotein (LDL) was observed to possess sphingomyelinase activity. Accordingly, the formation of ceramide was catalyzed by LDL at 37 degrees C using tertiary liposomes composed of sphingomyelin (mole fraction (x) = 0.2), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (x = 0.7), 1, 2-dimyristoyl-sn-glycero-3-phospho-rac-glycerol (x = 0.1), and either the fluorescent sphingomyelin analog Bodipy-sphingomyelin or [(14)C]sphingomyelin as substrates. However, this activity was not present in either very low density lipoprotein or the high density lipoprotein subfractions HDL(2) and HDL(3). Oxidation of LDL abrogated its sphingomyelinase activity. Aggregation of the liposomes upon incubation with LDL was evident from the light scattering measurements. Microinjection of LDL to the surface of giant liposomes composed of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), N-palmitoyl-d-sphingomyelin (C16:0-sphingomyelin), and Bodipy-sphingomyelin as a fluorescent tracer (0.75:- 0.20:0.05, respectively) revealed the induction of vectorial budding of vesicles, resembling endocytosis.

Dimyristoylphosphatidylcholine/C16:0-ceramide binary liposomes studied by differential scanning calorimetry and wide- and small-angle x-ray scattering.

Ceramide has recently been established as a central messenger in the signaling cascades controlling cell behavior. Physicochemical studies have revealed a strong tendency of this lipid toward phase separation in mixtures with phosphatidylcholines. The thermal phase behavior and structure of fully hydrated binary membranes composed of dimyristoylphosphatidylcholine (DMPC) and N-palmitoyl-ceramide (C16:0-ceramide, up to a mole fraction X(cer) = 0.35) were resolved in further detail by high-sensitivity differential scanning calorimetry (DSC) and x-ray diffraction. Both methods reveal very strong hysteresis in the thermal phase behavior of ceramide-containing membranes. A partial phase diagram was constructed based on results from a combination of these two methods. DSC heating scans show that with increased X(cer) the pretransition temperature T(p) first increases, whereafter at X(cer) > 0.06 it can no longer be resolved. The main transition enthalpy DeltaH remains practically unaltered while its width increases significantly, and the upper phase boundary temperature of the mixture shifts to approximately 63 degrees C at X(cer) = 0.30. Upon cooling, profound phase separation is evident, and for all of the studied compositions there is an endotherm in the region close to the T(m) for DMPC. At X(cer) >/= 0.03 a second endotherm is evident at higher temperatures, starting at 32.1 degrees C and reaching 54.6 degrees C at X(cer) = 0.30. X-ray small-angle reflection heating scans reveal a lamellar phase within the temperature range of 15-60 degrees C, regardless of composition. The pretransition is observed up to X(cer) < 0.18, together with an increase in T(p). In the gel phase the lamellar repeat distance d increases from approximately 61 A at X(cer) = 0. 03, to 67 A at X(cer) = 0.35. In the fluid phase increasing X(cer) from 0.06 to 0.35 augments d from 61 A to 64 A. An L(beta')/L(alpha) (ripple/fluid) phase coexistence region is observed at high temperatures (from 31 to 56.5 degrees C) when X(cer) > 0.03. With cooling from temperatures above 50 degrees C we observe a slow increase in d as the coexistence region is entered. A sudden solidification into a metastable, modulated gel phase with high d values is observed for all compositions at approximately 24 degrees C. The anomalous swelling for up to X(cer) = 0.30 in the transition region is interpreted as an indication of bilayer softening and thermally reduced bending rigidity.

Vectorial budding of vesicles by asymmetrical enzymatic formation of ceramide in giant liposomes.

Sphingomyelin is an abundant component of eukaryotic membranes. A specific enzyme, sphingomyelinase can convert this lipid to ceramide, a central second messenger in cellular signaling for apoptosis (programmed cell death), differentiation, and senescence. We used microinjection and either Hoffman modulation contrast or fluorescence microscopy of giant liposomes composed of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), N-palmitoyl-sphingomyelin (C16:0-SM), and Bodipy-sphingomyelin as a fluorescent tracer (molar ratio 0.75:0.20:0.05, respectively) to observe changes in lipid lateral distribution and membrane morphology upon formation of ceramide. Notably, in addition to rapid domain formation (capping), vectorial budding of vesicles, i.e., endocytosis and shedding, can be induced by the asymmetrical sphingomyelinase-catalyzed generation of ceramide in either the outer or the inner leaflet, respectively, of giant phosphatidylcholine/sphingomyelin liposomes. These results are readily explained by 1) the lateral phase separation of ceramide enriched domains, 2) the area difference between the adjacent monolayers, 3) the negative spontaneous curvature, and 4) the augmented bending rigidity of the ceramide-containing domains, leading to membrane invagination and vesiculation of the bilayer.

Mechanisms of initiation of membrane fusion: role of lipids.

Main emphasis in studies on the mechanisms of fusion of cellular membranes has been in the roles of various proteins, with far less interest in the properties of lipids. Yet, on a molecular level fusion involves the merging of lipid bilayers. Studies so far have revealed lipids forming inverted non-lamellar phases to be important in controlling membrane fusion. However, the underlying molecular level mechanisms have remained controversial. While this review is focused on presenting one possible mechanism, involving so-called extended lipid conformation, we are also advocating the view, that in order to obtain a more complete understanding of this process it is necessary to merge the relevant physicochemical properties of lipids with the models describing the specific functions of proteins. To this end, taking into account the central importance of fusion in a wide range of cellular processes, we may anticipate its control to open novel possibilities also for therapeutic intervention.

Fluorescent phosphoinositide derivatives reveal specific binding of gelsolin and other actin regulatory proteins to mixed lipid bilayers.

Fluorescent derivatives of phosphatidyl inositol (PtdIns)-(4,5)-P2 were synthesized and used to test the effects of the PtdIns-(4, 5)-P2-regulated proteins gelsolin, tau, cofilin, and profilin on labeled PtdIns-(4,5)-P2 that was either in micellar form or mixed with phosphatidylcholine (PtdCho) in bilayer vesicles. Gelsolin increased the fluorescence of 7-nitrobenz-2-oxa-1,3-diazole (NBD)- or pyrene-labeled PtdIns-(4,5)-P2 and NBD-PtdIns-(3,4,5)-P3. Cofilin and profilin produced no detectable change at equimolar ratios to PtdIns-(4,5)-P2, while tau decreased NBD-PtdIns-(4,5)-P2 fluorescence. Fluorescence enhancement by gelsolin of NBD-PtdIns-(4, 5)-P2 in mixed lipid vesicles depended on the mole fraction of PtdIns-(4,5)-P2 in the bilayer. Specific enhancement of 3% NBD-PtdIns-(4,5)-P2 : 97% PtdCho was much lower than that of 10% PtdIns-(4,5)-P2 : 90% PtdCho, but the enhancement of 3% NBD-PtdIns-(4,5)-P2 could be increased by addition of 7% unlabeled PtdIns-(4,5)-P2. The gelsolin-dependent increase in NBD-PtdIns-(4, 5)-P2 fluorescence was reversed by addition of Ca2+ or G-actin. Significant, but weaker, fluorescence enhancement was observed with the gelsolin N-terminal domain (residues 1-160) and a peptide comprised of gelsolin residues 150-169. Fluorescence energy transfer from gelsolin to pyrene-PtdIns-(4,5)-P2 was much stronger with intact gelsolin than the N-terminal region of gelsolin containing the PtdIns-(4,5)-P2 binding sites, suggesting that PtdIns-(4,5)-P2 may bind near a site formed by the juxtaposition of the N- and C-terminal domains of gelsolin.

Purification and characterization of the assembly factor P17 of the lipid-containing bacteriophage PRD1.

Assembly factors, proteins assisting the formation of viral structures, have been found in many viral systems. The gene encoding the assembly factor P17 of bacteriophage PRD1 has been cloned and expressed in Escherichia coli. P17 acts late in phage assembly, after capsid protein folding and multimerization, and sorting of membrane proteins has occurred. P17 has been purified to near homogeneity. It is a tetrameric protein displaying a rather high heat stability. The protein is largely in an alpha-helical conformation and possesses a putative leucine zipper which is not essential for protein function, as judged by in vitro mutagenesis and complementation analysis. Although heating does not cause structural changes in the conformation of the protein, the dissociation of the tetramer into smaller units is evident as diminished self-quenching of the fluorescently labeled P17. Similarly, dissociation of the tetramer is also obtained by dialysis of the protein against 6-M guanidine hydrochloride (GdnHCl) or 1% SDS. The reassembly of these smaller units upon cooling is evident from resonance energy transfer.

Evidence for the extended phospholipid conformation in membrane fusion and hemifusion.

Molecular-level mechanisms of fusion and hemifusion of large unilamellar dioleoyl phosphatidic acid/phosphocholine (DOPA/DOPC, 1:1 molar ratio) vesicles induced by millimolar Ca2+ and Mg2+, respectively, were investigated using fluorescence spectroscopy. In keeping with reduction of membrane free volume Vf, both divalent cations increased the emission polarization for 1,6-diphenyl-1,3, 5-hexatriene (DPH). An important finding was a decrease in excimer/monomer emission intensity ratio (Ie/Im) for the intramolecular excimer-forming probe 1, 2-bis[(pyren-1-)yl]decanoyl-sn-glycero-3-phosphocholine (bis-PDPC) in the course of fusion and hemifusion. Comparison with another intramolecular excimer-forming probe, namely, 1-[(pyren-1)-yl]decanoyl-2-[(pyren-1)-yl]tetradecanoyl-sn-gl ycero-3-p hosphocholine (PDPTPC), allowed us to exclude changes in acyl chain alignment to be causing the decrement in Ie/Im. As a decrease in Vf should increase Ie/Im for bis-PDPC and because contact site between adhering liposomes was required we conclude the most feasible explanation to be the adoption of the extended conformation (P.K.J., Chem. Phys. Lipids 63:251-258) by bis-PDPC. In this conformation the two acyl chains are splaying so as to become embedded in the opposing leaflets of the two adhered bilayers, with the headgroup remaining between the adjacent surfaces. Our data provide evidence for a novel mechanism of fusion of the lipid bilayers.

Sphingomyelinase induces lipid microdomain formation in a fluid phosphatidylcholine/sphingomyelin membrane.

The behaviors of two chemically well-defined sphingolipids, N-palmitoyl-sphingomyelin (C16:0-SM) and the corresponding ceramide (C16:0-Cer), in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) matrix were compared. Minor attenuation of lateral diffusion upon increasing the mole fraction of C16:0-SM (XSM, up to 0.25) was indicated by the slight decrement in the excimer/monomer intensity ratio (Ie/Im) for a trace amount (mole fraction X = 0.01) of a pyrene-labeled ceramide analogue (N-[(pyren)-1-yl]decanoyl-sphingosine, PDCer) in keeping with the miscibility of C16:0-SM in POPC. Increasing membrane order was revealed by the augmented polarization P for diphenylhexatriene (DPH). In contrast, when C16:0-Cer was substituted for C16:0-SM an approximately 1.6-fold increase in Ie/Im for PDCer was evident upon increasing Xcer, with parallel increment in DPH polarization. In agreement with our recent data on natural ceramides in dimyristoylphosphatidylcholine (DMPC) bilayers [Holopainen et al. (1997) Chem. Phys. Lipids 88, 1-13], we conclude that C16:0-Cer becomes enriched into microdomains in the fluid POPC membrane. Interestingly, enhanced formation of microdomains by ceramide was observed when the total sphingolipid content in tertiary alloys with POPC was maintained constant (Xcer + XSM = 0.25) and the SM/Cer stoichiometry was varied. Finally, when ceramide was generated enzymatically in POPC/C16:0-SM (3:1, molar fraction) LUVs by sphingomyelinase (SMase, Bacillus cereus), maximally approximately 85% of hydrolysis of sphingomyelin was measured within <3 min at 30 degreesC. The formation of ceramide was accompanied by a closely parallel increase in DPH polarization. There was also an increase in Ie/Im for PDCer; however, these changes in Ie/Im were significantly slower, requiring approximately 105 min to reach a steady state. These data show that the rapid enzymatic formation of ceramide under these conditions is followed by much slower reorganization process, resulting in the formation of microdomains enriched in this lipid.