Neutral lipids induce critical behavior in interfacial monolayers of pulmonary surfactant

We have shown previously that lateral compression of pulmonary surfactant monolayers initially induces separation of two phases but that these remix when the films become more dense (1). In the studies reported here, we used fluorescence microscopy to examine the role of the different surfactant constituents in the remixing of the separated phases. Subfractions containing only the purified phospholipids (PPL), the surfactant proteins and phospholipids (SP&PL), and the neutral and phospholipids (N&PL) were obtained by chromatographic separation of the components in extracted calf surfactant (calf lung surfactant extract, CLSE). Compression of the different monolayers produced nonfluorescent domains that emerged for temperatures between 20 and 41 degreesC at similar surface pressures 6-8 mN/m higher than values observed for dipalmitoyl phosphatidylcholine (DPPC), the most prevalent component of pulmonary surfactant. Comparison of the different preparations showed that the neutral lipid increased the total nonfluorescent area at surface pressures up to 25 mN/m but dispersed that total area among a larger number of smaller domains. The surfactant proteins also produced smaller domains, but they had the opposite effect of decreasing the total nonfluorescent area. Only the neutral lipids caused remixing. In images from static monolayers, the domains for N&PL dropped from a maximum of 26 +/- 3% of the interface at 25 mN/m to 4 +/- 2% at 30 mN/m, similar to the previously reported behavior for CLSE. During continuous compression through a narrow range of pressure and molecular area, in N&PL, CLSE, and mixtures of PPL with 10% cholesterol, domains became highly distorted immediately prior to remixing. The characteristic transition in shape and abrupt termination of phase coexistence indicate that the remixing caused by the neutral lipids occurs at or close to a critical point.     

Protein-lipid interactions and enzyme requirements for light subtype generation on cycling reconstituted surfactant

Surfactant convertase is required for conversion of heavy density (H) natural surfactant to light density (L) subtype during cycling in vitro, a technique that reproduces surfactant metabolism. To study mechanisms of H to L conversion, we prepared liposomes of dipalmitoylphosphatidylcholine (DPPC) and phosphatidylglycerol (PG), or the phospholipids (PL) in combination with either surfactant protein A (SP-A), surfactant protein B (SP-B), or both SP-A and SP-B. Phospholipids alone showed time-dependent conversion from heavy to light subtype on cycling in the absence of convertase, which was decreased by adding SP-B, but not SP-A, to phospholipids (p < 0.01 for PL+SP-B, or PL+SP-A+SP-B vs. PL, or PL+SP-A). The ultrastructure, surface activity, buoyant density, and L subtype generation on cycling PL+SP-A+SP-B with partially purified convertase or with phospholipase D were similar to those of natural TM. In conclusion, a reconstituted surfactant mimics the behavior of natural surfactant on cycling, and reveals that interaction of SP-B with phospholipids decreases L subtype generation. In addition, esterase/ phospholipase D activity is required for conversion of heavy to light subtype on cycling.     

Interaction of pulmonary surfactant protein SP-A with DPPC/egg-PG bilayers

In the mixture of lipids and proteins which comprise pulmonary surfactant, the dominant protein by mass is surfactant protein A (SP-A), a hydrophilic glycoprotein. SP-A forms octadecamers that interact with phospholipid bilayer surfaces in the presence of calcium. Deuterium NMR was used to characterize the perturbation by SP-A, in the presence of 5 mM Ca(2+), of dipalmitoyl phosphatidylcholine (DPPC) properties in DPPC/egg-PG (7:3) bilayers. Effects of SP-A were uniformly distributed over the observed DPPC population. SP-A reduced DPPC chain orientational order significantly in the gel phase but only slightly in the liquid-crystalline phase. Quadrupole echo decay times for DPPC chain deuterons were sensitive to SP-A in the liquid-crystalline mixture but not in the gel phase. SP-A reduced quadrupole splittings of DPPC choline beta-deuterons but had little effect on choline alpha-deuteron splittings. The observed effects of SP-A on DPPC/egg-PG bilayer properties differ from those of the hydrophobic surfactant proteins SP-B and SP-C. This is consistent with the expectation that SP-A interacts primarily at bilayer surfaces.     

Method of purification affects some interfacial properties of pulmonary surfactant proteins B and C and their mixtures with dipalmitoylphosphatidylcholine

Two methods were employed for preparation of lipid extracts from porcine lung surfactant. Pulmonary surfactant proteins SP-B and SP-C were isolated from the extracts using gel-exclusion chromatography on LH-60 with chloroform:methanol acidified with hydrochloric acid. Monolayers of pure SP-B or SP-C isolated from butanol lipid extracts spread at the air-water interface showed larger molecular areas than those determined in films of SP-B or SP-C isolated from chloroform surfactant extracts. Aqueous dispersions of dipalmitoylphosphatidylcholine (DPPC) supplemented with 2.5 and 5.0 wt% of SP-B or SP-C obtained from butanol extracts adsorbed faster to the air-water interface than their counterparts reconstituted with proteins isolated from chloroform extracts. Surface pressure-area characteristics of spread monolayers of DPPC plus SP-B or SP-C did not depend on the method of isolation of the proteins. The diagrams of the mean molecular areas vs. composition for the monolayers of DPPC plus SP-B or SP-C showed positive deviations from the additivity rule, independently of the procedure used for preparation of lipid extract surfactant. Matrix-assisted laser desorption/ionization spectrometry of the proteins isolated from different extraction solvents was consistent with some differences in the chemical compositions of SP-Bs. Butylation of SP-B during extraction of surfactant pellet with butanol may account for the differences observed in the molecular masses of SP-Bs isolated by the two different extraction protocols. The study suggests that the method of purification of SP-B and SP-C may modify their ability to enhance the adsorption rates of DPPC/protein mixtures, and this may be relevant to the formulation of protein-supplemented lipids for exogenous treatment of pulmonary surfactant insufficiency.     

NT-3 regulates expression of Brn3a but not Brn3b in developing mouse trigeminal sensory neurons

To further elucidate the nature of the molecular interactions of surfactant apoprotein B (SP-B) with phospholipid (PL) membranes, we studied the binding of SP-B to PL membranes and the lipid-dependency of its subsequent effects on leakage and fusion of membranes. SP-B binding to membranes was studied by labeling the protein with the fluorophore 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD) and measuring the fluorescence of the labeled protein in the presence of varying amounts of dipalmitoylphosphatidylcholine-egg phosphatidylglycerol (DPPC-eggPG; 7-3). Leakage of contents from liposomes made of DPPC and varying molar fraction of egg phosphatidylcholine (eggPC) or eggPG was assessed by measuring the fluorescence of entrapped water-soluble probes ANTS and DPX. Fusion of membranes was assessed by measuring the fluorescence of membrane-bound NBD-phosphatidylethanolamine (NBD-PE) and rhodamine-PE (RHO-PE). We found that SP-B bound to PL membranes with high affinity and appeared to irreversibly cluster at the membrane surface, leading to graded release of the vesicle contents and eventually fusion of the membranes with increasing protein-lipid ratios. All lipid mixtures tested were susceptible to the membrane disruptive effects of SP-B, but DPPC-eggPG membranes displayed a biphasic response to increasing molar fractions of eggPG, whereas increasing fractions of eggPC elicited a monotonic response.     

Dioleylphosphatidylglycerol inhibits the expression of type II phospholipase A2 in macrophages

We have recently shown that modified natural pulmonary surfactant Curosurf inhibits the synthesis of type II phospholipase A2 (sPLA2-II) by cultured guinea-pig alveolar macrophages (AM). The goal of the present study was to identify the surfactant components and the mechanisms involved in this process. We show that protein-free artificial surfactant (AS) mimicked the inhibitory effect of Curosurf, suggesting that phospholipid components of surfactant play a role in the inhibition of sPLA2-II expression. Among surfactant phospholipids, dioleylphosphatidylglycerol (DOPG) was the most effective in inhibiting the synthesis of sPLA2-II. By contrast, the concentrations of platelet-activating factor (PAF)-acetylhydrolase and lysophospholipase activities remained unchanged, indicating that inhibition of sPLA2-II synthesis was caused by a specific effect of surfactant. The effect of DOPG on sPLA2-II synthesis was concentration-dependent and was accompanied by a rapid and time-dependent uptake of DOPG by AM whereas dipalmitoylphosphatidylcholine (DPPC) was only marginally taken up. Curosurf, AS, and DOPG inhibited tumor necrosis factor-alpha (TNF-alpha) secretion, a key step in the induction of sPLA2-II synthesis by AM, in contrast to DPPC which had only a marginal effect. We conclude that phospholipid components, especially DOPG, play a major role in the inhibition of sPLA2-II synthesis by surfactant and that this effect can be explained, at least in part, by an impairment of TNF-alpha secretion.     

Interfacial effects of surface-active agents under zinc pressure leach conditions

Liquid sulfur-zinc sulfate solution interfacial tensions and liquid sulfur-zinc sulfate solution-zinc sulfide (marmatite) contact angles were measured in the absence and presence of surface-active agents. Interfacial tensions measured varied between 54 ± 1 mN/m in the surfactant-free system and 20 ± 1 mN/m in the presence of a surfactant. The liquid sulfur-zinc sulfide mineral-zinc sulfate solution contact angle varies between 80 ± 5 deg, in the absence of any surfactant, and 148 ± 5 deg, depending on the surfactant used. The surface-active agents were used as dispersants for sulfur in bench-scale zinc pressure-leaching experiments. The observed extent of zinc extraction depends on the surfactant and varies from 40 to 96 pct.     

Effects of a cationic and hydrophobic peptide, KL4, on model lung surfactant lipid monolayers

We report on the surface behavior of a hydrophobic, cationic peptide, [lysine-(leucine)4]4-lysine (KL4), spread at the air/water interface at 25 degrees C and pH 7.2, and its effect at very low molar ratios on the surface properties of the zwitterionic phospholipid 1,2-dipalmitoylphosphatidylcholine (DPPC), and the anionic forms of 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) and palmitic acid (PA), in various combinations. Surface properties were evaluated by measuring equilibrium spreading pressures (pi(e)) and surface pressure-area isotherms (pi-A) with the Wilhelmy plate technique. Surface phase separation was observed with fluorescence microscopy. KL4 itself forms a single-phase monolayer, stable up to a surface pressure pi of 30 mN/m, and forms an immiscible monolayer mixture with DPPC. No strong interaction was detected between POPG and KL4 in the low pi region, whereas a stable monolayer of the PA/KL4 binary mixture forms, which is attributed to ionic interactions between oppositely charged PA and KL4. KL4 has significant effects on the DPPC/POPG mixture, in that it promotes surface phase separation while also increasing pi(e) and pi(max), and these effects are greatly enhanced in the presence of PA. In the model we have proposed, KL4 facilitates the separation of DPPC-rich and POPG/PA-rich phases to achieve surface refinement. It is these two phases that can fulfill the important lung surfactant functions of high surface pressure stability and efficient spreading.     

Formation and structure of surface films: captive bubble surfactometry

The adsorption model for soluble surfactants has been modified for suspensions of pulmonary surfactant. The dynamic adsorption behavior may be governed by a two-step process: (1) the transfer of molecules between the surface layer and the subsurface layer, which has a thickness of a few molecular diameters only; (2) the exchange of molecules between the subsurface and the bulk solution. The first step is an adsorption process and the second step is a mass transfer process. Between the subsurface and the bulk solution is an undisturbed boundary layer where mass transport occurs by diffusion only. The thickness of this boundary layer may be reduced by stirring. Rapid film formation by adsorption bursts from lipid extract surfactants, as observed in the captive bubble system, suggests that the adsorption process as defined above is accompanied by a relatively large negative change in the free energy. This reduction in the free energy is provided by a configurational change in the association of the specific surfactant proteins and the surfactant lipids during adsorption. The negative change in the free energy during film formation more than compensates for the energy barrier related to the film surface pressure. In the traditional view, the extracellular alveolar lining layer is composed of two parts, an aqueous subphase and a surfactant film, believed to be a monolayer, at the air-water interface. The existence and continuity of the aqueous subphase has recently been demonstrated by Bastacky and coworkers, and a continuous polymorphous film has recently been shown by Bachofen and his associates, using perfusion fixation of rabbit lungs with slight edema. In the present chapter, we have described a fixation technique using a non-aqueous fixation medium of perfluorocarbon and osmium tetroxide to fix the peripheral airspaces of guinea pig lungs. A continuous osmiophilic film which covers the entire alveolar surface, including the pores of Kohn, is demonstrated. By transmission electron microscopy, the surface film frequently appears multilaminated, not only in the alveolar corners or crevices, but also at the thin air-blood barrier above the capillaries. Disk-like structures or multilamellar vesicles appear partially integrated into the planar multilayered film. In corners and crevices, tubular myelin appears closely associated with the surface film. Tubular myelin, however, is not necessary for the generation of a multilaminated film. This is demonstrated in vitro by the fixation for electron microscopy of a film formed from lipid extract surfactant on a captive bubble. Films formed from relatively high surfactant concentration (1 mg/ml of phospholipid) are of variable thickness and frequent multilayers are seen. In contrast, at 0.3 mg/ml, only an amorphous film can be visualized. Although near zero minimum surface tensions can be obtained for both surfactant concentrations, film compressibility and mechanical stability are substantially better at the higher concentrations. This appears to be related to the multilaminated structure of the film formed at the higher concentration.     

Interaction of indomethacin and naproxen with gastric surface-active phospholipids: a possible mechanism for the gastric toxicity of nonsteroidal anti-inflammatory drugs (NSAIDs)

The possibility that the molecular mechanism underlying the topical gastric irritancy of nonsteroidal anti-inflammatory drugs (NSAIDs) may involve alterations in the surface-active properties of gastric phospholipids was investigated. Indomethacin and naproxen were intragastrically administered to rats and the hydrophobicity of the luminal surface of the stomach wall was assessed by contact angle analysis. Both NSAIDs have the ability to attenuate the phospholipid-related hydrophobic properties of the gastric mucosa by more than 80-85% in a dose-dependent fashion. Potential molecular interactions between both NSAIDs and surface-active phospholipids were analyzed using fluorescent probes. Indomethacin has the ability to displace, in a dose-dependent manner, ANS (1-anilino-8-naphthalene sulphonate), a fluorescent anionic probe previously bound to the head group of phosphatidylcholine molecules. Estimations of the resonance fluorescence transfer between naproxen and the surface probe ANS or the hydrophobic probe, pyrene, bound to dipalmitoylphosphatidylcholine (DPPC) vesicles revealed that naproxen diffuses within the phospholipid bilayers. The dynamic of the gastric lipid material extracted from the surface scraping material (SSM) of the mucosa was altered by the NSAID as shown by the increase in the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) (at 25 degrees, rSSM = 0.106+/-0.006, rssM + indomethacin = 0.137+/-0.005, and rSSM + naproxen = 0.133+/-0.007, P < 0.001). The thermodynamic behavior of a model bilayer containing DPPC was also perturbed by the NSAIDs tested. These results provide evidence that NSAIDs may reduce the ability of gastric surface-active phospholipids to form a hydrophobic protective layer.     

Effect of nitroglycerin and nicorandil on regional poststenotic quantitative coronary blood flow in coronary artery disease: a combined digital quantitative angiographic and intracoronary doppler study

The biophysical activity of lung surfactant depends, to a large extent, on the presence of the hydrophobic surfactant proteins B (SP-B) and C (SP-C). The role of these proteins in lipid adsorption and lipid squeeze-out under dynamic conditions simulating breathing is not yet clear. Therefore, the aim of this study was to investigate the interaction of spread hydrophobic surfactant proteins with phospholipids in a captive-bubble surfactometer during rapid cyclic area changes (6 cycles/min). We found that SP-B and SP-C facilitated the rapid transport of lipids into the air-water interface in a concentration-dependent manner (threshold concentration > or = 0.05:0.5 mol% SP-B/SP-C). Successive rapid cyclic area changes did not affect the concentration-dependent lipid adsorption process, suggesting that SP-B and SP-C remained associated with the surface film.     

Restoration of lung compliance with calf lung surfactant extract and a surfactant analog in an in situ model of surfactant deficiency in rats

We have developed a standardized in situ lung surfactant deficiency model in the rat by using a single bronchoalveolar lavage (BAL). The purpose of this study was to assess the usefulness of surfactants and surfactant analogs in terms of their in vivo physiological properties. Calf lung surfactant extract (CLSE) was shown to improve lung compliance in a dose-dependent manner in this surfactant deficiency model when administered intratracheally immediately after BAL. In addition, CLSE formulated with a diether (palmityl) phosphonolipid surfactant analog significantly improved the compliance post-BAL as compared to CLSE alone. We propose that this in situ bioassay may be useful for the assessment of physiological capabilities of surfactants, surfactant analogs and surfactant formulations.     

Lyme disease--on the basis of our observations

We biologically assessed functions of several reconstituted surfactants with the same minimum surface tension (2-3 mN/m) as "complete" porcine pulmonary surfactant (natural surfactant) but with longer surface adsorption times. Administration of natural surfactant (adsorption time 0.29 s) into the lungs of surfactant-deficient immature rabbits brought a tidal volume of 16.1 +/- 4.4 (SD) ml/kg during mechanical ventilation with 40 breaths/min and 20 cmH2O insufflation pressure. In static pressure-volume recordings, these animals showed a lung volume of 62.4 +/- 9.7 ml/kg at 30 cmH2O airway pressure and maintained 55% of this volume when the pressure decreased to 5 cmH2O. With two reconstituted surfactants consisting of synthetic lipids or isolated lipids from porcine lungs plus surfactant-associated hydrophobic proteins (adsorption times 0.57 and 0.78 s, respectively), tidal volumes were < 38% of that with natural surfactant (P < 0.05), but static pressure-volume recordings were not different. Care is therefore needed in estimating the in vivo function of surfactant preparations from minimum surface tension or static pressure-volume measurements.     

Chronic K depletion inhibits renal brush border membrane Na/sulfate cotransport

Conversion of the highly surface-active subtype of pulmonary surfactant known as large surfactant aggregates (LA) to small aggregates (SA) with poor surface activity has recently been shown to occur upon cyclic changes of the air-liquid interface area in vitro. By subjecting pooled rabbit bronchoalveolar lavage fluid (BALF) to this maneuver, we found that conversion of LA to SA was accompanied by a marked decline in the ability of the remaining LA fraction to reduce surface tension by adsorption and during film compression on a pulsating bubble surfactometer. SA obtained by centrifugation of noncycled rabbit BALF had a similar phospholipid (PL) but different neutral lipid (NL) composition than did the LA. Upon cycling, the increased formation of SA obliterated this difference. No substantial difference in the PL, NL, or fatty acid profile of LA was noted before and after cycling. In contrast, the content of surfactant apoprotein-B (SP-B) in the LA decreased dramatically to nearly undetectable levels during the cycling maneuver, and this decline in SP-B content was closely correlated with the decrease in proportional appearance of LA and loss of surface activity of this fraction. Reconstitution of LA with intact SP-B after cycling virtually fully restored the surface activity of this surfactant subtype. When comparing lavage samples from adults with acute respiratory distress syndrome (ARDS; n = 10) with samples from healthy controls (n = 11), we noted a marked reduction of SP-B in the LA fraction. There was a significant correlation between the SP-B content of the LA fraction and the relative percentage of LA in BALF or the lower surface activity of this surfactant subtype. We conclude that an SP-B-related loss of LA integrity and function may substantially contribute to the decline of this surfactant subtype and the loss of its surface activity during cycling in vitro and in clinical ARDS.     

Differential effects in vivo of thyroid hormone on the expression of surfactant phospholipid, surfactant protein mRNA and antioxidant enzyme mRNA in fetal rat lung

Antenatal administration of triiodo-L-thyronine (T3) to late gestation rats resulted in decreased lung antioxidant enzyme (AOE) activity but increased surfactant phospholipids. In fetal rat lung explant cultures, T3 decreased the expression of surfactant proteins (SP) A and B. There have been no reported studies of the simultaneous in vivo developmental influence of T3 on both pulmonary AOE and SP gene expression. We hypothesized that antenatal T3 treatment would cause differential regulation of surfactant phospholipid, SP, and AOE genes in the late gestation fetal rat. Timed pregnant rats received intramuscular injections of either T3 (7 mg/kg) or placebo on days 19 and 20 of gestation and fetuses were delivered on day 21. Fetal lung SP-A, SP-B, SP-C, and AOE mRNA levels were studied by Northern analysis. AOE mRNA levels were further quantitated by solution hybridization. Total lung phospholipids (TPL) and disaturated phosphatidylcholine (DSPC) content were quantitated by a phosphorus assay. T3 significantly increased TPL and DSPC content, and significantly decreased the expression of SP-A, SP-C, CuZnSOD, and catalase genes. Because of a crucial interplay of these factors for normal lung function at the time of birth, the molecular mechanisms by which these apparently opposing changes are accomplished warrant further investigation.     

Depth profiles of pulmonary surfactant protein B in phosphatidylcholine bilayers, studied by fluorescence and electron spin resonance spectroscopy

Pulmonary surfactant-associated protein B (SP-B) has been isolated from porcine lungs and reconstituted in bilayers of dipalmitoylphosphatidylcholine (DPPC) or egg yolk phosphatidylcholine (PC) to characterize the extent of insertion of the protein into phospholipid bilayers. The parameters for the interaction of SP-B with DPPC or PC using different reconstitution protocols have been estimated from the changes induced in the fluorescence emission spectrum of the single protein tryptophan. All the different reconstituted SP-B-phospholipid preparations studied had similar Kd values for the binding of the protein to the lipids, on the order of a few micromolar. The depth of penetration of SP-B into phospholipid bilayers has been estimated by the parallax method, which compares the relative efficiencies of quenching of the protein fluorescence by a shallow or a deeper spin-labeled phospholipid probe. SP-B tryptophan was found to be located 10-13 A from the center of bilayers, which is consistent with a superficial location of SP-B in phosphatidylcholine membranes. Parallax experiments, as well as resonance energy transfer from SP-B tryptophan to an acceptor probe located in the center of the bilayer, indicate that there are significant differences in the extent of insertion of the protein, depending on the method of reconstitution. SP-B reconstituted from lipid/protein mixtures in organic solvents is inserted more deeply in PC or DPPC bilayers than the protein reconstituted by addition to preformed phospholipid vesicles. These differences in the extent of insertion lead to qualitative and quantitative differences in the effect of the protein on the mobility of the phospholipid acyl chains, as studied by spin-label electron spin resonance (ESR) spectroscopy, and could represent different functional stages in the surfactant cycle.     

A comparison of exposures to refractory ceramic fibres over multiple work shifts

The effect of gamma-radiation on aqueous solutions of saturated phospholipids, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 1-palmitoyl-2-lyso-sn-glycero-3-phosphocholine (lysoPC), and bovine brain sphingomyelin (SM) has been investigated. It is shown that the phospholipids with an OH group in beta-position to the P-O bond (DPPG and lysoPC), or to the amide bond (SM), undergo a free radical fragmentation. As a result of such fragmentation, stearoylamide, palmitoxyacetone and phosphatidic acid are formed from SM, lysoPC and DPPG, respectively. In parallel with the formation of hydrophobic fragments, an accumulation of hydrophilic species such as oxyacetone and phosphocholine in the irradiated DPPG and lysoPC dispersions was observed. On the basis of the data obtained for free radical transformation of phospholipids and their simplest analogs, such as glycero-1-phosphate, triacetin and 1,2-isopropylidene glycerol, it is suggested that the fragmentation of the radicals derived from the above compounds proceed by a concerted mechanism through a five-membered transition state. The accumulation of hydrophobic fragments in phospholipid membranes is shown to influence the temperature and co-operativity of the 'gel-to-liquid crystal' phase transition. An assumption is made that the fragmentation of phospholipids caused by free radical attack on the hydrophilic moiety, along with lipid peroxidation, may constitute principal mechanisms of radiation-induced damage of biological membranes.     

Structure and orientation of lung surfactant SP-C and L-alpha-dipalmitoylphosphatidylcholine in aqueous monolayers

SP-C, a pulmonary surfactant-specific protein, aids the spreading of the main surfactant phospholipid L-alpha-dipalmitoylphosphatidylcholine (DPPC) across air/water interfaces, a process that has possible implications for in vivo function. To understand the molecular mechanism of this process, we have used external infrared reflection-absorption spectroscopy (IRRAS) to determine DPPC acyl chain conformation and orientation as well as SP-C secondary structure and helix tilt angle in mixed DPPC/SP-C monolayers in situ at the air/water interface. The SP-C helix tilt angle changed from approximately 24 degrees to the interface normal in lipid bilayers to approximately 70 degrees in the mixed monolayer films, whereas the acyl chain tilt angle of DPPC decreased from approximately 26 degrees in pure lipid monolayers (comparable to bilayers) to approximately 10 degrees in the mixed monolayer films. The protein acts as a "hydrophobic lever" by maximizing its interactions with the lipid acyl chains while simultaneously permitting the lipids to remain conformationally ordered. In addition to providing a reasonable molecular mechanism for protein-aided spreading of ordered lipids, these measurements constitute the first quantitative determination of SP-C orientation in Langmuir films, a paradigm widely used to simulate processes at the air/alveolar interface.     

Orientation of anthracyclines in lipid monolayers and planar asymmetrical bilayers: a surface-enhanced resonance Raman scattering study

The interaction of anthracyclines (daunorubicin and idarubicin) with monolayers of zwitterionic palmitoyloleoylphosphatidylcholine (POPC) and anionic dipalmitoylphosphatidic acid (POPC-DPPA 80-20 mol%) was studied by surface pressure measurements and compared with previous results obtained with other anthracyclines (pirarubicin and adriamycin). These anthracycline/phospholipid monolayers were next transferred by a Langmuir-Blodgett technique onto planar supports and studied by surface-enhanced resonance Raman scattering (SERRS), which gave information about the orientation of anthracycline in the monolayers. On the whole, the adsorption of anthracyclines in zwitterionic monolayers increases with the anthracycline hydrophobic/hydrophilic balance, which underlines the role of the hydrophobic component of the interaction. On the contrary, the anthracyclines remain adsorbed on the polar headgroups of the phospholipids in the presence of DPPA and form a screen that limits a deeper penetration of other anthracycline molecules. To study by SERRS measurements the crossing of pirarubicin through a phospholipid bilayer used as a membrane model, asymmetrical POPC-DPPA/POPC or POPC/POPC-DPPA bilayers were transferred by the Langmuir-Sch?fer method, thanks to a laboratory-built set-up, and put in contact with a pirarubicin aqueous solution. It has been shown that the presence of anionic DPPA in the first monolayer in contact with pirarubicin would limit its crossing. This limiting effet is not observed if the first monolayer is zwitterionic.     

Palmitoylation of lung surfactant protein SP-C alters surface thermodynamics, but not protein secondary structure or orientation in 1,2-dipalmitoylphosphatidylcholine langmuir films

Pulmonary surfactant-specific protein, SP-C, isolated from porcine lung lavage, has been deacylated to investigate the role of the two thioester linked palmitoyl chains located near the N-terminus. Surface thermodynamic properties, secondary structure, and orientation of native and deacylated SP-C in 1, 2-dipalmitoylphosphatidylcholine (DPPC) monolayers has been characterized by combined surface pressure-molecular area (pi-A) isotherms and infrared reflection-absorption spectroscopy (IRRAS) measurements. The isotherms indicate that deacylation of SP-C produces more fluid monolayers at pressures less than 30 mN m-1. The helical secondary structure and tilt angle (70-80 degrees relative to the surface normal) of SP-C remained essentially unchanged upon deacylation in DPPC monolayers at a surface pressure approximately 30 mN m-1. The results are consistent with a model that acylation of SP-C may influence the rapid protein-aided spreading of a surface-associated surfactant reservoir, but not the structure of DPPC or SP-C in the monolayer at higher surface pressures.