A method for S- and O-palmitoylation of peptides: synthesis of pulmonary surfactant protein-C models.
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A method for O- and S-palmitoylation of non-protected peptides has been developed. The peptides are treated with excess of palmitoyl chloride in 100% trifluoroacetic acid for 10 min at room temperature. The acidic conditions prevent acylation of amino groups, which is only significant after prolonged treatment (hours to days). The tripeptides Gly-Cys-Phe and Gly-Ser-Phe were converted into the respective S- and O-palmitoylated compounds, and the hydrophobic pulmonary surfactant protein-C model peptides, LRIPCCPVNLKRLLVVV [SP-C(1-17)] and FGIPSSPVLKRLLILLLLLLLILLLILGALLMGL [SP-C(Leu)] were converted into their respective S,S- and O,O-dipalmitoylated peptides. The reactions were virtually quantitative, and the palmitoylated peptides were isolated in about 75-80% yield after reversed-phase HPLC purification. CD spectroscopy showed that S, S-dipalmitoylation of SP-C(1-17) affects the peptide secondary structure (substantial increase in the alpha-helix content) in dodecylphosphocholine micelles. (+info)
Structural characterization of human and bovine lung surfactant protein D.
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Human and bovine surfactant proteins D (SP-D) were purified from late amniotic fluid and bronchioalveolar lavage on the basis of its Ca(2+)-dependent affinity for maltose. The molecular mass of a trimeric subunit was determined by matrix-assisted laser desorption ionization MS to lie in the range 115-125 kDa for human SP-D and 110-123 kDa for bovine SP-D. A single polypeptide chain was determined at 37-41 and 36-40 kDa for the human and bovine species respectively. The major parts of the primary structures of both SP-D molecules were determined by a combination of MS and Edman degradation. The heterogeneity in SP-D was caused mainly by a high number of post-translational modifications in the collagen-like region. Proline and lysine residues were partly hydroxylated and lysine residues were further O-glycosylated with the disaccharide galactose-glucose. A partly occupied N-linked glycosylation site was characterized in human SP-D. The carbohydrate was determined as a complex type bi-antennary structure, with a small content of mono-antennary and tri-antennary structures. No sialic acid residues were present on the glycan, but some had an attached fucose and/or an N-acetylglucosamine residue linked to the core. Bovine SP-D was determined as having a similar structure. (+info)
Keratinocyte growth factor-induced hyperplasia of rat alveolar type II cells in vivo is resolved by differentiation into type I cells and by apoptosis.
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Keratinocyte growth factor (KGF) is a potent mitogen of alveolar epithelial type II cells (AEII). AEII hyperplasia is resolved within several days following intratracheal instillation of KGF by unknown mechanism(s). AEII hyperplasia was induced in rat lungs by intrabronchial instillation of 5 mg recombinant human (rh)KGF x kg body weight(-1) or an equivalent amount of diluent. Epithelial architecture, cell proliferation, transformation of AEII into type I cells (AEI) and apoptosis were investigated by means of immunohistochemistry, stereology, double immunofluorescence microscopy, electron microscopy and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labelling (TUNEL) technique in lungs fixed 1, 2, 3 and 7 days after treatment. After 1 day of rhKGF instillation, an increase was observed in the nuclear antigen Ki-67, a proliferation marker detected by the antibody MIB-5-expressing surfactant protein (SP)-B, -C, -D-positive AEII. The incidence of mitosis was increased by day 2, resulting in AEII micropapillae with intense basolateral expression of the exon 6 containing isoform (v6) of CD446 (CD44v6), a marker for AEII. By day 3, monolayers of AEII exhibiting lateral CD44v6 covered 45% of the alveolar surface. After 7 days, there were numerous intermediate AEII/AEI cells characterized by a flat elongated shape, staining for SP-D, apical appearance of AEI marker Lycopersicon esculentum lectin and lateral staining for AEII marker CD44v6. Increased numbers of TUNEL-positive epithelial cells were seen at days 2-7. In conclusion, restoration of normal alveolar epithelium after instillation of recombinant human keratinocyte growth factor is accomplished by terminal differentiation and apoptosis of hyperplastic alveolar epithelial type II cells in vivo. (+info)
Surfactant abnormalities in idiopathic pulmonary fibrosis, hypersensitivity pneumonitis and sarcoidosis.
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Bronchoalveolar lavage fluids (BALF) from patients with idiopathic pulmonary fibrosis (IPF; n=36), hypersensitivity pneumonitis (HP; n=32) and sarcoidosis (n=44) were investigated for their surfactant properties and compared to healthy control subjects (n=29). The phospholipid (PL) and protein concentration, the PL:protein ratio, PL subclasses, and the surfactant apoproteins (SP)A and SP-B were quantified in BALF. Large surfactant aggregates (LSA) were measured by means of ultracentrifugation and assayed for surface activity using the pulsating bubble surfactometer. As compared to controls, SP-A concentrations, LSA content and PL:protein ratios were significantly decreased in all groups, whereas PL and SP-B concentrations remained unchanged. Changes in the phospholipid profile, with reduced percentages of phosphatidylcholine (not significant) and phosphatidylglycerol and increased fractions of phosphatidylinositol and sphingomyelin (p<0.05), occurred more in IPF than in HP, and not in sarcoidosis. Surface activity was found to be severely impaired in IPF (minimum surface tension (gamma min) approximately 15-20 mN x m(-1)), but only modestly affected in HP and sarcoidosis (gamma min approximately 5 mN x m(-1)) compared to controls (gamma min approximately 0 mN x m(-1)). Reconstitution of pelleted surfactant material with soluble BALF proteins further increased gamma min values. In conclusion, moderate changes in biochemical and physical surfactant properties are encountered in hypersensitivity pneumonitis and sarcoidosis, but pronounced disturbances occur in idiopathic pulmonary fibrosis. (+info)
Porcine pulmonary surfactant preparations contain the antibacterial peptide prophenin and a C-terminal 18-residue fragment thereof.
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Surfactant preparations obtained from porcine lungs by extraction with chloroform/methanol followed by chromatography over Lipidex-5000 are used for treatment of respiratory distress syndrome in preterm infants. These preparations contain about 98% phospholipids and 1-2% of the hydrophobic pulmonary surfactant-associated proteins B and C (SP-B and SP-C). Separation of the proteins in the surfactant preparation by reversed-phase high performance liquid chromatography revealed, in addition to SP-B and SP-C, the presence of three peptides derived from the cathelicidin family of antibacterial peptides. The 79-residue proline-rich peptide prophenin (identical to that isolated from leukocytes), an 80-residue prophenin with an N-terminal pyroglutamic acid residue, and a C-terminal 18-residue fragment of prophenin were found in approximate molar ratios of 1:20:5. A synthetic version of the C-terminal 18-residue peptide exhibits salt-dependent antibacterial activity (higher activity in the absence of salt) against the Gram-positive bacterium Bacillus megaterium Bm11 and, to a lesser extent, against Gram-negative Escherichia coli D21 cells. It appears possible that the presence of prophenin peptides may contribute to the antibacterial properties of surfactant preparations. (+info)
Cellular activation by Ca2+ release from stores in the endoplasmic reticulum but not by increased free Ca2+ in the cytosol.
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Ca(2+) release from intracellular stores and/or transmembrane influx can increase the cytosolic free Ca(2+) concentration ([Ca(2+)](i)). Such changes in [Ca(2+)](i) might transduce signals regulating transcription, motility, secretion, and so on. Surfactant secretagogues such as ATP and ionomycin stimulate the release and transmembrane influx of Ca(2+), both of which increase [Ca(2+)](i). The addition of surfactant protein A (SP-A) or depleting cellular Ca(2+) inhibited both surfactant secretion and Ca(2+) transients. Current results suggest that Ca(2+) signalling stimulates surfactant secretion by type II pneumocytes, but not via increased [Ca(2+)](i). Treatment of cells with a Ca(2+) chelator, bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid acetoxymethyl ester (BAPTA-AM), stimulated secretion but decreased [Ca(2+)](i). Adding SP-A or depleting Ca(2+) inhibited BAPTA-AM-induced secretion. When studied directly, Ca(2+) in the endoplasmic reticulum store ([Ca(2+)](l)) decreased in response to BAPTA, ionomycin and thapsigargin, and increased in response to SP-A. Phorbol ester (PMA) induced surfactant secretion without altering [Ca(2+)](i) or [Ca(2+)](l) and was unaffected by Ca(2+) depletion. The addition of PMA to Ca(2+)-releasing secretagogues increased secretion, but combining two Ca(2+)-releasing secretagogues did not. These results suggest that (1) Ca(2+) signalling of type II cell surfactant secretion reflects changes in [Ca(2+)](l), not [Ca(2+)](i), (2) PMA elicits secretion differently from Ca(2+)-releasing secretagogues, and (3) SP-A inhibits secretion by enhancing Ca(2+) sequestration within endoplasmic reticulum stores. Whether other cell types signal via changes in [Ca(2+)](l) is unknown. (+info)
Surfactant protein A binds to the fusion glycoprotein of respiratory syncytial virus and neutralizes virion infectivity.
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Collectins are a family of calcium-dependent collagenous lectins that appear to be important in innate host defense. We investigated the ability of three human collectins, namely, lung surfactant proteins A (SP-A) and D (SP-D) and the serum mannose-binding protein (MBP), to bind to the surface glycoproteins of respiratory syncytial virus (RSV). SP-A was shown to bind to the F (fusion) glycoprotein but not to the viral G (attachment) glycoprotein, and binding was completely abrogated in the presence of EDTA. Neither SP-D nor MBP bound to either glycoprotein. SP-A also neutralized RSV in a calcium dependent fashion. These results support a role for SP-A in the defense of infants against infection with RSV and indicate a possible mechanism for its protective activity. (+info)
An alternative view of the role(s) of surfactant and the alveolar model.
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Currently, the study of surfactant proteins is much in vogue, but, in the early days, the physics underlying surfactant function was treated somewhat superficially, leaving assumptions that have become culturally embedded, such as the "bubble" model of the alveolus. This review selectively reexamines these assumptions, comparing each combination of alveolar model and role of surfactant for compatibility with the major features of pulmonary mechanics and alveolar stability, morphology, and fluid balance. (+info)