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Biomedical subjects

W M Hull

Publications and source records attributed to W M Hull.

33 records · Page 2Linked to original sources
PubMed1991

Lung calcium-dependent phospholipid-binding proteins: structure and function.

Distinct peptide maps of two rabbit lung Ca2(+)-dependent phospholipid-binding proteins (PLBPs), 36,000 and 33,000, were generated by cyanogen bromide (CNBr) cleavage, trypsin or Staphylococcus aureus V8 proteinase digestion. The amino acid sequence of a CNBr-cleaved peptide of the 36,000 PLBP was aligned to the amino terminus of human lipocortin I with more than 77% identity, but had no identity with the known amino terminal sequence of other known annexins. Partial amino acid sequence of a 33,000 PLBP peptide demonstrated a close (56%) relationship to endonexin II, human placental anticoagulant protein, and porcine intestine protein II, but shared only 32% identity with lipocortin I, 30% with lipocortin II. Antiserum generated against purified 36,000 PLBP reacted strongly with the 33,000 PLBP, but did not react with any other rabbit lung cytosolic proteins. Both PLBPs inhibited the phospholipase A2 reaction when dioleoyl phosphatidylcholine and phosphatidylglycerol vesicles or monolayers were used as substrates. In the vesicle assay, the phospholipase A2 reaction was inhibited at lower substrate phospholipid concentrations but not at nearly saturating substrate concentrations. In the monolayer assay, the phospholipid-binding proteins did not inhibit phospholipase A2 at a low phospholipid surface concentration of 3.8.10(-3) molecules/A2, but they did at higher surface concentrations between 1.1 x 10(-2) and 3.8 x 10(-2) molecules/A2. The inhibition of phospholipase A2 by rabbit lung phospholipid-binding proteins is most likely due to the prevention of penetration by phospholipase A2 into the interface, a requirement for the enzyme to act on the substrate.

Amino Acid Sequence↗
PubMed1991

Ontogeny of surfactant proteins A and B in human amniotic fluid as indices of fetal lung maturity.

Surfactant proteins A and B (SP-A and SP-B) were measured in human amniotic fluid by ELISA and correlated with lecithin to sphingomyelin ratio (L/S), phosphatidylglycerol (PG), and perinatal outcome. Amniotic fluid SP-A, SP-B, and L/S increased with advancing gestation. SP-A was detected at 19 wk gestation and increased dramatically in the 3rd trimester of pregnancy. SP-B was first detectable at 31 wk gestation and increased significantly to term. SP-A was a more specific predictor of nonrespiratory distress syndrome (RDS) than L/S or SP-B; however, the sensitivity of SP-A in predicting RDS was less than L/S less than 2.0 (26.3 versus 82.3%, respectively). In 209 pregnancies assessed within 48 h of delivery, the sensitivity of SP-B in predicting RDS (nondetectable SP-B) was comparable to the L/S, however, SP-B = 0 was frequently observed in mature infants, limiting its specificity for prediction of RDS. The greatest sensitivity and specificity were achieved with the measurement of L/S less than 2.0 and negative PG, which correctly predicted 100% of the infants with RDS and 94% of those who did not develop the disorder. Measurement of SP-A or SP-B did not improve the prediction of RDS. SP-A, SP-B, and L/S were not affected by infant sex, Apgar score, rupture of membranes, size for gestational age, maternal diabetes, hypertension, or exposure to medications. SP-A, SP-B, and L/S were significantly elevated in amniotic fluid from black mothers. SP-A was significantly elevated in amniotic fluid from mothers who smoked during pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)

Amniotic Fluid↗
PubMed1991

Failure to detect surfactant protein-specific antibodies in sera of premature infants treated with survanta, a modified bovine surfactant.

Serum from premature infants enrolled in either single-dose or multidose surfactant replacement studies with bovine lung-based exogenous surfactant (Survanta) were analyzed for antibodies reactive with mixtures of bovine surfactant proteins SP-B and SP-C. Sera from 404 premature infants enrolled in single-dose studies and 1024 premature infants enrolled in multidose studies were analyzed, representing a total of 987 samples and 2743 serum samples, respectively. The sera were obtained from treated and control infants at the time of treatment and 1 week, 4 weeks, and 6 months thereafter. Polyclonal antisera generated in rabbits against the small molecular weight proteins were uniformly reactive with the bovine surfactant test antigens; however, antibodies reacting with the surfactant proteins were never detected by immunoblot analysis with the infant sera. Antibodies against common human viral antigens were readily detected in infant serum samples. The horseradish-peroxidase conjugated second antibodies (antihuman immunoglobin G or antihuman immunoglobins A, G, and M) used in the studies were highly reactive with both immunoglobin G and immunoglobin M classes of human antibodies. Therefore there was failure to detect specific immunological responses to the bovine surfactant proteins present in Survanta after single or multiple doses of exogenous surfactant administered in the perinatal period.

Antibodies↗
PubMed1990

Phorbol ester inhibits surfactant protein SP-A and SP-B expression.

Effects of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), on expression of pulmonary surfactant proteins, SP-A and SP-B, were determined in a human pulmonary adenocarcinoma cell line (H441-4). TPA decreased cellular SP-A content in association with decreased de novo synthesis of SP-A as assessed by [35S]methionine incorporation. Effects of TPA were time (0-72 h) and dose (IC50 0.5-1.0 nM)-dependent. Phorbol 12,13-dibutyrate (PDBu), and adenosine 5'-O-(3-thiotriphosphate), and 1-oleoyl-2-acetyl-sn-glycerol also decreased SP-A content in these cells. Characteristics of inhibition of SP-A content by PDBu were similar to those of [3H]PDBu binding to H441-4 cells. Inhibitory effects of TPA on SP-A synthesis were associated with concomitant decreases in SP-A mRNA. Expression of a distinct surfactant protein, SP-B, was also markedly decreased after exposure to TPA. SP-A and SP-B mRNA contents decreased more rapidly after treatment with TPA than after actinomycin D. Actinomycin D completely blocked the rapid decrease in SP-A and SP-B mRNAs caused by the phorbol ester, consistent with the concept that the inhibitory effect of TPA on the surfactant protein mRNAs required continued gene transcription and was not mediated solely by changes in SP-A or SP-B transcription. Inhibitory effects of phorbol esters on SP-A and SP-B synthesis support the concept that protein kinase C modulates surfactant protein expression in this cell.

Adenocarcinoma↗
PubMed1989

Glucocorticoids regulate surfactant protein synthesis in a pulmonary adenocarcinoma cell line.

Synthesis of pulmonary surfactant proteins SP-A, SP-B, and SP-C was demonstrated in a cell line derived from a human adenocarcinoma of the lung. The cells contained numerous lamellar inclusion bodies and formed organized groups of cells containing well-developed junctional complexes and apical microvillous membranes. Synthesis of SP-A was detected in the cells by enzyme-linked immunoabsorbent assay and by immunoprecipitation of [35S]methionine-labeled protein. SP-A was identified as an Mr 31,000-36,000 polypeptide containing asparagine-linked carbohydrate. Northern blot analysis detected SP-A mRNA of 2.2 kb. Dexamethasone (1-10 nM) enhanced the relative abundance of SP-A mRNA. Despite stimulation of SP-A mRNA, intracellular SP-A content was unaltered or inhibited by dexamethasone. SP-B and SP-C mRNAs and synthesis of the SP-B and SP-C precursors were markedly induced by dexamethasone. ProSP-B was synthesized and secreted primarily as an Mr 42,000-46,000 polypeptide. Proteolysis of the proSP-B resulted in the generation of endoglycosidase F-sensitive Mr = 19,000-21,000 and 25,000-27,000 peptides, which were detected both intra- and extracellularly. SP-C proprotein of Mr = 22,000 and smaller SP-C fragments were detected intracellularly but were not detected in the media. Mature forms of SP-B (Mr = 8,000) and SP-C (Mr = 4,000) were not detected. Glucocorticoids directly enhance the relative synthesis and mRNA of the surfactant proteins SP-A, SP-B, and SP-C. Discrepancies among SP-A mRNA, its de novo synthesis, and cell content suggest that glucocorticoid may alter both pre- and posttranslational factors modulating SP-A expression.

Adenocarcinoma↗
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↗
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

Surfactant associated protein (SAP-35) in amniotic fluid from diabetic and nondiabetic pregnancies.

We have recently shown that, with the current management of insulin-dependent diabetes during pregnancy, infants of diabetic mothers are at no greater risk of respiratory distress syndrome (RDS) than an appropriately matched control population. A previous study suggested a selective inhibition of surfactant associated protein of 35,000 daltons (SAP-35) in the amniotic fluid of diabetic pregnancies. In order to determine whether a selective inhibition of SAP-35 occurs in well controlled, insulin-dependent diabetic pregnancies, we compared SAP-35 concentration and lecithin/sphingomyelin (L/S) ratios in amniotic fluid from 30 well controlled, insulin-dependent women with 30 nondiabetic pregnant women pair-matched for gestational age, race, and indication for amniocentesis. Gestational ages ranged from 30-43 weeks, with a mean of 36.5 +/- 2.5 weeks, in both groups. Surfactant associated protein-35 was measured by an enzyme-linked capture immunoassay specific for SAP-35 and its oligomers. Mean +/- SEM SAP-35 was 3.7 +/- 0.4 micrograms/mL (N = 30) in the diabetic group, not significantly different from 5.0 +/- 1.1 micrograms/mL (N = 30) in the control group (P greater than .05). Mean L/S ratios were also not different: 2.4 +/- 0.1 (diabetic) compared with 2.3 +/- 0.1 (control); P greater than .05. The rate of RDS was similar in both groups. We conclude that in well controlled diabetic pregnancies, fetal lung maturation, as assessed by the L/S ratio, SAP-35 concentration, and outcome, is not adversely affected.

Amniotic Fluid↗
PubMed1986

In vitro acetylation of rat pulmonary surfactant-associated glycoprotein(s) A primary translation products.

The primary translation products of pulmonary surfactant-associated glycoprotein(s) A, the major apolipoprotein in mammalian surfactants, exhibit extensive charge heterogeneity. After in vitro translation of poly(A)+ mRNA from rat lung, the primary translation products of glycoprotein(s) A were identified as a charge train of five proteins of 26 kDa (pI 4.6-5.0), the predominant forms being the more acidic members (pI less than 4.8). Inhibition of acetylation during in vitro translation of rat lung poly(A)+ mRNA resulted in a predominance of the more basic isoforms (pI greater than or equal to 4.8). Intracellular forms of glycoprotein(s) A were immunoprecipitated from rat Type II epithelial cells after treatment with tunicamycin or after deglycosylation with endoglycosidase H. Five intracellular precursors consisting primarily of acidic members of the charge train were identified, this being consistent with the intracellular acetylation of the protein. In contrast, canine glycoprotein(s) A translation products consisted of only three proteins of 26 kDa (pI 4.8-5.0), in which most of the radiolabel was concentrated in the more basic components. Acetylation may account for some, but not all, of the charge heterogeneity in the primary translation products and processed forms of surfactant-associated glycoprotein(s) A in the rat.

Acetylation↗
PubMed1986

Immunologic identification of a pulmonary surfactant-associated protein of molecular weight = 6000 daltons.

Hydrophobic, small molecular weight, surfactant-associated protein of Mr = 6000 (SAP-6) was isolated from bovine, canine, and human alveolar lavage and identified by silver staining after sodium dodecyl sulfate polyacrylamide gel electrophoresis gels. Lesser amounts of protein of Mr = 14,000, 20,000, and 26,000 daltons also copurified with SAP-6, likely representing oligomers of the Mr = 6,000 dalton protein. In the absence of sulfhydryl-reducing agents, increased amounts of the larger forms of the protein were observed. Antisera generated against bovine SAP-6 were used to further characterize the protein and distinguish it from the more abundant surfactant-associated glycoprotein of Mr = 35,000 (SAP-35) present in mammalian surfactants. Rabbit antisera generated against the bovine hydrophobic protein recognized SAP-6 and lesser amounts of the proteins of Mr = 14,000, 20,000, and 26, 000 daltons. The SAP-6 antisera were reactive against the hydrophobic proteins from human, bovine, and canine surfactants as assessed by immunoblot analysis after sodium dodecyl sulfate polyacrylamide gel electrophoresis. SAP-6 antisera did not detect bovine SAP-35 the abundant surfactant-associated glycoprotein, by immunoblot analysis; however, some reactivity of the anti-SAP-6 was detected against purified bovine SAP-35 by a sensitive enzyme-linked immune-adsorbant assay. Anti-SAP-6-did not react with bovine serum components either by immunoblot or by enzyme-linked immune-adsorbant assay. Monospecific antisera generated against bovine SAP-35 did not detect SAP-6 by immunoblot analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Intracellular and oligomeric forms of surfactant-associated apolipoproteins(s) A in the rat.

Sulfhydryl-dependent oligomeric forms of the surfactant-associated apolipoprotein(s) A, obtained from particulate preparations of adult rat lung lavage, were characterized by immunoblot analysis and by silver staining of proteins separated by one- and two-dimensional SDS-polyacrylamide gel electrophoresis. Under non-reducing conditions, these proteins migrated as oligomers, Mr approx. 50-70, 115, 160 kDa and greater. The large oligomers were reduced to the apolipoprotein(s) A subunits by treatment with beta-mercaptoethanol; Mr 38 (A3), 32 (A2) and 26 kDa (A1), pI 4.2-4.8. Mr 50 kDa protein was composed of sulfhydryl-dependent homo-dimers of protein(s) A1 (Mr 26 kDa). 55 kDa protein was a hetero-dimer composed primarily of A1 and A2 (Mr 26 and 32 kDa). 62 kDa protein was composed of hetero-dimers of A3 and apolipoprotein A2 (Mr 38 and 32 kDa). 70 kDa protein was a homodimer composed of apolipoprotein A3 A3 (38 kDa). Larger molecular forms were composed primarily of 38 and 32 kDa and lesser amounts of 26 kDa. Treatment with endoglycosidase F reduced A2 and A3 to 26 kDa. Apolipoprotein A1 co-migrated with a protein of Mr 26 kDa immunoprecipitated from [35S]methionine-labelled Type II epithelial cells. Chymotryptic-tryptic peptide maps of apolipoproteins A1, A2 and A3 were identical, suggesting that apolipoproteins A3 and A2 arise through extensive glycosylation of apolipoprotein A1.

Animals↗
PubMed1985

Identification of canine pulmonary surfactant-associated glycoprotein A precursors.

Surfactant-associated glycoproteins A were identified by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude surfactant from canine alveolar lavage: an unglycosylated form (protein A1), 27,000-28,000 daltons; glycoprotein A2, 32,000-34,000 daltons; and glycoprotein A3, 37,000-38,000 daltons; pH at isoelectric point (pI) 4.5-5.0. Glycoproteins A2 and A3 were electroeluted and used to prepare a monospecific antiserum that identified proteins A1, A2, and A3 in immunoblots of crude surfactant obtained from dog lung lavage. This antiserum precipitated several proteins from in vitro translated canine lung poly(A)+ mRNA; proteins of 27,000 daltons, pI 5.0, and 28,000 daltons, pI 4.8-5.0, which precisely comigrated with proteins A1 from canine surfactant. Cotranslational processing of the primary translation products by canine pancreatic microsomal membranes resulted in larger proteins of 31,000-34,000 daltons, pI 4.8-5.0. Treatment of these processed forms of glycoprotein A with endoglycosidase F, to remove N-linked carbohydrate, resulted in proteins of 27,000-28,000 daltons which precisely comigrated with surfactant protein A1. These observations demonstrate that the polypeptide precursors to the glycoproteins A complex are extensively modified by addition of asparagine N-linked complex carbohydrate and are subsequently secreted as glycoproteins A2 and A3.

Animals↗
PubMed1985

Activation of cAMP dependent protein kinase during surfactant release from type II pneumocytes.

Release of surfactant from pulmonary type II epithelial cells was stimulated by the beta-adrenergic agonist terbutaline and the diterpene forskolin. Cytosolic cyclic adenosine monophosphate (cAMP) concentrations increased significantly following exposure to terbutaline or forskolin and reached maximal levels within 5 min after treatment. Terbutaline and forskolin had a synergistic effect on cytosolic cAMP levels when added simultaneously. cAMP-dependent protein kinase activity was identified in cytosolic preparations of type II pneumocytes by phosphorylation of the peptide substrate Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) and binding of 3H-cAMP to the regulatory components of cAMP-dependent protein kinase. Type I and type II regulatory subunits of the cANP-dependent kinase were present in approximately equal concentrations in type II cell cytosol. Activation ratio of cAMP-dependent protein kinase in cultured type II cells increased significantly in the presence of terbutaline, forskolin, or terbutaline plus forskolin. Activation ratios increased from 0.45 +/- 0.03 for control cells to 0.96 +/- 0.06 for cells exposed to terbutaline (10 microM) plus forskolin (5 microM) for 20 min. Release of 3H-phosphatidylcholine was also stimulated by terbutaline and forskolin. Effects of terbutaline and forskolin on surfactant release were approximately additive. Our results demonstrated increased cytosolic cAMP levels, increased cAMP-dependent protein kinase activation ratios, and subsequent augmented surfactant release from isolated type II pneumocytes in response to terbutaline and forskolin. These data support a role for activation of cAMP-dependent protein kinase as a mediator of surfactant release and document the utility of forskolin for study of cAMP-mediated effects in isolated type II cells.

Animals↗
PubMed1980

Measurement of histamine: a quality control study.

This study evaluated the ability of various laboratories to accurately quantitate histamine in samples of plasma and buffered saline that contained known amounts of histamine. Histamine was dissolved in buffered saline and in plasma and these solutions were lyophilized glass ampules. Sealed ampules were sent to laboratories for analysis of their histamine content by one or more of the following methods: (1) the double-isotope dilution enzymatic method, (2) single-isotope enzymatic method, and (3) manual and automated fluorometry. We analyzed the same solutions in our laboratory by the double-isotope dilution enzymatic method over a 6-mo period; we found a coefficient of variation averaging 26% for these samples over that period of time and our values agreed with theoretical values within 12%. Twelve laboratories analyzed histamine by the double-isotope dilution enzymatic assay. The results revealed a marked variation among laboratories both for the determination of histamine in buffer and, more strikingly, for the determination of histamine in plasma. Three laboratories determined histamine by the single-isotope dilution enzymatic method and one reported results rather close to the standard, while the others reported results that were clearly different from the standards. Five laboratories measured histamine by automated fluorometry and three by manual fluorometry; again, there was marked variation among the results. Overall, these findings indicate that measurements of histamine by different laboratories vary greatly. Thus, absolute values for histamine in biologic specimens in the literature must be regarded with caution.

Chemical Phenomena↗