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E Crouch

Publications and source records attributed to E Crouch.

At least 37 records · Page 2Linked to original sources
PubMed1991

Human surfactant protein D: SP-D contains a C-type lectin carbohydrate recognition domain.

Lung surfactant protein D (SP-D) shows calcium-dependent binding to specific saccharides, and is similar in domain structure to certain members of the calcium-dependent (C-type) lectin family. Using a degenerate oligomeric probe corresponding to a conserved peptide sequence derived from the amino-terminus of the putative carbohydrate binding domain of rat and bovine SP-D, we screened a human lung cDNA library and isolated a 1.4-kb cDNA for the human protein. The relationship of the cDNA to SP-D was established by several techniques including amino-terminal microsequencing of SP-D-derived peptides, and immunoprecipitation of translation products of transcribed mRNA with monospecific antibodies to SP-D. In addition, antibodies to a synthetic peptide derived from a predicted unique epitope within the carbohydrate recognition domain of SP-D specifically reacted with SP-D. DNA sequencing demonstrated a noncollagenous carboxy-terminal domain that is highly homologous with the carboxy-terminal globular domain of previously described C-type lectins. This domain contains all of the so-called "invariant residues," including four conserved cysteine residues, and shows high homology with the mannose-binding subfamily of C-type lectins. Sequencing also demonstrated an amino-terminal collagenous domain that contains an uninterrupted sequence of 59 Gly-X-Y triplets and that also contains the only identified consensus for asparagine-linked oligosaccharides. The studies demonstrate that SP-D is a member of the C-type lectin family, and confirm predicted structural similarities to conglutinin, SP-D, and the serum mannose binding proteins.

Amino Acid Sequence↗
PubMed1991

Primary translation products of pulmonary surfactant protein D.

Surfactant protein D (SP-D) is a collagenous, surfactant-associated, carbohydrate-binding protein that is synthesized by alveolar type II epithelial cells. To further characterize SP-D, we isolated RNA from adult rat lungs and rat type II cells and translated mRNAs in vitro. [35S]methionine-labeled translation products were precipitated with antibodies to rat SP-D, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and visualized by fluorography. Immune precipitates of translation reactions for rat lung or rat type II cells demonstrated a single collagenous polypeptide (39.3 kDa) that was smaller than surfactant-associated SP-D (43 kDa, reduced) but larger than the mature secreted form of rat SP-A. This component was not identified in translation reactions of rat liver, gut, brain, mammary gland, or rat L2 cell RNA. There was a fivefold enrichment of SP-D mRNA in freshly isolated type II cells relative to lung; however, the levels of translatable SP-D mRNA decreased rapidly during the first 24 h of cell culture. The SP-D translation product migrated faster than the major cellular form of SP-D but approximately 1 kDa slower than cellular SP-D synthesized in the presence of 2,2'-dipyridyl plus tunicamycin. Translation in the presence of canine pancreatic microsomes gave a single glycosylated, endoglycosidase F-sensitive form (40.6 kDa) and demonstrated cleavage of a small signal peptide. These results indicate that SP-D is a secretory product of differentiated type II epithelial cells and that SP-D is secreted in a mature form that does not undergo further proteolytic processing in vivo.

Animals↗
PubMed1991

Developmental expression of pulmonary surfactant protein D (SP-D).

Surfactant protein D (SP-D) is a collagenous, surfactant-associated, carbohydrate-binding protein that is synthesized by pulmonary epithelial cells. In the present studies, we examined the expression of SP-D and SP-D mRNA during late fetal (day 17, 19, 21) and early postnatal (day 5) rat lung development using immunochemical, cell-free translation, and Northern hybridization assays. SP-D mRNA and immunoreactive SP-D protein were first detected in guanidine extracts of whole rat lung at 21 days of gestation and reached even higher concentrations during the postnatal period. Likewise, immunoperoxidase studies of rat lung using affinity-purified antibodies to SP-D showed no staining at day 17 or 19, but demonstrated strong cytoplasmic staining of cuboidal epithelial cells lining immature airspaces at day 21 and strong cytoplasmic staining of type II and nonciliated bronchiolar cells in adult lung. SP-D also appeared in amniotic fluid by day 21 and was partially purified by affinity chromatography on maltosyl-agarose under conditions used for the isolation of rat lung SP-D. These studies indicate that the production of SP-D is increased shortly prior to birth, and that the increases in total lung SP-D and SP-D mRNA are temporally correlated with SP-D secretion and the appearance of SP-D in amniotic fluid.

Amino Acid Sequence↗
PubMed1991

Surfactant protein D. Increased accumulation in silica-induced pulmonary lipoproteinosis.

Surfactant protein D (SP-D) (CP4) is a collagenous surfactant-associated carbohydrate binding protein that is synthesized and secreted by alveolar epithelial cells. Previous studies have shown that intratracheal administration of crystalline silica to rats elicits a marked increase in the alveolar accumulation of surfactant lipids and surfactant protein A (SP-A). The authors examined the accumulation of SP-D using this animal model of alveolar proteinosis. Immunoperoxidase localization of SP-D studies at 2 weeks after silica instillation showed intense staining of intra-alveolar exudates, and cytoplasmic staining of hypertrophic type II cells. Immunoelectron microscopy showed that airspace SP-D was specifically associated with granular material, but not tubular myelin or other membranous structures. SP-D was quantified in bronchoalveolar lavage by immunoassay using antibodies specific for SP-D, and by reversephase HPLC after affinity purification of SP-D on maltosyl-agarose. Within 2 weeks after silica instillation, there was a greater than 45-fold increase in lavage SP-D per lung compared with saline controls, including an almost ten-fold increase in the insoluble or surfactant-associated protein. These studies indicate that the extracellular accumulation of SP-D is markedly increased in silica-induced lipoproteinosis, and that SP-D is associated with amorphous components identified by electron microscopy.

Animals↗
PubMed1990

Surfactant protein D is a divalent cation-dependent carbohydrate-binding protein.

Surfactant protein D (SP-D, CP4) is a collagenous surfactant-associated glycoprotein synthesized by lung type II epithelial cells. SP-D can be selectively and efficiently eluted from isolated rat surfactant with glucose, maltose, and certain other saccharides. We therefore examined the ability of the purified protein to interact with carbohydrates in vitro. Saccharide-substituted bovine serum albumins (BSA neoglycoproteins) were adsorbed to plastic wells, and binding of purified SP-D was quantified with monospecific antibodies to SP-D using an indirect immunoassay. SP-D showed specific calcium-dependent binding to alpha-D-glucosidophenyl isothiocyanate-BSA and maltosyl-BSA, but negligible binding to beta-D-glucosidophenyl isothiocyanate-BSA or unconjugated BSA. The most efficient inhibitors of SP-D binding were alpha-glucosyl-containing saccharides (e.g. isomaltose, maltose, malotriose). SP-D showed quantitative binding to maltosyl-agarose and was specifically eluted with maltose or EDTA. High affinity binding to maltosyl-BSA was also demonstrated using a solution-phase polyethylene glycol precipitation assay. These studies demonstrate that SP-D is a calcium dependent lectin-like protein and that the association of SP-D with surfactant is mediated by carbohydrate-dependent interactions with specificity for alpha-glucosyl residues.

Animals↗
PubMed1990

Pathobiology of pulmonary fibrosis.

Pulmonary fibrosis is characterized by an increase in lung matrix and alterations in the numbers and spatial relationships of lung parenchymal cells. The increase in matrix results from a proliferation and "activation" of fibroblasts (FB) with increased production and deposition of matrix macromolecules at sites of lung injury. Connective tissue cell activation is associated with increased gene expression of collagens, fibronectin, proteoglycans and other matrix components; cytoskeletal alterations; and probably also with changes in the expression of matrix receptors and matrix-degrading enzymes and inhibitors. The fibroproliferative reaction involves the participation of a variety of cytokines and inflammatory mediators by resident and inflammatory cells at sites of lung injury. Thickening of the alveolar wall can result secondary to matrix deposition within the interstitium and as a result of "mural incorporation" of organized airspace exudate. However, marked structural remodeling of the gas-exchange tissues, with the development of honeycomb lung, involves airspace fibrosis and alveolar collapse. The latter processes lead to areas of airspace obliteration secondary to airspace filling, and to fibrous adhesion of collapsed septa. The extent of airspace obliteration is determined largely by the severity or extent of epithelial injury. Although lung fibrosis is usually irreversible, the activated state is reversible after clearance of exudate and reepithelialization. A continuing and seemingly autonomous fibroproliferative reaction can result in the face of ongoing injury and delayed repair.

Animals↗
PubMed1989

Purification and biochemical characterization of CP4 (SP-D), a collagenous surfactant-associated protein.

CP4 is a collagenous glycoprotein (43 kDa, reduced) synthesized by rat type II pulmonary epithelial cells in primary culture (Persson et al., 1988). In order to better characterize this protein, CP4 was isolated from rat bronchoalveolar lavage and EDTA extracts of lung surfactant by adsorption to barium sulfate and elution with sodium citrate followed by reverse-phase HPLC. Amino acid analysis of purified CP4 demonstrated 4-hydroxyproline (Hyp), hydroxylysine (Hyl), and acid-labile components coeluting with Hyl glycosides. In addition, gas-phase amino-terminal microsequencing of two CP4 CNBr peptides demonstrated nonoverlapping collagenous sequences comprised of nine and six Gly-X-Y triplets, containing a total of four residues of Hyp and two of Hyl. There was less than 50% sequence homology of these peptides with the cDNA-derived sequence of the collagenous domain of rat SP-A. Two-dimensional IEF/SDS-PAGE resolved the protein into a charge train of basic isoforms (pI approximately 6-8), similar to those of newly synthesized CP4 and the class D surfactant proteins (Phelps & Taeusch, 1985). Gel filtration of nondenatured CP4 on 4% agarose showed a high apparent molecular mass complex comprised of disulfide-bonded trimers of the 43-kDa subunits. Antibodies to purified lavage CP4 showed specific binding to newly synthesized and surfactant-associated CP4. We propose that CP4 be designated "surfactant protein D" (SP-D) in accordance with an accepted nomenclature for surfactant-associated proteins.

Amino Acid Sequence↗
PubMed1989

On the relationship between carcinogenicity and acute toxicity.

Parodi et al. and Zeise et al. found a surprising statistical correlation (or association) between acute toxicity and carcinogenic potency. In order to shed light on the questions of whether or not it is a causal correlation, and whether or not it is a statistical or tautological artifact, we have compared the correlations for the NCI/NTP data set with those for chemicals not in this set. Carcinogenic potencies were taken from the Gold et al. database. We find a weak correlation with an average value of TD50/LD50 = 0.04 for the non-NCI data set, compared with TD50/LD50 = 0.15 for the NCI data set. We conclude that it is not easy to distinguish types of carcinogens on the basis of whether or not they are acutely toxic.

Animals↗
PubMed1989

An immunohistochemical study of architectural remodeling and connective tissue synthesis in pulmonary fibrosis.

Fibroblasts in healthy adult lung are quiescent, synthesizing little collagen. We studied lung biopsies from 30 patients with pulmonary fibrosis, using immunohistochemistry with monoclonal antibodies against the propeptides of type I collagen to localize fibroblasts actively synthesizing collagen. Adjacent sections were stained with antibodies to type III and IV collagen, fibrin, cytokeratin, plasma fibronectin, or EDIIIa-containing "cellular" fibronectin (cFN). In rapid pulmonary fibrosis, including the proliferative phase of diffuse alveolar damage, organizing pneumonia, and subacute idiopathic fibrosis, collagen-synthesizing cells were numerous in organizing exudate filling airspaces but were also seen in the interstitium of the alveolar walls, interlobular septa, and walls of blood vessels. The new matrix deposited in the airspaces also contained type III collagen and EDIIIa-containing fibronectin. In chronic pulmonary fibrosis, more than half of the biopsies showed foci of collagen synthesis and cFN deposition near the air-tissue interface. The foci were consistently localized outside remnants of basal lamina and therefore within airspaces. The results indicate that (1) fibrosis in chronic idiopathic pulmonary fibrosis results mainly from organization of exudate within airspaces, just as it does after acute lung injury, and (2) during this process, fibroblasts increase their synthesis of collagen and fibronectin coordinately. Foci of active matrix deposition provide evidence for the progressive nature of chronic pulmonary fibrosis.

Antibodies, Monoclonal↗
PubMed1988

CP4: a pneumocyte-derived collagenous surfactant-associated protein. Evidence for heterogeneity of collagenous surfactant proteins.

Type II pneumocytes secrete pulmonary surfactant and are known to synthesize SP-35, a collagenous surfactant-associated protein. Freshly isolated type II cells also synthesize other bacterial collagenase-sensitive and hydroxyproline-containing proteins, including a glycoprotein designated CP4. CP4 was isolated from rat pneumocyte culture medium by immune precipitation with polyclonal antibodies to rat surfactant proteins or by DEAE chromatography and reverse-phase or gel permeation HPLC. CP4 did not cross-react with polyclonal antibodies to SP-35 and was completely resolved from SP-35 by SDS-PAGE (Mr 43K reduced) or isoelectric focusing. Unlike SP-35, which consists of acidic isoforms assembled as disulfide-bonded dimers and multimers, CP4 was secreted as basic isoforms assembled as disulfide-bonded trimers. Differences in primary structure were demonstrated by CNBr and V8 protease peptide mapping. The secretion of both proteins was inhibited by 2,2'-dipyridyl, an inhibitor of posttranslational prolyl and lysyl hydroxylation and collagen triple helix formation. CP4 was isolated from EDTA extracts of rat surfactant. These studies provide evidence for the heterogeneity of pneumocyte-derived collagenous surfactant-associated proteins.

Animals↗
PubMed1987

Collagen-binding proteins secreted by type II pneumocytes in culture.

The alveolar epithelial basement membrane is believed to play important roles in lung development, in maintaining normal alveolar architecture, and in guiding repair following lung injury. However, little is known about the formation of this structure, or of the mechanisms which mediate interactions between the epithelium and specific matrix macromolecules. Since type IV collagen is a major structural component of basement membranes, we investigated the production of type IV collagen-binding proteins by primary cultures of rat lung type II pneumocytes. Cultures were labeled for up to 24 h with 3H-labeled amino acids or [3H]mannose. Soluble collagen-binding proteins which accumulated in the culture medium were isolated by chromatography on collagen-Sepharose and examined by SDS-polyacrylamide gel electrophoresis. The major type IV collagen-binding protein (CBP1) was identified as fibronectin. We also identified a novel disulfide-bonded collagen-binding glycoprotein (CBP2; Mr = 45,000, reduced). This protein was not recognized by polyclonal antibodies to fibronectin, and showed no detectable binding to denatured type I collagen. The protein was resolved from fibronectin and partially purified by sequential chromatography on gelatin and type IV collagen-Sepharose. We suggest that type II pneumocyte-derived collagen-binding proteins contribute to the formation of the epithelial basement membrane and/or mediate the attachment of these cells to collagenous components of the extracellular matrix.

Animals↗
PubMed1987

Tautology or not tautology?

It has been suggested that the good correlations found between carcinogenic potency in mice and in rats could be an artifact. The artifact suggested arises because there are four correlations to consider--interspecies correlations of toxicity, interspecies correlations of carcinogenic potency, and two intraspecies correlations between toxicity and carcinogenic potency--and the existence of any three implies the fourth. It was argued that the intraspecies correlations between toxicity and potency were due to criteria for data selection. Here we discuss the correlations in detail and show that they are principally due to the experimental observation that there are few cases where most or all animals in a bioassay get cancer. We conclude that the correlations between carcinogenic potency are valid.

Animals↗
PubMed1987

Differentiation of proximal bronchogenic carcinoma from postobstructive lobar collapse by magnetic resonance imaging. Comparison with computed tomography.

MRI and CT studies in 18 patients with proximal bronchogenic carcinoma and postobstructive lobar collapse were analyzed retrospectively. The relative abilities of these imaging techniques to identify central tumor by a contour abnormality and to distinguish tumor mass from collapsed lung by CT attenuation values and MRI signal intensities were compared. MRI and CT were equivalent in their ability to identify a contour abnormality, both succeeding in 13 of 18 (72%) patients. CT was more successful than MRI in differentiating tumor mass from collapsed lung. Dynamic computed tomography scanning differentiated tumor from collapsed lung in eight of ten (80%) patients. MRI demonstrated different signal intensities of tumor and collapsed lung in 8 of 18 (44%) patients. T2-weighted images more often separated tumor from collapsed lung than other imaging sequences.

Aged↗
PubMed1986

Synthesis of type IV procollagen in lung explants.

Pulmonary basement membranes are believed to play important roles in lung morphogenesis, in maintenance of lung alveolar architecture, and in repair after pulmonary injury. However, very little is known about the synthesis and matrix deposition of lung basement membrane macromolecules. Accordingly, we have investigated the synthesis of type IV procollagen, the major collagenous component of basement membranes, in slices of adult rat lung. After a 4-h labeling with [3H]proline, type IV procollagen chains were extracted from lung homogenates with 2 M guanidine-HCl and purified by salt fractionation and ion exchange chromatography. The chains comigrated with authentic type IV chains by SDS-PAGE and were selectively coprecipitated with antibodies to type IV collagen. Peptide mapping confirmed their identity as pro alpha 1(IV) and pro alpha 2(IV), and suggested that the chains are predominantly assembled as heteropolymers. There was no evidence for proteolytic processing of the newly synthesized type IV procollagen even though type I and III procollagens were rapidly processed to lower molecular weight intermediates and collagen. Type IV procollagen accounted for approximately 40% of the extracted radiolabeled collagen, suggesting that there may be a relatively high turnover of lung basement membrane collagen in vivo.

Animals↗
PubMed1986

Biosynthesis of interstitial and basement membrane collagens in pulmonary fibrosis.

We have investigated the synthesis and matrix deposition of interstitial and basement membrane collagens in a rat model of bleomycin-induced pulmonary fibrosis. Rats weighing 175 to 200 g were injected intratracheally with bleomycin sulfate (1.5 units) or saline. At 10 days, lungs from bleomycin-treated rats showed multifocal interstitial and intraalveolar fibrosis, a nearly 2-fold increase in total lung hydroxyproline, and a 3-fold increase in the incorporation of [3H]proline into bacterial collagenase-sensitive protein. For characterization of the newly synthesized collagens, lung slices were labeled for 4 h with [3H]proline, homogenized in 4.5 M NaCl, and extracted at 4 degrees C with 2 M guanidine-HCl and/or by reduction and alkylation. Extracts contained type IV procollagen, types I, III, and V collagen, and greater than 95% of the nondialyzable [3H]hydroxyproline. At 10 days, bleomycin-treated rats showed a nearly 2-fold decrease in the proportion of type IV in extracts of the 4.5 M salt residue, a greater than 3-fold increase in incorporation of proline into interstitial collagens, and an increase in the ratio of type I to type III collagen. Total incorporation of proline into type IV procollagen was not significantly increased. These results suggest that the period of maximal collagen synthetic activity observed at 1 to 2 wk after bleomycin administration is associated with a selective increase in the synthesis and accumulation of interstitial collagens.

Animals↗