The role of sterols in the lipid vesicle response induced by the pore-forming agent nystatin

The influences of ergosterol and cholesterol on the activity of the nystatin were investigated experimentally in a POPC model membrane as well as theoretically. The behavior of giant unilamellar vesicles (GUVs) under osmotic stress due to the formation of transmembrane pores was observed on single vesicles at different nystatin concentrations using phase-contrast microscopy. A significant shift of the typical vesicle behavior, i.e., morphological alterations, membrane bursts, slow vesicle ruptures and explosions, towards lower nystatin concentrations was detected in the ergosterol-containing vesicles and a slight shift towards higher nystatin concentrations was detected in the cholesterol-containing membranes. In addition, the nystatin activity was shown to be significantly affected by the ergosterol membrane’s molar fraction in a non-proportional manner. The observed tension-pore behavior was interpreted using a theoretical model based on the osmotic phenomena induced by the occurrence of size-selective nystatin pores. The number of nystatin pores for different vesicle behavior was theoretically determined and the role of the different mechanical characteristics of the membrane, i.e., the membrane's expansivity and bending moduli, the line tension and the lysis tension, in the tension-pore formation process was quantified. The sterol-induced changes could not be explained adequately on the basis of the different mechanical characteristics, and were therefore interpreted mainly by the direct influences of the membrane sterols on the membrane binding, the partition and the pore-formation process of nystatin.     

Persistent fusion pores but transient fusion in alveolar type II cells

The release of vesicle contents following exocytotic fusion is limited by various factors including the size of the fusion pore. Fusion pores are channel-like, narrow structures after formation and proceed through semi-stable states ('fusion pore flickering'), unless they fully expand (full fusion) or close again (transient fusion). Partial release of vesicle contents may occur during transient fusion, which was described to last between milliseconds and seconds, depending on the size of the vesicle. We studied fusion pores in a slow-secreting lung epithelial cell (type II cell) using fluorescence staining of vesicle contents (surfactant) and fluorescence recovery after photobleaching (FRAP). Surfactant is a lipidic material, which is secreted into the alveolar lumen to reduce the surface tension in the lung. We found release of surfactant to be a slow process, which can last for hours. Accordingly, fusion pores in these cells are stable structures, which appear to be a barrier for release. FRAP measurements suggest that transient fusions occasionally take place in these long-lasting fusion pores, resulting in partial release of surfactant into the extracellular space. These data suggest that postfusion mechanisms may regulate the amount of secreted surfactant.     

Cascades of transient pores in giant vesicles: line tension and transport

Under ordinary circumstances, the membrane tension of a giant unilamellar vesicle is essentially nil. Using visible light, we stretch the vesicles, increasing the membrane tension until the membrane responds by the sudden opening of a large pore (several micrometers in size). Only a single pore is observed at a time in a given vesicle. However, a cascade of transient pores appear, up to 30-40 in succession, in the same vesicle. These pores are transient: they reseal within a few seconds as the inner liquid leaks out. The membrane tension, which is the driving force for pore opening, is relaxed with the opening of a pore and the leakage of the inner liquid; the line tension of the pore's edge is then able to drive the closure of a pore. We use fluorescent membrane probes and real-time videomicroscopy to study the dynamics of the pores. These can be visualized only if the vesicles are prepared in a viscous solution to slow down the leakout of the internal liquid. From measurements of the closure velocity of the pores, we are able to infer the line tension,. We have studied the effect of the shape of inclusion molecules on. Cholesterol, which can be modeled as an inverted cone-shaped molecule, increases the line tension when incorporated into the bilayers. Conversely, addition of cone-shaped detergents reduces. The effect of some detergents can be dramatic, reducing by two orders of magnitude, and increasing pore lifetimes up to several minutes. We give some examples of transport through transient pores and present a rough measurement of the leakout velocity of the inner liquid through a pore. We discuss how our results can be extended to less viscous aqueous solutions which are more relevant for biological systems and biotechnological applications.     

Video fluorescence microscopy studies of phospholipid vesicle fusion with a planar phospholipid membrane. Nature of membrane-membrane interactions and detection of release of contents

Video fluorescence microscopy was used to study adsorption and fusion of unilamellar phospholipid vesicles to solvent-free planar bilayer membranes. Large unilamellar vesicles (2-10 microns diam) were loaded with 200 mM of the membrane-impermeant fluorescent dye calcein. Vesicles were ejected from a pipette brought to within 10 microns of the planar membrane, thereby minimizing background fluorescence and diffusion times through the unstirred layer. Vesicle binding to the planar membrane reached a maximum at 20 mM calcium. The vesicles fused when they were osmotically swollen by dissipating a KCl gradient across the vesicular membrane with the channel-forming antibiotic nystatin or, alternatively, by making the cis compartment hyperosmotic. Osmotically induced ruptures appeared as bright flashes of light that lasted several video fields (each 1/60 s). Flashes of light, and therefore swelling, occurred only when channels were present in the vesicular membrane. The flashes were observed when nystatin was added to the cis compartment but not when added to the trans. This demonstrates that the vesicular and planar membranes remain individual bilayers in the region of contact, rather than melding into a single bilayer. Measurements of flash duration in the presence of cobalt (a quencher of calcein fluorescence) were used to determine the side of the planar membrane to which dye was released. In the presence of 20 mM calcium, 50% of the vesicle ruptures were found to result in fusion with the planar membrane. In 100 mM calcium, nearly 70% of the vesicle ruptures resulted in fusion. The methods of this study can be used to increase significantly the efficiency of reconstitution of channels into planar membranes by fusion techniques.     

Drunken Membranes: Short-Chain Alcohols Alter Fusion of Liposomes to Planar Lipid Bilayers

Although the effects of ethanol on protein receptors and lipid membranes have been studied extensively, ethanol’s effect on vesicles fusing to lipid bilayers is not known. To determine the effect of alcohols on fusion rates, we utilized the nystatin/ergosterol fusion assay to measure fusion of liposomes to a planar lipid bilayer (BLM). The addition of ethanol excited fusion when applied on the cis (vesicle) side, and inhibited fusion on the trans side. Other short-chain alcohols followed a similar pattern. In general, the inhibitory effect of alcohols (trans) occurs at lower doses than the excitatory (cis) effect, with a decrease of 29% in fusion rates at the legal driving limit of 0.08% (w/v) ethanol (IC₅₀ = 0.2% v/v, 34 mM). Similar inhibitory effects were observed with methanol, propanol, and butanol, with ethanol being the most potent. Significant variability was observed with different alcohols when applied to the cis side. Ethanol and propanol enhanced fusion, butanol also enhanced fusion but was less potent, and low doses of methanol mildly inhibited fusion. The inhibition by trans addition of alcohols implies that they alter the planar membrane structure and thereby increase the activation energy required for fusion, likely through an increase in membrane fluidity. The cis data are likely a combination of the above effect and a proportionally greater lowering of the vesicle lysis tension and hydration repulsive pressure that combine to enhance fusion. Alternate hypotheses are also discussed. The inhibitory effect of ethanol on liposome-membrane fusion is large enough to provide a possible biophysical explanation of compromised neuronal behavior.     

Indirect evidence of submicroscopic pores in giant unilamellar [correction of unilamelar] vesicles

Formation of pore-like structures in cell membranes could participate in exchange of matter between cell compartments and modify the lipid distribution between the leaflets of a bilayer. We present experiments on two model systems in which major lipid redistribution is attributed to few submicroscopic transient pores. The first kind of experiments consists in destabilizing the membrane of a giant unilamellar vesicle by inserting conic-shaped fluorescent lipids from the outer medium. The inserted lipids (10% of the vesicle lipids) should lead to membrane rupture if segregated on the outer leaflet. We show that a 5-nm diameter pore is sufficient to ease the stress on the membrane by redistributing the lipids. The second kind of experiments consists in forcing giant vesicles containing functionalized lipids to adhere. This adhesion leads to hemifusion (merging of the outer leaflets). In certain cases, the formation of pores in one of the vesicles is attested by contrast loss on this vesicle and redistribution of fluorescent labels between the leaflets. The kinetics of these phenomena is compatible with transient submicroscopic pores and long-lived membrane defects.     

Voltage-induced nonconductive pre-pores and metastable single pores in unmodified planar lipid bilayer

Electric fields promote pore formation in both biological and model membranes. We clamped unmodified planar bilayers at 150-550 mV to monitor transient single pores for a long period of time. We observed fast transitions between different conductance levels reflecting opening and closing of metastable lipid pores. Although mean lifetime of the pores was 3 +/- 0.8 ms (250 mV), some pores remained open for up to approximately 1 s. The mean amplitude of conductance fluctuations (approximately 500 pS) was independent of voltage and close for bilayers of different area (40,000 and 10 microm(2)), indicating the local nature of the conductive defects. The distribution of pore conductance was rather broad (dispersion of approximately 250 pS). Based on the conductance value and its dependence of the ion size, the radius of the average pore was estimated as approximately 1 nm. Short bursts of conductance spikes (opening and closing of pores) were often separated by periods of background conductance. Within the same burst the conductance between spikes was indistinguishable from the background. The mean time interval between spikes in the burst was much smaller than that between adjacent bursts. These data indicate that opening and closing of lipidic pores proceed through some electrically invisible (silent) pre-pores. Similar pre-pore defects and metastable conductive pores might be involved in remodeling of cell membranes in different biologically relevant processes.     

Single giant vesicle rupture events reveal multiple mechanisms of glass-supported bilayer formation

The formation of supported lipid bilayers (SLBs) on glass from giant unilamellar vesicles (GUVs) was studied using fluorescence microscopy. We show that GUV rupture occurs by at least four mechanisms, including 1), spontaneous rupture of isolated GUVs yielding almost heart-shaped bilayer patches (asymmetric rupture); 2), spontaneous rupture of isolated GUVs yielding circular bilayer patches (symmetric rupture); 3), induced rupture of an incoming vesicle when it contacts a planar bilayer edge; and 4), induced rupture of an adsorbed GUV when a nearby GUV spontaneously ruptures. In pathway 1, the dominant rupture pathway for isolated GUVs, GUVs deformed upon adsorption to the glass surface, and planar bilayer patch formation was initiated by rupture pore formation near the rim of the glass-bilayer interface. Expanding rupture pores led to planar bilayer formation in approximately 10-20 ms. Rupture probability per unit time depended on the average intrinsic curvature of the component lipids. The membrane leaflet adsorbed to the glass surface in planar bilayer patches originated from the outer leaflet of GUVs. Pathway 2 was rarely observed. We surmise that SLB formation is predominantly initiated by pathway 1 rupture events, and that rupture events occurring by pathways 3 and 4 dominate during later stages of SLB formation.     

Effects of Taurine–Magnesium Coordination Compound on Ionic Channels in Rat Ventricular Myocytes of Arrhythmia Induced by Ouabain

Taurine-magnesium coordination compound (TMCC) has anti-arrhythmic effects. The aim of the present study was to explore the targets of the anti-arrhythmic effect of TMCC and the electrophysiological effects of TMCC on ouabain-induced arrhythmias in rat ventricular myocytes. Sodium current (I(Na)), L-type calcium current (I(ca, L)), and transient outward potassium current (I(to)) were measured and analyzed using whole-cell patch-clamp recording technique in normal rat cardiac myocytes and rat ventricular myocytes of arrhythmia induced by ouabain. In isolated ventricular myocytes, I(Na) and I(to) were blocked by TMCC (100, 200, 400 μM) in a concentration-dependent manner, and the effects of TMCC (400 μM) were equal to that of amiodarone. However, I (ca, L) was moderately increased by TMCC (400 μM) while significantly decreased by amiodarone. Ouabain (5 μM) significantly decreased sodium, L-type calcium, and transient outward potassium currents. TMCC (100 μM) relieved abnormal sodium currents induced by ouabain through facilitation of steady-state inactivation. TMCC (200 and 400 μM) relieved abnormal L-type calcium currents induced by ouabain through facilitation of steady-state activation and retardation of steady-state inactivation. TMCC failed to further inhibit abnormal transient outward potassium currents induced by ouabain. However, amiodarone inhibited the decreasing sodium, L-type calcium, and transient outward potassium currents further. These data suggest that I(Na), I(ca, L), and I(to) may be the targets of the antiarrhythmic effect of TMCC, which can antagonize ouabain-induced changes of ionic currents in rat ventricular myocytes.     

Response of adenine and pyridine metabolism during germination and early seedling growth under arsenic stress in Brassica juncea

Adenine and pyridine nucleotides play vital roles in virtually all aspects of plant growth. This study analyzed the response of adenine and pyridine metabolism during germination and early seedling growth (ESG) of Brassica juncea exposed to two doses of arsenate (AsV), 100 and 250 μM, having non-significant or significant inhibitory effects, respectively, on germination and ESG. The ratio of NAD/NADP and NAD/NADH showed no significant change in control and 100 μM AsV, but increased significantly at 250 μM AsV during initial 24 h and also at 7th day. The activity of enzymes of NAD metabolism, viz. NAD kinase, NADP phosphatase, nicotinamidase and poly(ADP-ribose) polymerases showed significant change mostly at 250 μM AsV. Further, significant decrease was observed in the ratio of ATP/ADP and in the activities of adenylate kinase and apyrase at 250 μM AsV at 7th day. External supply of ATP (1 mM) to 100 and 250 μM AsV significantly improved germination percentage and germination strength of the seeds as compared to AsV treatments alone. The study concludes that with the increase in concentration of AsV, the balance of NAD/NADP, NAD/NADH and ATP/ADP and the activities of enzymes of adenine and pyridine metabolism were significantly altered and that these changes may be responsible for inhibitory effects of AsV on germination and ESG.     

Ultradeformable lipid vesicles can penetrate the skin and other semi-permeable barriers unfragmented. Evidence from double label CLSM experiments and direct size measurements

The stability of various aggregates in the form of lipid bilayer vesicles was tested by three different methods before and after crossing different semi-permeable barriers. First, polymer membranes with pores significantly smaller than the average aggregate diameter were used as the skin barrier model; dynamic light scattering was employed to monitor vesicle size changes after barrier passage for several lipid mixtures with different bilayer elasticities. This revealed that vesicles must adapt their size and/or shape, dependent on bilayer stability and elasto-mechanics, to overcome an otherwise confining pore. For the mixed lipid aggregates with highly flexible bilayers (Transfersomes®), the change is transient and only involves vesicle shape and volume adaptation. The constancy of ultradeformable vesicle size before and after pores penetration proves this. This is remarkable in light of the very strong aggregate deformation during an enforced barrier passage. Simple phosphatidylcholine vesicles, with less flexible bilayers, lack such capability and stability. Conventional liposomes are therefore fractured during transport through a semi-permeable barrier; as reported by other researchers, liposomes are fragmented to the size of a narrow pore if sufficient pressure is applied across the barrier; otherwise, liposomes clog the pores. The precise outcome depends on trans-barrier flux and/or on relative vesicle vs. pore size. Lipid vesicles applied on the skin behave accordingly. Mixed lipid vesicles penetrate the skin if they are sufficiently deformable. If this is the case, they cross inter-cellular constrictions in the organ without significant composition or size modification. To prove this, we labelled vesicles with two different fluorescent markers and applied the suspension on intact murine skin without occlusion. The confocal laser scanning microscopy (CLSM) of the skin then revealed a practically indistinguishable distribution of both labels in the stratum corneum, corroborating the first assumption. To confirm the second postulate, we compared vesicle size in the starting suspension and in the blood after non-invasive transcutaneous aggregate delivery. Size exclusion chromatograms of sera from the mice that received ultradeformable vesicles on the skin were undistinguishable from the results measured with the original vesicle suspension. Taken together, the results support our previous postulate that ultradeformable vesicles penetrate the skin intact, that is, without permanent disintegration.     

Effect of Membrane Structure on the Action of Polyenes II: Nystatin Activity along the Phase Diagram of Ergosterol- and Cholesterol-Containing POPC Membranes

Pores formed by the polyene antibiotic nystatin were studied in solvent-free lipid membranes. The membranes were formed by the tip-dip technique using 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) with different mol fractions (0–50%) of cholesterol or ergosterol. The effects of the mol fraction of sterol and of temperature variation (15–35°C) on the activity of the pores, their unitary conductances, lifetimes and time average conductances were studied. The results were used to analyze the behavior of nystatin channels along the phase diagrams previously reported for these lipid mixtures and to propose that membrane structure is the determinant factor for the known ergosterol/cholesterol selectivity.     

Photoperiod and elicitors increase steviol glycosides,phenolics, and flavonoid contents in root cultures of Stevia rebaudiana

The establishment of green root cultures of Stevia rebaudiana Bertoni, and the effect of elicitors such as hydrogen peroxide (H₂O₂) and methyl jasmonate (MeJA), is shown in the present study. Stevioside, rebaudioside A, and the isomers steviol/isosteviol were identified through DFI-ESI-IT-MSⁿ and UPLC-TOFMS spectrometric systems, in combination with solid-phase extraction. The accumulation of steviol glycosides increased by 2.4 times (compared to the control value of 22.35 μgSG per gDW), with the addition of 250 μM H₂O₂. The non-enzymatic antioxidant response, which resulted from production of phenolic and flavonoid compounds, was modified based on the elicitor and the dose used. The maximum accumulation of flavonoids was induced on the third day with the addition of H₂O₂ (250 or 500 μM), and with MeJA (250 or 500 μM); the increase was observed on the fifth day. The enzymatic antioxidant response of the catalase and peroxidase from the roots under elicitation confirmed the stress conditions.

     

The inhibitory effects of perfluoroalkyl substances on human and rat 11β-hydroxysteroid dehydrogenase 1

Perfluoroalkyl substances (PFASs) are man-made polyfluorinated compounds that are widely used and persistent in the environment. PFASs have potential effects on many biological systems including the development of lung. Glucocorticoids have been reported to promote fetal and neonatal lung development at the late stage, and 11β-hydroxysteroid dehydrogenase 1(11βHSD1) in the lung is critical for the generation of local active glucocorticoid cortisol (human) or corticosterone (rodents) from biologically inert 11keto-steroids. The purpose of the present study is to study the direct inhibitory effects of PFASs on 11βHSD1 activities and action modes. Microsomal 11βHSD1 was subjected to the exposure to various PFASs, including perfluorooctane sulfonic acid (PFOS), perfluorooctanoic acid (PFOA), potassium perfluorohexanesulfonate (PFHxS) and potassium perfluorobutane sulfonate (PFBS). PFOS and PFOA inhibited neonatal rat lung 11βHSD1 activity with IC(50)s of 3.45μM (95% Confidence Intervals, CI(95): 1.97-6.37μM) and 45.31μM (CI(95): 27.64-74.26μM), respectively, while PFHxS and PFBS did not inhibit the enzyme activity at 250μM. PFOS and PFOA inhibited human 11βHSD1 activity with IC(50)s of 7.56μM (CI(95): 2.86-19.97μM) and 37.61μM (CI(95): 24.49-57.75μM), respectively, while PFHxS and PFBS did not inhibit the enzyme activity at 250μM. PFASs showed competitive inhibition on both human and rat 11βHSD1. In conclusion, the present study shows that PFOS and PFOA are the inhibitors of 11βHSD1.     

MPTP modulates hippocampal synaptic transmission and activity-dependent synaptic plasticity via dopamine receptors

Parkinson's disease (PD)-like symptoms and cognitive deficits are inducible by 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP). Since cognitive abilities, including memory formations rely also on hippocampus, we set out to clarify the effects of MPTP on hippocampal physiology. We show that bath-application of MPTP (25?μM) to acute hippocampal slices enhanced AMPA receptor-mediated field excitatory postsynaptic potentials (AMPAr-fEPSPs) transiently, whereas N-methyl-D-aspartate (NMDA) receptor-mediated fEPSPs (NMDAr-fEPSPs) were facilitated persistently. The MPTP-mediated transient AMPAr-fEPSP facilitation was antagonized by the dopamine D2-like receptor antagonists, eticlopride (1?μM) and sulpiride (1 and 40?μM). In contrast, the persistent enhancement of NMDAr-fEPSPs was prevented by the dopamine D1-like receptor antagonist SCH23390 (10?μM). In addition, we show that MPTP decreased paired-pulse facilitation of fEPSPs and mEPSCs frequency. Regarding activity-dependent synaptic plasticity, 25?μM MPTP transformed short-term potentiation (STP) into a long-term potentiation (LTP) and caused a slow onset potentiation of a non-tetanized synaptic input after induction of LTP in a second synaptic input. This heterosynaptic slow onset potentiation required activation of dopamine D1-like and NMDA-receptors. We conclude that acute MPTP application affects basal synaptic transmission by modulation of presynaptic vesicle release and facilitates NMDAr-fEPSPs as well as activity-dependent homo- and heterosynaptic plasticity under participation of dopamine receptors.     

Evolution of the hemifused intermediate on the pathway to membrane fusion

The pathway to membrane fusion in synthetic and biological systems is thought to pass through hemifusion, in which the outer leaflets are fused while the inner leaflets engage in a hemifusion diaphragm (HD). Fusion has been proposed to be completed by lysis of the expanded HD that matures from a localized stalklike initial connection. However, the process that establishes the expanded HD is poorly understood. Here we mathematically modeled hemifusion of synthetic vesicles, where hemifusion and fusion are most commonly driven by calcium and membrane tension. The model shows that evolution of the hemifused state is driven by these agents and resisted by interleaflet frictional and tensile stresses. Predicted HD growth rates depend on tension and salt concentration, and agree quantitatively with experimental measurements. For typical conditions, we predict that HDs expand at ～30 μm(2)/s, reaching a final equilibrium area ～7% of the vesicle area. Key model outputs are the evolving HD tension and area during the growth transient, properties that may determine whether HD lysis occurs. Applying the model to numerous published experimental studies that reported fusion, our results are consistent with a final fusion step in which the HD ruptures due to super-lysis HD membrane tensions.     

Competitive binding of cholesterol and ergosterol to the polyene antibiotic nystatin. A fluorescence study

Competition studies between cholesterol and ergosterol were carried out to gain insight into the binding interactions between nystatin and these sterols. Lipid vesicles were prepared with mixtures of palmitoyloleoylphosphocholine/ergosterol/cholesterol, and both sterol molar ratio and total content were varied. The inhibitory effect of cholesterol toward the ergosterol ability to induce the formation of long-lived fluorescent antibiotic species was used to detect nystatin-cholesterol interactions. It was found that the key factor controlling nystatin photophysical properties in the ternary lipid mixtures was their ergosterol/cholesterol molar ratio and not their overall sterol content. Moreover, permeabilization studies showed that nystatin was able to form pores in all the mixed vesicles, but the initial rate of pore formation was also dependent on the ergosterol/cholesterol molar ratio. Our data show that ergosterol is displaced by competing cholesterol, indirectly confirming cholesterol's ability to coassemble with nystatin. The distinct spectroscopic properties emphasize the different molecular architecture adopted by nystatin-cholesterol and -ergosterol complexes, and therefore are relevant to understanding the interaction of the antibiotic with membranes.     

Effects of cadmium on meristem activity and nucleus ploidy in roots of Pisum sativum L. cv. Frisson seedlings

The effects of cadmium (Cd) administration on primary root growth, mitotic activity of apical meristems, mitotic aberrations and percentage of nucleus ploidy classes of differentiated roots were examined in Pisum sativum L. cv. Frisson. Cadmium caused a reduction of root length related to concentration, with an almost complete block of growth in plants treated with 250 μM Cd, from 24 h of treatment. Root lengthening is generally related to apical meristem activity, however, in the examined pea plants, mitotic activity was suppressed by 2.5 and 25 μM Cd treatment, while the highest Cd concentration, 250 μM, caused the occurrence of mitotic figures consisting almost exclusively of prophases. The lack of relation between root lengthening and mitotic activity was explained by the meristematic activity in the first period of treatment and by a different cell elongation. Lower (0.25, 0.5 and 1 μM), non-blocking Cd concentrations induced a number of mitotic aberrations, mainly consisting of sticky metaphases and anaphase bridges, whose frequency increased with Cd concentration. Besides, Cd induced variations of the percentages of nucleus populations in the differentiated roots, increasing the percentage of 4C nuclei and decreasing that of 2C. The mechanisms involved in the nuclear response to Cd, and the possible relations between Cd alteration of meristem cell activity and nuclear ploidy of differentiated cells are discussed.     

Effects of alcohol-induced lipid interdigitation on proton permeability in L-alpha-dipalmitoylphosphatidylcholine vesicles.

6-Carboxyfluorescein was employed to examine the effect of alcohol-induced lipid interdigitation on proton permeability in L-alpha-dipalmitoylphosphatidylcholine (DPPC) large unilamellar vesicles. Proton permeability was measured by monitoring the decrease of 6-carboxyfluorescein fluorescence after a pH gradient from 3.5 (outside the vesicle) to 8.0 (inside the vesicle) was established. At 20 degrees C and below 1.2 M ethanol, the fluorescence decrease is best described by a single exponential function. Above 1.2 M ethanol, the intensity decrease is better described by a two-exponential decay law. Using the fitted rate constants and the vesicle radii determined from light-scattering measurements, the proton permeability coefficient, P, in DPPC vesicles was calculated as a function of ethanol concentration. At 20 degrees C, P increases monotonically with increasing ethanol content up to 1.0 M, followed by an abrupt increase at 1.2 M. The vesicle size also exhibits a sudden increase at around 1.2 M ethanol, which has been shown to result from vesicle aggregation rather than vesicle fusion. The abrupt increases in P and in vesicle size occur at the concentration region close to the critical ethanol concentration for the formation of the fully interdigitated gel state of DPPC. At 14 degrees C, the abrupt change in P shifts to 1.9-2.0 M ethanol, completely in accordance with the ethanol-temperature phase diagram of interdigitated DPPC. Effects of methanol and benzyl alcohol on lipid interdigitation have also been examined. At 20 degrees C, DPPC large unilamellar vesicles exhibit a dramatic change in P at 3 M methanol and at 40 mM benzyl alcohol. These concentrations come close to the critical methanol and benzyl alcohol concentrations for the formation of fully interdigitated DPPC structures determined previously by others. It can be concluded that proton permeability increases dramatically as DPPC is transformed from the noninterdigitated gel to the fully interdigitated gel state by high concentrations of alcohol. This marked increase in proton permeability can be attributed to the combined effect of the changes in membrane thickness and surface charge density, due to the ethanol-induced lipid interdigitation. The possible effects of the increased proton permeability caused by ingested ethanol on gastric mucosal membranes are discussed.     

Mechanical properties of vesicles. I. Coordinated analysis of osmotic swelling and lysis.

To determine how transmembrane osmotic gradients perturb the structure and dynamics of biological membranes, we examined the effects of medium dilution on the structures of osmolyte-loaded lipid vesicles. Our preparations were characterized by dynamic light scattering (DLS) and nuclear magnetic resonance (NMR) spectroscopies. Populations of Escherichia coli phosphatidylethanolamine (PE) or dioleoylphosphatidylglycerol (DOPG) vesicles prepared by the pH jump technique were variable and polymodal in size distribution. Complex and variable structural changes occurred when PE vesicles were diluted with hypotonic buffer. Such vesicles could not be used as model systems for the analysis of membrane mechanical properties. NaCl-loaded, DOPG vesicles prepared by extrusion through 100 nm (diameter) pores were reproducible and monomodal in size distribution and unilamellar, whereas those prepared by extrusion through 200-, 400-, or 600-nm pores were variable and polymodal in size distribution and/or multilamellar. Time and pressure regimes associated with osmotic lysis of extruded vesicles were defined by monitoring release of carboxyfluorescein, a self-quenching fluorescent dye. Corresponding effects of medium dilution on vesicle structure were assessed by DLS spectroscopy. These experiments and the accompanying analysis (Hallett, F.R., J. Marsh, B.G. Nickel, and J.M. Wood. 1993. Biophys. J. 64:000-000) revealed conditions under which vesicles are expected to reside in a consistently strained state.