Formation of membrane lattice structures and their specific interactions with surfactant protein A

Nades Palaniyar1, Ross A. Ridsdale1, Stephen A. Hearn2, Fred Possmayer3, and George Harauz1

1 Department of Molecular Biology and Genetics, University of Guelph, Guelph N1G 2W1; 2 Department of Pathology, St. Joseph's Health Center, London N6A 4L6; and 3 Medical Research Council Group in Fetal and Neonatal Health and Development, Departments of Obstetrics and Gynaecology and Biochemistry, University of Western Ontario, London, Ontario, Canada N6A 5A5


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

tubular myelin; surfactant protein A-tubular myelin interaction; pulmonary surfactant; dipalmitoylphosphatidylcholine; phosphatidylcholine


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PULMONARY SURFACTANT is essential for lowering surface tension at the air-liquid interface of the alveolus, thereby ensuring proper breathing and enabling gas exchange (16, 25). Surfactant is composed of several lipids (~90%) and proteins (~10%) (16, 33). The lipid component of pulmonary surfactant exists as a monolayer at the air-liquid interface and as a bilayer and possibly other forms in the alveolar hypophase (5, 28). Lipid bilayers are secreted as lamellar bodies (LBs) and are thought to pass through the tubular myelin (TM) intermediate before reaching the surface to become monolayers (5). Surfactant protein (SP) A, SP-B, and SP-C are present in the LBs, although SP-A is also secreted directly into the aqueous hypophase (3, 12, 30). LBs appear to interact with the secreted SP-A and calcium to form TM (27, 32). TM contains membranes as square lattices in cross sections and as tubules in longitudinal sections (1, 25, 28, 29). SP-A appears to be located near the corners of the square membrane lattices of TM (1, 8, 17, 27-29).

SP-A, a glycoprotein member of the C-type (calcium-binding) collectin (collagen-like lectin) family, is involved in various biological processes including recognition of microbes for macrophage-mediated immunity and in surfactant processing (10, 31). SP-A is an inherent component of TM (25, 30), and transgenic mouse models show that TM is absent in SP-A null mutants (9). In addition, in vitro experiments show that SP-A is essential for the formation of TM structures (17, 24). These in vitro experiments further identified dipalmitoylphosphatidylcholine (DPPC), phosphatidylglycerol, SP-B, and calcium as the other components necessary for the formation of TM structures.

Recently, Palaniyar et al. (15) elaborated the structure of bovine SP-A and its abilities to form specific filaments using transmission electron microscopy (TEM). Palaniyar and colleagues (13, 15) and Ridsdale et al. (19) also showed that calcium and other metal cations altered SP-A quaternary structure and that the supraquaternary filamentous structure of SP-A was influenced by the presence of metal ions and protein concentration. These studies determined that both SP-A-SP-A interactions and SP-A-lipid interactions were affected by the types of lipids used. Therefore, the parameters such as protein concentration, cations, and type of lipid could directly affect the formation of TM and the interaction of SP-A with TM.

Although many studies have investigated the structural organization of TM, the manner in which these structures are formed is not clearly understood. Here, we show that DPPC-egg phosphatidylcholine (PC) bilayers spontaneously form lattice structures ~50 nm on each side. These structures specifically interact with SP-A in the presence of calcium. These lattice structures share certain similarities with TM structures found in thin sections of native surfactant. Our experiments also provided images of several structures that could act as intermediates of this latticelike lipid structure. Using this new information as well as previously published data by Palaniyar and colleagues (13-15) and Ridsdale et al. (19), we propose a model for the formation of TM. This model emphasizes the fact that the lipids are not passive structures but instead can play an important role in determining the three-dimensional organization of TM. Lipids may also play a similar role in the organization of specialized structures in other biological membranes.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

All experiments were performed literally as described in the companion paper (13). Briefly, SP-A was purified from bovine lung lavage with standard procedures involving delipidation by organic solvents and column chromatography (3). Lipids were purchased from Avanti Polar Lipids (Alabaster, AL) and dissolved in chloroform-hexane (1:1), and vesicles of heterogeneous sizes were made as previously described (22). Freshly prepared lipid vesicles were mixed with buffer containing SP-A and incubated at 37°C for 10-20 min. Control reactions involved buffer without SP-A. After time was allowed for the protein to associate with lipids, CaCl2 was added to each tube, and the incubation was continued. Control tubes received double-distilled H2O in place of CaCl2. Considering the time of CaCl2 addition as the beginning, samples were withdrawn at various times of incubation for TEM analysis. Generally, a 30- to 60-min incubation time sufficed to produce the structures that we present here. The SP-A-lipid vesicle samples were negatively stained before examination with TEM (22).

Fourier transform analysis of native TM structures. Image analysis was performed with the IMAGIC program system (26). Briefly, micrographs of native TM structures were scanned with a charge-coupled device camera and Winview software (Princeton Instruments, EMPIX, Mississauga, ON). The image was filtered to enhance the contrast, and a circular mask was placed at the center of the image. The diffraction pattern of this image was obtained by allowing the repeating information to pass through the filter. This information was used to reconstruct the final image.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Purified SP-A preparations do not contain detectable amounts of SP-B. To ascertain the purity of the SP-A preparations, we size fractionated the purified protein preparation on 14.5% polyacrylamide gels with SDS-PAGE (Fig. 1). Because SP-B is extremely hydrophobic, the addition of urea to this gel system was necessary. Loading of even 2-3 µg of the protein and subsequent sensitive silver staining did not show any protein band comparable to SP-B. Instead, the preparations showed the typical protein profile expected for SP-A (2). Because the detection limit of protein by silver staining is roughly 10 ng, SP-B can constitute no more than 0.5% of the total protein, if it is at all present. Therefore, the SP-A preparations used in our study are devoid of detectable and significant amounts of SP-B.


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Fig. 1.   Polyacrylamide gel showing purity of surfactant protein (SP) A preparation. Approximately 2 µg of protein from SP-A and SP-B preparations were size fractionated via SDS-PAGE on a 14.5% polyacrylamide gel in presence of 2 M urea. Gel was subsequently stained with silver. Lanes 1 and 4, molecular-mass markers (nos. on right); lane 2, SP-A preparation; lane 3, SP-B preparation. SP-A preparation did not contain any detectable amounts of SP-B (<0.5% of total protein).

Fusion of small vesicles leads to the formation of large, striated bilayers. To study the interaction of SP-A with lipid bilayers, we incubated SP-A with DPPC-egg PC vesicles in calcium-containing buffer, negatively stained the preparations, and analyzed them with TEM. This lipid combination generated some vesicles with corrugated surfaces (Fig. 2A). At lower magnification, it could be seen that SP-A-containing lipid preparations showed a high degree of vesicle aggregation (Fig. 2B). In some cases, large vesicles that interacted with many small vesicles and other lipid structures could be identified. These large complexes were up to 100 µm in length. However, most of the isolated vesicles were <1 µm in size. Often, small vesicles interacted with large vesicles composed of striated sheets (Fig. 2). Small vesicles were coaggregated in these complexes. To obtain details of the process involved, we analyzed the lipid and associated lipid structures in detail. Clearer information was obtained from the images where SP-A-lipid interactions were detectable. These images showed that the DPPC-egg PC mixture was capable of generating different types of bilayers, which appeared as either "smooth" or "striated" vesicles (Fig. 3A) (13). Although both of these types of vesicles were seen in the same fields of view, the predominant type had a smooth surface. Accurate quantitation of different types of vesicles was not feasible because of the presence of aggregates that were common in these types of experiments.


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Fig. 2.   Assembly of large lipid structures. A: micrograph showing presence of striated vesicles in dipalmitoylphosphatidylcholine (DPPC)-egg phosphatidylcholine (PC) lipid preparations. B: field of view showing assembly of various lipid structures in presence of SP-A. Several small and isolated vesicles have fused to form large and corrugated vesicles. Bars, 50 nm.


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Fig. 3.   Fusion of vesicles forms large lipid bilayers. A: representative micrograph showing fusion of small vesicles to form larger sheets of lipid bilayers. Sheet regions often exist with corrugated surfaces. B: SP-A molecules specifically interact with corrugations of bilayers (circles), often in a filamentous arrangement. Bars, 50 nm.

SP-As were most frequently associated with the striated vesicles compared with smooth-surfaced vesicles (Fig. 3B). But even in the absence of SP-A, some lipid vesicles contained striations (Fig. 2), showing that the formation of striated surfaces was not entirely dependent on SP-A. However, in the presence of SP-A, many of these small vesicles were aggregated (Figs. 2 and 3), and some small vesicles apparently fused to form large sheets of bilayers that often contained striated surfaces (Fig. 3A). Some of these areas were sparsely populated by SP-A, whereas others were completely devoid of it (Fig. 3). A detailed description of the association between the SP-A filaments or octadecamers and the corrugated surfaces of the lipid bilayers can be found in the companion paper (13).

These results allowed two conclusions to be made regarding the formation of lipid structures. First, because control vesicles also formed striated surfaces, the DPPC-egg PC lipid mixture was inherently capable of forming striated vesicles. Second, the presence of SP-A appeared to enhance but was not essential for the formation of striated vesicles.

Large corrugated sheets fold to form lattice structures. Another group of TEM images showed that the large lipid bilayer sheets could fold onto themselves in a U-turn manner (Fig. 4). The folding of these striated vesicles often occurred at 90° to the direction of the striations (Fig. 4, A and B), with corresponding square lattice arrangements. Roughly one-fourth of the vesicles folded at non-90° angles to the direction of the striations and showed an altered spacing on the surfaces of the folded vesicles (Fig. 4, C and D). Folded vesicles also contained a mixture of lattice structures and striations (Fig. 4A). These vesicles often contained apparently intact vesicles associated with their folds (Fig. 4). Irregular folding, furthermore, created unequal spacings in the lattice structures (Fig. 4D). In some cases, multiple U-turns appeared to occur on the same bilayer, leading to the multiple layers of folded vesicles (Fig. 4D). Recently, Siegel and Epand (21) observed very similar structures of different lipid vesicles undergoing fusion. They performed cryo-TEM of vesicles embedded in vitreous ice and thereby trapped in their native hydrated state. The congruence with our work here supports the observation that the structures that we observed were not merely drying artifacts.


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Fig. 4.   Folding of striated vesicles forms lattices. A: large vesicle folded at ~90° to striations. B: near-perfect square lattices formed by ~90° folding of bilayers. C and D: folding of lipid bilayers at non-90° angles generated imperfect square lattice structures. Bars, 100 nm.

These results (Fig. 4) revealed two features of vesicle folding. First, the large striated vesicles formed folds, and the folding occurred more frequently in the presence of SP-A. Second, the folding occurred via a U-turn, often at 90° to the direction of the striations. These folded structures generated square lattice structures in two-dimensional projection.

SP-A specifically interacts with junctions of the lattice structures. When we examined the interaction between SP-A and the folded lipid vesicles, we found that many of these vesicles contained SP-A at specific locations on the surface, mainly at the junctions of square lattice structures (Fig. 5). Some of the clearer examples shown in Fig. 5A showed that the SP-As were reorganized on the lipid surface created by vesicle folds. SP-A molecules on these completely folded structures did not exist as filaments as seen on corrugated vesicles (Fig. 3B) (13). To determine the relationship between the lattice structures and SP-A molecules, spacings of regularly arranged lattice structures and spacings between SP-A molecules were measured. The lengths (48.4 ± 7.5 nm; n = 28 measurements) and widths (45.3 ± 6.0 nm; n = 28 measurements) of individual lattice structures were not significantly different from each other (P < 0.05). Hence the lattices existed predominantly as square structures with a mean side dimension of 46.9 nm (Figs. 4, A and B, and 5C). The spacings between clearly spaced SP-A octadecamers were also measured (Fig. 5, B and C) and found to average 47.4 ± 6.7 nm (n = 64 measurements). The mean lattice spacings and the SP-A spacings were not significantly different (P < 0.05).


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Fig. 5.   Arrangement of SP-As on square lattices. A: folding striated vesicles contained clearly identifiable SP-A octadecamers at regular intervals. B: magnified view of region in box in A. SP-A octadecamers were regularly spaced on lipid bilayers. C: SP-A octadecamers were found at corners of square lattices. White circles, individual SP-A octadecamers; black circles, enlargement of individual SP-A octadecamers. Bars, 50 nm.

These results (Fig. 5) illustrate two parameters regarding the interaction of SP-A and the folded vesicles. First, SP-As were preferentially located in the junctions of the square lattices. Second, the spacings between the SP-A molecules and the spacings between the square lattices were the same, reflecting a strong association of SP-A with the corners of the square lattice structures.

TM structures found in the native surfactant preparations. To compare the results of the in vitro lipid vesicle-SP-A experiments with native structures, we studied native surfactant materials from bovine lungs. These were fixed (involving the use of absolute alcohol), acetone treated, embedded in resin, sectioned, and analyzed with TEM. Because our method generated very clear images of square lattice structures (Fig. 6, A-F), we performed Fourier transform analysis as for two-dimensional crystals (26). The results clearly demonstrated the regularity of the spacings of the lattice structures (45 ± 3.2 nm; n = 80 measurements; Fig. 6, E and F). Furthermore, "X"-shaped electron-dense materials were clearly identified (Fig. 6, E and F). These stained preparations were probed with SP-A-specific antibodies, and the locations of these complexes were identified by gold particles. Many of the gold particles were located near the corners of the lattice structures (Fig. 6G). The average distance between the closest lattice corner and the gold particle was 8.7 ± 4.1 nm (n = 158 measurements), whereas the average distance between the centers of the square lattices and the gold particles was 15.6 ± 5.9 nm (n = 148 measurements). These results showed that SP-A antibodies bound to SP-As near the corners of the lattice structure (Fig. 6G). Interestingly, gold particles were not detected in the centers of the lattices with the X-shaped structures. When these native lattice structures were compared with the lattice structures seen in the in vitro experiments (Figs. 4 and 5 vs. Fig. 6), the spacings were not significantly different from one another (P < 0.05).


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Fig. 6.   Structure of native surfactant lattices after delipidation. A: negatively stained square lattice structure. B: filtered image of A with a circular mask. C: diffraction pattern after a "holey" filter was applied to pass through only the repeating structural information. D: reconstructed image. E: magnified view of a portion of D. F: image in E with reverse contrast. G: immunogold detection of SP-A showing that protein is located primarily at corners. Bars, 50 nm.

The results from these experiments showed parallels between the in vitro system and the native surfactant system. First, the spacings of the native square lattice structures were identical to the square lattice spacings of the TM-like structures found in the in vitro experiments. Second, SP-As were primarily located near the corners of the native TM lattice structures, and the SP-As were found in the same location in the TM-like lattice structures seen in the in vitro experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Lipid membranes of TM and TM-like structures are unusual in their organization, and it is evident that SPs and lipid composition play critical roles in their formation (24, 30). To study TM and/or surfactant functions, researchers have previously used different combinations of lipids: DPPC, egg PC, and phosphatidylglycerol in different proportions (17, 24, 30). Because unsaturated PC (25-30%) and DPPC (35-45%) are the major phospholipid components of pulmonary surfactant (16, 33), we used these lipids in the ratio of 1:1 (wt/wt). Interestingly, either DPPC alone or this simple lipid combination was capable of forming corrugations (Figs. 2 and 3) (13), and some of the folded bilayers formed lattice structures with certain similarities to the native TM (Figs. 4-6). SP-A preferred to interact with such folded structures (Figs. 2 and 3). Although these structures were not the major types in the lipid vesicle-SP-A preparations, these intermediates could explain several key steps in the interconversion of LBs to TM. Note that detailed characterization of different surfactant lipid components (5) shows that TM contains only <5-10% of the total surfactant lipids.

The structures detected in our in vitro experiments (Figs. 4 and 5) show similarities in appearance to reconstituted native TM (Fig. 6) and to structures found in sections of fixed lung tissues (1, 28) and so could reflect the nature of pulmonary surfactant material in vivo. The TM structures detected in the native preparations did not always portray a perfectly square lattice; instead, various sizes and shapes were observed (Fig. 6) (8, 27-29). We also found similar structural variations in the in vitro system (Fig. 4). However, these lipid structures formed predominantly square lattices of 45-47 nm in size for each side and smaller and larger variants from this norm. The variants could be explained by the folding of bilayers at non-90° angles in relation to the striations (Fig. 4, C and D). A careful reconstruction of TM and LBs with serial sections and electron microscopy revealed that TM did, indeed, contain U-turns and that the lipid bilayers connected the LBs and TM (32). Sections of surfactant materials also revealed the presence of folded bilayers in vivo (23). Formation of such striated surfaces on the lipid bilayer from native surfactant has also been previously detected (20). These authors considered different lipid arrangements at the molecular level and suggested that the formation of striations was possible under a variety of conditions.

Many reports (17, 24, 30) envisaged the presence of SP-B as an essential component for the formation of TM structures in vitro. Our results showed that SP-A alone was capable of inducing the reorganization of phospholipid membranes into latticelike structures that, in part, resembled TM. To confirm that there was no SP-B in our SP-A preparations, we examined them by SDS-PAGE followed by silver staining and found no detectable amounts (Fig. 1). Furthermore, lipid vesicles made without any protein were also able to generate these latticelike structures (Fig. 2A). These observations are evidence that SP-B is not essential for the formation of latticelike structures, at least in the initial stages. SP-B probably plays a more important role in lipid mixing and/or fusion in the terminal stages of TM formation (17, 18). Because our in vitro preparations were thin and did not need to be examined with the sectioning procedure, the structures that we observed clearly represented an intermediate and not the true final TM product. Hence this approach greatly facilitated the detection and interpretation of intermediate structures during the formation of TM.

In previous publications, Palaniyar and colleagues (13-15) elaborated the interaction of SP-A octadecamers or filaments with various lipid surfaces. These studies showed that SP-A interacted with lipid bilayers via the head or neck region. In addition, we also showed that most or all of the SP-A headgroups could interact with depressions in the lipid bilayer. SP-As in the filamentous arrangements also appeared to interact with the lipid bilayers via the SP-A headgroups. Curiously, when SP-A interacted with striated vesicles, formation of extensive SP-A filamentous networks was prevented. Instead, SP-A formed filaments in a single direction. This observation suggested that when most or all of the headgroups in the lateral positions were interacting with lipids, the remaining headgroups were available to interact with other headgroups on adjacent SP-As to form elongated filaments. It may be that when lipid bilayers fold to create a "pocket" or a corner, all of the headgroups interact with the lipid surfaces and the continuity of the protein filaments is broken (Fig. 5). When the lipid structure becomes a complete tubular structure, with the four corners forming long tubes, then the preferred arrangement could be to arrange the SP-As in all of the corners of the tube as illustrated in Fig. 6 and previously reported by others (1, 27, 29).

Our system of native surfactant processing for microscopy greatly facilitated detection of X-shaped structures in the center of the lattice structures that had previously been reported by some workers (1, 28). The improvement presumably resulted from the acetone treatment to remove lipid components and the use of LR White resin. In addition, Fourier transform-filtered images clearly showed the presence of electron-dense materials observed as X-shaped structures, but the antibody labeling was exclusively found only near the corners (Fig. 6). Because the carbohydrate moiety at the head of the SP-A molecules is responsible for immunoreactivity (22), it is probable that SP-A heads interact near the lattice corners. This conclusion agrees with previous findings by Palaniyar and colleagues (13, 14) that SP-A can interact with corrugations or depressions on DPPC-egg PC lipid bilayers.

Model for the formation of TM. Considering all the different types of images that were observed under different conditions in this and the companion paper (13), we propose the following model to explain the formation of TM (Fig. 7). The lipid bilayers required for the formation of TM exist in different forms, primarily vesicular forms such as LBs. Smaller vesicles, possibly with different amounts of SPs, could fuse to form larger vesicles, especially in the presence of calcium. TM formation could also begin from the preformed bilayers of LBs. LBs are potentially a rich source of calcium because they have a fivefold higher calcium concentration than the surrounding alveolar environment (4). Outer layers of the LBs are more accessible to the alveolar environment and can act as targets for changes in the concentration of calcium and other ions and also for constitutively secreted SP-A. Lateral phase separation of phospholipids, especially DPPC in these bilayers, could lead to the alteration in the lipid surface structures. Such a phase separation or alteration of surfaces may occur by different mechanisms such as preferential binding of calcium to certain phospholipids or the formation of gel-liquid interfaces to generate corrugations. SP-A octadecamers or filaments specifically interact with these lipid structures, possibly at the gel-liquid interface. Folding of striated vesicles containing SP-A at 90° to form U-turns could lead to further specific SP-A-lipid bilayer interactions. Repeated lipid bilayer folding could generate multiple layers of TM-like lattice structures (Fig. 7). Presumably, the hydrophobic SP-B plays a role in lipid mixing and membrane-membrane fusion at this step (18), resulting in the formation of the complete tubular structures seen in native TM.


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Fig. 7.   Proposed mechanism of formation of tubular myelin (TM) structures. ULV, unilamellar vesicle; MLV, multilamellar vesicle; LB lamellar body; SP-A 18-mers, SP-A octadecamers, i.e., the 6 SP-A basic trimers assembled into the form of a bouquetlike structure.

This model for the formation of TM structures could explain many of the present findings satisfactorily (Fig. 7) and also accommodates the findings from previous experiments by Palaniyar and colleagues (13-15). Furthermore, the presence of calcium in this system would favor the formation of compact forms of SP-A molecules with clearly identifiable stems (14, 19). This could explain the differences in the detection of linear arrays of particles at the TM corners (1, 7, 29). Our results contradict one proposed arrangement for SP-A in TM structures where the stems of the SP-A octadecamers interact with TM lattice corners (1, 6) but are in agreement with many recent biochemical studies (10, 11, 34) that suggest that the carbohydrate recognition domain is responsible for SP-A-lipid interactions.


    ACKNOWLEDGEMENTS

We thank Bob Harris for technical assistance in electron microscopy and Dianne Moyles for sectioning native surfactant materials.


    FOOTNOTES

This work was supported in part by the Ontario Thoracic Society (G. Harauz), the Natural Sciences and Engineering Research Council of Canada (G. Harauz), and the Medical Research Council of Canada (F. Possmayer).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: G. Harauz, Dept. of Molecular Biology and Genetics, University of Guelph, Guelph, Ontario, Canada N1G 2W1 (E-mail: gharauz{at}uoguelph.ca).

Received 13 August 1998; accepted in final form 14 January 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Lung Cell Mol Physiol 276(4):L642-L649
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