Biomedical subjects

B J Benson

Publications and source records attributed to B J Benson.

At least 19 recordsLinked to original sources

PubMed1995

Association of surfactant protein C with isolated alveolar type II cells.

Surfactant protein C (SP-C) is a small hydrophobic protein that is synthesized and secreted by alveolar type II cells. The mechanism of clearance of SP-C from the alveolar airspace is not well understood, although previous studies demonstrated that recombinant SP-C instilled into the lungs of spontaneously breathing anaesthetized rats was taken up by type II cells and incorporated into lamellar bodies. The current investigation was undertaken to characterize the interaction of a complex of SP-C and surfactant-like lipids with freshly isolated rat alveolar type II cells under conditions in which the extracellular milieu can be regulated. SP-C was isolated from alveolar proteinosis lavage fluid and radiolabeled with 125I-Bolton-Hunter reagent. The radiolabeled protein retained its ability to facilitate adsorption of phospholipids to an air/liquid interface. Labeled human SP-C associated with isolated type II cells in a concentration-dependent manner that was also dependent upon temperature and time. The association of labeled SP-C with isolated type II cells did not saturate up to 150 micrograms/ml. SP-A significantly enhanced the association of SP-C with isolated type II cells. Under the experimental conditions tested, SP-C was not degraded to TCA-soluble products. These results are consistent with the hypothesis that association or uptake of SP-C by type II cells may be enhanced by SP-A and that like SP-A, SP-C is recycled by type II cells.

Animals↗

PubMed1994

Lung function, surfactant apoprotein content, and level of PEEP in prematurely delivered rabbits.

To study the in vivo activity of the surfactant apoproteins (SP-A, SP-B, SP-C), we administered surfactants with defined apoprotein compositions to prematurely delivered rabbit pups. Rabbits given simple phospholipid mixtures containing dipalmitoylphosphatidylcholine and phosphatidylglycerol supplemented with both SP-B and SP-C or either protein alone had significantly greater lung compliance during ventilation and lung expansion during a quasi-static pressure-volume maneuver than did saline-or lipid-treated controls. The response to the surfactants containing SP-B/C was markedly dependent on the level of end-expiratory pressure used during ventilation. When the rabbits were ventilated with a positive end-expiratory pressure (PEEP) of 4 cmH2O, lung function in the pups treated with SP-B/C was not significantly different from rabbit surfactant-treated controls. Addition of SP-A to the surfactants containing SP-B/C did not significantly further improve lung function if the pups were ventilated with a PEEP of 4 cmH2O. With a lower PEEP of 1 cmH2O, lung function in the pups given surfactants containing SP-B/C was no longer equivalent to the lung function of the rabbit surfactant-treated controls. At the lower PEEP, SP-A significantly improved lung function when it was added to surfactants containing SP-B and SP-C. No beneficial effect of SP-A was seen when the surfactant contained either SP-B or SP-C alone. We conclude that with assisted ventilation that includes a moderate level of PEEP, SP-B and SP-C significantly enhance the effect of a simple phospholipid mixture on the lung function of prematurely delivered rabbits. At lower levels of PEEP the effects of SP-B and SP-C on lung function are markedly reduced but can be restored by the addition of SP-A. Our results are consistent with the existence of cooperative protein-protein interactions in surfactant function in vivo and suggest that the response to a surfactant will be determined by both the ventilation strategy and the surfactant composition. composition.

Animals↗

PubMed1993

Effect of acylation on structure and function of surfactant protein C at the air-liquid interface.

Pulmonary surfactant protein C (SP-C) is a small hydrophobic peptide that is palmitoylated on 2 adjacent cysteine residues. SP-C enhances the adsorption of phospholipids into a monolayer. The function of the acylation is not clear yet. The experiments described in this article were carried out in order to investigate the function of SP-C acylation in (protein-catalyzed) lipid monolayer formation, and in bilayer interactions. Palmitoylated and nonpalmitoylated human recombinant SP-C were used. In addition, a nonacylated SP-C with a Cys-->Ser mutations was included in these studies. In Wilhelmy plate experiments using negatively charged, protein-containing phospholipid monolayers and negatively charged vesicles, CaCl2 was required to obtain a maximal insertion rate of lipids into the monolayer. If the negatively charged phospholipids in the monolayer were replaced by neutral phospholipids, CaCl2 was only required to show a maximal SP-C-catalyzed insertion rate (if the molecule is palmitoylated, but not if nonpalmitoylated proteins were added). In pressure area measurements, the palmitoylated protein showed a different change in pressure as a function of the surface area, as compared with the nonpalmitoylated proteins. Circular dichroism experiments showed that all three proteins had a high content of alpha-helix. All three proteins showed a preferential orientation at the air-water interface, but the palmitoylated protein has an orientation which is more parallel to the monolayer than that of the nonpalmitoylated proteins. It is concluded that acylation of SP-C alters structural and physical properties of this protein.

Acylation↗

PubMed1993

Uptake of pulmonary surfactant protein C into adult rat lung lamellar bodies.

Previous studies have provided evidence that a large proportion of secreted surfactant lipids is taken up from the alveolar air space by type II cells, incorporated into lamellar bodies, and resecreted. Our goal was to characterize the clearance of exogenously administered recombinant surfactant protein C (SP-C) and to determine if SP-C is taken up by type II cells and incorporated into lamellar bodies. SP-C was radiolabeled by alkylation with [3H]iodoacetic acid and retained its ability to enhance phospholipid adsorption to an air-liquid interface. A mixture of 100 micrograms phospholipid radiolabeled with [14C]dipalmitoylphosphatidylcholine and 10 micrograms SP-C was instilled into the lungs of spontaneously breathing anesthetized adult rats. At later times, the lungs were lavaged and subcellular organelles were isolated. The radioactivity of both phospholipids and SP-C (expressed as disintegrations per minute per microgram phospholipid) in lamellar body fractions increased up to 4 h postinstillation and began to decline after approximately 4 h. The results of this study suggest that SP-C and dipalmitoylphosphatidylcholine are taken up promptly from the alveolar air space and are incorporated into lamellar bodies with time courses that do not differ greatly.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1993

Genetically engineered human pulmonary surfactant.

Pulmonary surfactant is a complex array of phospholipids and proteins that specifically interact to lower the surface tension in the alveoli of the lungs. This article reviews pulmonary surfactant proteins A, B, and C. Also surfactant replacement in animal studies is discussed.

Animals↗

PubMed1993

Development and verification of the proximal/marginal plaque index.

The purpose of this study was to compare the ability of experienced and newly trained investigators to use the published Global Plaque Index and a newly developed Proximal/Marginal Index (PMI) that focuses on disclosed plaque at clearly defined proximal and marginal surfaces. Four independent clinical studies were performed, two with each index, on 11 subjects per study. In each study, examinations were conducted before and after brushing, to determine inter- and intra-examiner comparisons over a range of plaque levels. Inter-examiner reliability estimates for the PMI were at a level below Global Plaque index means. Intra-examiner agreement values were similar for both indices. Both scoring systems are considered sufficiently validated to detect product differences in clinical studies.

Clinical Competence↗

PubMed1993

Clinical plaque removal efficacy of three toothbrushes.

A single-blind, randomized clinical study compared plaque removal efficacy of three toothbrush designs under conditions simulating normal use. Ninety (90) subjects with substantially complete dentition used one of the three toothbrushes: Advanced Design Reach, Crest Complete and Oral-B 40. Subjects were examined for plaque before and after a single brushing using the Global Plaque Index to estimate plaque on the entire tooth surface, and the Proximal/Marginal Plaque Index (PMI), a new index, was used to estimate plaque at proximal and marginal surfaces of the teeth. The Advanced Design Reach toothbrush reduced plaque scores significantly more than did the other toothbrushes tested (p < 0.05) using either scoring method. At marginal and proximal sites, combined or separate, Advanced Design Reach toothbrush was significantly more effective than the other toothbrushes in plaque removal and produced significantly more plaque free sites than the other two toothbrushes. Evaluation of all anterior and posterior parts of the dentition with the Global Plaque Index indicated that Advanced Design Reach was superior in removing plaque in these regions. Both plaque indices were highly correlated (correlation coefficient 0.91) indicating excellent consistency by the dental examiner.

Adolescent↗

PubMed1993

Plaque removal efficacy of two children's toothbrushes: a one-month study.

Reach Wonder Grip toothbrush for children was evaluated for plaque removal efficacy and compared to the Colgate Plus Junior (extra soft). The subjects appeared at the test site with overnight (14-18 hours) plaque accumulation. After qualifying for the study, sixty-eight (68) subjects were randomly assigned one of the test brushes. They then participated in the test evaluation at the clinical site wherein plaque levels were measured before and after a one minute brushing. Plaque removal efficacy was evaluated by the Global Plaque Index (percent of tooth surface covered by stained deposit). The subjects then used the assigned toothbrush at home for one month and returned to the test site to repeat the baseline evaluations. After their final plaque evaluations, the subjects were given a new toothbrush from each brand to use at home for one week and the subject and the parent were asked to complete a preference questionnaire. Sixty-seven (67) subjects completed the one month study. After one month's use of twice per day brushing, neither of the test toothbrushes demonstrated any evidence of oral irritation that could be related to toothbrushing. The results indicated that Reach Wonder Grip for Children was significantly more effective (p < 0.05) than Colgate Plus Junior in removing overall (Global) plaque at the initial exam period. The preference questionnaire indicated that the Reach Wonder Grip toothbrush was preferred by both the children and their parents. It is concluded that the new Reach Wonder Grip toothbrush for children is safe and effective for plaque removal.

Child↗

PubMed1992

Reducing white spot lesions in orthodontic populations with fluoride rinsing.

A clinical study was conducted to determine if rinsing frequency with a neutral 0.05% sodium fluoride rinse influenced white spot lesion formation associated with orthodontic brackets. Patients from two private orthodontic practices participated. Each received home-care instructions and were told to use 10 ml of sodium fluoride rinse daily before bedtime. The rinse was supplied free of charge to determine if this affected compliance with its prescribed use. Compliance was measured by recording the number of bottles used by each patient. As assessment of oral hygiene status was conducted, and at the time of debonding, white spot lesions were recorded. Only 13% of the 206 participants fully complied with the rinse protocol; 42% of the subjects used 10 ml approximately every other day; and 45% used the rinse less frequently. A significant dose response relationship was noted in which those who rinsed at least once every other day had fewer lesions (21%) than those who rinsed less frequently (49%). It was concluded that a significant reduction in enamel white spot lesions can be achieved during orthodontic therapy through the use of a 10 ml neutral sodium fluoride rinse. The more closely patients complied with the prescribed use, the more likely they could expect a decrease in the occurrence of lesions.

Adolescent↗

PubMed1991

Lung surfactant protein SP-C from human, bovine, and canine sources contains palmityl cysteine thioester linkages.

Lung surfactant is a complex mixture of lipids and proteins that coats the alveoli to reduce surface tension and prevent airspace collapse. One of the principal protein constituents, surfactant protein C (SP-C), has been characterized following isolation from human, canine, and bovine sources. In each species, this highly hydrophobic protein is composed of 33-35 amino acids, the differences being due to NH2-terminal heterogeneity. A COOH-terminal leucine is conserved throughout. The cysteines in each species were found by fast atom bombardment mass spectrometry to be present as thioesters of palmitic acid. Acylation of recombinant SP-C with palmityl coenzyme A, followed by characterization before and after release of the acyl group with 1,4-dithiothreitol, provided corroborating evidence for the native structure.

Acylation↗

PubMed1990

Binding of calcium to SP-A, a surfactant-associated protein.

SP-A is a lung-specific pulmonary surfactant-associated protein containing a calcium-dependent carbohydrate recognition domain and collagen-like sequence. The protein is a major component of the extracellular form of surfactant known as tubular myelin. SP-A is thought to influence the surface properties of surfactant lipids and regulate the turnover of extracellular surfactant through interaction with a specific cell-surface receptor. These properties of SP-A are dependent on the presence of calcium. We have estimated calcium binding parameters for SP-A from binding data obtained by equilibrium dialysis and gel permeation chromatography. Our results suggest that each SP-A monomer binds two to three calcium ions in conditions chosen as similar to those found in the alveolar lumen. The binding data are best fit to a model incorporating two calcium binding sites with different affinities. Studies with a fragment of SP-A generated by limited proteolysis suggest the higher affinity site for calcium is located in the noncollagenous carboxy-terminal end of SP-A. This region of SP-A contains a carbohydrate recognition domain homologous to other C-type lectins. The binding of calcium to this region of SP-A causes a conformational change as assessed by a small change in the intrinsic fluorescence spectrum and a marked change in the susceptibility to proteolysis. At physiological calcium concentrations, intact SP-A aggregates in a reversible fashion, a property that may be relevant to the formation of tubular myelin.

Animals↗

PubMed1989

Studies of the structure of lung surfactant protein SP-A.

SP-A, a glycoprotein of pulmonary surfactant, consists of an NH2-terminal domain containing a collagen-like sequence and a COOH-terminal domain with sequence homology to several Ca2(+)-dependent lectins. We have compared the size, thermal stability, and secondary structure of recombinant SP-A, the product of a fibroblast line transfected with a single human gene encoding SP-A, with natural SP-A isolated from canine and human lungs. Our results suggest both recombinant and natural SP-A are assembled as large oligomers. More variability in the degree of oligomerization was observed with recombinant human SP-A than with natural canine SP-A. As shown by collagenase digestion, the full assembly of protein subunits was dependent on an intact collagen-like domain. The cysteines in the noncollagen domain of SP-A form intrachain bonds between residues 135-226 and 204-218. The circular dichroism spectra of both recombinant and natural SP-A were consistent with the presence of a collagen-like triple helix. As determined by the change in ellipticity at 205 nm, the thermal transition temperatures of canine, natural human, and recombinant SP-A were 51.5, 52.3, and 42.0 degrees C, respectively. These results suggest differences in the assembly and stability of the natural and recombinant proteins.

Amino Acid Sequence↗

PubMed1989

Regulation of messenger RNAs for the hydrophobic surfactant proteins in human lung.

The pulmonary surfactant proteins SP-B (8,000 D) and SP-C (4,000 D) accelerate surface film formation by surfactant phospholipids. We used cDNA probes to examine regulation of these proteins in human fetal lung. The mRNAs were detectable at 13 wk gestation and increased to approximately 50% (SP-B) and approximately 15% (SP-C) of adult levels at 24 wk. The mRNAs were detected only in lung of 11 dog tissues examined. When human fetal lung was cultured as explants without hormones, SP-B mRNA increased and SP-C mRNA decreased. Exposure for 48 h to glucocorticoids, but not other steroids, increased both SP-B mRNA (approximately 4-fold) and SP-C mRNA (approximately 30-fold) vs. controls. Half-maximal stimulation occurred with 1 nM dexamethasone and 300 nM cortisol for SP-B mRNA and at three- to fivefold higher concentrations for SP-C mRNA. Both stimulation and its reversal on removal of hormone were more rapid for SP-B than for SP-C. Terbutaline and forskolin increased SP-B mRNA but not SP-C mRNA. Levels of both mRNAs were much higher in type II cells than fibroblasts prepared from explants. Thus, the genes for SP-B and SP-C are expressed in vivo before synthesis of both SP-A (28,000-36,000 D) and surfactant lipids. Glucocorticoid induction of SP-B and SP-C mRNAs in type II cells appears to be receptor mediated but may involve different mechanisms.

Animals↗

PubMed1988

Transfer of phospholipids by a protein fraction obtained from canine pulmonary lavage.

Surfactant phospholipid exists in multicompartment pools within the subphase of the lung. Movement among these pools and back into type II alveolar cells may be catalyzed by a phospholipid transfer protein resident in the subphase. We demonstrate here that a protein fraction obtained from canine lung lavage catalyzes the intermembrane transfer of all the major surfactant phospholipids. The protein is probably not derived from serum and is unrelated to surfactant proteins that have already been described.

Animals↗

PubMed1988

Glucocorticoids both stimulate and inhibit production of pulmonary surfactant protein A in fetal human lung.

Pulmonary surfactant is a mixture of phospholipids and proteins which stabilizes lung alveoli and prevents respiratory failure. The surfactant-associated protein of Mr = 28,000-36,000 (SP-A) influences the structure, function (film formation), and metabolism of surfactant. We have characterized glucocorticoid regulation of SP-A and SP-A mRNA in explants of fetal human lung. The time course of response to dexamethasone was biphasic, with early stimulation and later inhibition of SP-A accumulation. Maximal induction of SP-A occurred with 3-10 nM dexamethasone and approximately 300 nM cortisol for 72 hr, and stimulation diminished at higher concentrations. SP-A mRNA accumulation was maximally stimulated at 24-48 hr of exposure to dexamethasone (10 nM) and was generally inhibited by 4-6 days. Stimulation was also observed with cortisone and corticosterone but not with sex steroids, suggesting a receptor-mediated process. When explants were exposed to cortisol for only 24 hr, SP-A content was transiently increased above the level in continuously treated tissue and subsequently was similar to control. The content of SP-A and its mRNA was also increased by dibromo-cAMP, terbutaline, and forskolin, and effects were approximately additive with those of dexamethasone. However, elevated in tracellular cAMP did not alter the biphasic time course or dose-response patterns of dexamethasone. We propose that glucocorticoids have both stimulatory and inhibitory effects on SP-A gene expression. This biphasic regulation is not consistent with generalized toxic effects, product-feedback inhibition, or receptor down-regulation, and it appears to be specific for SP-A among the various surfactant components.

Adult↗

PubMed1987

Divalent cation and hydrogen ion effects on the structure and surface activity of pulmonary surfactant.

The structure and surface activity of the extracellular fraction of pulmonary surfactant known as tubular myelin are Ca2+ dependent. Previous studies have demonstrated surfactant-specific proteins with monomeric molecular weights of 28,000-36,000 (SP28-36) are associated with this fraction. In reassembled lipoprotein mixtures, SP28-36 promotes the Ca2+-induced aggregation and surface activity of surfactant lipids, but the detailed interactions between Ca2+, SP28-36, and surfactant lipids have not been established. In this study, we investigated the effect of various cations on the aggregation of surfactant lipid liposomes in the presence of SP28-36. SP28-36 reduced the threshold ion concentration for liposome aggregation from greater than 10 to 0.5 mM for Ca2+, Ba2+, and Sr2+ but not Mg2+ or Mn2+. The liposome aggregation was reversed by ethylenediaminetetraacetic acid and not associated with leakage of carboxyfluorescein. SP28-36 promoted similar liposome aggregation at pH less than 5 in the absence of divalent cations. Surfactant lipids adsorbed slowly to an air-fluid interface in all ionic conditions unless SP28-36 was present. Both Ca2+ and H+ induced rapid lipid adsorption in the presence of SP28-36. The surface activity of native surfactant had a similar ion dependence. Electron micrographs of native surfactant showed typical tubular myelin structures at pH 7.4 only in the presence of Ca2+. At pH 4.4 in the absence of Ca2+, similar but not identical structures were seen. In the reconstituted system, SP28-36 in the presence of Ca2+ induced the formation of larger multilayered structures including parallel bilayers and small areas of squares and triangles with dimensions similar to structures found in the native material.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

The major lung surfactant protein, SP 28-36, is a calcium-dependent, carbohydrate-binding protein.

SP 28-36, a major protein of pulmonary surfactant, has striking amino acid sequence homology with soluble mannose-binding proteins isolated from rat liver and contains residues common to the carbohydrate-binding domains of other mammalian lectins. We have used carbohydrate-affinity chromatography to investigate carbohydrate-binding properties of SP 28-36 isolated from canine and human (alveolar proteinosis patients) lung lavage. SP 28-36 binds to immobilized D-mannose, L-fucose, D-galactose, and D-glucose. The protein binds only weakly to N-acetyl-D-galactosamine and N acetyl-D-glucosamine. Binding is Ca2+-dependent. The threshold Ca2+ concentration is 0.6 mM and maximal binding occurs with 1 mM Ca2+. Bound protein is quantitatively recovered by elution with 2 mM EDTA. Ba2+, Sr2+, and Mn2+, but not Mg2+, can substitute for Ca2+. Unlike some other mammalian lectins, SP 28-36 binds to carbohydrate at pH 5.0. Recombinant human SP 28-36 isolated from the media of Chinese hamster ovary cells, transfected with a DNA construct encoding SP 28-36, has similar carbohydrate-binding activity to the native proteins. Mannose affinity chromatography of the culture medium of Chinese hamster ovary cells results in an efficient purification of the secreted recombinant human SP 28-36.

Animals↗

PubMed1987

Nucleotide and amino acid sequences of pulmonary surfactant protein SP 18 and evidence for cooperation between SP 18 and SP 28-36 in surfactant lipid adsorption.

Pulmonary surfactant is a lipid-rich material that promotes alveolar stability by lowering the surface tension at the air-fluid interface in the peripheral air spaces. The turnover of surfactant phospholipids in the alveolar space is fast, and several lines of evidence suggest there is rapid formation and replenishment of the phospholipid surface film during normal respiration. Specific proteins may regulate these dynamic surface properties. The predominant surfactant protein is a well-characterized, lipid-associated glycoprotein, SP 28-36 (28-36 kDa). A second group of very hydrophobic proteins has recently been shown to affect the surface activity of surfactant phospholipids. We have isolated this group of hydrophobic proteins, herein called SP 5-18 (5-18 kDa), from canine surfactant and have shown by NH2-terminal sequence analysis that at least two proteins, SP 5-8 and SP 18, are present in this group. We have derived the full amino acid sequence of SP 18 from the nucleotide sequence of the cDNAs identified with oligonucleotide probes that were based on the NH2-terminal amino acids of SP 18. The protein isolated from extracellular surfactant appears to be a fragment of a much larger precursor protein (40 kDa). The amino acid sequence of SP 18 is markedly hydrophobic and contains two possible bilayer-spanning domains. We have shown that SP 18 and the glycoprotein SP 28-36 have a cooperative, calcium-dependent action in promoting the formation of phospholipid surface films.

Amino Acid Sequence↗