Dipalmitoylphosphatidylcholine and cholesterol in monolayers spread from adsorbed films of pulmonary surfactant

Pulmonary surfactant forms a surface film that consists of a monolayer and a monolayer-associated reservoir. The extent to which surfactant components including the main component, dipalmitoylphosphatidylcholine (DPPC), are adsorbed into the monolayer, and how surfactant protein SP-A affects their adsorptions, is not clear. Transport of cholesterol to the surface region from dispersions of bovine lipid extract surfactant [BLES(chol)] with or without SP-A at 37 degrees C was studied by measuring surface radioactivities of [4-(14)C]cholesterol-labeled BLES(chol), and the Wilhelmy plate technique was used to monitor adsorption of monolayers. Results showed that transport of cholesterol was lipid concentration dependent. SP-A accelerated lipid adsorption but suppressed the final level of cholesterol in the surface. Surfactant adsorbed from a dispersion with or without SP-A was transferred via a wet filter paper to a clean surface, where the surface radioactivity and surface tension were recorded simultaneously. It was observed that 1) surface radioactivity was constant over a range of dispersion concentrations; 2) cholesterol and DPPC were transferred simultaneously; and 3) SP-A limited transfer of cholesterol.These results indicate that non-DPPC components of pulmonary surfactant can be adsorbed into the monolayer. Studies in the transfer of [1-(14)C]DPPC-labeled BLES(chol) to an equal or larger clean surface area revealed that SP-A did not increase selective adsorption of DPPC into the monolayer. Evaluation of transferred surfactant with a surface balance indicated that it equilibrated as a monolayer. Furthermore, examination of transferred surfactants from dispersions with and without prespread BLES(chol) monolayers revealed a functional contiguous association between adsorbed monolayers and reservoirs.     

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.     

The Mechanism of Collapse of Heterogeneous Lipid Monolayers

Collapse of homogeneous lipid monolayers is known to proceed via wrinkling/buckling, followed by folding into bilayers in water. For heterogeneous monolayers with phase coexistence, the mechanism of collapse remains unclear. Here, we investigated collapse of lipid monolayers with coexisting liquid-liquid and liquid-solid domains using molecular dynamics simulations. The MARTINI coarse-grained model was employed to simulate monolayers of ∼80 nm in lateral dimension for 10–25 μs. The monolayer minimum surface tension decreased in the presence of solid domains, especially if they percolated. Liquid-ordered domains facilitated monolayer collapse due to the spontaneous curvature induced at a high cholesterol concentration. Upon collapse, bilayer folds formed in the liquid (disordered) phase; curved domains shifted the nucleation sites toward the phase boundary. The liquid (disordered) phase was preferentially transferred into bilayers, in agreement with the squeeze-out hypothesis. As a result, the composition and phase distribution were altered in the monolayer in equilibrium with bilayers compared to a flat monolayer at the same surface tension. The composition and phase behavior of the bilayers depended on the degree of monolayer compression. The monolayer-bilayer connection region was enriched in unsaturated lipids. Percolation of solid domains slowed down monolayer collapse by several orders of magnitude. These results are important for understanding the mechanism of two-to-three-dimensional transformations in heterogeneous thin films and the role of lateral organization in biological membranes. The study is directly relevant for the function of lung surfactant, and can explain the role of nanodomains in its surface activity and inhibition by an increased cholesterol concentration.     

Role of pulmonary surfactant components in surface film formation and dynamics

Pulmonary surfactant is a mixture of lipids and proteins which is secreted by the epithelial type II cells into the alveolar space. Its main function is to reduce the surface tension at the air/liquid interface in the lung. This is achieved by forming a surface film that consists of a monolayer which is highly enriched in dipalmitoylphosphatidylcholine and bilayer lipid/protein structures closely attached to it. The molecular mechanisms of film formation and of film adaptation to surface changes during breathing in order to remain a low surface tension at the interface, are unknown. The results of several model systems give indications for the role of the surfactant proteins and lipids in these processes. In this review, we describe and compare the model systems that are used for this purpose and the progress that has been made. Despite some conflicting results using different techniques, we conclude that surfactant protein B (SP-B) plays the major role in adsorption of new material into the interface during inspiration. SP-C's main functions are to exclude non-DPPC lipids from the interface during expiration and to attach the bilayer structures to the lipid monolayer. Surfactant protein A (SP-A) appears to promote most of SP-B's functions. We describe a model proposing that SP-A and SP-B create DPPC enriched domains which can readily be adsorbed to create a DPPC-rich monolayer at the interface. Further enrichment in DPPC is achieved by selective desorption of non-DPPC lipids during repetitive breathing cycles.     

Interaction of gentamicin with phosphatidylserine/phosphatidylcholine mixtures in adsorption monolayers and thin liquid films: morphology and thermodynamic properties

Gentamicin possesses strong adverse actions like oto and nephrotoxicity. The latter is a result of strong gentamicin–acid phospholipid interactions, resulting in cell fusion, fission, etc., ions as calcium interact with gentamicin and effectively deter its toxicity. In this work, the interactions of gentamicin and Ca²⁺ with phosphatidylserine/phosphatidylcholine (PS/PC) mixtures of different ratio are experimentally characterized. Special attention is paid to bridge thermodynamic and morphological properties of adsorption monolayers and thin liquid films (TLFs) composed of these lipid mixtures. Our results show that gentamicin decreases the stability of common black TLFs formed of pure PS coupled with suppression of lipid surface adsorption to the monolayers at the air–water interface; also, gentamicin reveals effects of lowering of lipid spreading on the interface and significant loss of material during monolayer cycling, increase of condensed phase, and organization of dense net-like domain monolayer texture. Gentamicin addition results in opposite effects for films formed of DPPC/PS (95:5) mixture. It increases the stability of Newton black TLFs formed by DPPC/PS correlated with faster and stronger surface adsorption and better surface spreading; also, gentamicin lowers the amount of condensed phase and organization of domains of smaller size. We also showed that Ca²⁺ itself decreases the stability of common black TLFs formed of PS accompanied with weaker surface adsorption, formation of higher amounts of condensed phase and organization of domains. In our experiments, Ca²⁺ softens, even deters, the effects of gentamicin on both PS and DPPC/PS films.     

The Mechanism of Collapse of Heterogeneous Lipid Monolayers

Collapse of homogeneous lipid monolayers is known to proceed via wrinkling/buckling, followed by folding into bilayers in water. For heterogeneous monolayers with phase coexistence, the mechanism of collapse remains unclear. Here, we investigated collapse of lipid monolayers with coexisting liquid-liquid and liquid-solid domains using molecular dynamics simulations. The MARTINI coarse-grained model was employed to simulate monolayers of ∼80 nm in lateral dimension for 10–25 μs. The monolayer minimum surface tension decreased in the presence of solid domains, especially if they percolated. Liquid-ordered domains facilitated monolayer collapse due to the spontaneous curvature induced at a high cholesterol concentration. Upon collapse, bilayer folds formed in the liquid (disordered) phase; curved domains shifted the nucleation sites toward the phase boundary. The liquid (disordered) phase was preferentially transferred into bilayers, in agreement with the squeeze-out hypothesis. As a result, the composition and phase distribution were altered in the monolayer in equilibrium with bilayers compared to a flat monolayer at the same surface tension. The composition and phase behavior of the bilayers depended on the degree of monolayer compression. The monolayer-bilayer connection region was enriched in unsaturated lipids. Percolation of solid domains slowed down monolayer collapse by several orders of magnitude. These results are important for understanding the mechanism of two-to-three-dimensional transformations in heterogeneous thin films and the role of lateral organization in biological membranes. The study is directly relevant for the function of lung surfactant, and can explain the role of nanodomains in its surface activity and inhibition by an increased cholesterol concentration.     

Hydroxyzine, promethazine and thioridazine interaction with phospholipid monomolecular layers at the air-water interface

In this work the interaction of Hydroxyzine, Promethazine and Thioridazine with Langmuir films of dipalmitoylphosphatidylcholine (dpPC) and dipalmitoylphosphatidic acid (dpPA), is studied. Temporal variations in lateral surface pressure (pi) were measured at different initial pi (pi(i)), subphase pH and drug-concentration. Drugs with the smallest (PRO) and largest (HYD) molecular size exhibited the lowest adsorption (k(a)) and the highest desorption (k(d)) rate constant values, respectively. The affinity binding constants (K(b)) obtained in monolayers followed the same profile (K(b,PRO) < K(b,HYD) < K(b,THI)) of the egg-PC/water partition coefficients (P) determined in bilayers. The drug concentration required to reach the half-maximal Deltapi at pi(i) = 14 mN/m (K(0.5)), was very sensitive to pH. The maximal increment in pi upon drug incorporation into the monolayer (deltapi(max)) will depend on the phospholipid collapse pressure (pi(c)), the monolayers's compressibility and drug's size, shape and charge. The higher pi(c) of dpPC lead to higher pi(cut-off) values (maximal pi allowing drug penetration), if compared with dpPA. In dpPC and dpPA pi(cut-off) decreased as a function of the molecular size of the uncharged drugs. In dpPA, protonated drugs became electrostatically trapped at the monolayer surface hence drug penetration, monolayer deformation and pi increase were impaired and the correlation between pi(cut-off) and drug molecular size was lost.     

Utilization of rice husks as a feedstock for preparation of activated carbon by microwave induced KOH and K2CO3 activation

Rice husk (RH), an abundant by-product of rice milling, was used for the preparation of activated carbon (RHAC) via KOH and K(2)CO(3) chemical activation. The activation process was performed at the microwave input power of 600 W for 7 min. RHACs were characterized by low temperature nitrogen adsorption/desorption, scanning electron microscopy and Fourier transform infrared spectroscopy. The adsorption behavior was examined using methylene blue as adsorbate. The K(2)CO(3)-activated sample showed higher yield and better pore structures and adsorption capacity development than the KOH-activated sample, with a BET surface area, total pore volume and monolayer adsorption capacity of 1165 m(2)/g, 0.78 cm(3)/g and 441.52 mg/g, respectively. The results revealed the feasibility of microwave heating for preparation of high surface area activated carbons from rice husks via K(2)CO(3) activation.     

Interaction of a nonspecific wheat lipid transfer protein with phospholipid monolayers imaged by fluorescence microscopy and studied by infrared spectroscopy.

The interaction of a nonspecific wheat lipid transfer protein (LTP) with phospholipids has been studied using the monolayer technique as a simplified model of biological membranes. The molecular organization of the LTP-phospholipid monolayer has been determined by using polarized attenuated total internal reflectance infrared spectroscopy, and detailed information on the microstructure of the mixed films has been investigated by using epifluorescence microscopy. The results show that the incorporation of wheat LTP within the lipid monolayers is surface-pressure dependent. When LTP is injected into the subphase under a dipalmytoylphosphatidylglycerol monolayer at low surface pressure (< 20 mN/m), insertion of the protein within the lipid monolayer leads to an expansion of dipalmytoylphosphatidylglycerol surface area. This incorporation leads to a decrease in the conformational order of the lipid acyl chains and results in an increase in the size of the solid lipid domains, suggesting that LTP penetrates both expanded and solid domains. By contrast, when the protein is injected under the lipid at high surface pressure (> or = 20 mN/m) the presence of LTP leads neither to an increase of molecular area nor to a change of the lipid order, even though some protein molecules are bound to the surface of the monolayer, which leads to an increase of the exposure of the lipid ester groups to the aqueous environment. On the other hand, the conformation of LTP, as well as the orientation of alpha-helices, is surface-pressure dependent. At low surface pressure, the alpha-helices inserted into the monolayers are rather parallel to the monolayer plane. In contrast, at high surface pressure, the alpha-helices bound to the surface of the monolayers are neither parallel nor perpendicular to the interface but in an oblique orientation.     

Molecular dynamics simulations of the insertion of two ideally amphipathic lytic peptides LK(15) and LK(9) in a 1,2-dimyristoylphosphatidylcholine monolayer.

We present here the results of 1-ns molecular dynamics (MD) simulations of two ideally amphipathic lytic peptides, namely LK(15) and LK(9), in a 1,2-dimyristoylphosphatidylcholine monolayer with two different cross-sectional areas per lipid of 80 A(2) (loose film) and 63 A(2) (tight standard film). These peptides are lytic, ideally amphipathic with a minimalist composition L(i)K(j) and the following sequences: H(2)N-KLLKLLLKLLLKLLK-CO-Ph for LK(15) and H(2)N-KLKLKLKLK-CO-Ph for LK(9). From experimental data, LK(15) exhibits an alpha-helical secondary structure, whereas LK(9) was found to form antiparallel beta-sheets at the interface of a DMPC monolayer. Whatever the specific lipid surface is, the two peptides exhibit very different behavior: the alpha-helix inserts deeply into the monolayer whereas the beta-sheeted peptide stays at the surface within the upper polar part of the monolayer. In all cases, a loose monolayer (80 A(2)) results in noticeable artifacts whereas a monolayer with standard specific surface leads to very reliable behavior well in accordance with experimental data. Despite their different insertion depth, the two peptides exhibit identical lytic efficiency. This is very likely a direct consequence of the very strong Van der Waals interactions between the fatty alkyl chains of the lipids and the highly lipophilic lower part of the peptide, resulting in an identical thinning of the two monolayers.     

Simulation study of methanol and ethanol adsorption on graphitized carbon black

GCMC simulations are applied to the adsorption of sub-critical methanol and ethanol on graphitized carbon black at 300 K. The carbon black was modelled both with and without carbonyl functional groups. Large differences are seen between the amounts adsorbed for different carbonyl configurations at low pressure prior to monolayer coverage. Once a monolayer has been formed on the carbon black, the adsorption behaviour is similar between the model surfaces with and without functional groups. Simulation isotherms for the case of low carbonyl concentrations or no carbonyls are qualitatively similar to the few experimental isotherms available in the literature for methanol and ethanol adsorption on highly graphitized carbon black. Isosteric heats and adsorbed phase heat capacities are shown to be very sensitive to carbonyl configurations. A maximum is observed in the adsorbed phase heat capacity of the alcohols for all simulations but is unrealistically high for the case of a plain graphite surface. The addition of carbonyls to the surface greatly reduces this maximum and approaches experimental data with carbonyl concentration as low as 0.09 carbonyls/nm².     

Long-period gratings in photonic crystal fiber as an optofluidic label-free biosensor

Using long-period gratings (LPG) inscribed in photonic crystal fiber (PCF) and coupling this structure with an optically aligned flow cell, we have developed an optofluidic refractive index transduction platform for label-free biosensing. The LPG-PCF scheme possesses extremely high sensitivity to the change in refractive index induced by localized binding event in different solution media. A model immunoassay experiment was carried out inside the air channels of PCF by a series of surface modification steps in sequence that include adsorption of poly(allylamine hydrochloride) monolayer, immobilization of anti-rat bone sialoprotein monoclonal primary antibody, and binding interactions with non-specific goat anti-rabbit IgG (H+L) and specific secondary goat anti-mouse IgG (H+L) antibodies. These adsorption and binding events were monitored in situ using the LPG-PCF by measuring the shift of the core-to-cladding mode coupling resonance wavelength. Steady and significant resonance changes, about 0.75 nm per nanometer-thick adsorbed/bound bio-molecules, have been observed following the sequence of the surface events with monolayer sensitivity, suggesting the promising potential of LPG-PCF for biological sensing and evaluation.     

Kinetics of phospholipid insertion into monolayers containing the lung surfactant proteins SP-B or SP-C

The lung surfactant proteins SP-B and SP-C are pivotal for fast and reversible lipid insertion at the air/liquid interface, a prerequisite for functional lung activity. We used a model system consisting of a preformed monolayer at the air/liquid interface supplemented with surfactant protein SP-B or SP-C and unilamellar vesicles injected into the subphase of a film balance. The content of SP-B or SP-C was similar to that found in lung lavage. In order to elucidate distinct steps of lipid insertion, we measured the time-dependent pressure increase as a function of the initial surface pressure, the temperature and the phosphatidylglycerol content by means of surface tension measurements and scanning force microscopy (SFM). The results of the film balance study are indicative of a two-step mechanism in which initial adsorption of vesicles to the protein-containing monolayer is followed by rupture and integration of lipid material. Furthermore, we found that vesicle adsorption on a preformed monolayer supplemented with SP-B or SP-C is strongly enhanced by negatively charged lipids as provided by DPPG and the presence of Ca2+ ions in the subphase. Hence, long-range electrostatic interactions are thought to play an important role in attracting vesicles to the surface, being the initial step in replenishment of lipid material. While insertion into the monolayer is independent of the type of protein SP-B or SP-C, initial adsorption is faster in the presence of SP-B than SP-C. We propose that the preferential interaction between SP-B and negatively charged DPPG leads to accumulation of negative charges in particular regions, causing strong adhesion between DPPG-containing vesicles and the monolayer mediated by Ca2+ ions, which eventually causes flattening and rupture of attached liposomes as observed by in situ SFM.     

Adsorption equilibria between liposome membrane formed of phosphatidylcholine and aqueous sodium chloride solution as a function of pH

The effect has been studied of the adsorption of ions (H(+), Na(+), OH(-), Cl(-)) which are present in solution upon the electric charge of the liposome membrane formed of phosphatidylcholine (PC). The surface charge density of the membrane was determined as a function of pH and electrolyte concentration from electrophoretic mobility measurements. The measurements were carried out by the laser-Doppler microelectrophoresis method. A four-equilibria model has been proposed to describe the phenomena occurring on the membrane surface. The equilibria in which the adsorption of other ions on the liposome membrane surface was involved were assumed to exist beside the equilibria in which the H(+) and OH(-) ions were engaged. The idea was confirmed by mathematical calculations. Association constants of the liposome membrane surface with ions of solution (K(AH), K(ANa), K(BOH), K(BCl)) were determined. The proposed model has been proved to be correct by comparing the resulting theoretic charge variation curves of the lecithin membrane with the experimental data.     

Analysis of epithelial cell surface polarity with monoclonal antibodies

The hybridoma technique of K?hler and Milstein was utilized to isolate hybrid cell lines secreting monoclonal antibodies against cell surface proteins on the Madin-Darby canine kidney (MDCK) epithelial cell line. These antibodies were employed as high-affinity ligands to study the development and maintenance of epithelial cell polarity in MDCK cells and for the identification of nephron segment-specific proteins. Using standard procedures, we were able to immunoprecipitate glycoproteins with molecular weights of 25,000 ( 25K ), 35,000 ( 35K ), and 50,000 (50K). Immunofluorescence and immunoelectron microscopy of MDCK demonstrated that the 35K and 50K proteins could be localized on both the apical and basolateral membranes of subconfluent cells but primarily on the basolateral membranes of confluent cells. By determining the cell surface distribution of the 35K and 50K proteins on MDCK cells during growth into a confluent monolayer, and after the experimental disruption of tight junctions, evidence was obtained that the polarized distribution of these cell surface glycoproteins required the presence of tight junctions. We propose that confluent MDCK cells have a mechanism that is responsible for the establishment and maintenance of epithelial apical and basolateral membranes as distinct cell surface domains. These monoclonal antibodies were also used to localize the 25K and 35K glycoproteins in the kidney. The distribution of these proteins was mapped by immunofluorescence and immunoelectron microscopy and was determined to be on the basolateral membranes of epithelial cells in only certain tubular segments of the nephron. The possible functional implications of these distributions are discussed.     

Effect of diolein on hydrolysis of phosphatidylcholine by phospholipase C from Clostridium perfringens.

The activity of phospholipase C from Clostridium perfringens on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) as a monolayer at an air/water interface was examined. With a pure POPC monolayer, sharp cut-off of the enzyme activity was observed on increase in surface pressure. However, this cut-off disappeared on addition of a 0.3 molar fraction of 1,2-dioleoylglycerol (1,2-DO) to the monolayer. An abrupt change in the enzyme activity was observed with molar fractions of between 0.2 and 0.3 1,2-DO in the POPC monolayer at an initial surface pressure of 35 mN/m. For examination of the effect of 1,2-DO on the phospholipase C activity, the quantity of [125I]phospholipase C adsorbed to the surface was determined. The enzyme was found to be adsorbed nonspecifically to all lipid films except that of POPC only. The adsorption of enzyme was not affected by the presence or absence of Ca2+ and Zn2+. The rate constant for enzyme adsorption to a 1,2-DO film was 4.5 times that for its adsorption to a POPC film. The adsorption decreased linearly with increase in the surface concentration of POPC, and increased with increase in the surface concentration of 1,2-DO. These data suggest that 1,2-DO (a reaction product) regulates the interaction of phospholipase C with films containing substrate and may also regulate the enzyme activity.     

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.     

Determination of hydrophobicity on bacterial surfaces by nonionic surfactants.

The hydrophobicity of the bacterial cell surface was determined by using nonionic surfactants. The method is based on the adsorption of nonionic surfactants at the hydrophobic sites of the cell surface. Among many nonionic surfactants, C18H37O(CH2CH2O)13H was preferred. The surfactant was added in excess to a bacterial suspension, and the suspension was mixed by sonication or mechanical stirring. The amount of surfactant remaining in the supernatant after centrifugation was determined spectrophotometrically by measuring the absorbance of tetrabromophenolphthalein ethylester. Effective dispersion of bacterial cells such as Staphylococcus aureus and Mycobacterium smegmatis was achieved by sonication in the presence of the nonionic surfactant. Adsorption measurements coincided with Langmuir's equation, indicative of monolayer adsorption. The method is useful for the determination of the hydrophobicity of various bacterial cell surfaces.     

Influence of lipid saturation grade and headgroup charge: a refined lung surfactant adsorption model

Rapid adsorption of surfactant material to the air/liquid interface of the lung is essential for maintaining normal lung function. The detailed mechanism of this process, however, remains unclear. In this study, we elucidate the influence of lipid saturation grade and headgroup charge of surface layer lipids on surfactant protein (SP)-induced vesicle insertion into monolayers spread at the air/water interface of a film balance. We used dipalmitoylphosphatidlycholine (DPPC),1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) as monolayer lipids doped with either hydrophobic surfactant-specific protein SP-B or SP-C (0.2 and 0.4 mol %, respectively). Vesicles consisting of DPPC/DPPG (4:1, mol ratio) were injected into a stirred subphase to quantify adsorption kinetics. Based on kinetic film balance and fluorescence measurements, a refined model describing distinct steps of vesicle adsorption to surfactant monolayers is presented. First, in a protein-independent step, lipids from vesicles bridged to the interfacial film by Ca²⁺ ions are inserted into defects of a disordered monolayer at low surface pressures. Second, in a SP-facilitated step, active material insertion involving an SP-B- or SP-C-induced flip-flop of lipids occurs at higher surface pressures. Negatively charged lipids obviously influence the threshold pressures at which this second protein-mediated adsorption mechanism takes place.