Recombinant SP-D carbohydrate recognition domain is a chemoattractant for human neutrophils

Guang-Zuan Cai1, Gail L. Griffin2, Robert M. Senior2, William J. Longmore1, and Michael A. Moxley1

1 Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis 63104; and 2 Division of Pulmonary and Critical Care Medicine, Department of Medicine, Washington University School of Medicine at Barnes-Jewish Hospital, St. Louis, Missouri 63110

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Human pulmonary surfactant protein D (SP-D) is a collagenous C-type lectin with high binding specificity to alpha -D-glucosyl residues. It is composed of four regions: a short NH2-terminal noncollagen sequence, a collagenous domain, a short linking domain ("neck" region), and a COOH-terminal carbohydrate recognition domain (CRD). Previous studies demonstrated that SP-D is chemotactic for inflammatory cells. To test which domain of SP-D might play a role in this function, a mutant that contains only neck and CRD regions was expressed in Escherichia coli and purified by affinity chromatography on maltosyl-agarose. A 17-kDa recombinant SP-D CRD was identified by two antibodies (antisynthetic SP-D COOH-terminal and neck region peptides) but not by synthetic SP-D NH2-terminal peptide antibody. The recombinant SP-D CRD was confirmed by amino acid sequencing. Gel-filtration analysis found that 84% of CRD was trimeric and the rest was monomeric. Analysis of the chemotactic properties of the trimeric CRD demonstrated that the CRD was chemotactic for neutrophils (polymorphonuclear leukocytes), with peak activity at 10-10 M equal to the positive control [formyl-Met-Leu-Phe (fMLP) at 10-8 M]. The chemotactic activity was abolished by 20 mM maltose, which did not suppress the chemotactic response to fMLP. The peak chemotactic activity of the CRD is comparable to the activity of native SP-D, although a higher concentration is required for peak activity (10-10 vs. 10-11 M). The chemotactic response to CRD was largely prevented by preincubation of polymorphonuclear leukocytes with SP-D, and the response to SP-D was prevented by preincubation with CRD. These preincubations did not affect chemotaxis to fMLP. These results suggest that trimeric CRD accounts for the chemotactic activity of SP-D.

surfactant protein D; collectins; host defense proteins; chemotaxis

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

PULMONARY SURFACTANT PROTEIN (SP) D is a member of a family of collagenous C-type lectins (13). The protein is synthesized and secreted by alveolar epithelial type II cells and by nonciliated bronchiolar or Clara cells (4, 31). SP-D has been shown to be a calcium-dependent, carbohydrate-binding protein (20) that interacts with one of the lipid components of surfactant (19, 21) and with microorganisms (11, 16), leukocytes (7), and macrophages (15, 18). Four distinct regions of the protein can be identified: a short NH2-terminal noncollagen sequence, a collagenous domain, a short linking domain ("neck" or "hinge" region), and a COOH-terminal carbohydrate recognition domain (CRD). Electron-microscopic studies (6) show that SP-D molecules are assembled as tetramers of trimeric subunits (12 mers). A previous study (7) showed that SP-D is chemotactic for polymorphonuclear leukocytes (PMNs) and monocytes. This finding fits with a role for SP-D in host defense. Because both maltose and antibodies to the COOH-terminal domain of SP-D diminish the chemotactic response of human PMNs to SP-D (7), this domain may be the domain responsible for the chemotactic activity of SP-D. To test for the chemotactic activity of this domain, a mutant that contains only the neck and CRD regions (neck-CRD) of SP-D was expressed in Escherichia coli and purified by affinity chromatography on maltosyl-agarose. The chemotactic response of human PMNs to this protein was determined.

    METHODS
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Methods
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Construction and expression. A human lung cDNA of SP-D, which was kindly supplied by E. C. Crouch (Washington University, St. Louis, MO) was used as a template for a PCR. Two oligonucleotides were synthesized on the basis of the published cDNA sequence (8), engineering Sal I and Nde I restriction sites at the 5'-end of the neck region oligonucleotide (5'-GACAGTCGACCATATGGATGTTGCTTCTCTGAGG-3') and BamH I and a stop codon at the 5'-end of the CRD oligonucleotide (5'-CCAAGGATCCCTATCAGAACTCGCAGACCAC-3'). The PCR product was purified with a Gene Clean kit (Bio101, Vista, CA) after electrophoresis on a 1.5% SeaPlaque agarose gel (FMC Bioproducts, Rockland, ME). The DNA fragment was digested with Sal I and BamH I and ligated into the Sal I and BamH I side of pBluescript IIKS with T4 ligase. The ligation mixture was transformed into competent cells of E. coli strain DH5 for color selection. The correct clone was digested with BamH I and Nde I and ligated into pET/3xa vector under the control of the T7 promoter. The target plasmid was transformed into E. coli strain BL21(DE3) for expression.

E. coli was grown in Luria-Bertani broth (Difco, Detroit, MI) containing 200 mg/l of ampicillin at 37°C. Expression of the CRD was achieved by adding 1 mM isopropyl-beta -D-thiogalactopyranoside (IPTG; Sigma, St. Louis, MO) at an optical density of 0.9 at 600 nm. Cells were harvested 2 h after IPTG induction by centrifugation at 1,500 g for 5 min.

Sequencing. Plasmids isolated from single colonies were subjected to double-stranded sequencing with the T7 sequencing kit (Amersham Life Science, Arlington Heights, IL).

Purification of neck-CRD. The purification protocol was adapted from Weis et al. (33). Briefly, the pellet of the induced cells was washed with 10 mM Tris, pH 7.8, sonicated (30 s, 6×), and centrifuged at 10,000 g for 15 min at 4°C. The pellet was extracted with 6 M guanidine HCl (GuHCl) in 100 mM Tris containing 0.01% 2-mercaptoethanol for 2 h on ice. The mixture was centrifuged at 100,000 g for 60 min at 4°C. The supernatant was dialyzed extensively against loading buffer (1.25 M NaCl, 25 mM CaCl2, and 25 mM Tris, pH 7.8) and again centrifuged at 100,000 g for 60 min at 4°C. The supernatant of the resulting solution was further purified by maltosyl-agarose affinity chromatography (20). Briefly, the supernatant was loaded onto the maltosyl-agarose column, and the column was washed with the loading buffer. The protein was then eluted from the column with 1.24 M NaCl, 25 mM EDTA, and 25 mM Tris, pH 7.8. After the protein content of each fraction was determined (1.5 ml/fraction), 1 µg of each fraction was loaded onto an SDS-tricine polyacrylamide gel to identify CRD-containing fractions.

The trimeric neck-CRD was isolated by HPLC isocratically on a Progel TSK-GEL G3000SW (TSK; 30 cm × 7.5-mm ID) size-exclusion column (Supelco, Bellefonte, PA) with a mobile phase of buffer A (100 mM NaH2PO4, 100 mM Na2SO4, and 0.05% NaN3, pH 6.8). Thyroglobulin, BSA, ovalbumin, and myoglobin (Sigma) were used to calibrate the TSK column.

Antibody preparations and characterization. Three polyclonal antibodies to different regions of human SP-D were prepared in rabbits with synthetic peptides conjugated to thyroglobulin. The three synthetic peptides used for immunization were the COOH terminus of human SP-D (amino acids 281-293), the NH2 terminus of human SP-D (amino acids 37-50), and the neck or hinge region of SP-D (amino acids 169-183). The antisera were purified on a protein A-Sepharose 4B column (Sigma). The antibodies were characterized by Western blot with both specific peptide-thyroglobulin conjugates and human SP-D as antigens. Human SP-D was isolated from human alveolar proteinosis lavage fluid (5). Each antibody detected its specific antigen, and each was capable of detecting human SP-D (data not shown). For Western blot analysis of the CRD, the three antibodies were used in a 1:250 dilution. A 1:3,000 dilution of goat anti-rabbit IgG alkaline phosphate conjugate was used as the secondary antibody. Color was developed with 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium as substrate.

Denaturation and renaturation of neck-CRD. The purified neck-CRD was subjected to denaturation by GuHCl to demonstrate possible effects of the extraction procedure on the conformation of the CRD. Two samples of purified trimeric neck-CRD (final concentrations of 1 and 0.5 µM in buffer A) were mixed with varying amounts of GuHCl to give final GuHCl concentrations ranging from 0 to 2 M. Samples were equilibrated at 20°C for 1 h. The denaturation was monitored by fluorescence at 20°C. The emission spectra were recorded from 300 to 450 nm in 0.5-nm wavelength increments, with an excitation wavelength of 295 nm (2). The renaturation of the neck-CRD was accomplished by removing GuHCl from the samples by extensively dialyzing the denaturation mixture against buffer A at 4°C.

Chemotaxis assay. Neutrophil (PMN) chemotaxis to recombinant neck-CRD and native SP-D was examined in modified Boyden chamber assays at 37°C with a membrane sandwich technique (25). PMNs were isolated from human peripheral venous blood by density gradient centrifugation with Histopaque (1) and resuspended in DMEM with 0.5 mg/ml of human serum albumin. PMN migration was expressed as cells per high-power grid by light microscopy (×400). In each experiment, the data points represent the means of cell counts of five high-power fields on each of three membrane pairs from the Boyden chambers (n = 15 cell counts). The lower compartment of each chamber was filled with 240 µl of test solution or basal medium, and the upper compartment contained 360 µl of cell suspension. Negative controls consisting of medium only in the lower compartment, and positive controls of medium containing fMLP (10-8 M) in the lower compartment were included in each experiment. All cell counts were corrected for medium blanks. In experiments involving maltose, 20 mM maltose was added to the upper and lower compartments. To determine further whether CRD accounts for the chemotactic activity of SP-D for PMNs, we preincubated the PMNs with CRD (15 min, room temperature) and then tested for residual chemotactic responsiveness to SP-D (24). In other desensitization experiments, cells were preincubated with SP-D and then exposed to CRD.

Other procedures. Protein concentrations were measured by the modified method of Lowry (22) with BSA as a standard. SDS-PAGE was performed according to the method of Laemmli (17). Immunoblotting of proteins was performed by the method of Towbin et al. (26). Digital images of the gels and blots were captured and notated with a Macintosh-based imaging system utilizing a charge-coupled device camera (Digital Imagers, Elburn, IL) and National Institutes of Health Image 1.60 software.

    RESULTS
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Introduction
Methods
Results
Discussion
References

Expression and purification. Two colonies from the BL21(DE3)/pET-3xa-CRD plate were incubated at 37°C in Luria-Bertani broth containing 200 mg/l of ampicillin until the optical density of the medium reached 0.9 at 600 nm. After 1 ml of culture medium was taken as a negative control, 1 mM IPTG was added to the medium and the incubation continued for another 2 h. Cells were harvested and analyzed by SDS-PAGE and Western blot. Figure 1 shows the results of silver staining and Western blotting of a 16% SDS-PAGE, which indicated a dominant band 17 kDa in size and was recognized by COOH-terminal peptide antibody. Noninduced cell extracts were negative. The higher molecular-weight species apparent on the gel represent undenatured trimer as determined by molecular-weight analysis with National Center for Supercomputer Applications GelReader 2.0. The NH2-terminal amino acid sequencing confirmed that the 17-kDa protein starts with the neck region (Asp-Val-Ala-Ser-Leu-Arg).


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Fig. 1.   Expression of carbohydrate recognition domain (CRD). Two colonies were incubated at 37°C until optical density at 600 nm equaled 0.9. Colonies were then induced by adding 1 mM isopropyl-beta -D-thiogalactopyranoside (IPTG). (In each case, 1 ml of cells was saved as a negative control.) Lanes 1, 2, 7, and 8, negative controls that were noninduced cell extracts; lanes 3, 4, 5, and 6, induced cell extracts. Lanes 1-4 were stained with silver. Lanes 5-8 were blotted with antibody raised against COOH-terminal peptide of surfactant protein (SP) D. +, With; -, without. Nos. at left, molecular mass.

The GuHCl extraction yielded 10 mg of the recombinant neck-CRD per liter of culture. The neck-CRD was eluted in a single peak by maltosyl-agarose affinity chromatography (Fig. 2). The eluted fraction was subjected to Western blot analysis with antibodies generated against the COOH terminus, the NH2 terminus, and the neck or hinge region of SP-D. The CRD was recognized by the neck region and COOH-terminal antisera but not by NH2-terminal and nonimmune sera (Fig. 3).


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Fig. 2.   Purification of CRD by guanidine HCl (GuHCl) extraction and maltosyl-Sepharose chromatography (silver-stained polyacrylamide gel). Protein was extracted from a sonicated cell pellet as described in METHODS. Dialyzed extract was loaded on a maltosyl-agarose 4B column and eluted as described in METHODS. Lane 1, molecular-mass markers; lane 2, extract dialyzed against loading buffer before loading on column; lane 3, proteins that did not bind to maltose column; lane 4, column wash; lanes 5-7, CRD eluted from column. Nos. at left, molecular mass.


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Fig. 3.   Western blot of CRD with SP-D antibodies. Lane 1, molecular-mass markers stained with amido black; lanes 2-7, 1 µg of CRD loaded in each and run on SDS-PAGE under reducing conditions. Lanes 2, 4, and 6 were reacted with nonimmune serum; lane 3 was reacted with antibody raised against COOH terminus of SP-D; lane 5 was reacted with antibody against neck region; and lane 7 was reacted with antibody against NH2 terminus of SP-D. Nos. at left, molecular mass.

Separation of trimeric neck-CRD. To determine whether the recombinant CRD was isolated as a monomer or as a trimer, we subjected the protein to size-exclusion chromatography. Five micrograms of the CRD purified from a maltosyl-agarose column were injected onto the TSK column that was calibrated by thyroglobulin, BSA, ovalbumin, and myoglobin (Fig. 4A). Analysis of two preparations showed that an average of 84% of the recombinant neck-CRD was found to assemble into the trimeric form (Fig. 4, B and C). These results demonstrate that our isolation procedure allows us to isolate the CRD in a conformation similar to its conformation in native SP-D.


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Fig. 4.   Recorder output of size-exclusion chromatography of maltose affinity-purified CRD. A: Progel TSK-GEL G3000SW column was calibrated with thyroglobulin (1), BSA (2), ovalbumin (3), and myoglobin (4). B: 76% of CRD is present as trimer (T). M, monomer. C: 93% is present as trimer. B and C represent 2 separate preparations.

Denaturation and renaturation. To determine whether the extraction procedure had any effect on the conformation of the CRD, denaturation-renaturation experiments were carried out. Fluorescence emission spectra were used to monitor the denaturation of neck-CRD at 20°C. Figure 5 shows that the trimeric neck-CRD was completely dissociated by GuHCl at 1 M. Renaturation was accomplished by extensively dialyzing the neck-CRD denatured by 2 M GuHCl to remove GuHCl at 4°C. Again monitored by fluorescence emission spectra, the intensity of the fluorescence returned to normal. As shown in Fig. 5, the intensity of the renatured CRD was 98% of that of the starting material.


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Fig. 5.   Denaturation of CRD by GuHCl. CRD was exposed to increasing concentrations of GuHCl. Fluorescence intensity decreased as denaturation increased. At 1 M GuHCl, CRD was completely denatured. After extensive dialysis to remove GuHCl, fluorescence intensity returned to control values (data not shown).

Chemotactic activity. Purified recombinant neck-CRD showed dose-dependent effects on human PMN migration, with a maximal response at a concentration of 2 ng/ml (~10-10 M; Fig. 6). At this concentration, neck-CRD was as active as the optimal concentration of fMLP, a potent leukocyte chemoattractant.


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Fig. 6.   CRD is chemotactic for human polymorphonuclear leukocytes and chemotactic activity is abolished by maltose. Peak chemotactic activity is at 10-10 M and approximated activity of formyl-Met-Leu-Phe (fMLP) at 10-8 M. Chemotaxis was determined as described in METHODS. Maltose at 20 mM does not affect chemotactic response to fMLP. Data are means ± SD from a representative experiment in triplicate, with 5 high-power fields counted for each membrane pair (n = 15 cell counts). HPG, high-power grid.

Maltose (20 mM), an inhibitor of SP-D binding to various saccharide ligands (16, 20), reduced the chemotactic activity of neck-CRD by 98% at all concentrations of the CRD (Fig. 6). In contrast, as observed previously (7), maltose had no effect on the chemotactic activity of fMLP.

In two separate experiments, each done in triplicate, preincubation of PMNs with CRD reduced the chemotactic activity of SP-D at its optimal chemotactic activity of 10-11 M by 75 and 91% (Fig. 7B), without inhibiting the response of the cells to fMLP. In two other experiments, also in triplicate, cells preincubated with SP-D displayed 83 and 68% reductions in chemotaxis to CRD (Fig. 7A). These findings suggest that the neck-CRD contains the chemotactically active domain of SP-D.


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Fig. 7.   Effect of preincubations with SP-D or CRD on neutrophil chemotaxis to CRD and SP-D. See METHODS for details. A: CRD chemotactic activity after cells were preincubated with SP-D. B: SP-D chemotactic activity after cells were preincubated with CRD. Data are means ± SD from a representative experiment as noted in Fig. 6. In each case, preincubations reduced chemotactic activities of both CRD and SP-D without having an effect on chemotactic activity of fMLP.

    DISCUSSION
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Introduction
Methods
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Pulmonary surfactant is a complex mixture of lipids and proteins lining the alveolar surface. The major function of the complex is to prevent the alveoli from collapsing at end expiration. The major active component of surfactant is disaturated phosphatidylcholine (3, 23), but both hydrophilic and hydrophobic proteins are required (12). The two hydrophilic proteins SP-A and SP-D are produced by alveolar type II cells and nonciliated bronchiolar or Clara cells (4, 31). These two proteins are members of the family of Ca2+-dependent, collagenous carbohydrate-binding proteins known as C-type lectins or collectins (13). These pulmonary proteins have been suggested to have a role in nonimmune host defense (13, 27). SP-A interacts with specific saccharide ligands (10) and with various microorganisms and stimulates the uptake of bacteria and viruses by macrophages (29, 30). SP-D also interacts with specific carbohydrate ligands (20) and binds to carbohydrates on the surface of a variety of microorganisms (11, 16) as well as macrophages (15) and leukocytes (7). SP-A has been shown to be chemotactic for macrophages (34), and SP-D has been shown to elicit a chemotactic response in human neutrophils (7). The chemotactic response of macrophages to SP-A appears to be at least partially dependent on the collagenous nature of SP-A (34). However, the response of human PMNs to SP-D may be dependent on the lectin nature of the protein because it is inhibited by an antibody to the COOH-terminal domain and by maltose (7). This study was undertaken to further assess directly the lectin domain-dependent nature of the chemotactic response to SP-D.

The recombinant CRD was constructed and expressed as a peptide containing both the CRD and the neck or hinge region of SP-D. This was done because it was considered that such a structure would be more likely to trimerize and thus be analogous to the native structure of SP-D. Wang et al. (32) demonstrated that a recombinant conglutinin polypeptide composed of the CRD and neck region of that molecule self-associated into homotrimers, and Kishore et al. (14) recently demonstrated that the neck region is required for homotrimer formation of a recombinant SP-D CRD derived from a fusion protein. Our method of preparation differs somewhat from that described by Kishore et al. in that the peptide is not prepared as a fusion protein, thus eliminating the requirement for a cleavage step followed by further purification. In addition, lysis of the cells and extraction of the peptide utilized the same medium, eliminating the denaturation and refolding step described by Wang et al. (32). It also differs from the preparation described by Eda et al. (9) in that it does not contain any portion of the collagen-like domain of SP-D. Figure 1 shows that the peptide was successfully expressed in E. coli and was recognized by an antibody to the COOH terminus of SP-D. NH2-terminal amino acid sequencing confirmed that the 17-kDa product started with the neck region (data not shown). To purify the protein, we took advantage of its lectin nature and specificity for alpha -D-glucosyl residues (20). The peptide was successfully purified by maltosyl-Sepharose affinity chromatography (Fig. 2) and is recognized by antibodies to the neck region and the COOH terminus but not by an antibody to the NH2-terminal domain (Fig. 3). These results were predicted because the recombinant protein was constructed to lack the NH2-terminal region of the lectin. Size-exclusion chromatography indicated that the majority of the isolated peptide was present in the trimeric form. Denaturation with GuHCl followed by renaturation indicated that the isolation procedure did not permanently affect the conformation of the isolated peptide.

The recombinant CRD was shown to elicit a chemotactic response in human PMNs. Significant chemotaxis was demonstrated at 10-12 M CRD, with a maximum response at 10-10 M. This is an order of magnitude higher than the maximally effective concentration of native SP-D. The differing chemotactic response to native SP-D and the CRD may indicate that the tetrameric conformation of native SP-D with four available CRDs elicits a greater response than a single recombinant consisting of a single trimeric subunit of the tetramer. However, it must be acknowledged that these studies do not completely eliminate the possible contribution of the NH2-terminal and collagen domain regions to the chemotactic activity of SP-D. The maximum response is comparable to that elicited by fMLP at 10-8 M. The dependence of the response on the lectin nature of the peptide was demonstrated by the response of the cells to the peptide in the presence of a known carbohydrate ligand for SP-D. Maltose at 20 mM inhibited the chemotactic response at all concentrations of the CRD up to 10-7 M (Fig. 6). This is in agreement with the demonstrated effect of maltose on the chemotactic activity of SP-D for PMNs (7).

Desensitization studies (Fig. 7) supported the probable role of the CRD in the chemotactic response to SP-D. Cells preincubated with SP-D (10-8 M) were much less responsive to 10-10 M CRD, and cells preincubated with CRD were likewise much less responsive to SP-D. Preincubation with either protein had no effect on the response of PMNs to fMLP. These results suggest that the chemotactic response of the cells to SP-D is largely dependent on the CRD domain of the protein. The precise sequence or conformation of the recombinant neck-CRD was not determined; however, trimeric conformation is required for high-affinity binding to carbohydrates (14). Whether this high-affinity binding is required for chemotaxis or whether the monomeric form of the CRD can function as a chemoattractant remains to be determined. The fact that native SP-D and the recombinant neck-CRD show similar chemoattractant activity suggests that the trimeric form may be required.

These results and others (7, 15, 16) indicate that the proposed role of SP-D as a host defense protein is multifaceted. As noted above, the protein has been shown to interact with a variety of microorganisms, and, at higher concentrations than in the present study, it has been shown to stimulate oxygen radical production in rat macrophages (28). The demonstrated ability of the protein to function as a chemoattractant suggests that it could play a role in the accumulation of inflammatory cells in lung injury. It is of interest to note that the chemoattractant activity of SP-D (7) or its CRD (present study) is demonstrable at concentrations several orders of magnitude lower than that required to show chemoattractant activity of SP-A for macrophages, suggesting that SP-D is a more potent chemoattractant in vivo.

    ACKNOWLEDGEMENTS

We thank Thomas Broekelmann and Robert Mecham for sequencing the recombinant peptide.

    FOOTNOTES

This investigation was supported by National Heart, Lung, and Blood Institute Grants HL-13405 and HL-29594.

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: M. A. Moxley, 1402 South Grand Blvd., Saint Louis Univ. School of Medicine, Edward A. Doisy Dept. of Biochemistry and Molecular Biology, St. Louis, MO 63104-1079.

Received 6 July 1998; accepted in final form 7 October 1998.

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Discussion
References

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Am J Physiol Lung Cell Mol Physiol 276(1):L131-L136
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