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The dramatic impact of COVID-19 on humans worldwide has initiated an extraordinary search for effective treatment approaches. One of these is the administration of exogenous surfactant, which is being tested in ongoing clinical trials.Exogenous surfactant is a life-saving treatment for premature infants with neonatal respiratory distress syndrome. This treatment has also been tested for acute respiratory distress syndrome (ARDS) with limited success possibly due to the complexity of that syndrome. The 60-year history of successes and failures associated with surfactant therapy distinguishes it from many other treatments currently being tested for COVID-19 and provides the opportunity to discuss the factors that may influence the success of this therapy.Clinical data provide a strong rationale for using exogenous surfactant in COVID-19 patients. Success of this therapy may be influenced by the mechanical ventilation strategy, the timing of treatment, the doses delivered, the method of delivery and the preparations utilized. In addition, future development of enhanced preparations may improve this treatment approach. Overall, results from ongoing trials may not only provide data to indicate if this therapy is effective for COVID-19 patients, but also lead to further scientific understanding and improved treatment strategies.

Due to its branching structure, drug delivery to the peripheral areas of the lung is a major challenge. Consequently, most pulmonary therapies utilize large systemic dosing, with the potential for adverse side effects. One proposed strategy to overcome this challenge is to use exogenous surfactant, a material capable of distributing throughout the lung, as a pulmonary drug delivery vehicle. The objective was to develop and test an in vitro system to rapidly assess surfactant based therapies prior to animal studies. The Wet Bridge Transfer System consisted of two connected wells in which drugs were instilled into a delivery well and function was tested in a remote well which mimicked the remote areas of the lung where drug activity would be required. The system was used to assess surfactant as a carrier for antibiotics (Gentamicin, Ciprofloxacin, and Colistin) by measuring their ability to kill Pseudomonas aeruginosa bacteria in the remote well. Anti-inflammatory agents (Budesonide and a host defense peptide, CATH-2) with and without exogenous surfactant were examined using stimulated macrophages in the remote well and IL-6 concentration as an outcome. The results showed that being paired with surfactant, Gentamicin and Ciprofloxacin, but not Colistin, had significantly greater bacterial killing in the remote wells. Similarly, when combined with a surfactant, both Budesonide and CATH-2 significantly lowered IL-6 concentrations. We conclude that the wet-bridge system can be used to rapidly screen surfactant-based therapies prior to their assessment in vivo. Furthermore, exogenous surfactant was an effective delivery vehicle for several antimicrobial and anti-inflammatory therapeutics.

Hydrolysis of surfactant phospholipids (PL) by secretory phospholipases A2(sPLA2) contributes to surfactant damage in inflammatory airway diseases such as acute lung injury/acute respiratory distress syndrome. We and others have reported that each sPLA2exhibits specificity in hydrolyzing different PLs in pulmonary surfactant and that the presence of hydrophilic surfactant protein A (SP-A) alters sPLA2-mediated hydrolysis. This report tests the hypothesis that hydrophobic SP-B also inhibits sPLA2-mediated surfactant hydrolysis. Three surfactant preparations were used containing varied amounts of SP-B and radiolabeled tracers of phosphatidylcholine (PC) or phosphatidylglycerol (PG): 1) washed ovine surfactant (OS) (pre- and postorganic extraction) compared with Survanta (protein poor), 2) Survanta supplemented with purified bovine SP-B (1–5%, wt/wt), and 3) a mixture of dipalmitoylphosphatidylcholine (DPPC), 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG) (DPPC:POPC:POPG, 40:40:20) prepared as vesicles and monomolecular films in the presence or absence of SP-B. Hydrolysis of PG and PC by Group IB sPLA2(PLA2G1A) was significantly lower in the extracted OS, which contains SP-B, compared with Survanta ( P = 0.005), which is SP-B poor. Hydrolysis of PG and PC in nonextracted OS, which contains all SPs, was lower than both Survanta and extracted OS. When Survanta was supplemented with 1% SP-B, PG and PC hydrolysis by PLA2G1B was significantly lower ( P < 0.001) than in Survanta alone. When supplemented into pure lipid vesicles and monomolecular films composed of PG and PC mixtures, SP-B also inhibited hydrolysis by both PLA2G1B and Group IIA sPLA2 (PLA2G2A). In films, PLA2G1B hydrolyzed surfactant PL monolayers at surface pressures ≤30 mN/m ( P < 0.01), and SP-B lowered the surface pressure range at which hydrolysis can occur. These results suggest the hydrophobic SP, SP-B, protects alveolar surfactant PL from hydrolysis mediated by multiple sPLA2in both vesicles (alveolar subphase) and monomolecular films (air-liquid interface).

Pulmonary surfactant facilitates breathing by forming a surface tension reducing film at the air–liquid interface of the alveoli. The objective was to characterize the structure of surfactant films using endogenous rat surfactant. Solid-support surfactant films, at different surface pressures, were obtained using a Langmuir balance and were analyzed using atomic force microscopy. The results showed a lipid film structure with three distinct phases: liquid expanded, liquid ordered and liquid condensed. The area covered by the liquid condensed domains increased as surface pressure increased. The presence of liquid ordered phase within these structures correlated with the cholesterol content. At a surface pressure of 50 mN/m, stacks of bilayers appeared. Several structural details of these films differ from previous observations made with goat and exogenous surfactants. Overall, the data indicate that surfactant films demonstrate phase separation at low surface pressures and multilayer formation at higher pressure, features likely important for normal surfactant function.

Pulmonary surfactant is a complex lipid-protein mixture whose main function is to reduce the surface tension at the air-liquid interface of alveoli to minimize the work of breathing. The exact mechanism by which surfactant monolayers and multilayers are formed and how they lower surface tension to very low values during lateral compression remains uncertain. We used time-of-flight secondary ion mass spectrometry to study the lateral organization of lipids and peptide in surfactant preparations ranging in complexity. We show that we can successfully determine the location of phospholipids, cholesterol and a peptide in surfactant Langmuir-Blodgett films and we can determine the effect of cholesterol and peptide addition. A thorough understanding of the lateral organization of PS interfacial films will aid in our understanding of the role of each component as well as different lipid-lipid and lipid-protein interactions. This may further our understanding of pulmonary surfactant function. © 2010 Elsevier B.V. All rights reserved.

This review is focused on the evolution and function of alveolar proteins The lung faces physical and environmental challenges, due to changing pressures/volumes and foreign pathogens, respectively The pulmonary surfactant system is integral in protecting the lung from these challenges via two groups of surfactant proteins - the small molecular weight hydrophobic SPs. SP-B and -C, that regulate interfacial adsorption of the lipids, and the large hydrophilic SPs. SP-A and -D, which are surfactant collectins capable of inhibiting foreign pathogens Further aiding pulmonary host defence are non-surfactant collectins and antimicrobial peptides that are expressed across the biological kingdoms Linking to the first symposium session, which emphasised molecular structure and biophysical function of surfactant lipids and proteins, this review begins with a discussion of the role of temperature and hydrostatic pressure in shaping the evolution of SP-C in mammals Transitioning to the role of the alveolus in innate host defence we discuss the structure, function and regulation of antimicrobial peptides, the defensins and cathelicidins. We describe the recent discovery of novel avian collectins and provide evidence for their role in preventing influenza infection This is followed by discussions of the roles of SP-A and SP-D in mediating host defence at the alveolar surface and in mediating inflammation and the allergic response of the airways Finally we discuss the use of animal models of lung disease including knockouts to develop an understanding of the role of these proteins in initiating and/or perpetuating disease with the aim of developing new therapeutic strategies (C) 2010 Elsevier B V All rights reserved

Studies on the interaction of cholesterol (CHOL) with palmitoyl-oleoyl phosphatidylcholine (POPC) and sphingomyelin (SPM) are considered to be important because of the occurrence of strong interact...

PbS nanocrystals (NCs) are synthesized in aqueous phase within a temperature range of 40-80°C in the presence of native and denatured states of bovine serum albumen (BSA) as the capping/stabilizing agent The NCs are characterized with the help of field-emission scanning electron microscopy, high-resolution transmission electron microscopy, X-ray diffraction, and energy-dispersive X-ray analysis. At40°C, large ball-shaped NCs (145 ± 37 nm) with small surface protrusions are formed when 1 x 10 -4 g mL -1 BSA is used. As the reaction temperature is increased towards 80°C, the size of NCs decreases and they acquire somewhat cubic geometries (49.1 ± 7.0 nm) due to a change in the capping behavior of BSA between its native and denatured states. The native and denatured states of BSA are simultaneously studied by fluorescence spectroscopy using tryptophan emission, and pH measurements with respect to time and temperature. Gel electrophoresis is used to determine the polarity ofthe BSA capped NCs. Only the small sized NCs conjugated with relatively larger amounts of BSA show a displacement towards the positively charged electrode in comparison to larger NCs with lower amounts of BSA capping. It was concluded that the denatured state of BSA is more effective in controlling the crystal growth of PbS than its native state especially in the low concentration range.

Lead selenide (PbSe), cadmium selenide (CdSe), and selenium (Se) nano- and microcrystals were synthesized by using respective metal acetate salts along with sodium selenite as the Se source in the presence of bovine serum albumen (BSA) as the capping/stabilizing agent. Aqueous phase hydrazine reduction at 85 °C produced fine crystalline morphologies within 48 h. Both PbSe and CdSe reactions produced Se microrods (MRs) as reaction byproduct. The concentrations of metal acetate and sodium selenite used were always 1:1 (i.e., 1.25 mM in each case) and that of hydrazine was fixed at 0.78 M. The amount of BSA was changed systematically from 1−10 × 10−4 g/mL to determine its influence on the crystal growth of these chalcogenides. Their morphologies and chemical compositions were determined with FESEM, TEM, and EDX analysis. A selective and precise EDX analysis of a single particle helped us to elucidate its shape and chemical composition. Such analyses lead to the finding that both reactions produced Se rods, t...

Pulmonary surfactant (PS) dysfunction because of the leakage of serum proteins into the alveolar space could be an operative pathogenesis in acute respiratory distress syndrome. Albumin-inhibited PS is a commonly used in vitro model for studying surfactant abnormality in acute respiratory distress syndrome. However, the mechanism by which PS is inhibited by albumin remains controversial. This study investigated the film organization of albumin-inhibited bovine lipid extract surfactant (BLES) with and without surfactant protein A (SP-A), using atomic force microscopy. The BLES and albumin (1:4w/w) were cospread at an air-water interface from aqueous media. Cospreading minimized the adsorption barrier for phospholipid vesicles imposed by preadsorbed albumin molecules, i.e., inhibition because of competitive adsorption. Atomic force microscopy revealed distinct variations in film organization, persisting up to 40mN/m, compared with pure BLES monolayers. Fluorescence confocal microscopy confirmed that albumin remained within the liquid-expanded phase of the monolayer at surface pressures higher than the equilibrium surface pressure of albumin. The remaining albumin mixed with the BLES monolayer so as to increase film compressibility. Such an inhibitory effect could not be relieved by repeated compression-expansion cycles or by adding surfactant protein A. These experimental data indicate a new mechanism of surfactant inhibition by serum proteins, complementing the traditional competitive adsorption mechanism.

Gold (Au) nanoribbons were synthesized in aqueous phase under ambient conditions by using dimethylene bis(tetradecyldimethyl-ammonium bromide) (14-2-14) as a capping agent as well as a soft template. A two steps seed-growth (S-G) method was used. The first step of S-G method mainly gave nanorods of high aspect ratio along with nanoparticles of other shapes, but the next step produced mainly fine Au nanoribbons of several micrometers long, 50–150 nm wide, and ≈5 nm thick. They were characterized by transmission electron microscopy, X-ray photoelectron spectroscopy, energy dispersive X-ray, and X-ray diffraction analysis. Isotropic liquid crystalline phase of 14-2-14 provided a soft template effect and derived nanorods of high aspect ratios toward nanoribbon formation at room temperature.

Monolayers of a functional pulmonary surfactant (PS) can reach very low surface tensions well below their equilibrium value. The mechanism by which PS monolayers reach such low surface tensions and maintain film stability remains unknown. As shown previously by fluorescence microscopy, phospholipid phase transition and separation seem to be important for the normal biophysical properties of PS. This work studied phospholipid phase transitions and separations in monolayers of bovine lipid extract surfactant using atomic force microscopy. Atomic force microscopy showed phospholipid phase separation on film compression and a monolayer-to-multilayer transition at surface pressure 40–50mN/m. The tilted-condensed phase consisted of domains not only on the micrometer scale, as detected previously by fluorescence microscopy, but also on the nanometer scale, which is below the resolution limits of conventional optical methods. The nanodomains were embedded uniformly within the liquid-expanded phase. On compression, the microdomains broke up into nanodomains, thereby appearing to contribute to tilted-condensed and liquid-expanded phase remixing. Addition of surfactant protein A altered primarily the nanodomains and promoted the formation of multilayers. We conclude that the nanodomains play a predominant role in affecting the biophysical properties of PS monolayers and the monolayer-to-multilayer transition.

A seed-mediated approach was applied to synthesize gold (Au) nanoparticles (NP) by using twin tail alkylammonium cationic surfactants such as 12-6-12 and 12-0-12 as capping agents in aqueous phase at ambient conditions. The growth of Au NP was monitored by changing the amount of seed. Spherical NP (10–50nm) and nanorods (aspect ratio = 2–3) were obtained in the presence of 12-6-12 as capping agent; their shape and size systematically deformed because of anisotropic growth with a decrease in the amount of seed. In contrast, when 12-0-12 was used as a capping agent, no anisotropic growth was observed. An effective liquid/solid interfacial adsorption of 12-0-12 prevented anisotropic growth which led to precise morphologies. This was not observed in the case of 12-6-12 because of the presence of a spacer which restricted an effective interfacial adsorption because of the steric factors. XPS and FTIR studies clearly indicated the presence of a surfactant film on the surface of Au NP, while XRD analysis demonst...

Lead sulfide nanocrystals (NCs) along with nanothreads and nanosheets were synthesized in the presence of water soluble polymers such as poly(styrene sulfonate) sodium salt (PSS) and poly(vinyl pyrolidone) (PVP) in aqueous phases at relatively mild temperature of 80 °C. The amount of each polyelectrolyte was systematically varied between 1 and 4 mM (monomolar based). The presence of PSS produced roughly cubic geometries at low concentrations, while nanothreads and nanosheets were produced at high concentrations. This was not so in the presence of equivalent amounts of PVP, where only fine cubes and nanobars were obtained. The shape and structure of all kinds of geometries were characterized by TEM, FESEM, EDX, and XRD analyses. All results demonstrated that the different preferential adsorption of polyelectrolyte PSS and neutral PVP for different crystal planes of the fcc geometry of PbS controlled the final morphology.

Innate neuroinflammatory changes, increased oxidative stress and disorders of glutathione metabolism may be involved in the pathophysiology of autism spectrum disorders (ASD). Propionic acid (PPA) is a dietary and gut bacterial short chain fatty acid which can produce brain and behavioral changes reminiscent of ASD following intraventricular infusion in rats. Adult Long-Evans rats were given intraventricular infusions of either PPA (.26M, 4µl animal -1 ) or phosphate buffered saline (PBS) vehicle, twice daily for 7 days. Immediately following the second daily infusion, the locomotor activity of each rat was assessed in an automated open field (Versamax) for 30 min. PPA-treated rats showed significant increases in locomotor activity compared to PBS vehicle controls. Following the last treatment day, specific brain regions were assessed for neuroinflammatory or oxidative stress markers. Immunohistochemical analyses revealed reactive astrogliosis (GFAP), activated microglia (CD68, Iba1) without apoptotic cell loss (Caspase 3' and NeuN) in hippocampus and white matter (external capsule) of PPA treated rats. Biomarkers of protein and lipid peroxidation, total glutathione (GSH) as well as the activity of the antioxidant enzymes superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione S-transferase (GST) were examined in brain homogenates. Some brain regions of PPA treated animals (neocortex, hippocampus, thalamus, striatum) showed increased lipid and protein oxidation accompanied by decreased total GSH in neocortex. Catalase activity was decreased in most brain regions of PPA treated animals suggestive of reduced antioxidant enzymatic activity. GPx and GR activity was relatively unaffected by PPA treatment while GST was increased, perhaps indicating involvement of GSH in the removal of PPA or related catabolites. Impairments in GSH and catalase levels may render CNS cells more susceptible to oxidative stress from a variety of toxic insults. Overall, these findings are consistent with those found in ASD patients and further support intraventricular PPA administration as an animal model of ASD.

Aqueous phase synthesis at 80 °C was carried out to synthesize lead sulfide (PbS) nanocrystals (NC) and microcrystals (MC) by using cationic twin-tail surfactants (TTS) such as 12-0-12, 10-2-10, 12-2-12, and 14-2-14 as capping agents in the concentration range from 0.1 −to 2 mM. The effect of hydrophobicity on the shape and size of PbS NC was evaluated by choosing DTAB as a reference surfactant for all TTS. TEM micrographs of PbS MC synthesized in the presence of DTAB indicated the formation of star-shaped MC with sizes between 3 −and 5 μm. An increase in the hydrophobicity, by introducing another tail in the basic structure of DTAB to make 12-0-12, significantly controlled the shape and size and lead to the formation of well-defined nanocubes and spheres 50−100 nm in size. Similarly, the effect of the hydrocarbon tail length on the shape controlled synthesis of PbS NC was systematically evaluated. Pyrene fluorescence measurements were used to determine the variation in the degree of hydrophobicity with r...

A seed-growth (S-G) method has been used to synthesize gold (Au) and Au−silver (Ag) bimetallic pear-necklace-type nanoparticles (NP) as bioconjugate materials by using a series of phospho-glycerol (PG) and phospho-choline (PC) lipids as capping agents. All PG lipids produce a fine pear-necklace arrangement of Au NP with dimensions between 10 and 20 nm. Addition of Ag converts this arrangement into Au−Ag bimetallic NP with a diameter essentially close to that of Au NP. Use of PC lipids does not show this arrangement but promotes a significant anisotropic growth especially in the presence of Ag. This difference has been attributed to a difference in the capping ability of PG and PC lipids because of their anionic and zwitterionic nature, respectively. XPS results have demonstrated the presence of adsorbed PG and PC lipids on Au or Au−Ag bimetallic surfaces in their respective samples. The results also indicate a decrease in the capping amount of a lipid with an increase in the growth of Au−Ag bimetallic NP....

Langmuir film behavior of bovine lipid extract surfactant (BLES), mixed with cholesterol (CHOL) and cholesterol palmitate (CHOLP), has been studied by surface pressure ( π )–area ( A ) measurements. Associative interactions, observed for both systems, were less favored at lower BLES content. The presence of unsaturated phospholipids and surfactant proteins in BLES favored the association. Miscibility of BLES was better with CHOLP than with CHOL at all compositions, indicating more compact packing of the BLES–CHOLP than of the BLES–CHOL system. The most stable mixtures were found at 30–40 mol% CHOL and at low π and at 20–25 mol% CHOLP but at higher π . These results suggest that BLES–CHOL miscibility is better at low π and low CHOL concentrations, while BLES–CHOLP miscibility is better at high π and high CHOLP concentrations.

Gold (Au) nanowires (NW) have been synthesized by using mixtures of l-α-dipalmitoylphosphatidylcholine (DPPC) and various conventional surfactants (CS) of different polarities. The capping ability of the surfactant mixture has been quantitatively evaluated by using UV−vis, TEM, XPS, FTIR, and XRD studies. The synthesis has been carried out by using a total [DPPC+CS] in the range of 0.125 to 0.5 mM. The results clearly demonstrate a surfactant selective synthesis that significantly depends on the polarity of the CS head group. An anionic surfactant like sodium dodecylsulfate (SDS) along with DPPC leads to a network of NW formation with ≈50 nm thickness at different total surfactant concentrations while cationic surfactant, cetyltrimethylammonium bromide (CTAB), predominantly controls the spherical Au nanoparticle (NP) formation at high concentration. At low [DPPC+CTAB], the NW is achieved by end-to-end fusion of spindle shaped Au NP with an aspect ratio of ≈2.15. The chemical composition of the adsorbed ca...

A seed-growth method has been applied to synthesize the gold (Au) nanoparticles (NP) in the presence of 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (DMPG) and hexamethylene-1,6-bis(dodecyldimethylammonium bromide)(12-6-12) as capping agents at ambient conditions. The systematic addition of CuSO4 leads to anisotropic growth of Au NP at low CuSO4 concentration in the presence of DMPG while this was not observed in the case of 12-6-12 where mostly spherical Au NP were obtained. At high CuSO4 concentration, small Cu NP appeared which arranged themselves around large Au NP in a typical pseudo-core−shell type arrangement. This was achieved by the fusion of lipid bilayers of lipid-capped Au and Cu NP. The cause of anisotropic growth of Au NP in the presence of DMPG has been discussed on the basis of its poor capping ability in comparison to 12-6-12.

Extracellular nucleotides, released in response to mechanical or inflammatory stimuli, signal through P2 receptors in many cell types, including osteoblasts. P2X7 receptors are ATP-gated cation channels that can induce formation of large membrane pores. Disruption of the gene encoding the P2X7 receptor leads to decreased periosteal bone formation and insensitivity of the skeleton to mechanical stimulation. Our purpose was to investigate signaling pathways coupled to P2X7 activation in osteoblasts. Live cell imaging showed that ATP or 2 ',3 '-O-(4-benzoylbenzoyl)-ATP (BzATP), but not UTP, UDP, or 2-methylthio-ADP, induced dynamic membrane blebbing in calvarial osteoblasts. Blebbing was observed in calvarial cells from wildtype but not P2X7 knock-out mice. P2X7 receptors coupled to activation of phospholipase D and A2, inhibition of which suppressed BzATP-induced blebbing. Activation of these phospholipases leads to production of lysophosphatidic acid (LPA). LPA caused dynamic blebbing in osteoblasts from both wild-type and P2X7 knock-out mice, similar to that induced by BzATP in wildtype cells. However, LPA-induced blebbing was more rapid in onset and was not affected by inhibition of phospholipase D or A2. Blockade or desensitization of LPA receptors suppressed blebbing in response to LPA and BzATP, without affecting P2X7-stimulated pore formation. Thus, LPA functions downstream of P2X7 receptors to induce membrane blebbing. Furthermore, inhibition of Rho-associated kinase abolished blebbing induced by both BzATP and LPA. In summary, we propose a novel signaling axis that links P2X7 receptors through phospholipases to production of LPA and activation of Rho-associated kinase. This pathway may contribute to P2X7-stimulated osteogenesis during skeletal development and mechanotransduction.

Bovine surfactant proteins B (SP-B) and C (SP-C) were analyzed by nano-electrospray ionization mass spectrometry (nano-ESI-MS). The observed molecular masses showed discrepancies compared to the calculated molecular masses using the published amino acid sequences. The number of cysteine residues in the published bovine SP-B amino acid sequences also failed to match the observed mass shift upon reduction of the SP-B dimer. To determine the amino acid sequences of two proteins, SP-B was first digested with trypsin and analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS), while SP-C was analyzed by MS/MS in its intact form. The amino acid sequence of bovine SP-B determined here matches the observed molecular mass. The sequence is almost identical to the sheep SP-B except for two amino acid residues, consistent with the proximity of the two species. The correct sequence contains seven cysteine residues. Bovine SP-B exists as dimers and all cysteines are oxidized to form disulfide bonds in physiological conditions, which is in agreement with the observed mass shift upon reduction of the SP-B dimer. These cysteine residues are completely conserved across all species indicating their importance for the biological functions of this surfactant protein. The sequence of SP-C determined here also reveals an L to V substitution at its position 22 compared with the published bovine SP-B sequence.

Time of flight secondary ion mass spectrometry (ToF-SIMS) provides the capability to image the distribution of molecular ions and their associated fragments that are emitted from monolayer films. ToF-SIMS can be applied to the analysis of monolayers of complex lipid mixtures that act as a model to understand the organization of cell membranes into solid-like domains called lipid rafts. The ability to determine the molecular distribution of lipids using ToF-SIMS in monolayer films is also important in studies of the function of pulmonary surfactant. One of the limitations of the use of ToF-SIMS to studies of complex lipid mixtures found in biological systems, arises from the similarity of the mass fragments that are emitted from the components of the lipid mixture. The use of selectively deuterated components in a mixture overcomes this limitation and results in an unambiguous assignment of specific lipids to particular surface domains. The use of deuterium labeling to identify specific lipids in a multi-component mixture can be done by the deuteration of a single lipid or by the addition of more than one lipid with selectively deuterated components. The incorporation of deuterium into the lipid chains does not alter the miscibility or phase behavior of these systems. The use of deuterium labeling to identify lipids and determine their distribution in monolayer films will be demonstrated using two biological systems. Principal components analysis (PCA) is used to further analyze these deuterated systems checking for the origin of the various mass fragments present.

Exposing bovine lipid extract surfactant (BLES), a clinical surfactant, to reactive oxygen species arising from hypochlorous acid or the Fenton reaction resulted in an increase in lipid (conjugated dienes, lipid aldehydes) and protein (carbonyls) oxidation products and a reduction in surface activity. Experiments where oxidized phospholipids (PL) were mixed with BLES demonstrated that this addition hampered BLES biophysical activity. However the effects were only moderately greater than with control PL. These results imply a critical role for protein oxidation. BLES oxidation by either method resulted in alterations in surfactant proteins SP-B and SP-C, as evidenced by altered Coomassie blue and silver staining. Western blot analyses showed depressed reactivity with specific antibodies. Oxidized SP-C showed decreased palmitoylation. Reconstitution experiments employing PL, SP-B, and SP-C isolated from control or oxidized BLES demonstrated that protein oxidation was more deleterious than lipid oxidation. Furthermore, addition of control SP-B can improve samples containing oxidized SP-C, but not vice versa. We conclude that surfactant oxidation arising from reactive oxygen species generated by air pollution or leukocytes interferes with surfactant function through oxidation of surfactant PL and proteins, but that protein oxidation, in particular SP-B modification, produces the major deleterious effects.

In this study we sought to determine the effect of brief repeated umbilical cord occlusions (rUCO) on surfactant protein (SP) mRNA levels in the fetal sheep lung at two different gestational ages.Fourteen fetuses at 112 to 115 days' gestation (control n = 7, rUCO n = 7) and 15 fetuses at 130 to 133 days' gestation (control n = 7, rUCO n = 8) were studied over 4 successive days with rUCO of 90 seconds duration performed every 30 minutes for 3 to 5 hours each day in the rUCO animals. Blood samples were collected for corticotrophin (ACTH) and cortisol measurements. Animals were killed within 1 hour of the final cord occlusion. SP-A, -B, -C, and -D mRNA levels were determined in lung tissue using a ribonuclease protection assay.Cord occlusions resulted in temporary increases in circulating ACTH on day 1 with both gestational ages, but the elevations were blunted by day 4. Plasma cortisol levels increased transiently with the larger effect being observed on day 4, in particular with the near-term group. With advancing gestational age there was a significant (P.05) increase in the level of SP-A (control 112-115 days: 0.01 +/- 0.01 vs control 130-133 days: 0.07 +/- 0.02 fmol/mg RNA), SP -B (control 112-115 days: 0.02 +/- 0.01 vs control 130-133 days: 0.07 +/- 0.01 fmol/mg RNA) and SP-C (control 112-115 days: 0.13 +/- 0.09 vs control 130-133 days: 0.51 +/- 0.10 fmol/mg RNA), but not SP-D mRNA levels (control 112-115 days: 0.002 +/- 0.002 vs control 130-133 days: 0.01 +/- 0.002 fmol/mg RNA). At 112 to 115 days, there was no significant change in any of the SP mRNA levels following rUCO compared to controls. However, the same regime of rUCO at 130 to 133 days resulted in an 85% reduction in SP-A and SP-B mRNA content and a 66% reduction in SP-C mRNA levels compared to controls.The surprising decrease in SP-A and SP-B mRNA levels, which contrasts with other studies, suggests intermittent asphyxial episodes impact differently on surfactant apoprotein mRNA expression than does prolonged hypoxia.

Surfactant ion selective membranes, prepared using different sets of catanionic species, have been found superior to the membranes prepared earlier in terms of sensitivity and durability. The aggregates (or coacervates) of cetyltrimethylammonium (CTA + ) ion with dodecylsulfate (DS − ), dodecylbenzenesulfonate (DBS − ), dioctylsulfosuccinate (AOT − ), deoxycholate (DC − ), and taurochenodeoxycholate (TCDC − ) ions in 1:1 molar ratio were used as the carrier complexes in the membrane for estimating anionic surfactants whereas carrier complexes of DBS − with CTA + , tetradecyltrimethylammonium (TTA + ) and dodecyltrimethylammonium (DTA + ) cations (1:1 molar ratio) were employed for estimating the cationic surfactants. Electrochemical behavior of membrane electrodes was, although, not always Nernstian, the cell emf versus log[surfactant] plots were linear with sharp breaks corresponding to the critical micelle concentration (CMC). Film behavior of complex aggregates (coacervates) was studied at the air–water interface by surface pressure ( π )–area ( A ) measurements. Limiting area per molecule, maximum attainable surface pressure, film compressibility, etc., were evaluated from the experimental data. Formation of probe-excluded regions of compressed monolayers, doped with a fluorescent probe (NBD-PC) was observed by epifluorescence microscopy.

Mixing of long-chain saturated fatty acids (FAs), from dodecanoic (lauric) to octadecanoic (stearic) acid with a disaturated phospholipid, dipalmitoylphosphatidylcholine (DPPC), and the lipid-extracted materials from bovine lung surfactant (BLES), a clinical pulmonary surfactant (PS) formulation, have been studied at the air–water interface using Langmuir surface balance. BLES is a natural mixture of both saturated (solid) and unsaturated (fluid) lipids having DPPC as its major constituent; besides it contains two hydrophobic proteins SP-B and -C that help in better surface activity. Surface pressure–area behavior of DPPC and BLES, mixed with different proportions of FA, were studied. While DPPC–FA mixtures showed positive deviations from ideality, BLES–FA mixtures showed negative deviations irrespective of the chain length of FAs. Minimum repulsion was observed for palmitic acid in DPPC–FA mixtures while maximum association occurred with palmitic acid for BLES–FA mixtures, especially at higher mole fractions of FAs, as revealed from the excess free energy or free energy of mixing. The miscibilities are discussed in terms of the chain lengths of the FAs, the size of lipid and the composition of BLES.

Objective: To evaluate the effects of hyperoxia exposure on lung function and the endogenous surfactant system in spontaneously breathing adult rats. Design: Analysis of the pulmonary surfactant system isolated from adult rats following exposure to >90% oxygen or room air for 48 or 72 hrs. Setting: A basic science research laboratory in a university setting. Subjects: Sixty pathogen-free Sprague-Dawley rats. Interventions: Exposure to >90% oxygen for 72 hrs. Measurements and Main Results: Exposure to >90% oxygen for 72 hrs resulted in significant lung dysfunction and an increase in neutrophils and total protein concentrations within the airspace compared with animals exposed to room air or 48 hrs of 90% oxygen exposure. Total alveolar surfactant and large aggregate pool sizes were increased after 72 hrs of hyperoxia compared with the other groups, and there was evidence of lipid peroxidation within these large aggregate subfractions. The biophysical function of large aggregate isolated from 72-hr hyperoxia-exposed animals was also impaired and converted into small aggregate forms faster than large aggregate from normoxia-exposed animals when assessed using in vitro surface area cycling techniques. Conclusions: Oxidative stress on the endogenous surfactant system may represent an important mechanism contributing to the surfactant dysfunction and abnormal surfactant metabolism associated with hyperoxia-induced lung injury.

The lung contains two distinct forms of phosphatidic acid phosphatase (PAP). PAP1 is a cytosolic enzyme that is activated through fatty acid-induced translocation to the endoplasmic reticulum, where it converts phosphatidic acid (PA) to diacylglycerol (DAG) for the biosynthesis of phospholipids and neutral lipids. PAP1 is Mg2+dependent and sulfhydryl reagent sensitive. PAP2 is a six-transmembrane-domain integral protein localized to the plasma membrane. Because PAP2 degrades sphingosine-1-phosphate (S1P) and ceramide-1-phosphate in addition to PA and lyso-PA, it has been renamed lipid phosphate phosphohydrolase (LPP). LPP is Mg2+independent and sulfhydryl reagent insensitive. This review describes LPP isoforms found in the lung and their location in signaling platforms (rafts/caveolae). Pulmonary LPPs likely function in the phospholipase D pathway, thereby controlling surfactant secretion. Through lowering the levels of lyso-PA and S1P, which serve as agonists for endothelial differentiation gene receptors, LPPs regulate cell division, differentiation, apoptosis, and mobility. LPP activity could also influence transdifferentiation of alveolar type II to type I cells. It is considered likely that these lipid phosphohydrolases have critical roles in lung morphogenesis and in acute lung injury and repair.

Mechanical ventilation is an essential but potentially harmful therapeutic intervention for patients with acute lung injury. The objective of this study was to investigate the effects of mechanical ventilation on large-aggregate surfactant (LA) structure and function. Isolated rat lungs were randomized to either a nonventilated control group, a relatively noninjuriously ventilated group [1 h, 10 ml/kg tidal volume, 3 cmH2O positive end-expiratory pressure (PEEP)], or an injuriously ventilated group (1 h, 20 ml/kg tidal volume, 0 cmH2O PEEP). Injurious ventilation resulted in significantly decreased lung compliance compared with the other two groups. LA structure, as determined by electron microscopy, revealed that LA from the injurious group had significantly lower amounts of organized lipid-protein structures compared with LA obtained from the other groups. Analysis of the biophysical properties by using a captive bubble surfactometer demonstrated that adsorption and surface tension reduction were significantly impaired with LA from the injuriously ventilated lungs. We conclude that the injurious mechanical ventilation impairs LA function and that this impairment is associated with significant morphological alterations.

The effects of surfactant treatment on surfactant subtype conversion after lung injury were examined. Dogs were subjected to hyperventilation for 8 h with or without surfactant treatment. Lungs were stored for 17 h, and the right lung was transplanted and reperfused for 6 h. Conversion of large aggregate (LA) surfactant to small aggregates was investigated using in vitro surface area cycling. LA from transplanted lungs (Transplant-LA) from the nontreated group converted more rapidly than Transplant-LA from the treated group. Transplant-LA from both groups converted more rapidly than LA from normal lungs. Calculations based on [3H]dipalmitoylphosphatidylcholine in the administered surfactant [bovine lipid extract surfactant (BLES)] showed that the endogenous component of Transplant-LA converted more rapidly than the exogenous component. This indicates exogenous BLES did not equilibrate completely with endogenous surfactant. LA from hyperventilated, stored donor right lungs and from the recipients' native lungs from the nontreated group converted more rapidly than corresponding LA in the BLES-treated group. Similar relative conversions were observed with exogenous components from all lungs. Relative conversion of endogenous component from Transplant-LA was more rapid than that from LA from donor's stored right lung or from the recipient's native right lung. Low levels of phenylmethylsulfonyl fluoride inhibited conversion of Transplant-LA to a greater extent than normal LA. LA from all experimental groups had similar protein levels. These studies show acute lung injury, transplant, ischemia-reperfusion, and surfactant treatment have major effects on surfactant subtype integrity.

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°C was studied by measuring surface radioactivities of [4-14C]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 [1-14C]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. —Yu, S-H., and F. Possmayer. Dipalmitoylphosphatidylcholine and cholesterol in monolayers spread from adsorbed films of pulmonary surfactant.

Pattle, who provided some of the initial direct evidence for the presence of pulmonary surfactant in the lung, was also the first to show surfactant was susceptible to proteases such as trypsin. Pattle concluded surfactant was a lipoprotein. Our group has investigated the roles of the surfactant proteins (SP-) SP-A, SP-B, and SP-C using a captive bubble tensiometer. These studies show that SP-CSP-BSP-A in enhancing surfactant lipid adsorption (film formation) to the equilibrium surface tension of approximately 22-25 mN/m from the 70 mN/m of saline at 37 degrees C. In addition to enhancing adsorption, surfactant proteins can stabilize surfactant films so that lateral compression induced through surface area reduction results in the lowering of surface tension (gamma) from approximately 25 mN/m (equilibrium) to values near 0 mN/m. These low tensions, which are required to stabilize alveoli during expiration, are thought to arise through exclusion of fluid phospholipids from the surface monolayer, resulting in an enrichment in the gel phase component dipalmitoylphosphatidylcholine (DPPC). The results are consistent with DPPC enrichment occurring through two mechanisms, selective DPPC adsorption and preferential squeeze-out of fluid components such as unsaturated phosphatidylcholine (PC) and phosphatidylglycerol (PG) from the monolayer. Evidence for selective DPPC adsorption arises from experiments showing that the surface area reductions required to achieve gamma near 0 mN/m with DPPC/PG samples containing SP-B or SP-A plus SP-B films were less than those predicted for a pure squeeze-out mechanism. Surface activity improves during quasi-static or dynamic compression-expansion cycles, indicating the squeeze-out mechanism also occurs. Although SP-C was not as effective as SP-B in promoting selective DPPC adsorption, this protein is more effective in promoting the reinsertion of lipids forced out of the surface monolayer following overcompression at low gamma values. Addition of SP-A to samples containing SP-B but not SP-C limits the increase in gamma(max) during expansion. It is concluded that the surfactant apoproteins possess distinct overlapping functions. SP-B is effective in selective DPPC insertion during monolayer formation and in PG squeeze-out during monolayer compression. SP-A can promote adsorption during film formation, particularly in the presence of SP-B. SP-C appears to have a superior role to SP-B in formation of the surfactant reservoir and in reinsertion of collapse phase lipids.

Objective: We hypothesized that the proinflammatory response to intra-amniotic endotoxin would induce lung maturation in preterm lambs. Study Design: Ewes were randomly assigned to receive 20 mg Escherichia coli endotoxin by intra-amniotic injection, maternal betamethasone (0.5 mg/kg), or sodium chloride solution. Preterm lambs were delivered at 125 days' gestation and underwent ventilation to assess lung function. Lung gas volume, surfactant concentrations, and inflammation were subsequently evaluated, with data analyzed by analysis of variance. Results: Fetal endotoxin exposure 6 days before delivery increased compliance by 59%, increased lung gas volume 2.3-fold, increased concentrations of surfactant lipids, increased surfactant A and B protein levels, and increased messenger ribonucleic acid expressions for surfactant proteins (all P Conclusion: A single fetal exposure to endotoxin resulted in large improvements in postnatal lung function and increases in surfactant concentrations after preterm delivery. These effects were qualitatively larger than those achieved with betamethasone. (Am J Obstet Gynecol 2000;182:401-8.)

Glucocorticoid treatment increases content of surfactant protein (SP) A and SP-B in lung tissue and lavage fluid of preterm lambs. To investigate this process, we determined the ontogeny and glucocorticoid induction of SP mRNAs. In separate treatment protocols, each with its own controls, sheep were injected with betamethasone 15 h, 48 h, or weekly for 1–4 doses before preterm delivery. Using ovine SP cDNAs, we found an increase equal to or more than threefold in basal levels of all three SP mRNAs between 125 days and term. After betamethasone treatment, SP-B and SP-C mRNA levels increased by 15 h and all SP mRNAs were elevated after 24 h (≥2-fold); mRNA levels in fetuses delivered 1–3 wk after betamethasone were not different from control. We conclude that in vivo betamethasone rapidly induces a coordinated increase in SP mRNAs, which is fully reversible within 7 days despite repetitive doses of betamethasone. Similar increases in mRNA and protein contents for SP-A and SP-B suggest that glucocorticoid regulation of these SPs in vivo is primarily pretranslational.

Pulmonary surfactant stabilizes the alveoli by lining the air-fluid interface with films that reduce surface tension to near 0 mN/m (γmin). Surfactant protein B (SP-B) enhances the surface activity of surfactant phospholipids. A captive bubble tensiometer (CBT) was used to study the properties of adsorbed films of dipalmitoylphosphatidylcholine (DPPC) with acidic 1-palmitoyl-2-oleoyl- sn-glycero-3-[phospho- rac-(1-glycerol)] (POPG) or neutral 1-palmitoyl-2-oleoyl- sn-glycerol-3-phosphocholine with (7:3) and without 1% dimeric SP-B. SP-B enhanced the adsorption rate of DPPC-containing neutral or acidic lipid suspensions (1 mg/ml) to a similar extent. Quasi-static cycling of these films revealed that SP-B significantly decreased the film area reduction required to reach γminfor the acidic but not for the neutral system. The results obtained with DPPC-phosphatidylglycerol (PG)-SP-B were consistent with selective DPPC adsorption into the surface monolayer during film formation. Film area reduction required to reach γminwith this system (with and without calcium) approached that of pure DPPC, suggesting selective DPPC insertion and PG squeeze-out. Dynamic cycling of such films showed that larger film area reductions were required to reach γminfor the neutral than for acidic system, even after 20 cycles. Fluorescence microscopy of solvent-spread DPPC-POPG-SP-B planar films revealed highly condensed structures at ∼25 mN/m, although no specific PG phase-segregated structures could be identified. The study suggests that specific interactions of SP-B with acidic phospholipids of surfactant may be involved in the generation and maintenance of DPPC-rich films in the alveoli.

Pulmonary surfactant stabilizes the lung by reducing surface tension at the air-water interface of the alveoli. Surfactant is present in the lung in a number of morphological forms, including tubular myelin (TM). TM is composed of unusual 40 x 40 nm square elongated proteolipid tubes. Atomic force microscopy (AFM) was performed on polymer-embedded Lowicryl and London Resin-White (LR-White) unstained thin sections. AFM was used in imaging regions of the sections where TM was detected by transmission electron microscopy (EM) of corresponding stained sections. Tapping- and contact-mode AFM imaging of the unstained sections containing TM indicated a highly heterogeneous surface topography with height variations ranging from 10 to 100 nm. In tapping-mode AFM, tubular myelin was seen as hemispherical protrusions of 30-70 nm in diameter, with vertical dimensions of 5-8 nm. In contact-mode AFM and with phase imaging using a sharper (>10 nm nominal radius) probe, square open-ended tubes which resembled typical electron micrographs of such regions were observed. The cross-hatch structures observed inside the tubes using EM were not observed using AFM, although certain multilobe structures and topographic heterogeneity were detected inside some tubes. Other regions of multilamellar bodies and some regions where such bilayer lamella appear to fuse with the tubes were found in association with TM using AFM. EM of acetone-delipidated tubes in LR-White revealed rectangular tubular cores containing cross-hatched structures, presumably protein skeletons. AFM surface topography of these regions showed hollow depressions at positions at which the protein was anticipated instead of the protrusions seen in the lipid-containing sections. Gold-labeled antibody to surfactant protein A was found associated somewhat randomly within the regions containing the protein skeletons. The topography of the gold particles was observed as sharp peaks in contact-mode AFM. This study suggests a method for unambiguous detection of three-dimensional nanotubes present in low abundance in a biological macromolecular complex. Only limited detection of proteins and lipids in surfaces of embedded tubular myelin was possible. EM and AFM imaging of such unusual biological structures may suggest unique lipid-protein associations and arrangements in three dimensions.

Pulmonary surfactant protein A (SP-A) is an oligomeric glycoprotein that binds dipalmitoylphosphatidylcholine (DPPC). Interactions of rat SP-A and recombinant SP-As with pure and binary monolayers of DPPC and cholesterol were studied using a rhomboid surface balance at 37°C. A marked inflection at equilibrium surface tension (23 mN/m) in surface tension-area isotherm of a pure DPPC film was abolished by rat SP-A. The inflection was decreased and shifted to 18 mN/m with wild-type recombinant SP-A (SP-Ahyp). Both rat SP-A and SP-Ahyp decreased surface area reduction required for pure DPPC films to reach near zero surface tension from 30 to 25%. SP-Ahyp,E195Q,R197D, mutated in carbohydrate recognition domain (CRD) known to be essential for SP-A–vesicle interactions, conveyed a detrimental effect on DPPC surface activity. SP-AΔG8-P80, with deletion of collagen-like domain, had little effect. Both SP-Ahyp,C6S (Ser substitution for Cys6) and SP-Ahyp,ΔN1-A7 (N-terminal segment deletion) which appear mainly as monomers on non-reducing SDS-PAGE analysis, increased required surface area reduction for minimal surface tension. All SP-As reduced collapse surface tension of a pure cholesterol film from 27 to 23 mN/m in the presence of Ca2+. When mixed films were formed by successive spreading of DPPC/SP-A/cholesterol, rat SP-A, SP-Ahyp, or SP-AΔG8-P80 blocked the interaction of cholesterol with DPPC; SP-Ahyp,E195Q,R197D could not impede the interaction; SP-Ahyp,C6S or SP-Ahyp,ΔN1-A7 only partially blocked the interaction, and cholesterol appeared to stabilize SP-Ahyp,C6S–DPPC association. These results demonstrate the importance of CRD and N-terminal dependent oligomerization in SP-A–phospholipid associations. The findings further indicate that SP-A–cholesterol interactions differ from SP-A–DPPC interactions and may be nonspecific.—Yu, S-H., F. X. McCormack, D. R. Voelker, and F. Possmayer. Interactions of pulmonary surfactant protein SP-A with monolayers of dipalmitoylphosphatidylcholine and cholesterol: roles of SP-A domains. J. Lipid Res. 1999. 40: 920–929.

Biological membranes exist in many forms, one of which is known as tubular myelin (TM). This pulmonary surfactant membranous structure contains elongated tubes that form square lattices. To understand the interaction of surfactant protein (SP) A and various lipids commonly found in TM, we undertook a series of transmission-electron-microscopic studies using purified SP-A and lipid vesicles made in vitro and also native surfactant from bovine lung. Specimens from in vitro experiments were negatively stained with 2% uranyl acetate, whereas fixed native surfactant was delipidated, embedded, and sectioned. We found that dipalmitoylphosphatidylcholine-egg phosphatidylcholine (1:1 wt/wt) bilayers formed corrugations, folds, and predominantly 47-nm-square latticelike structures. SP-A specifically interacted with these lipid bilayers and folds. We visualized other proteolipid structures that could act as intermediates for reorganizing lipids and SP-As. Such a reorganization could lead to the localization of SP-A in the lattice corners and could explain, in part, the formation of TM-like structures in vivo.

Components of the airspace-lining material may contribute to the local regulation of immune function within the lung. We report here that recombinant rat pulmonary surfactant-associated protein D (SP-D) inhibits the lectin- and anti-CD3-stimulated proliferation of human PBMCs. Inhibition was associated with a decreased production of IL-2, and the addition of human rIL-2 blocked the inhibitory action of SP-D. These effects were not inhibited by maltose, indicating that the inhibitory activity was not dependent upon the lectin activity of SP-D. Studies employing mutant SP-D lacking N-linked sugars or defective in multimerization further indicated that inhibition was not dependent upon cellular interactions with the N-linked oligosaccharide on SP-D or the oligomerization of trimeric SP-D subunits. Although a peptide containing an inverted DGR showed similar IL-2-dependent effects on anti-CD3-stimulated proliferation, deletion of the conserved DGRDGR sequence near the amino-terminal end of the collagen domain did not decrease the suppressive activity of SP-D. We hypothesize that SP-D can dampen lymphocyte responses to exogenous stimuli and protect the lung against collateral immune-mediated damage.

We have found by transmission electron microscopy that bovine surfactant protein A (SP-A) formed extended fibers in the presence of calcium. On phosphatidylcholine or especially dipalmitoylphosphatidylcholine monolayers, SP-A at roughly 0.005 mg/ml formed large numbers of fibers and elaborate fibrous networks. This observation suggested that the weak protein:protein interactions amongst free SP-As could be stabilized by phospholipids. In the presence of glycolipid GM1-ganglioside, SP-A's globular headgroup regions appeared enlarged and only small non-fibrous clusters were observed.