Surfactant Proteins SP-B Research Articles

Protein modeling and molecular dynamic studies of two new surfactant proteins

Surfactant proteins are of major importance for the stability and flexibility of lipid systems like the lung surfactant or the tear film. They can support the adsorption of phospholipids into a layer or specifically influence the surface tension of a lipid surface [1]. To fulfil these functions, they are described to be highly posttranslationally modified. Furthermore, immunological functions were described for some of the already known surfactant proteins SP-A, SP-B, SP-C and SP-D [2]. For that reason, they are of great interest in the investigation of diseases like the acute respiratory distress syndrome (ARDS) or the dry eye syndrome (DES). Recently sequences of two putative surfactant proteins, called SP-G and SP-H, were identified. To get insights into the function of SP-G and SP-H, protein structure models were generated, including predictions for posttranslational modifications. These models successfully guided the design of antibodies for the localization of SP-G and SP-H in different human tissues, including lung and ocular system. Both protein models were transferred into a lung surfactant model system consisting of a dipalmitoyl¬phosphatidyl¬choline monolayer. MD simulations over 50 ns were performed with GROMACS starting from different orientations of the protein models. During these simulations, it was possible to track the accumulation of the proteins to the lipid layer and observe the interactions between protein surface and lipid head groups on an atomic scale. The results show that SP-G and SP-H indeed are capable of surface-regulatory properties. Furthermore, it is evident that the interaction between proteins and lipid systems is highly dependent on the present posttranslational modifications. In combination with further experimental work, these simulations can help to determine the functions of SP-G and SP-H in vivo and may support the development of new therapies for ARDS and DES.

Effect of Cholesterol and Palmitoylation on the Structure, Orientation and Lipid-Protein Interactions of Pulmonary Surfactant Protein SP-C

Pulmonary surfactant is a specialized lipid-protein complex, which essential function is to stabilize the gas exchange surface of the respiratory epithelium against physical forces tending to its collapse. Hydrophobic surfactant proteins SP-B and SP-C are critical to promote very rapid adsorption of phospholipids into the interface and to stabilize the surface active film along the compression-expansion respiratory cycles. The composition of pulmonary surfactant varies slightly between species but in general it consists of 90% lipids by weight and 8-10% proteins, including the specific surfactant proteins SP-A, SP-B, SP-C and SP-D. The main feature of protein SP-C (35 amino acids, 3.7kDa) is its extreme hidrophobicity. Structurally it consists of a transmembrane α-helical stretch and a short N-terminal segment. The positions 5 and 6 of SP-C sequence are in most species occupied by two cysteines that are palmitoylated. It has been proposed that SP-C interacts with cholesterol-rich phases of surfactant membranes and films, contributing to modulate the interfacial behavior of surfactant in the alveoli but evidence remains uncertain.Our aim in this work was to elucidate how palmitoylation influences the interaction of SP-C with lipids in different surfactant-mimetic systems, in the presence or absence of cholesterol. To this end, we have compared the secondary structure, tilting and lipid-protein interactions of native palmitoylated SP-C purified from porcine lungs with the behavior of non-palmitoylated recombinant versions of SP-C produced in bacteria, by means of ATR-FTIR. This technique is sensitive to the orientation, structure and interactions of SP-C in membranes, which can be correlated with the contribution of SP-C to stabilize compressed cholesterol-containing surfactant films.

Hydrophobic Pulmonary Surfactant Proteins SP-B and SP-C Induce Pore Formation in Planar Lipid Membranes: Evidence for Proteolipid Pores

Pulmonary surfactant is a complex mixture of lipids and specific surfactant proteins, including the hydrophobic proteins SP-B and SP-C, in charge of stabilizing the respiratory surface of mammalian lungs. The combined action of both proteins is responsible for the proper structure and dynamics of membrane arrays in the pulmonary surfactant network that covers the respiratory surface. In this study, we explore the possibility that proteins SP-B and SP-C induce the permeabilization of phospholipid membranes via pore formation. To this end, electrophysiological measurements have been carried out in planar lipid membranes prepared with different lipid/protein mixtures. Our main result is that channel-like structures are detected in the presence of SP-B, SP-C, or the native mixture of both proteins. Current traces show a high variety of conductance states (from pS to nS) that are dependent both on the lipid composition and the applied potential. We also show that the type of host lipid crucially determines the ionic selectivity of the observed pores: the anionic selectivity observed in zwitterionic membranes is inverted to cationic selectivity in the presence of negatively charged lipids. All those results suggest that SP-B and SP-C proteins promote the formation of proteolipid channels in which lipid molecules are functionally involved. We propose that proteolipidic membrane-permeabilizing structures may have an important role to tune ionic and lipidic flows through the pulmonary surfactant membrane network at the alveolar surfaces.

Membrane-Perturbing Activities of KL4-Related Surfactant Peptides

KL4 is a 21-residue peptide proposed as a potential substitute of pulmonary surfactant protein SP-B in synthetic surfactants, intended for the treatment of respiratory pathologies. The peptide, composed by leucines interrupted by lysine every four residues, was synthesized to simulate C-terminal amphipathic helical segments of SP-B. Once incorporated into lipid-protein complexes, KL4 promotes formation of interfacial films that produce and maintain surface tensions below 5 mN/m during compression-expansion cycling. Although KL4 was designed as an amphipathic helix at the membrane surface, the data on orientation and interactions of the peptide in membranes are contradictory. In the present work the surface activity and interaction with membranes of KL4 was compared with the behavior of two other peptides, KL2A2 and KL4PQ, to gain information about the properties defining the potential of KL4 as surfactant additive.The surface activity of the different peptides in lipid suspensions mimicking surfactant composition (DPPC/POPC/POPG 50:25:15 w/w/w), was analyzed in a captive bubble surfactometer (CBS). Only lipid suspensions containing KL4 adsorbed to the air-liquid interface with kinetics comparable to those containing SP-B, with KL4PQ and specially KL2A2 showing less activity. The interaction of the three peptides with membranes was analyzed by DSC, Laurdan fluorescence and ATR-FTIR. Perturbations induced by peptides on the structure and permeability of giant unilamellar vesicles (GUVs) were also analyzed and compared with the behavior of natural SP-B.The results indicate that the extent of penetration of the peptides in bilayers and their ability to perturb membrane structure correlate with their different interfacial activity.

2P215 肺サーファクタントタンパク質SP-Bによるリン脂質膜の構造変化(13B.生体膜・人工膜:ダイナミクス,ポスター,日本生物物理学会年会第51回(2013年度))

2P215 肺サーファクタントタンパク質SP-Bによるリン脂質膜の構造変化(13B.生体膜・人工膜:ダイナミクス,ポスター,日本生物物理学会年会第51回(2013年度))

Structure–function correlations of pulmonary surfactant protein SP-B and the saposin-like family of proteins

Pulmonary surfactant is a lipid-protein complex secreted by the respiratory epithelium of mammalian lungs, which plays an essential role in stabilising the alveolar surface and so reducing the work of breathing. The surfactant protein SP-B is part of this complex, and is strictly required for the assembly of pulmonary surfactant and its extracellular development to form stable surface-active films at the air-liquid alveolar interface, making the lack of SP-B incompatible with life. In spite of its physiological importance, a model for the structure and the mechanism of action of SP-B is still needed. The sequence of SP-B is homologous to that of the saposin-like family of proteins, which are membrane-interacting polypeptides with apparently diverging activities, from the co-lipase action of saposins to facilitate the degradation of sphingolipids in the lysosomes to the cytolytic actions of some antibiotic proteins, such as NK-lysin and granulysin or the amoebapore of Entamoeba histolytica. Numerous studies on the interactions of these proteins with membranes have still not explained how a similar sequence and a potentially related fold can sustain such apparently different activities. In the present review, we have summarised the most relevant features of the structure, lipid-protein and protein-protein interactions of SP-B and the saposin-like family of proteins, as a basis to propose an integrated model and a common mechanistic framework of the apparent functional versatility of the saposin fold.

Topology and lipid selectivity of pulmonary surfactant protein SP-B in membranes: Answers from fluorescence

Contradictory results have been reported with respect to the depth of penetration and the orientation of pulmonary surfactant protein SP-B in phospholipid membranes and its relative selectivity to interact with anionic over zwitterionic phospholipid species. In the present study we have re-evaluated lipid–protein interactions of SP-B by analysing Förster resonance energy transfer (FRET) efficiencies, obtained from time-resolved measurements, from the single tryptophan in SP-B to different fluorescently labelled phospholipids in matrix bilayers made of either pure phosphatidylcholine (POPC) or the full lipid extract obtained from purified surfactant. In the background of POPC membranes SP-B exhibits a certain level of selectivity for anionic fluorescent phospholipids over the corresponding zwitterionic analogues, but apparently no preference for phosphatidylglycerol over other anionic species such as phosphatidylserine. No selectivity was detected in membranes made of full surfactant lipids, indicating that specific lipid–protein binding sites could already be occupied by endogenous anionic phospholipids. Furthermore, we have analysed the fit of two different models of how SP-B could be orientated with respect to phospholipid membrane surfaces to the FRET data. The FRET results are consistent with topology models in which the protein has a superficial orientation, with no regions of exclusion by the protein to the access of phospholipids, both in POPC membranes and in membranes made of the whole surfactant lipid fraction. This discards a deep penetration of the protein into the core of bilayers and suggests that most hydrophobic segments of SP-B could participate in protein–protein instead of lipid–protein interactions.

Structural and Functional Characterization of Native Complexes of Pulmonary Surfactant Proteins Purified with Detergents

Pulmonary surfactant is a specialized lipid-protein complex, which essential function is to stabilize the gas exchange surface of the respiratory epithelium against physical forces tending to its collapse. Hydrophobic surfactant proteins SP-B and SP-C are critical to promote very rapid adsorption of phospholipids into the interface and to stabilize the surface active film along the compression-expansion respiratory cycles. Mature SP-B is a homodimeric protein of approximately 18kDa (two 79 amino-acid polypeptides), while SP-C is an apparently monomeric lipopeptide of 35 residues (3.7kDa).To date, the classical method to extract and purify these proteins from surfactant membranes involves the use of organic solvents. This method can disrupt native supramolecular protein complexes. We have optimized the purification of surfactant membrane proteins upon solubilization of surfactant membranes by the zwitterionic detergent CHAPS. Different fractions have been purified from the solubilised material by gradient centrifugation and exclusion-size chromatography, showing the presence of two types of protein complexes in terms of size and density, heterogeneously composed of different oligomeric states of surfactant proteins.An ionic-exchange chromatography of solubilized surfactant yielded a fraction of cationic protein complexes consisting mainly of surfactant protein SP-B. Their structural characterization by circular dichroism and fluorescence emission revealed a structure entirely analogous to that of SP-B purified in organic solvents. However, detergent-purified SP-B showed a much more oligomeric structure, as analyzed by blue-native electrophoresis, analytical centrifugation or electron microscopy. This SP-B complex has been reconstituted into surfactant phospholipids and the resulting lipid/protein complexes showed in the captive bubble surfactometer (CBS) a similar surface active behaviour than provided by SP-B purified using the classic chloroform/methanol-based method.

Effects of Hidrophobic Surfactant Proteins SP-B and SP-C on the Mechanical Properties and Structural Stability of Phospholipid Bilayers

The respiratory surface is stabilized by pulmonary surfactant, a complex mixture of lipids and proteins, whose main function is reducing surface tension at the alveolar air-liquid interface in order to facilitate the work of breathing. It is composed by around 90% lipids and 8-10% specific proteins, including the hydrophobic polypeptides SP-B and SP-C. A combined action of both proteins is essential for a proper organization of functional membrane arrays in surfactant complexes.SP-B and SP-C have dramatical effects on membrane structure and dynamics. In the present study we compare some structural, mechanical and dynamical properties of model POPC vesicles in the absence and presence of them, either in their physiological combined proportion or each protein by itself. Structural effects caused by hydrophobic surfactant proteins were noticed both by optical microscopy of giant proteolipid vesicles and by electron microscopy of 100 nm diameter extruded vesicles. Also, impermeable POPC membranes became permeable when supplemented with any of these proteins. Significant differences were noticed between the effect of SP-B and SP-C on giant vesicles: suspensions containing only SP-B were stable but those containing only SP-C were quite dynamic, undergoing frequent fluctuations, reorganizations and ruptures as observed under the microscope. In order to investigate the physical explanation of these phenomena, mechanical studies have been carried out with giant phospholipid and proteolipid vesicles.The differences found between the effect of each protein separately and their physiological mixture support the concept that SP-B and SP-C mutually modulate their membrane-perturbing properties, which may be crucial for the structure, arrangement and dynamics of pulmonary surfactant membranes.

Activin-A Overexpression in the Murine Lung Causes Pathology That Simulates Acute Respiratory Distress Syndrome

Activin-A is up-regulated in various respiratory disorders. However, its precise role in pulmonary pathophysiology has not been adequately substantiated in vivo. To investigate in vivo the consequences of dysregulated Activin-A expression in the lung and identify key Activin-A-induced processes that contribute to respiratory pathology. Activin-A was ectopically expressed in murine lung, and functional, structural, and molecular alterations were extensively analyzed. The validity of Activin-A as a therapeutic target was demonstrated in animals overexpressing Activin-A or treated with intratracheal instillation of LPS. Relevancy to human pathology was substantiated by demonstrating high Activin-A levels in bronchoalveolar lavage (BAL) samples from patients with acute respiratory distress syndrome (ARDS). Overexpression of Activin-A in mouse airways caused pulmonary pathology reminiscent of acute lung injury (ALI)/ARDS. Activin-A triggered a lasting inflammatory response characterized by acute alveolar cell death and hyaline membrane formation, sustained up-regulation of high-mobility group box 1, development of systemic hypercoagulant state, reduction of surfactant proteins SpC, SpB, and SpA, decline of lung compliance, transient fibrosis, and eventually emphysema. Therapeutic neutralization of Activin-A attenuated the ALI/ARDS-like pathology induced either by ectopic expression of Activin-A or by intratracheal instillation of LPS. In line with the similarity of the Activin-A-induced phenotype to human ARDS, selective up-regulation of Activin-A was found in BAL of patients with ARDS. Our studies demonstrate for the first time in vivo the pathogenic consequences of deregulated Activin-A expression in the lung, document novel aspects of Activin-A biology that provide mechanistic explanation for the observed phenotype, link Activin-A to ALI/ARDS pathophysiology, and provide the rationale for therapeutic targeting of Activin-A in these disorders.

Influence of Hydrophobic Alkylated Gold Nanoparticles on the Phase Behavior of Monolayers of DPPC and Clinical Lung Surfactant

The effect of hydrophobic alkylated gold nanoparticles (Au NPs) on the phase behavior and structure of Langmuir monolayers of dipalmitoylphosphatidylcholine (DPPC) and Survanta, a naturally derived commercial pulmonary surfactant that contains DPPC as the main lipid component and hydrophobic surfactant proteins SP-B and SP-C, has been investigated in connection with the potential implication of inorganic NPs in pulmonary surfactant dysfunction. Hexadecanethiolate-capped Au NPs (C(16)SAu NPs) with an average core diameter of 2 nm have been incorporated into DPPC monolayers in concentrations ranging from 0.1 to 0.5 mol %. Concentrations of up to 0.2 mol % in DPPC and 16 wt % in Survanta do not affect the monolayer phase behavior at 20 °C, as evidenced by surface pressure-area (π-A) and ellipsometric isotherms. The monolayer structure at the air/water interface was imaged as a function of the surface pressure by Brewster angle microscopy (BAM). In the liquid-expanded/liquid-condensed phase coexistence region of DPPC, the presence of 0.2 mol % C(16)SAu NPs causes the formation of many small, circular, condensed lipid domains, in contrast to the characteristic larger multilobes formed by pure lipid. Condensed domains of similar size and shape to those of DPPC with 0.2 mol % C(16)SAu NPs are formed by compressing Survanta, and these are not affected by the C(16)SAu NPs. Atomic force microscopy images of Langmuir-Schaefer-deposited films support the BAM observations and reveal, moreover, that at high surface pressures (i.e., 35 and 45 mN m(-1)) the C(16)SAu NPs form honeycomb-like aggregates around the polygonal condensed DPPC domains. In the Survanta monolayers, the C(16)SAu NPs were found to accumulate together with the proteins in the liquid-expanded phase around the circular condensed lipid domains. In conclusion, the presence of hydrophobic C(16)SAu NPs in amounts that do not influence the π-A isotherm alters the nucleation, growth, and morphology of the condensed domains in monolayers of DPPC but not of those of Survanta. Systematic investigations of the effect of the interaction of chemically defined NPs with the lipid and protein components of lung surfactant on the physicochemical properties of surfactant films are pertinent to understanding how inhaled NPs impact pulmonary function.

A combined action of pulmonary surfactant proteins SP-B and SP-C modulates permeability and dynamics of phospholipid membranes

Proteins SP-B and SP-C are essential to promote formation of surface-active films at the respiratory interface, but their mechanism of action is still under investigation. In the present study we have analysed the effect of the proteins on the accessibility of native, quasi-native and model surfactant membranes to incorporation of the fluorescent probes Nile Red (permeable) and FM 1-43 (impermeable) into membranes. We have also analysed the effect of single or combined proteins on membrane permeation using the soluble fluorescent dye calcein. The fluorescence of FM 1-43 was always higher in membranes containing SP-B and/or SP-C than in protein-depleted membranes, in contrast with Nile Red which was very similar in all of the materials tested. SP-B and SP-C promoted probe partition with markedly different kinetics. On the other hand, physiological proportions of SP-B and SP-C caused giant oligolamellar vesicles to incorporate FM 1-43 from the external medium into apparently most of the membranes instantaneously. In contrast, oligolamellar pure lipid vesicles appeared to be mainly labelled in the outermost membrane layer. Pure lipidic vesicles were impermeable to calcein, whereas it permeated through membranes containing SP-B and/or SP-C. Vesicles containing only SP-B were stable, but prone to vesicle-vesicle interactions, whereas those containing only SP-C were extremely dynamic, undergoing frequent fluctuations and ruptures. Differential structural effects of proteins on vesicles were confirmed by electron microscopy. These results suggest that SP-B and SP-C have different contributions to inter- and intra-membrane lipid dynamics, and that their combined action could provide unique effects to modulate structure and dynamics of pulmonary surfactant membranes and films.

Pulmonary surfactant proteins and polymer combinations reduce surfactant inhibition by serum

Acute respiratory distress syndrome (ARDS) is an inflammatory condition that can be associated with capillary leak of serum into alveoli causing inactivation of surfactant. Resistance to inactivation is affected by types and concentrations of surfactant proteins, lipids, and polymers. Our aim was to investigate the effects of different combinations of these three components. A simple lipid mixture (DPPC/POPG) or a more complex lipid mixture (DPPC/POPC/POPG/cholesterol) was used. Native surfactant proteins SP-B and SP-C obtained from pig lung lavage were added either singly or combined at two concentrations. Also, non-ionic polymers polyethylene glycol and dextran and the anionic polymer hyaluronan were added either singly or in pairs with hyaluronan included. Non-ionic polymers work by different mechanisms than anionic polymers, thus the purpose of placing them together in the same surfactant mixture was to evaluate if the combination would show enhanced beneficial effects. The resulting surfactant mixtures were studied in the presence or absence of serum. A modified bubble surfactometer was used to evaluate surface activities. Mixtures that included both SP-B and SP-C plus hyaluronan and either dextran or polyethylene glycol were found to be the most resistant to inhibition by serum. These mixtures, as well as some with either SP-B or SP-C with combined polymers were as or more resistant to inactivation than native surfactant. These results suggest that improved formulations of lung surfactants are possible and may be useful in reducing some types of surfactant inactivation in treating lung injuries.

Lung Surfactant Protein SP-B Promotes Formation of Bilayer Reservoirs from Monolayer and Lipid Transfer between the Interface and Subphase

We investigated the possible role of SP-B proteins in the function of lung surfactant. To this end, lipid monolayers at the air/water interface, bilayers in water, and transformations between them in the presence of SP-B were simulated. The proteins attached bilayers to monolayers, providing close proximity of the reservoirs with the interface. In the attached aggregates, SP-B mediated establishment of the lipid-lined connection similar to the hemifusion stalk. Via this connection, a lipid flow was initiated between the monolayer at the interface and the bilayer in water in a surface-tension-dependent manner. On interface expansion, the flow of lipids to the monolayer restored the surface tension to the equilibrium spreading value. SP-B induced formation of bilayer folds from the monolayer at positive surface tensions below the equilibrium. In the absence of proteins, lipid monolayers were stable at these conditions. Fold nucleation was initiated by SP-B from the liquid-expanded monolayer phase by local bending, and the proteins lined the curved perimeter of the growing fold. No effect on the liquid-condensed phase was observed. Covalently linked dimers resulted in faster kinetics for monolayer folding. The simulation results are in line with existing hypotheses on SP-B activity in lung surfactant and explain its molecular mechanism.

Interaction of the propeptide NH 2 -terminal of surfactant protein SP-B with phospholipid membranes

Interaction of the propeptide NH 2 -terminal of surfactant protein SP-B with phospholipid membranes

Effect of Hydrophobic Surfactant Proteins SP-B and SP-C on the Permeability of Phospholipid Membranes

Pulmonary surfactant is a complex mixture of lipids and proteins whose main function is to reduce surface tension at the alveolar air-liquid interface in order to avoid alveolar collapse at the end of expiration and facilitate the work of breathing. It is composed by around 90% lipids and 8-10% specific proteins, including the hydrophobic SP-B and SP-C. In this study, we have analyzed the effect of hydrophobic surfactant proteins on the permeability of phospholipid membranes by two different approaches: fluorescence microscopy of giant vesicles (GV) and electroconduction in planar lipid membranes. The effect of surfactant proteins on the permeability of GV membranes was assessed under the microscope using the fluorescent water-soluble probes FM®1-43 and calcein. Membrane-sensitive FM®1-43 only labels the external leaflet of membranes, and calcein emits green fluorescence in aqueous media. Neither can permeate through pure lipid membranes. In the presence of physiological amounts of SP-B and SP-C, giant oligolamellar POPC vesicles incorporated FM®1-43 in every single membrane when added to the external medium and were also permeable to calcein. These results suggest the existence of direct connections between aqueous compartments of GV in the presence of these proteins. On the other hand, planar lipid membranes (PLM) have been widely used to study ionic permeation through phospholipid bilayers mediated by membrane proteins. Permeability of bilayers incorporating small amounts of hydrophobic surfactant proteins has been analyzed in PLMs prepared by the dual monolayer technique. Conductance and selectivity experiments as well as noise analysis of the signals were performed. All our measurements indicate the formation of channel-like structures with defined properties in terms of ionic conductance and selectivity. Possible implications of membrane-permeabilizing structures in the biological context of the pulmonary surfactant system will be discussed.

Palmitoylation of Pulmonary Surfactant Protein SP-C Is Critical for Its Functional Cooperation with SP-B to Sustain Compression/Expansion Dynamics in Cholesterol-Containing Surfactant Films

Recent data suggest that a functional cooperation between surfactant proteins SP-B and SP-C may be required to sustain a proper compression-expansion dynamics in the presence of physiological proportions of cholesterol. SP-C is a dually palmitoylated polypeptide of 4.2 kDa, but the role of acylation in SP-C activity is not completely understood. In this work we have compared the behavior of native palmitoylated SP-C and recombinant nonpalmitoylated versions of SP-C produced in bacteria to get a detailed insight into the importance of the palmitic chains to optimize interfacial performance of cholesterol-containing surfactant films. We found that palmitoylation of SP-C is not essential for the protein to promote rapid interfacial adsorption of phospholipids to equilibrium surface tensions (∼22 mN/m), in the presence or absence of cholesterol. However, palmitoylation of SP-C is critical for cholesterol-containing films to reach surface tensions ≤1 mN/m at the highest compression rates assessed in a captive bubble surfactometer, in the presence of SP-B. Interestingly, the ability of SP-C to facilitate reinsertion of phospholipids during expansion was not impaired to the same extent in the absence of palmitoylation, suggesting the existence of palmitoylation-dependent and -independent functions of the protein. We conclude that palmitoylation is key for the functional cooperation of SP-C with SP-B that enables cholesterol-containing surfactant films to reach very low tensions under compression, which could be particularly important in the design of clinical surfactants destined to replacement therapies in pathologies such as acute respiratory distress syndrome.

Combined and Independent Action of Proteins SP-B and SP-C in the Surface Behavior and Mechanical Stability of Pulmonary Surfactant Films

The hydrophobic proteins SP-B and SP-C are essential for pulmonary surfactant function, even though they are a relatively minor component (<2% of surfactant dry mass). Despite countless studies, their specific differential action and their possible concerted role to optimize the surface properties of surfactant films have not been completely elucidated. Under conditions kept as physiologically relevant as possible, we tested the surface activity and mechanical stability of several surfactant films of varying protein composition in vitro using a captive bubble surfactometer and a novel (to our knowledge) stability test. We found that in the naturally derived surfactant lipid mixtures, surfactant protein SP-B promoted film formation and reextension to lower surface tensions than SP-C, and in particular played a vital role in sustaining film stability at the most compressed states, whereas SP-C produced no stabilization. Preparations containing both proteins together revealed a slight combined effect in enhancing film formation. These results provide a qualitative and quantitative framework for the development of future synthetic therapeutic surfactants, and illustrate the crucial need to include SP-B or an efficient SP-B analog for optimal function.

Folding of lipid monolayers containing lung surfactant proteins SP-B 1–25 and SP-C studied via coarse-grained molecular dynamics simulations

To explore the role of lung surfactant proteins SP-B and SP-C in storing and redelivering lipid from lipid monolayers during the compression and re-expansion occurring in lungs during breathing, we simulate the folding of lipid monolayers with and without these proteins. We utilize the MARTINI coarse-grained force field to simulate monolayers containing pure dipalmitoylphosphatidylcholine (DPPC) and DPPC mixed with palmitoyloleoylphosphatidylglycerol (POPG), palmitic acid (PA), and/or peptides. The peptides considered include the 25-residue N-terminal fragment of SP-B (SP-B 1 – 25), SP-C, and several SP-B 1 – 25 mutants in which charged and hydrophilic residues are replaced by hydrophobic ones, or vice-versa. We observe two folding mechanisms: folding by the amplification of undulations and folding by nucleation about a defect. The first mechanism is observed in monolayers containing either POPG or peptides, while the second mechanism is observed only with peptides present, and involves the lipid-mediated aggregation of the peptides into a defect, from which the fold can nucleate. Fold nucleation from a defect displays a dependence on the hydrophobic character of the peptides; if the number of hydrophobic residues is decreased significantly, monolayer folding does not occur. The addition of POPG or peptides to the DPPC monolayer has a fluidizing effect, which assists monolayer folding. In contrast, the addition of PA has a charge-dependent condensing affect on DPPC monolayers containing SP-C. The peptides appear to play a significant role in the folding process, and provide a larger driving force for folding than POPG. In addition to promoting fold formation, the peptides also display fusogenic behavior, which can lead to surface refining.

Surfactant Replacement Therapy in Influenza A H1N1

To the Editors: A 22-month-old boy was admitted to the Clinic for Infective Diseases, University Clinical Center, Tuzla (Bosnia and Herzegovina), because of fever, cough, loss of appetite, and vomiting. An influenza virus type A H1N1 infection quick test (QuickVue, Influenza A+B—QuickVue Influenza A+B—bioMérieux) had a positive result, and 48 hours later infection with Influenza type A H1N1 was confirmed by polymerase chain reaction (multiplex real-time PCR, firma Roche Diagnostics, Laval, Quebec, Canada) at the University Clinical Center, Sarajevo. A chest radiograph showed bilateral pneumonia. During the first 4 days, he had attacks of hypoxemia, which finally became irremediable on oxygen therapy via nasal cannula. The boy was afebrile, unconscious, periodically agitated, cyanotic, dehydrated, pale, tachycardic and tachydyspnoic with clear signs of cardial decompensation, and worsening of the chest radiograph. He was transferred to the pediatric intensive care unit (PICU, Clinic for Pediatric Disease, University Clinic Center in Tuzla) for mechanical support. After 4 hours of mechanical ventilation with low lung compliance, it was decided to give bovine surfactant (Survanta, Abbott Laboratories) endotracheally. After 10 minutes, there was a significant improvement in lung compliance, oxygen saturation, and normalization of the heart rate. Therapy with surfactant was repeated every 8 hours. After 48 hours, auscultatory examination and chest radiograph of the boy were considerably improved. The role of surfactant in treating respiratory failure is doubtful in children, except in the newborn, even though surfactant can improve oxygenation and significantly decrease mortality in infants, children, and adolescents with acute lung injury and respiratory distress syndrome.1 That kind of therapy has been found ineffective in adults.2 Some investigations have shown that pulmonary surfactant proteins SP-A and SP-D can act as opsonins and neutralize influenza virus.3 In treatment of this child, we used medication that consisted of only surfactant proteins SP-B and SP-C. The medication led to significant improvement in lung function and the general condition, which was probably the result of lower alveolar surface tension and better lung compliance, but it is not possible to exclude a neutralizing effect of surfactant on influenza virus type A H1N1 by opsonization. Fahrija Skokić, MD, PhD Clinic of Pediatrics Igor Hudić, MD, PhD Clinic of Gynecology and Obstetrics Nešad Hotić, MD, PhD Belkisa Cǒlić, MD, PhD Clinic of Pediatrics Mirsada Prašo, MD, PhD Clinic of Anesthesiology and Reanimation Dubravka Bačaj, MD, PhD Clinic of Pediatrics University Clinical Center Tuzla, Bosnia and Herzegovina