Effect of chain unsaturation on bilayer response to pressure.

Using wide-line deuterium NMR, the effects of pressure on saturated-chain orientational order and gel-to-liquid-crystal phase transition temperature were observed in bilayers of 16:0-18:1 PC-d31 (POPC-d31) and 16:0-18:2 PC-d31 (PLPC-d31). Spectra were recorded for a range of pressures at selected temperatures and for a range of temperatures at selected pressures up to 193 MPa. For 16:0-18:1 PC-d31, the main transition temperature increased by approximately 0.18 K/MPa, a rate that is similar to what is found for bilayers of disaturated PC's. For 16:0-18:2 PC-d31, the increase in transition temperature with pressure was slightly smaller at approximately 0.13 K/MPa. To investigate the isothermal response of chain orientational order to pressure, spectra for each lipid were obtained for three pressures (ambient, 55 MPa, and 110 MPa) near room temperature (approximately 25 degrees C) and for three pressures (ambient, 110 MPa, and 193 MPa) at higher temperature (approximately 40 degrees C). These temperatures were chosen such that the difference between the higher observation temperature and the main transition of 16:0-18:1 PC-d31 would be similar to the difference between the lower observation temperature and the main transition of 16:0-18:2 PC-d31. Application of a given pressure was found to raise the orientational order for all methylene groups on the saturated chain of a particular lipid by roughly similar amounts. For comparable pressure differences, the pressure-induced ordering of the 16:0-18:1 PC-d31 saturated chain at approximately 40 degrees C was greater than that of the corresponding chain in 16:0-18:2 PC- d31 at approximately 25 degrees C. These observations suggest that increasing levels of chain unsaturation may reduce the sensitivity of bilayer order to variations in pressure at corresponding temperatures relative to their ambient pressure transitions.

Thermal stability and DPPC/Ca2+ interactions of pulmonary surfactant SP-A from bulk-phase and monolayer IR spectroscopy.

Surfactant protein A (SP-A), the most abundant pulmonary surfactant protein, is implicated in multiple biological functions including surfactant homeostasis, biophysical activity, and host defense. SP-A forms ternary complexes with lipids and Ca2+ which are important for protein function. The current study uses infrared (IR) transmission spectroscopy to investigate the bulk-phase interaction between SP-A, 1,2-dipalmitoylphosphatidylcholine (DPPC), and Ca2+ ions along with IR reflection-absorption spectroscopy (IRRAS) to examine protein secondary structure and lipid orientational order in monolayer films in situ at the air/water interface. The amide I contour of SP-A reveals two features at 1653 and 1636 cm(-1) arising from the collagen-like domain and a broad feature at 1645 cm(-1) suggested to arise from the carbohydrate recognition domain (CRD). SP-A secondary structure is unchanged in lipid monolayers. Thermal denaturation of SP-A in the presence of either DPPC or Ca2+ ion reveals a sequence of events involving the initial melting of the collagen-like region, followed by formation of intermolecular extended forms. Interestingly, these spectral changes were inhibited in the ternary system, showing that the combined presence of both DPPC and Ca2+ confers a remarkable thermal stability upon SP-A. The ternary interaction was revealed by the enhanced intensity of the asymmetric carboxylate stretching vibration. The IRRAS measurements indicated that incorporation of SP-A into preformed DPPC monolayers at a surface pressure of 10 mN/m induced a decrease in the average acyl chain tilt angle from 35 degrees to 28 degrees. In contrast, little change in chain tilt was observed at surface pressures of 25 or 40 mN/m. These results are consistent with and extend the fluorescence microscopy studies of Keough and co-workers [Ruano, M. L. F., et al. (1998) Biophys. J. 74, 1101-1109] in which SP-A was suggested to accumulate at the liquid-expanded/liquid-condensed boundary. Overall these experiments reveal the remarkable stability of SP-A in diverse, biologically relevant environments.

Inactivation of pulmonary surfactant and the treatment of acute lung injuries.

Inactivation of pulmonary surfactant may be important in acute lung injury and acute respiratory distress syndrome. Treatment of surfactant dysfunction by instilling exogenous surfactants may improve gas exchange and pulmonary mechanics. Surfactants used for treatment vary in their attributes and effects, so when various surfactants are considered for therapy, resistance to inactivation is an important consideration. Animal models of acute lung injury exist in which the relative merits of surfactants can be compared. We hypothesize that the surfactants most resistant to inactivation in vitro will be the ones that are most effective in treatment of animal models of acute lung injury. Surfactants with higher concentrations of surfactant proteins specificallly A, B, and C) are more resistant to inactivation. Nonionic polymers mimic surfactant proteins in preventing surfactant inactivation under some conditions. Adding nonionic polymers to surfactant containing minimal amounts of SP-B and SP-C markedly improves lung function of animals with lung injury. Making surfactants more "inactivation-proof" may improve surfactant therapy of acute lung injuries.

Intrinsic structural differences in the N-terminal segment of pulmonary surfactant protein SP-C from different species.

Predictive studies suggest that the known sequences of the N-terminal segment of surfactant protein SP-C from animal species have an intrinsic tendency to form beta-turns, but there are important differences on the probable location of these motifs in different SP-C species. Our hypothesis is that intrinsic structural determinants of the sequence of the N-terminal region of SP-C could define conformation, acylation and perhaps surface properties of the mature protein. To test this hypothesis we have synthesized peptides corresponding to the 13-residue N-terminal sequence of porcine and canine SP-C, and studied their structural behaviour in solution and in phospholipid bilayers and monolayers. In these peptides, leucine at position 1 of both sequences has been replaced by tryptophan in order to allow their study by fluorescence spectroscopy. Far-u.v. circular dichroism spectra of the peptides in aqueous and organic solutions and in phospholipid micelles or vesicles are consistent with predicted conformational differences between the porcine and the canine sequences. Both families of peptides showed changes in their fluorescence emission spectra in the presence of phospholipids that were consistent with spontaneous lipid/peptide interactions. Both canine and porcine peptides were able to form monolayers at air-liquid interfaces, the canine peptides occupying lower area/molecule and being compressible to higher pressures than the porcine sequences. The peptides also shifted the isotherms and perturbed the packing of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) monolayers, the effects being always higher in anionic than in zwitterionic lipids, and also substantially higher in films containing canine peptide in comparison to porcine peptide. Acylation of cysteines at the N-terminal end of SP-C may modulate these intrinsic conformational features and the changes induced could be important for the development of its surface activity.

Putting the "affirm" into affirmative action: preferential selection and academic performance.

Two studies explored the relation between academic performance and preferential selection. In Study 1, female participants were led to believe that they had been selected to be leaders in a team problem-solving task because of their gender, because of their gender and ability, or at random. Results showed that women who believed they had been selected because of their gender performed significantly worse on a subsequent problem-solving test than women who believed they had been selected at random and women who believed they were selected because of both their gender and their ability. In Study 2, students' suspicion of having benefited from race-based preferences in college admissions was negatively related to their grade point average (GPA). Furthermore, this suspicion partially mediated the GPA gap between academically stigmatized (Black and Latino) and nonstigmatized (Caucasian and Asian) students.

Pulmonary surfactant protein A interacts with gel-like regions in monolayers of pulmonary surfactant lipid extract.

Epifluorescence microscopy was used to investigate the interaction of pulmonary surfactant protein A (SP-A) with spread monolayers of porcine surfactant lipid extract (PSLE) containing 1 mol % fluorescent probe (NBD-PC) spread on a saline subphase (145 mM NaCl, 5 mM Tris-HCl, pH 6.9) containing 0, 0.13, or 0.16 microg/ml SP-A and 0, 1.64, or 5 mM CaCl(2). In the absence of SP-A, no differences were noted in PSLE monolayers in the absence or presence of Ca(2+). Circular probe-excluded (dark) domains were observed against a fluorescent background at low surface pressures (pi approximately 5 mN/m) and the domains grew in size with increasing pi. Above 25 mN/m, the domain size decreased with increasing pi. The amount of observable dark phase was maximal at 18% of the total film area at pi approximately 25 mN/m, then decreased to approximately 3% at pi approximately 40 mN/m. The addition of 0.16 microg/ml SP-A with 0 or 1.64 mM Ca(2+) in the subphase caused an aggregation of dark domains into a loose network, and the total amount of dark phase was increased to approximately 25% between pi of 10-28 mN/m. Monolayer features in the presence of 5 mM Ca(2+) and SP-A were not substantially different from those spread in the absence of SP-A, likely due to a self-association and aggregation of SP-A in the presence of higher concentrations of Ca(2+). PSLE films were spread on a subphase containing 0.16 microg/ml SP-A with covalently bound Texas Red (TR-SP-A). In the absence of Ca(2+), TR-SP-A associated with the reorganized dark phase (as seen with the lipid probe). The presence of 5 mM Ca(2+) resulted in an appearance of TR-SP-A in the fluid phase and of aggregates at the fluid/gel phase boundaries of the monolayers. This study suggests that SP-A associates with PSLE monolayers, particularly with condensed or solid phase lipid, and results in some reorganization of rigid phase lipid in surfactant monolayers.

Differential effects of surfactant protein A on regional organization of phospholipid monolayers containing surfactant protein B or C.

Epifluorescence microscopy combined with a surface balance was used to study monolayers of dipalmitoylphosphatidylcholine (DPPC)/egg phosphatidylglycerol (PG) (8:2, mol/mol) plus 17 wt % SP-B or SP-C spread on subphases containing SP-A in the presence or absence of 5 mM Ca(2+). Independently of the presence of Ca(2+) in the subphase, SP-A at a bulk concentration of 0.68 microg/ml adsorbed into the spread monolayers and caused an increase in the molecular areas in the films. Films of DPPC/PG formed on SP-A solutions showed a pressure-dependent coexistence of liquid-condensed (LC) and liquid-expanded (LE) phases. Apart from these surface phases, a probe-excluding phase, likely enriched in SP-A, was seen in the films between 7 mN/m < or = pi < or = 20 mN/m. In monolayers of SP-B/(DPPC/PG) spread on SP-A, regardless of the presence of calcium ions, large clusters of a probe-excluding phase, different from probe-excluding lipid LC phase, appeared and segregated from the LE phase at near-zero surface pressures and coexisted with the conventional LE and LC phases up to approximately 35 mN/m. Varying the levels of either SP-A or SP-B in films of SP-B/SP-A/(DPPC/PG) revealed that the formation of the probe-excluding clusters distinctive for the quaternary films was influenced by the two proteins. Concanavalin A in the subphase could not replace SP-A in its ability to modulate the textures of films of SP-B/(DPPC/PG). In films of SP-C/SP-A/(DPPC/PG), in the absence of calcium, regions consisting of a probe-excluding phase, likely enriched in SP-A, were detected at surface pressures between 2 mN/m and 20 mN/m in addition to the lipid LE and LC phases. Ca(2+) in the subphase appeared to disperse this phase into tiny probe-excluding particles, likely comprising Ca(2+)-aggregated SP-A. Despite their strikingly different morphologies, the films of DPPC/PG that contained combinations of SP-B/SP-A or SP-C/SP-A displayed similar distributions of LC and LE phases with LC regions occupying a maximum of 20% of the total monolayer area. Combining SP-A and SP-B reorganized the morphology of monolayers composed of DPPC and PG in a Ca(2+)-independent manner that led to the formation of a separate potentially protein-rich phase in the films.

Microstructure and dynamic surface properties of surfactant protein SP-B/dipalmitoylphosphatidylcholine interfacial films spread from lipid-protein bilayers.

Suspensions of dipalmitoylphosphatidylcholine (DPPC) bilayers containing 5, 10 or 20% (w/w) surfactant protein SP-B have been reconstituted and spread at air-liquid interfaces. Compression isotherms of DPPC/SP-B monolayers spread from these preparations were qualitatively comparable to the isotherms of the corresponding DPPC/SP-B monolayers spread from solvents. SP-B was squeezed-out at higher pressures from vesicle-spread films than from solvent-spread monolayers. SP-B caused a marked decrease on the rate of relaxation of DPPC collapse phases to equilibrium pressures in all the lipid/protein films assayed. This stabilizing effect was higher in vesicle-spread than in solvent-spread monolayers. Inclusion in the films of traces of the fluorescent probe NBD-PC (1 mol%) and use of a fluorescent derivative of SP-B labeled with a rhodamine derivative, Texas Red, allowed for direct observation of protein and lipid domains at the interface by epifluorescence microscopy. Upon compression, SP-B altered the packing of phospholipids in the bilayer-spread films, observed as a SP-B-induced reduction of the area of liquid-condensed domains, in a way similar to its effect in solvent-spread monolayers. SP-B was not associated with condensed regions of the films. Fluorescence images from vesicle-spread films showed discrete fluorescent aggregates that could be consistent with the existence of lipid-protein vesicles in close association with the monolayer. Both the retention of SP-B at higher surface pressures and the greater stability of collapse phases of DPPC/SP-B films prepared by spreading from liposomes in comparison to those spread from solvents can be interpreted as a consequence of formation of complex bilayer-monolayer interacting systems.

Adsorption of pulmonary surfactant protein SP-A to monolayers of phospholipids containing hydrophobic surfactant protein SP-B or SP-C: potential differential role for tertiary interaction of lipids, hydrophobic proteins, and SP-A.

Surface balance techniques were used to study the interactions of surfactant protein SP-A with monolayers of surfactant components preformed at the air-water interface. SP-A adsorption into the monolayers was followed by monitoring the increase in the surface pressure Deltapi after injection of SP-A beneath the films. Monolayers of dipalmitoylphosphatidylcholine (DPPC):egg phosphatidylglycerol (PG) (8:2, mol/mol) spread at initial surface pressure pi(i) = 5 mN/m did not promote the adsorption of SP-A at a subphase concentration of 0.68 microg/mL as compared to its adsorption to the monolayer-free surface. Surfactant proteins, SP-B or SP-C, when present in the films of DPPC:PG spread at pi(i) = 5 mN/m, enhanced the incorporation of SP-A in the monolayers to a similar extent; the Deltapi values being dependent on the levels of SP-B or SP-C, 3-17 wt %, in the lipid films. Calcium in the subphase did not affect the intrinsic surface activity of SP-A but reduced the Deltapi values produced by the adsorption of the protein to all the preformed films independently of their compositions and charges. The divalent ions likely modified the interaction of SP-A with the monolayers through their effects on the conformation, self-association, and charge state of SP-A. Values of Deltapi produced by adsorption of SP-A to the films of DPPC:PG with or without SP-B or SP-C were a function of the initial surface pressure of the films, pi(i). In the range of pressures 5

Elevated levels of urine angiostatin and plasminogen/plasmin in cancer patients.

Previously, a specific angiogenesis inhibitor, angiostatin, discovered in urine and serum of tumor-bearing mice, was reported to potently block tumor growth and metastasis in animal models. Detection of angiostatin and its precursor proteins in urine from cancer patients has not been reported. Now, we report the development of an antibody-based analysis system that allows us to detect angiostatin and plasminogen/plasmin (Pgn/plasmin) in the urine of cancer patients. The detection system is a combination of a novel lysine-ELISA assay and Western immunoblot analysis using a specific antibody to human angiostatin and Pgn/plasmin. High levels of Pgn/plasmin were detected in the urine from various cancer patients, whereas healthy individuals showed relatively low levels of urine Pgn/plasmin. Of interest, angiostatin is detectable in urine samples of patients with various cancers, including acute lymphoblastic leukemia, suggesting that angiogenesis may play an important role in the development and progression of leukemia. Our data for the first time show that angiostatin and Pgn/plasmin are present at relatively high levels in the urine of human cancer patients. Detection of urine angiostatin in cancer patients helps us not only to understand the role of this angiogenesis inhibitor in cancer development and progression but also allows us to develop tools of cancer diagnosis and prognosis. Thus angiostatin has both therapeutic and diagnostic implications in cancer disease.

Interesterification (acidolysis) of butterfat with conjugated linoleic acid in a batch reactor.

Six commercial lipases, in free or immobilized form, were tested for their ability to catalyze acyl exchange between conjugated linoleic acid and anhydrous butterfat under solvent-free conditions. Immobilized Candida antarctica lipase exhibited the best activity. Experiments were conducted for this lipase in butterfat to conjugated linoleic acid ratios of 10:1 (vol/vol), temperatures from 30 to 70 degrees C, enzyme concentrations of 50 to 200 mg/g of reaction mixture, and water contents of 0.15 to 2% (wt/wt). At the maximum enzyme concentration used, equilibrium was reached within the first 24 h of reaction. The optimum temperature was 50 degrees C. The triacylglycerol profile of the product butterfat reflected changes in the relative proportions of fatty acid residues as the reaction proceeded, with increases in those triacylglycerols containing 46 to 54 carbon atoms and concomitant decreases in those triacylglycerols containing 34 to 42 carbon atoms.

Insulin-induced vascular endothelial growth factor expression in retina.

PURPOSE: Clinical studies have demonstrated that intensive insulin therapy causes a transient worsening of retinopathy. The mechanisms underlying the initial insulin-induced deterioration of retinal status in patients with diabetes remain unknown. Vascular endothelial growth factor (VEGF) is known to be operative in the pathogenesis of diabetic retinopathy. The current study was conducted to characterize the effect of insulin on retinal VEGF gene expression in vitro and in vivo. METHODS: The effect of insulin on VEGF expression in vivo was examined by in situ hybridization studies of rat retinal VEGF transcripts. To examine the mechanisms by which insulin regulates VEGF expression, human retinal pigment epithelial (RPE) cells were exposed to insulin, and VEGF mRNA levels were quantified with RNase protection assays (RPAs). Conditioned media from insulin-treated RPE cells were assayed for VEGF protein and capillary endothelial cell proliferation. The capacity of insulin to stimulate the VEGF promoter linked to a luciferase reporter gene was characterized in transient transfection assays. RESULTS: Insulin increased VEGF mRNA levels in the ganglion, inner nuclear, and RPE cell layers. In vitro, insulin increased VEGF mRNA levels in human RPE cells and enhanced VEGF promoter activity without affecting transcript stability. Insulin treatment also increased VEGF protein levels in conditioned RPE cell media in a dose-dependent manner with a median effective concentration of 5 nM. The insulin-conditioned RPE cell media stimulated capillary endothelial cell proliferation, an effect that was completely blocked by anti-VEGF neutralizing antibody. CONCLUSIONS: Insulin increases VEGF mRNA and secreted protein levels in RPE cells through enhanced transcription of the VEGF gene. Intensive insulin therapy may cause a transient worsening of retinopathy in patients with diabetes through increased retinal VEGF gene expression.

Interactions of pulmonary surfactant protein A with phospholipid monolayers change with pH.

The interaction of pulmonary surfactant protein A (SP-A) labeled with Texas Red (TR-SP-A) with monolayers containing zwitterionic and acidic phospholipids has been studied at pH 7.4 and 4.5 using epifluorescence microscopy. At pH 7.4, TR-SP-A expanded the pi-A isotherms of film of dipalmitoylphosphatidylcholine (DPPC). It interacted at high concentration at the edges of condensed-expanded phase domains, and distributed evenly at lower concentration into the fluid phase with increasing pressure. At pH 4.5, TR-SP-A expanded DPPC monolayers to a slightly lower extent than at pH 7.4. It interacted primarily at the phase boundaries but it did not distribute into the fluid phase with increasing pressure. Films of DPPC/dipalmitoylphosphatidylglycerol (DPPG) 7:3 mol/mol were somewhat expanded by TR-SP-A at pH 7.4. The protein was distributed in aggregates only at the condensed-expanded phase boundaries at all surface pressures. At pH 4.5 TR-SP-A caused no expansion of the pi-A isotherm of DPPC/DPPG, but its fluorescence was relatively homogeneously distributed throughout the expanded phase at all pressures studied. These observations can be explained by a combination of factors including the preference for SP-A aggregates to enter monolayers at packing dislocations and their disaggregation in the presence of lipid under increasing pressure, together with the influence of pH on the aggregation state of SP-A and the interaction of SP-A with zwitterionic and acidic lipid.

Elevated serum vascular endothelial growth factor in children and young adults with Crohn's disease.

Vascular endothelial growth factor (VEGF) is a cytokine released by fibroblasts, epithelial cells, and leukocytes that potentiates vascular permeability and growth of new capillaries. Because of these multiple effects, VEGF has been postulated to play a role in the pathogenesis of autoimmune disease, as well as in wound healing. We hypothesized that VEGF was potentially important in mediating the vascular permeability and angiogenesis seen in Crohn's disease, and therefore that VEGF would be increased in the serum of children with Crohn's disease. Serum was obtained from 73 children and young adults with Crohn's disease, 47 with ulcerative colitis, and 29 controls. VEGF levels were measured by enzyme-linked immunosorbent assay. Mean VEGF levels were significantly higher in patients with Crohn's disease (436.4 +/- 37.2 pg/ml) than in ulcerative colitis (306 +/- 41.1 pg/ml) or control (167.8 +/- 29.6 pg/ml) patients. Serum VEGF also correlated significantly with disease activity, being elevated in patients with moderate/severe Crohn's disease and ulcerative colitis. We conclude that serum VEGF is released by inflamed tissues in children with Crohn's disease. This multifunctional cytokine could promote inflammation by increasing vascular permeability or promote wound healing by mediating capillary growth.

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.

Zinc-binding of endostatin is essential for its antiangiogenic activity.

Endostatin is a potent angiogenesis inhibitor in vitro and in vivo. We used the yeast Pichia pastoris to express and purify soluble endostatin. It was discovered that metal chelating agents can induce N-terminal degradation of endostatin. We theorized that a metal was removed from endostatin which changed the conformation and allowed a contaminating protease to degrade the N-terminus. Atomic absorption and amino acid analysis of endostatin purified from Pichia pastoris and mammalian cells showed a 1:1 molar ratio of Zn2+ to protein. Ding et al. have shown that histidines 1, 3, 11, and aspartic acid 76 coordinate the Zn2+ atom (1). An H1/3A double, an H11A, and a D76A single mutant of endostatin were not able to regress Lewis lung carcinoma. We conclude that the ability of endostatin to bind Zn2+ is essential for its antiangiogenic activity.

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

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

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.