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Biomedical subjects

T E Weaver

Publications and source records attributed to T E Weaver.

At least 55 records · Page 3Linked to original sources
PubMed1993

Expression of the cyclin-dependent kinase cdk4 in perinatal and adult rat lung.

The identification of numerous cyclin-dependent kinases (cdk) and G1 cyclins suggests that cell cycle progression through G1/S may be controlled in a tissue-specific manner by various cdk/cyclin complexes. In situ hybridization was used to characterize expression of the cyclin-dependent kinase cdk4 in prenatal and postnatal rat lung and other tissues and to determine whether cdk4 expression is limited to proliferating cells, identified by BrdU incorporation and cdk1 mRNA expression. cdk4 co-localized with cdk1 in proliferating cells of both prenatal and postnatal lung and other tissues, consistent with an SPF function that is not tissue-specific. The distribution of cdk1 and cdk4 expression was identical in fetal rat tissues and was detected in lung parenchyma and throughout the airway. Pulmonary cell proliferation declined with increasing postnatal age and could be found only in focal areas of day 21 terminal and respiratory bronchiolar epithelium. Proliferation was undetectable in adult lung. Postnatal cdk4 expression was not restricted to cells expressing cdk1: cdk4 was evenly distributed in bronchiolar epithelium and was present throughout the airway and alveolar septae of day 21 lung. Expression of cdk4 was also maintained in adult bronchiolar epithelium. These studies demonstrate that although the expression of cdk1 is tightly correlated with proliferative capacity, the expression of cdk4 is not limited to proliferating cells, suggesting that cdk4 may have additional cell-specific functions unrelated to cell cycle progression.

Animals↗
PubMed1992

Surfactant protein C precursor is palmitoylated and associates with subcellular membranes.

Surfactant protein C (SP-C) is a 3.7 kDa, hydrophobic protein that enhances the adsorption of phospholipids in pulmonary surfactant. SP-C is generated by proteolytic processing of a 21 kDa precursor. Murine fetal lung explant cultures and a Chinese hamster ovary cell line expressing recombinant human SP-C gene (CHO/SPC) were used to determine the subcellular location and post-translational modification(s) of proSP-C. After in vitro translation, proSP-C of Mr = 21,000 was generated. ProSP-C was associated with canine pancreatic microsomes during co-translation and was partially protected from digestion with proteinase K, supporting the concept that proSP-C enters but does not completely traverse the membrane of the endoplasmic reticulum (ER). Association of proSP-C isoforms of 21 and 26 kDa with intracellular membranes was demonstrated by subcellular fractionation of CHO/SPC cells. Pulse/chase experiments demonstrated that the 21 kDa SP-C proprotein was synthesized first and after 15 min was modified to produce a 26 kDa isoform in CHO/SPC cells or a 24 kDa isoform in murine fetal lung. Both the 21 and 26 kDa proSP-C isoforms were detected after labelling CHO/SPC cells with [3H]palmitic acid. The formation of the 26 kDa proSP-C isoform in CHO/SPC cells and the 24 kDa proSP-C isoform in murine fetal lung was blocked by cerulenin, an inhibitor of fatty acid synthesis. In conclusion, proSP-C is associated with subcellular membranes. ProSP-C is palmitoylated and undergoes additional post-translational modification that is blocked by an inhibitor of fatty acid synthesis.

Animals↗
PubMed1992

Binding, uptake, and localization of surfactant protein B in isolated rat alveolar type II cells.

This study reports the ability of rat alveolar type II cells to internalize mature bovine surfactant protein B (SP-B) in vitro. Isolated type II cells were incubated with labeled SP-B, and binding and internalization were studied biochemically and morphologically. Biochemical analyses demonstrated a time-dependent association of 125I-labeled SP-B with type II cells; binding steadily increased through 4 h and then remained constant through 20 h of incubation. The association of [3H]SP-B with type II cells was characterized via light and electron microscopic autoradiography. Significant quantities of [3H]SP-B were found at the plasma membrane, in the endocytic pathway, and in lamellar bodies. The pathway of SP-B internalization was not altered by the presence of whole rat surfactant; however, the quantity of SP-B internalized into lamellar bodies was increased. 3[H]SP-B was not associated with coated pits and colocalized with horseradish peroxidase (HRP), consistent with receptor-independent internalization. Cell-associated SP-B was not degraded and was detected in lamellar bodies undergoing exocytosis. These results suggest that SP-B may follow a recycling pathway similar to that previously reported for surfactant phospholipids.

Animals↗
PubMed1992

Processing of surfactant protein B proprotein by a cathepsin D-like protease.

Surfactant protein B (SP-B) is a hydrophobic peptide of relative molecular weight (M(r)) = 8,000 that is associated with pulmonary surfactant phospholipids. SP-B is synthesized by the alveolar type II epithelial cell as a proprotein of M(r) = 42,000 which requires at least two proteolytic cleavages to generate the 79 residue mature SP-B peptide. We have previously reported that cleavage of the NH2-terminal propeptide, to generate a processing intermediate of M(r) = 25,000, occurs in close temporal approximation to secretion. In the present study we demonstrate that SP-B proprotein, isolated from stably transfected Chinese hamster ovary cells, is processed to M(r) = 25,000 by a crude type II cell membrane fraction but not by intact type II cells or type II cell conditioned media. In vitro processing of the proprotein by the type II cell membrane preparation resulted in release of a single peptide of M(r) = 16,000-17,000, which was detected by antiserum directed against antigenic epitopes in propeptide of the precursor. SP-B processing activity was extracted by Na2CO3 lysis of the type II cell membrane preparation, had a pH optimum of 5.0-6.0, and was inhibited by 10(-7) M pepstatin A, suggesting that the NH2-terminal peptide of the precursor is cleaved by an aspartyl protease. Consistent with this hypothesis, processing of SP-B by a crude type II cell membrane preparation was blocked by antiserum directed against the aspartyl protease cathepsin D; further, purified cathepsin D efficiently processed the SP-B precursor to M(r) = 25,000. Collectively these results suggest that cleavage of the NH2-terminal propeptide of the SP-B precursor is mediated by cathepsin D or a cathepsin D-like protease localized within the secretory pathway of the type II epithelial cell.

Animals↗
PubMed1992

Intracellular processing of pulmonary surfactant protein B in an endosomal/lysosomal compartment.

The hydrophobic surfactant protein B (SP-B) is synthesized in alveolar type II cells as a 40-kDa precursor protein that is processed via 39- and 23-kDa intermediates to the mature 8-kDa size. To determine the site of SP-B processing, the subcellular distribution of the precursor and mature forms of SP-B was investigated on ultrathin cryosections of human lung using two polyclonal antibodies that discriminate between precursor forms and the mature form of SP-B. An antibody against the human lysosomal membrane glycoprotein CD63 was used to identify organelles in the endosomal/lysosomal pathway. Precursor SP-B was present in the endoplasmic reticulum, the Golgi complex, and multivesicular bodies but was absent from lamellar bodies and plasma membrane. Mature SP-B was present in multivesicular bodies and lamellar bodies but almost completely absent in the endoplasmic reticulum and the Golgi complex. Semiquantitative evaluation of the immunogold labeling by counting the gold particles representing precursor or mature SP-B in the different compartments showed that the mature-to-precursor ratio was low in the endoplasmic reticulum and the Golgi complex (0.13 and 0.11, respectively), increased in the multivesicular bodies to 3.4, and was very high (65) in lamellar bodies. Multivesicular bodies and lamellar bodies contain the lysosomal membrane marker CD63 and are therefore part of the lysosomal pathway. These data strongly suggest that precursor SP-B is proteolytically processed to its mature 8-kDa form intracellularly in an endosomal/lysosomal compartment, most probably in multivesicular bodies.

Antigens, CD↗
PubMed1991

Increased expression of pulmonary surfactant proteins in oxygen-exposed rats.

Exposure of adult rats to 85% ambient oxygen increased the content of surfactant proteins SP-A, SP-B, and SP-C recovered from alveolar lavage. The surfactant proteins increased during 1 to 7 d of oxygen exposure. The increased surfactant protein was associated with increased relative abundance of mRNA encoding each of the proteins in lung tissue. Exposure to hyperoxia progressively increased the amounts of the surfactant proteins in alveolar lavage fluid as estimated by immunoblot analysis after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The mRNAs encoding SP-A (1.7 and 1.0 kb), SP-B (1.6 kb), and SP-C (0.9 kb) increased significantly after oxygen exposure for 5 d. The present findings support the concept that oxygen exposure mediates surfactant protein expression at a pretranslational level.

Animals↗
PubMed1989

In vitro translation, post-translational processing and secretion of pulmonary surfactant protein B precursors.

Surfactant proteolipid SP-B is a hydrophobic protein of Mr = 8000 identified in organic solvent extracts of pulmonary surfactant. Analysis of the human SP-B RNA predicts that the active surfactant peptide is derived by proteolysis of an Mr = 40,000 precursor. In the present work, characteristics of synthesis, secretion and processing of SP-B were demonstrated in a pulmonary adenocarcinoma cell line by immunoprecipitation of radiolabelled precursors. Treatment of cells with tunicamycin resulted in synthesis and secretion of unglycosylated proSP-B of Mr = 39,000. Immunoprecipitation of protein produced by in vitro translation of human lung poly(A)+ RNA detected an Mr = 40,000 protein; the size discrepancy is likely related to cleavage of a leader signal sequence. Endoglycosidase-H-sensitive precursors of Mr = 41,000-43,000, pI = 5.1-5.4 were the first isoforms detected within the cells and were processed to endoglycosidase-H-resistant isoforms and secreted. Neuraminidase and endoglycosidase-F-sensitive forms of proSP-B were first detected in the media at 60 min as Mr = 42-46,000 isoforms with pI = 4.6-5.1. Proteolytically processed isoforms of proSP-B were detected primarily in the media and were generated by cleavage of an amino-terminal Mr = 16,000 peptide resulting in Mr = 27,000-33,000 isoforms (pH = 5.6-6.8). The Mr = 27,000-33,000 isoforms were sensitive to neuraminidase, resulting in isoforms with pH = 6.0-6.8. Digestion of the Mr = 27,000-33,000 peptide with endoglycosidase-F resulted in isoforms of Mr = 23,000, pH = 6.0-6.8. The endoglycosidase-F-resistant peptide of Mr = 16,000, pI = 4.2-4.4 was identified with an antiserum generated against synthetic peptides derived from the amino-terminal domain, as deduced from the SP-B DNA sequence. Further proteolytic processing of the Mr = 27,000-33,000 isoforms to the Mr = 8000 peptide detected in surfactant was not observed in this cell line. Thus, in the H441-4 cells (a cell line with morphologic features of Clara cells), SP-B is synthesized as a preproprotein which undergoes cleavage of a signal sequence and addition of asparagine-linked carbohydrate; proSP-B is secreted by processes which are independent of glycosylation. SP-B peptides of Mr = 27,000-33,000 and Mr = 16,000, representing carboxy and amino-terminal domains, accumulate in the media.

Electrophoresis, Polyacrylamide Gel↗
PubMed1989

Processing of hydrophobic pulmonary surfactant protein B in rat type II cells.

The amino acid sequence of surfactant protein B (SP-B), derived from human genomic and cDNA sequences, indicates that the active polypeptide is contained within the sequence of a preproprotein of 381 residues. Synthesis of mature SP-B, which requires proteolytic processing at both the NH2- and COOH-termini of the proprotein, was studied in primary cultures of rat alveolar type II epithelial cells. Type II cells were pulse labeled with [35S]methionine-cysteine for 15-30 min and chased for 0-18 h. SP-B proprotein (Mr = 42,000) accumulated in the medium at early time points but declined at later time points suggesting extracellular proteolysis of the proprotein. In contrast, surfactant protein A (SP-A), another surfactant protein, continued to accumulate extracellularly during this time period. A proteolytic fragment of SP-B (Mr = 25,000) accumulated in the medium with a slightly slower time course, consistent with extracellular proteolysis of the proprotein. Intracellular processing of SP-B was also detected. SP-B polypeptides of Mr 8,000 and 12,000 were detected intracellularly and in the medium at late time points. These forms of SP-B (Mr = 8,000 and 12,000) comigrated in two-dimensional isoelectric-focusing sodium dodecyl sulfate-polyacrylamide gel electrophoresis with mature SP-B isolated from rat alveolar lavage fluid. The mature form of SP-B (Mr = 8,000), but not the Mr 42,000 and 25,000 SP-B precursors, was also detected in lamellar bodies isolated from rat lung homogenates. These experiments demonstrate complete proteolytic processing of prepro-SP-B to the alveolar Mr 8,000 form by type II epithelial cells in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

cDNA, deduced polypeptide structure and chromosomal assignment of human pulmonary surfactant proteolipid, SPL(pVal).

In hyaline membrane disease of premature infants, lack of surfactant leads to pulmonary atelectasis and respiratory distress. Hydrophobic surfactant proteins of Mr = 5,000-14,000 have been isolated from mammalian surfactants which enhance the rate of spreading and the surface tension lowering properties of phospholipids during dynamic compression. We have characterized the amino-terminal amino acid sequence of pulmonary proteolipids from ether/ethanol extracts of bovine, canine, and human surfactant. Two distinct peptides were identified and termed SPL(pVal) and SPL(Phe). An oligonucleotide probe based on the valine-rich amino-terminal amino acid sequence of SPL(pVal) was utilized to isolate cDNA and genomic DNA encoding the human protein, termed surfactant proteolipid SPL(pVal) on the basis of its unique polyvaline domain. The primary structure of a precursor protein of 20,870 daltons, containing the SPL(pVal) peptide, was deduced from the nucleotide sequence of the cDNAs. Hybrid-arrested translation and immunoprecipitation of labeled translation products of human mRNA demonstrated an Mr = 22,000 precursor protein, the active hydrophobic peptide being produced by proteolytic processing to Mr = 5,000-6,000. Two classes of cDNAs encoding SPL(pVal) were identified. mRNA of approximately 900 bases was identified on Northern analysis of fetal and adult RNA. Human SPL(pVal) mRNA was more abundant in the adult than in fetal lung. The SPL(pVal) gene locus was assigned to chromosome 8.

Amino Acid Sequence↗
PubMed1988

Identification of surfactant proteolipid SP-B in human surfactant and fetal lung.

Surfactant proteolipid (SP-B) is one of several hydrophobic peptides detected in organic extracts of pulmonary surfactant and associated with the dramatic surface-active properties of surfactant phospholipids. In the present study human SP-B was identified as a protein with a relative molecular weight (Mr) of 7,500-8,000 under reducing conditions; protein of Mr 18,000 was detected under nonreducing conditions by immunoblot analysis of organic extracts of bovine and human surfactant utilizing an antiserum directed against a 60-amino acid synthetic SP-B peptide. This peptide antiserum was subsequently used to identify SP-B in explant cultures of 18- to 23-wk gestation human fetal lung. Immunoprecipitation of explants labeled with [35S]methionine after 48 h of culture identified proteins of Mr 40,000-42,000, 25,000, and 18,000 after electrophoresis under nonreducing conditions. The Mr 18,000 form was reduced to Mr 7,500-8,000 in the presence of beta-mercaptoethanol. These molecular forms likely represent the SP-B precursor protein, a proteolytic intermediate, and the mature SP-B peptide, respectively. Immunocytochemistry with the peptide antiserum localized SPL(Phe) in granular inclusions in the apical region of type II-like epithelial cells, a pattern of staining similar to that observed for the major surfactant-associated protein of Mr 26,000-38,000 (SP-A). SP-B is a novel pulmonary surfactant-associated protein that is synthesized by the human alveolar type II epithelial cell as an Mr 40,000-42,000 precursor that is subsequently proteolytically processed to Mr 7,500-8,000.

Bronchoalveolar Lavage Fluid↗
PubMed1988

Structure and function of pulmonary surfactant proteins.

In summary, the isolation and characterization of three surfactant proteins have considerably changed our understanding of the nature of pulmonary surfactant and its metabolism. The isolation of the cDNAs and genes encoding the proteins and the elucidation of their structure now makes possible the generation of surfactant proteins for further study and for therapy of surfactant deficient states. Artificial surfactant consisting of appropriately modified human proteins can be produced by recombinant DNA technology. Mixed with appropriate synthetic phospholipids, these proteins may be useful for the treatment of infants with hyaline membrane disease. Significant progress has been made in identifying and characterizing these three more abundant proteins; however, many questions regarding the functions of numerous other proteins present in surfactant (whether produced by Type II cells, Type I cell, Clara cell, or others) remain unanswered. Knowledge of the factors controlling developmental expression of surfactant phospholipids and protein as well as the molecular basis of the interactions among the surfactant proteins and phospholipids may lead to new strategies for therapy of hyaline membrane disease. Major questions regarding the site and nature of posttranslational modification of the surfactant proteins, their sites of assembly with lipids, and their precise roles in the metabolism of surfactant in vitro remain to be clarified. It is hoped that answers to these questions will facilitate the identification and design of appropriate therapy of infants and adults with pulmonary disease associated with surfactant deficiency.

Animals↗
PubMed1987

Glucocorticoid enhances surfactant proteolipid Phe and pVal synthesis and RNA in fetal lung.

Two newly described surfactant proteolipids (SPL), Phe and pVal, are produced by proteolytic processing of distinct precursors of Mr = 40,000 and 22,000, respectively. These proteins are structurally related and intimately associated with surfactant phospholipids. We now demonstrate the expression of both SPL(Phe) and SPL(pVal) in explants of human fetal lung from 16-24 weeks of gestation. Content, synthesis, and mRNA for the proteolipids were low prior to organ culture of fetal lung. Induction of synthesis of the proteolipids occurred rapidly in explant culture in the absence of exogenous hormones and was enhanced by addition of dexamethasone. Increased synthesis of the proteolipids was detected by enzyme-linked immunosorbent assay and by [35S]methionine incorporation into the glycosylated Mr = 40,000-43,000 SPL (Phe) precursor. The response to dexamethasone occurred rapidly and contrasted with effects of dexamethasone on the expression of surfactant-associated protein- (SAP) 35, a distinct surfactant glycoprotein. 8-Br-cAMP did not significantly increase proteolipid content but markedly increased synthesis of SAP-35 in identical cultures. Increased proteolipid content was associated with increased mRNA for each protein as determined by the Northern blot analysis. Proteolipid RNA was also increased by 8-Br-cAMP, however, not to the extent observed with the glucocorticoid. Immunohistochemical analysis of fetal lung with anti-proteolipid antiserum confirmed that the dexamethasone-enhanced synthesis of the proteins by Type II epithelial cells. The time and hormone dependence of the regulation of expression of both SPL(Phe) and SPL(pVal) precursors were distinct from that of SAP-35. Expression of the surfactant proteolipids increased during explant culture of human fetal lung and was further enhanced by glucocorticoid. Developmental and hormonal regulation of the surfactant proteolipids may be important factors in surfactant function at birth.

Electrophoresis, Polyacrylamide Gel↗
PubMed1987

In vitro sulfation of pulmonary surfactant-associated protein-35.

Surfactant-associated protein-35 consists of a group of phospholipid-associated proteins of 26-36 kDa isolated from pulmonary alveolar surfactant. In the rat, surfactant-associated protein-35 is synthesized from 26-kDa primary translation products which are cotranslationally acetylated and glycosylated to heterogeneous 30 and 34 kDa forms. High-mannose oligosaccharide-containing precursors of surfactant-associated protein-35 are processed in the rough endoplasmic reticulum and Golgi to complex-type oligosaccharides, resulting in a mature glycoprotein which exhibits extensive charge heterogeneity in two-dimensional isoelectric focusing SDS-polyacrylamide gel electrophoresis. Much of this charge heterogeneity is related to terminal sialylation of the two asparagine-linked oligosaccharides. In the present study, we report that surfactant-associated protein-35 is also sulfated. Sulfation of the 30 and 34 kDa forms of surfactant-associated protein-35 was clearly detected in primary cultures of rat Type II epithelial cells. These sulfated isoforms were sensitive to endoglycosidase F digestion, but resistant to neuraminidase, suggesting that sulfation occurred at oligosaccharide residues other than sialic acid. The lack of sulfation of the 26 kDa forms of surfactant-associated protein-35 and the resistance of the sulfated isoforms to endoglycosidase H digestion are consistent with Golgi-associated sulfation of the complex type oligosaccharides of surfactant-associated protein-35. Thus, sulfation is another component of the complex post-translational processing of surfactant-associated protein-35, which includes acetylation, hydroxylation, glycosylation, sialylation, sulfhydryl-dependent oligomerization and sulfation.

Animals↗
PubMed1987

Differential effects of epidermal growth factor and transforming growth factor-beta on synthesis of Mr = 35,000 surfactant-associated protein in fetal lung.

Differentiation of pulmonary Type II epithelial cells in late gestation is associated with the synthesis of pulmonary surfactant required for adaptation to air breathing at birth. In the present work, induction of synthesis of a Type II epithelial cell protein, surfactant-associated glycoprotein of Mr = 35,000 (SAP-35) was studied in human fetal lung tissue obtained at 15-24 weeks of gestation. SAP-35 content increased during organ culture in the absence of exogenous hormones. Epidermal growth factor or triiodothyronine stimulated the induction of SAP-35 synthesis during culture. Stimulation by epidermal growth factor (EGF) was detected as early as 2 days and persisted for up to 5 days in culture. Response to EGF was dose-dependent (0.01-10 ng/ml) and was associated with enhanced incorporation of [35S]methionine into immunoprecipitable SAP-35. Increased SAP-35 synthesis was associated with increased SAP-35 RNA as assessed by Northern blot and hybridization assays with human SAP-35 cDNA. Effects of EGF were comparable to the induction of SAP-35 synthesis by 8-bromo-cAMP. In contrast to the stimulatory effect of EGF and triiodothyronine, SAP-35 content was inhibited by transforming growth factor-beta. Both the stimulatory and inhibitory effects of these agents on SAP-35 content were associated with concomitant changes in SAP-35 synthesis. These findings demonstrate multihormonal control of SAP-35 expression and strongly implicate both EGF and transforming growth factor-beta in the regulation of surfactant apoprotein synthesis.

DNA↗
PubMed1987

Induction of surfactant protein in fetal lung. Effects of cAMP and dexamethasone on SAP-35 RNA and synthesis.

Synthesis of surfactant-associated glycoprotein of Mr = 30,000-35,000 (SAP-35) was induced in explant culture of human fetal lung obtained from 8 to 24 weeks of gestation. SAP-35 synthesis and content increased markedly during 1-5 days in organ culture in association with the morphologic maturation of Type II epithelial cells and the appearance of lamellar bodies. [35S] Methionine labeling of the explants and subsequent immunoprecipitation of SAP-35 demonstrated distinct high-mannose precursors and sialylated SAP-35 forms as early in culture as SAP-35 synthesis was detectable. The increase in SAP-35 synthesis was associated with increased SAP-35 RNA of 2.1 kilobases as assessed by hybridization assay with [32P]cDNA specific for human SAP-35. Specific SAP-35 RNA increased during organ culture and both SAP-35 content and SAP-35 RNA increased in the absence of exogenous hormones in 2% carbon-stripped fetal calf serum. SAP-35 content and synthesis was stimulated by 8-Br-cAMP. Addition of 100 microM 8-Br-cAMP, enhanced both the concentration of SAP-35 protein and the SAP-35 RNA as assessed by hybridization assay. In contrast, treatment of the explants with dexamethasone was associated with decreased SAP-35 protein synthesis, SAP-35 content, and decreased SAP-35 RNA levels compared to untreated explants. Inhibition by dexamethasone occurred at all gestational ages tested, was dose-dependent, and detectable within 24-48 h during organ culture. Dexamethasone significantly inhibited both basal and cAMP-induced SAP-35 synthesis. Induction of pulmonary surfactant protein (SAP-35) synthesis during organ culture of human fetal lung was associated with increased SAP-35 RNA. SAP-35 synthesis and SAP-35 RNA were inhibited by dexamethasone and enhanced cAMP.

8-Bromo Cyclic Adenosine Monophosphate↗