Coupling of spectrin and polylysine to phospholipid monolayers studied by specular reflection of neutrons.

The technique of specular reflection of neutrons is applied for the first time to study the charge-dependent interaction of the protein spectrin and the polypeptide poly-L-lysine with model phospholipid monolayers in the condensed phase state. We first established the structure of a pure monolayer of dimyristolyphosphatidylcholine (DMPC) in both the expanded and condensed fluid phase states without protein in the subphase. The thickness of the hydrocarbon chains increases from 11.4 +/- 1.5 A in the expanded state to 15.8 +/- 1.5 A in the condensed state, whereas the head group region is approximately 10 A thick for both phase states. When spectrin is present in the subphase, the dimensions of DMPC in the condensed state are not significantly affected, but there is approximately 0.09 volume fraction spectrin in the head group region. Lipid-spectrin coupling is enhanced by electrostatic interaction, as the volume fraction of spectrin in the head group region increases to 0.22 in a mixed monolayer of DMPC and negatively charged dimyristolyphosphatidylglycerol in the condensed state. In contrast to spectrin, polylysine does not penetrate the head group region, but forms a layer electrostatically adsorbed to the charged head groups.     

Epifluorescence microscopic studies of monolayers containing mixtures of dioleoyl- and dipalmitoylphosphatidylcholines.

Monolayers of dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), and some mixtures of these lipids were investigated using an epifluorescence microscopic surface balance. Monolayers were visualized at 23 +/- 1 degree C through the fluorescence of 1 mol% of two different fluorescent probes, 1-palmitoyl-2-(12-[(7-nitro-2-1,3-benzoxadizole-4- yl)amino]dodecanoyl)phosphatidylcholine (NBD-PC), which partitions into the liquid expanded (LE) or disordered lipid phase and 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO-C18), which preferentially associates with the liquid condensed (LC) phase or lipid with ordered chains. LC domains were observed in pure DPPC monolayers at relatively low surface pressures (pi), and these domains grew with increasing surface pressure. Only liquid expanded phase was observed in pure DOPC monolayers up to the point of monolayer collapse. In monolayers containing 29:70:1, 49:50:1, and 69:30:1 (mol/mol/mol) of DPPC:DOPC:probe the domains of LC phase were smaller than those seen in DPPC monolayers at equivalent surface pressures. Quantitative analysis of the visual fields shown by the mixed monolayers showed a distribution of sizes of condensed domains at any given pi. At pi = 30 mN m-1, liquid-expanded, or fluid, regions occupied more than 70% of the total monolayer area in all three mixtures studied, whereas DPPC monolayers were more than 75% condensed or solid at that pressure. For monolayers of DPPC:DOPC:NBD-PC 49:50:1 and 69:30:1 the average domain size and the percentage of the total area covered with LC, or rigid, areas increased to a maximum at pi around 35 mN m-1 followed by a decrease at higher pi.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of long-chain nicotinates with dipalmitoylphosphatidylcholine

The interaction of four long-chain nicotinates, compounds that are of interest as potential chemopreventive agents, with dipalmitoylphosphatidylcholine (DPPC) was investigated in monolayers at the air-water interface and in fully hydrated bilayers. For the monolayer studies, the compression isotherms of mixtures of the respective nicotinate with DPPC were recorded at various compositions on a hydrochloric acid subphase (pH 1.9-2.1, 37 +/- 2 degrees C). The headgroup of the nicotinates (24-29 A2/molecule) is larger than that of the hydrophobic tail (20 A2/molecule). The pure nicotinates exhibit a temperature- and chain length-dependent transition from an expanded to a condensed phase. Analysis of the concentration dependence of the average molecular area at constant film pressure and the concentration dependence of the breakpoint of the phase transition from the expanded to the condensed state suggests that all four DPPC-nicotinate mixtures are partially miscible at the air-water interface. Although a complex phase behavior with several phase transitions was observed, differential scanning calorimetry studies of the four mixtures are also indicative of the partial miscibility of DPPC and the respective nicotinate. Overall, the complex phase behavior most likely results from the head-tail mismatch of the nicotinates and the geometric packing constraints in the two-component lipid bilayer.     

Interfacial structure and lipase action. Characterization of taurodeoxycholate-didecanoylglycerol monolayers by physical and kinetic methods.

Surface pressure-area isotherms for 1,3-didecanoyl-glycerol (dicaprin) were determined as a function of the concentration of taurodeoxycholate in the subphase. Analysis of these curves indicates that, from 0.05 to 0.80 mM bile salt, surface structure is dependent only on the surface concentration of the diglyceride. The limiting areas for dicaprin in the presence and absence of bile salt were about 38 A2/molecule. Subjecting the monolayers to hydrolysis by pancreatic lipase yielded kinetic data which, together with the physical studies, support a model for monolayer glyceride molecules undergoing discrete changes of state. In the absence of bile salt, the relatively expanded state exhibits an area of 75 A2/diglyceride molecule and is not a substrate for pancreatic lipase B. The more condensed state exhibits an area of 38 A2/diglyceride molecule and is hydrolyzed at a rate proportional to its concentration in the monolayer. Taurodeoxybholate at 0.05 to 0.60 mM shifts the apparent area of the expanded state to 360 A2/diglyceride molecule.     

Phase transitions in films of lung surfactant at the air-water interface.

Pulmonary surfactant maintains a putative surface-active film at the air-alveolar fluid interface and prevents lung collapse at low volumes. Porcine lung surfactant extracts (LSE) were studied in spread and adsorbed films at 23 +/- 1 degrees C using epifluorescence microscopy combined with surface balance techniques. By incorporating small amounts of fluorescent probe 1-palmitoyl-2-nitrobenzoxadiazole dodecanoyl phosphatidylcholine (NBD-PC) in LSE films the expanded (fluid) to condensed (gel-like) phase transition was studied under different compression rates and ionic conditions. Films spread from solvent and adsorbed from vesicles both showed condensed (probe-excluding) domains dispersed in a background of expanded (probe-including) phase, and the appearance of the films was similar at similar surface pressure. In quasistatically compressed LSE films the appearance of condensed domains occurred at a surface pressure (pi) of 13 mN/m. Such domains increased in size and amounts as pi was increased to 35 mN/m, and their amounts appeared to decrease to 4% upon further compression to 45 mN/m. Above pi of 45 mN/m the LSE films had the appearance of filamentous materials of finely divided dark and light regions, and such features persisted up to a pi near 68 mN/m. Some of the condensed domains had typical kidney bean shapes, and their distribution was similar to those seen previously in films of dipalmitoylphosphatidylcholine (DPPC), the major component of surfactant. Rapid cyclic compression and expansion of LSE films resulted in features that indicated a possible small (5%) loss of fluid components from such films or an increase in condensation efficiency over 10 cycles. Calcium (5 mM) in the subphase of LSE films altered the domain distribution, decreasing the size and increasing the number and total amount of condensed phase domains. Calcium also caused an increase in the value of pi at which the maximum amount of independent condensed phase domains were observed to 45 mN/m. It also induced formation of large amounts of novel, nearly circular domains containing probe above pi of 50 mN/m, these domains being different in appearance than any seen at lower pressures with calcium or higher pressures in the absence of calcium. Surfactant protein-A (SP-A) adsorbed from the subphase onto solvent-spread LSE films, and aggregated condensed domains in presence of calcium. This study indicates that spread or adsorbed lung surfactant films can undergo expanded to condensed, and possibly other, phase transitions at the air-water interface as lateral packing density increases. These phase transitions are affected by divalent cations and SP-A in the subphase, and possibly by loss of material from the surface upon cyclic compression and expansion.     

Mixed monolayers of straight-chain/branched-chain phospholipids. I. Mixed monolayers of distearoyl phosphatidylcholine and diisoeicosanoyl phosphatidylcholine

The temperature dependence of the force/area isotherms of monolayer of distearoyl phosphatidylcholine (DSPC), diisoeicosanoyl phosphatidylcholine (DIEPC) and a complete mixed compositional range of these two lecithins are reported. The isotherms for DSPC closely resemble those previously reported for dipalmitoyl phosphatidylcholine but are shifted to higher temperatures by 16 degrees C. The isotherms of DIEPC, an iso-branched lecithin, show differences from these obtained for similar straight-chain lecithins in that the full condensed isotherms are more expanded, the fully expanded isotherms are more condensed and therefore the liquid expanded (LE)/liquid condensed (LC) intermediate region is significantly reduced. This means that the condensed state is more disordered and the expanded state is less disordered than the corresponding states in straight-chain lecithins. Data for the mixed films are interpreted in terms of surface pressure/mole fraction phase diagrams and both energies and entropies of compression associated with the LE/LC transition. The phase diagrams at 34.1 degrees C, 35.8 degrees C and 38.5 degrees C are all of the negative azeotropic type with the surface pressure minimum point shifting with temperature. The thermodynamic analysis indicates that from 34.1 degrees C to 38.5 degrees C the driving force for mixing changes from the entropy to the energy of the transition. It would seem that at the lower temperature the packing of the distearoyl lecithin is perturbed by the diisoeicosanoyl lecithin, while at higher temperatures the very high entropy of pure or nearly pure diisoeicosanoyl lecithin results in other mixtures having less entropy than would be expected on an ideal mixing basis.     

Anaesthetic — phospholipid interaction

Binding of the positively charged drug chlorpromazine to phospholipid monolayers was investigated. A preferential uptake was observed near the phase transtion of the corresponding lipid. Cholesterol considerably diminishes the chlorpromazine uptake, again particularly near a lipid phase transition. The binding properties depend on the chlorpromazine concentration in the subphase. A critical concentration is 5·10^-5M, where higher uptake occurs in the liquid condensed than in the liquid expanded state of the monolayer at pressures of about 10 mN/m. Dipalmitoylphosphatidylcholine monolayers spread on a subphase containing chlorpromazine are comparable to monolayers at higher temperature but in the absence of chlorpromazine. These data are in agreement with previous fluorescence and electron paramagnetic resonance experiments on lipid bilayer membranes (Luxnat and Galla 1986).Abbreviations CPZ chlorpromazine - DPPC dipalmitoylphosphatidylcholine - DMPC dimyristoylphosphatidylcholine - LE liquid expanded - LC liquid condensed     

The detrimental effect of serum albumin on the re-spreading of a dipalmitoyl phosphatidylcholine Langmuir monolayer is counteracted by a fluorocarbon gas

We have recently reported that fluorocarbon gases exhibit an effective fluidizing effect on Langmuir monolayers of dipalmitoyl phosphatidylcholine (DPPC), preventing them from crystallizing up to surface pressures of approximately 40 mN m(-1), i.e. well above the DPPC's equilibrium surface pressure. We now report that gaseous perfluorooctyl bromide (gPFOB) promotes the re-spreading of DPPC Langmuir monolayers compressed on a bovine serum albumin (BSA)-containing sub-phase. The latter protein is known to maintain a concentration-dependent surface pressure that can exceed the re-spreading pressure of collapsed monolayers. This phenomenon was proposed to be responsible for lung surfactant inactivation. Compression/expansion isotherms and fluorescence microscopy experiments were carried out to assess the monolayers' physical state. We have found that, during expansion under gPFOB-containing air, the surface pressure of a DPPC monolayer on a BSA-containing sub-phase decreased to much lower values than when the DPPC monolayer was expanded in the presence of BSA under air ( approximately 0 mN m(-1) vs. approximately 7.5 mN m(-1) at 120 A(2), respectively). Moreover, fluorescence images showed that, during expansion, the BSA-coupled DPPC monolayers, in contact with gPFOB, remained in the liquid-expanded state for surface pressures lower than 10 mN m(-1), whereas they were in a liquid-condensed semi-crystalline state, even at large molecular areas (120 A(2)), when expanded under air. The re-incorporation of the PFOB molecules in the DPPC monolayer during expansion thus competes with the re-incorporation of BSA, thus preventing the latter from penetrating into the DPPC monolayer. We suggest that combinations of DPPC and a fluorocarbon gas may be useful in the treatment of lung conditions resulting from a deterioration of the native lung surfactant function due to plasma proteins, such as in the acute respiratory distress syndrome.     

Direct evidence for the formation of a monolayer from a bilayer. An ellipsometric study at the nitrogen-water interface.

Direct evidence for the formation of a monolayer from a bilayer was measured by ellipsometry after spreading unilamellar vesicles of dioleoyl phosphatidylcholine (DOPC) at the nitrogen-water interface. The ellipsometric isotherms of DOPC vesicles and DOPC spread from an organic solvent were compared and found similar. From the observed ellipsometric angle (delta delta) in the plateau region (-1.04 degrees) and literature data for refractive indices of an anisotropic film similar to DOPC, we have calculated a thickness of 20 +/- 1 A. These results strongly suggest that, similarly to DOPC spread from an organic solvent, DOPC vesicles form a monolayer when spread at the nitrogen-water interface.     

Effect of hydrophobic surfactant protein SP-C on binary phospholipid monolayers. Molecular machinery at the air/water interface

Fluorescent and modified dark-field microscopies were used to investigate the phase behavior of physiologically relevant lipid/protein monomolecular films containing surfactant protein C(SP-C). Synthetic human SP-C(1-34) was labeled at its N-terminus using the fluorescent probe 6-(((4(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)phenoxy)acetyl)amino)hexanoic acid (BODIPY/TR-X). Using dual fluorescent labeling (lipid and protein) in the monolayers, we have correlated (at physiologically small concentrations of the protein) the lipid phase separation and protein distribution in situ. A comparison of the lipid and protein dye fluorescent micrographs indicates that SP-C(1-34) is preferentially associated with the disordered lipid phase. Three concepts arise from our results. (1) The presence of SP-C alone does not result in the complete dissolution of condensed phase domains in a fashion that we have previously reported for the entire hydrophobic surfactant protein (SP-B/C) fraction (Biophys. J. 77 (1999) 903). Rather, the use of relatively high amounts ( approximately 10 wt.%) of the labeled SP-C protein is needed to reproduce the fluorescence monolayer morphology previously observed for small concentrations ( approximately 1 wt.%) of the natural SP-B/C mixture. (2) Scattered light, dark-field microscopy performed using grazing angle laser illumination reveals the presence of surface-associated, three-dimensional (3D) structures of micrometer-sized dimensions when the labeled BODIPY/TR-X:SP-C(1-34) protein is included in the monolayer, as previously observed with the naturally isolated SP-B/C mixture. The 3D structures are associated exclusively with the presence of the SP-C protein in disordered monolayer phases. (3) To explain these results, we have derived a molecular model accounting for the structure and physico-chemical properties of the SP-C protein in terms of its energetics. The molecular events involved in the SP-C-mediated production of the 3D surface particles are explained using the analogy of a simple molecular machine, namely a loaded spring. This interpretation is supported by an energetic analysis that suggests the major factor contributing to the formation of the 3D particles is the energy liberated by re-expansion of the surrounding phospholipid film into the area vacated by the SP-C protein as it re-orients away from the surface.     

Octyl-beta-D-glucopyranoside partitioning into lipid bilayers: thermodynamics of binding and structural changes of the bilayer.

The interaction of the nonionic detergent octyl-beta-D-glucopyranoside (OG) with lipid bilayers was studied with high-sensitivity isothermal titration calorimetry (ITC) and solid-state 2H-NMR spectroscopy. The transfer of OG from the aqueous phase to lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) can be investigated by employing detergent at concentrations below the critical micellar concentration; it can be defined by a surface partition equilibrium with a partition coefficient of K = 120 +/- 10 M-1, a molar binding enthalpy of delta H degrees D = 1.3 +/- 0.15 kcal/mol, and a free energy of binding of delta G degrees D = -5.2 kcal/mol. The heat of transfer is temperature dependent, with a molar heat capacity of delta CP = -75 cal K-1 mol-1. The large heat capacity and the near-zero delta H are typical for a hydrophobic binding equilibrium. The partition constant K decreased to approximately 100 M-1 for POPC membranes mixed with either negatively charged lipids or cholesterol, but was independent of membrane curvature. In contrast, a much larger variation was observed in the partition enthalpy. delta H degrees D increased by about 50% for large vesicles and by 75% for membranes containing 50 mol% cholesterol. Structural changes in the lipid bilayer were investigated with solid-state 2H-NMR. POPC was selectively deuterated at the headgroup segments and at different positions of the fatty acyl chains, and the measurement of the quadrupolar splittings provided information on the conformation and the order of the bilayer membrane. Addition of OG had almost no influence on the lipid headgroup region, even at concentrations close to bilayer disruption. In contrast, the fluctuations of fatty acyl chain segments located in the inner part of the bilayer increased strongly with increasing OG concentration. The 2H-NMR results demonstrate that the headgroup region is the most stable structural element of the lipid membrane, remaining intact until the disordering of the chains reaches a critical limit. The perturbing effect of OG is thus different from that of another nonionic detergent, octaethyleneglycol mono-n-dodecylether (C12E8), which produces a general disordering at all levels of the lipid bilayer. The OG-POPC interaction was also investigated with POPC monolayers, using a Langmuir trough. In the absence of lipid, the measurement of the Gibbs adsorption isotherm for pure OG solutions yielded an OG surface area of AS = 51 +/- 3 A2. On the other hand, the insertion area AI of OG in a POPC monolayer was determined by a monolayer expansion technique as AI = 58 +/- 10 A2. The similar area requirements with AS approximately AI indicate an almost complete insertion of OG into the lipid monolayer. The OG partition constant for a POPC monolayer at 32 mN/m was Kp approximately 320 M-1 and thus was larger than that for a POPC bilayer.     

Physical and surface properties of insect apolipophorin III

Apolipophorin III (apoLp-III) from Manduca sexta has a molecular weight of 18,100. Based on its hydrodynamic properties (sedimentation and diffusion coefficients, frictional ratio, intrinsic viscosity) and its behavior during gel permeation chromatography, we concluded that apoLp-III is a prolate ellipsoid with an axial ratio of about 3. The circular dichroic spectrum of apoLp-III suggests that the protein contains approximately 50% alpha-helix. At the air-water interface, apoLp-III forms a monolayer which is gaseous at surface pressures less than or equal to 1 dyne/cm. The isotherm of this phase yields an excluded molecular area of 3800 A2/molecule (23 A2/amino acid). At a surface pressure of 22.1 dynes/cm, the monolayer undergoes a phase transition reminiscent of a first-order phase transition of pure lipids. The monolayer can be compressed in this surface pressure range to an area per molecule of 480 A2 (2.9 A2/amino acid). Since a globular protein of molecular weight 18,100 could occupy an area of only about 2000 A2 when bound to a surface, it is suggested that in the expanded state, apoLp-III must unfold on the surface, whereas in the compressed state, the molecule is oriented with its minor axis parallel to the water surface. ApoLp-III binds with high affinity (Kd = 1.9 X 10(-7)M) to both phosphatidylcholine- and diacylglycerol-coated polystyrene beads. All of these results are consistent with the proposal that apoLp-III plays a key role in increasing the capacity of the insect lipoprotein, lipophorin, to transport diacylglycerol by stabilizing the increment of lipid-water interface that results from diacylglycerol uptake.     

Thermodynamic and dynamic characteristics of hydroxypropylmethylcellulose adsorbed films at the air-water interface

Surface pressure isotherms and structural and surface dilatational properties of three hydroxypropylmethycelluloses (HPMCs, called E4M, E50LV, and F4M) adsorbed films at the air-water interface were determined. In this work we present evidence that HPMC molecules are able to diffuse and saturate the air-water interface at very low concentrations in the bulk phase. As bulk concentration increased, structural changes at a molecular level occurred at the interface. These changes corresponded to transition from an expanded structure (structure I) to a condensed one (structure II). When the surface concentration of HPMC was high enough, the collapse of the monolayer was observed. The three HPMCs formed very elastic films at the air-water interface, even at low surface pressures. E4M showed features that make it unique. For instance it showed the highest surface activity, mainly at low bulk concentrations (<10(-4) wt %). The differences observed in surface activity may be attributed to differences in the hydroxypropyl molar substitution and molecular weight of HPMC. All three HPMCs formed films of similar viscoelasticity and elastic dilatational modulus, which can be accounted for by their similar degree of methyl substitution.     

Generation of a scaffold free cartilage-like implant from a small amount of starting material

INTRODUCTION: An autologous cellular based treatment of a traumatic cartilage injury requires a procedure whereby a biopsy of healthy cartilage is removed from the patient and the cells isolated and expanded by monolayer passage. This increases the cell number to required levels but also leads to a de-differentiation of the cells. We aim to produce a scaffold-free, de-novo implant from a biopsy of cartilage. METHODS: Bovine chondrocytes were isolated from a small biopsy and expanded. The chondrocytic phenotype of the monolayer expanded cells was recovered during a period of culture in alginate and the effect of factors such as IGF1, TFGbeta1 and dexamethasone was investigated. RESULTS: During the alginate culture period a pre-treatment with IGF1 and dexamethasone was shown to have little effect. IGF1 however increased the glycosaminoglycan/DNA (GAG/DNA) content on day 14 to 84.95+/-5 ng/ng compared with 37.3+/-1.8 ng/ng in the controls (P<0.001). 35S labeling demonstrated an increased GAG synthesis in the presence of IGF1 (P<0.001). IGF1 also induced a increase of DNA content 1383+/-314 ng/bead compared to 512+/-19 ng/bead in the controls (P<0.001).The cells were released from the alginate and cultured in a silicon mould for a further 14 days to obtain a three dimensional implant. Releasing the cells from the alginate and casting in a mould produced an implant of defined shape which contained no foreign material. After 31 days of culture the implants contained 152.4+/-13.14 ng/ng GAG/DNA and 42.93+/-10.23 ng/ng collagen II. DISCUSSION: We believe alginate released chondrocytes provide a real alternative to artificial scaffolds.     

Merocyanine 540 as a probe to monitor the molecular packing of phosphatidylcholine: a monolayer epifluorescence microscopy and spectroscopy study.

The characteristics of the fluorescent dye, merocyanine 540 (MC-540), incorporated in monolayers of 1,2-dipalmitoyl-phosphatidylcholine (DPPC), and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) were studied in different states of molecular packing. Conditions for phase separation in these monolayers were defined by their pressure/area (pi-A) isotherms. Within the liquid expanded (LE) and the liquid condensed (LC) coexisting phases of DPPC monolayers, low light level epifluorescence microscopy revealed 'dark' discoid domains embedded in a 'bright' matrix. Under the same conditions, and at temperatures as low as 12 degrees C, the pi-A isotherms of POPC demonstrate the existence of a single phase, and no fluorescent domains were observed. Fluorescence spectra of MC-540 labelled monolayers, recorded in different structural states, reveal three distinct emission peaks: a 572 nm peak, present for monolayer packing conditions at low surface pressures; a 585 nm peak, similar to that obtained from dye molecules in fluid phase lipid bilayers, and observed here within the respective area/molecule ranges of 54-62 A2 and 62-69 A2 for monolayers of DPPC and POPC with diminishing intensity at increasing surface pressure; and finally, a peak at 560 nm, which predominates in densely packed POPC monolayers. Our results are interpreted on the basis of dye partitioning between monolayer and subphase, and different orientations of the dye with respect to the monolayer in various structural states. The usefulness of MC-540 to differentiate lipid packing in cell membranes is discussed.     

Interpretation of the X-ray scattering profiles of chromatin at various NaCl concentrations by a simple chain model.

In order to interpret the change in the X-ray scattering profiles from rat thymus chromatin, extensive model calculation was carried out. Chromatin is modelled as a string of subunits (nucleosomes) in which disorder is introduced into the positions of adjacent subunits. Disposition parameters characterizing the arrangement of subunits were estimated for various states of chromatin, so that the main feature of the scattering profiles is described. The result indicated that the structure of chromatin changes, as the NaCl concentration increases, from the extended "beads-on-a string" structure to the condensed helical structure. The latter has an outer diameter of about 26 nm with 3-4 nucleosomes per turn. In the intermediate state, it has a loose helical structure. The estimation of disorder suggested that the arrangement of subunits is appreciably disordered even in the condensed helical filament at 50 mM NaCl. Our model for chromatin condensation seems to support models of the "crossed linker" type.     

Spread mixed monolayers of deoxycholic and dehydrocholic acids at the air-water interface, effect of subphase pH. Characterization by axisymmetric drop shape analysis

Bile acids (deoxycholic and dehydrocholic acids) spread mixed monolayers behavior at the air/water interface were studied as a function of subphase pH using a constant surface pressure penetration Langmuir balance based on the Axisymmetric Drop Shape Analysis (ADSA). We examined the influence of electrostatic, hydrophobic and hydration forces on the interaction between amphiphilic molecules at the interface by the collapse area values, the thermodynamic parameters and equation of state virial coefficients analysis. The obtained results showed that at neutral (pH=6.7) or basic (pH=10) subphase conditions the collapse areas values are similar to that of cholanoic acid and consistent with the cross-sectional area of the steroid nucleus (approximately 40 A(2)). The Gibbs energy of mixing values (DeltaG(mix)<0) and the first virial coefficients of the equation of state (b(0)<1) indicated that a miscible monolayer with laterally structured microdomains existed. The aggregation number (1/b(0)) was estimated within the order of 6 (pH=6.7) and 3 (pH=10). At pH=3.2, acidic subphase conditions, no phase separation occurs (DeltaG(mix)<0) but a high expanded effect of the monolayer could be noted. The mixed monolayer behavior was no ideal and no aggregates were formed (b(0)> or =1). Such behavior indicates that the polar groups of the molecules interacts each other more strongly by repulsive electrostatic forces than with the more hydrophobic part of the molecule.     

Nuclear magnetic resonance and calorimetric study of the structure, dynamics, and phase behavior of uranyl ion/dipalmitoylphosphatidylcholine complexes.

The interaction of UO2(2+) with dipalmitoylphosphatidylcholine (DPPC) has been studied as a function of temperature and composition using nuclear magnetic resonance (NMR) spectroscopy, differential scanning calorimetry (DSC), and monolayer studies. Computer simulations of the 31P-NMR powder spectra of DPPC dispersions in the presence of various concentrations of UO2(2+) are consistent with the binding stoichiometry of [UO2(2+)]/[DPPC] = 1:4 at [UO2(2+)]/[DPPC] less than 0.3. This complex undergoes a phase transition to the liquid crystalline phase at T'm = 50 +/- 3 degrees C with a breadth delta T'm = 7 +/- 3 degrees C. This broad transition gradually disappears at higher UO2(2+) concentrations, suggesting the presence of yet another UO2(2+)/DPPC complex (or complexes) whose NMR spectra are indistinguishable from those of the 1:4 UO2(2+)/DPPC species. The temperature-dependent 13C powder spectra of 2(1-13C) DPPC dispersions in the presence of 1.2 mol ratio of UO2(2+) show that this higher order complex (complexes) also undergoes a phase transition to the liquid crystalline state at T'm +/- = 58 +/- 3 degrees C with a breadth delta T"m = 15 +/- 5 degrees C. The NMR spectra indicate that exchange among these various UO2(2+)/DPPC complexes is slow. In addition, computer simulations of the 31P-, 13C-, and 2H-NMR powder spectra show that axial diffusion of the DPPC molecules about their long axes is quenched by addition of UO2(2+) and acyl chain isomerization is the dominant motional mode. The isomerization is best described as two-site hopping of the greater than C-D bond at a rate of approximately 10(6) s-1, a motional mode which is expected for a kink diffusion.     

Atypical Langmuir adsorption of inhalation anesthetics on phospholipid monolayer at various compressional states: difference between alkane-type and ether-type anesthetics

Adsorption of chloroform, halothane, enflurane and diethyl ether on the air/water interface was compared with adsorption on the dipalmitoylphosphatidylcholine monolayer, spread on the air/water interface, at four compressional states; 88.5, 77.0, 66.5 and 50.5 A2 surface area per phosphatidylcholine molecule. Anesthetics were administered from the gas phase. The affinities of these agents to the phosphatidylcholine monolayer varied according to the state of the monolayer. Chloroform and halothane showed a stronger affinity to the highly compressed phosphatidylcholine monolayer (50.5 A2) than to the expanded monolayer (88.5 A2) or to the air/water interface without the monolayer. Diethyl ether behaved in reverse; a stronger affinity to the expanded monolayer was exhibited than to the compressed monolayer. Enflurane showed the highest affinity to the intermediately compressed monolayer (77.0 A2). The adsorption isotherm of anesthetics to the monolayer was characterized by atypical Langmuir-type, in which available number of binding sites changed when anesthetics were adsorbed. The mode of adsorption onto the monolayer was dissimilar to adsorption onto air/water interface, where adsorption followed the Gibbs surface excess. A theory is presented to explain the above differences. The adsorbed anesthetic molecules do not stick to phosphatidylcholine molecules but penetrate into the monolayer lattice and occupy the phosphatidylcholine sites at the interface. Quantitative agreement between the theory and the experimental data was excellent. For the monolayer at 50.5 A2 compression, the changes in the transfer free energy accompanying the anesthetic adsorption from the gas phase to the monolayer were in the order of chloroform greater than halothane greater than enflurane greater than diethyl ether, in agreement with the clinical potencies.     

Aggregation of puroindoline in phospholipid monolayers spread at the air-liquid interface

Puroindolines, cationic and cystine-rich low molecular weight lipid binding proteins from wheat seeds, display unique foaming properties and antimicrobial activity. To unravel the mechanism involved in these properties, the interaction of puroindoline-a (PIN-a) with dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) monolayers was studied by coupling Langmuir-Blodgett and imaging techniques. Compression isotherms of PIN-a/phospholipid monolayers and adsorption of PIN-a to lipid monolayers showed that the protein interacted strongly with phospholipids, especially with the anionic DPPG. The electrostatic contribution led to the formation of a highly stable lipoprotein monolayer. Confocal laser scanning microscopy and atomic force microscopy showed that PIN-a was mainly inserted in the liquid-expanded phase of the DPPC, where it formed an aggregated protein network and induced the fusion of liquid-condensed domains. For DPPG, the protein partitioned in both the liquid-expanded and liquid-condensed phases, where it was aggregated. The extent of protein aggregation was related both to the physical state of phospholipids, i.e., condensed or expanded, and to the electrostatic interactions between lipids and PIN-a. Aggregation of PIN-a at air-liquid and lipid interfaces could account for the biological and technological properties of this wheat lipid binding protein.