Biomedical subjects

R J Mason

Publications and source records attributed to R J Mason.

At least 145 records · Page 8Linked to original sources

PubMed1988

Idiopathic pulmonary fibrosis. Abnormalities in bronchoalveolar lavage fluid phospholipids.

Bronchoalveolar lavage has been used to sample cells and proteins in the distal lung. One of the major secretory products of the alveolar type II epithelial cells, pulmonary surfactant, can be recovered by lavage. Abnormalities in alveolar type II cells are found in biopsies of patients with idiopathic pulmonary fibrosis (IPF), and abnormalities of pulmonary surfactant phospholipids have been reported after diffuse lung injury in animals and in humans. Therefore, we questioned if abnormalities in lavage phospholipids might also occur in IPF, a chronic inflammatory disease of the alveolar epithelium and interstitium, and, if present, would these abnormalities reflect histopathologic changes or predict responsiveness to therapy. Fifteen untreated patients with IPF, diagnosed by open lung biopsy, were studied and were found to have less than half the amount of bronchoalveolar lavage phospholipid as that recovered from healthy volunteers (p less than 0.05). In addition, patients with IPF had a lower proportion of phosphatidylglycerol and a higher proportion of phosphatidylinositol in the recovered phospholipids than did healthy volunteers (p less than 0.05). The severity of these alterations in phospholipid composition correlated with more advanced fibrotic histopathologic changes. Patients with less depression of total phospholipids in lavage improved with corticosteroid therapy, whereas the patients with more severely decreased total phospholipid recovered in lavage did not.

Adult↗

PubMed1988

Pulmonary lysozyme--a secretory protein of type II pneumocytes in the rat.

A 14 kDa basic protein isolated from rat lung lavage was demonstrated to be lysozyme by its amino acid sequence analysis. An antiserum to rat lysozyme stained type II pneumocytes and alveolar macrophages. In rat lungs, no staining of the airway cells was noted. Lysozyme was detectable in type II pneumocytes by immunocytochemistry and by a quantitative immunoassay of lung homogenates of fetal lungs at Day 20 of gestation. An increase in the lysozyme content of the lung with increasing gestational and postnatal age of the rat was noted. In adult animals, lysozyme accounts for about 169.0 micrograms/g of wet lung weight and 0.3% of the soluble proteins in lung homogenate. Lysozyme constitutes about 6.6% of the total soluble proteins in rat lung lavage. Metabolic labeling and immunoprecipitation were used to demonstrate that rat type II pneumocytes synthesize and secrete lysozyme in vitro. However, in human lungs, lysozyme was identified in serous submucosal glands but not in alveolar type II pneumocytes. The results demonstrate differential distribution of a secretory protein in rodent and human lungs and indicate that in the rat lysozyme could be used as an immunohistologic marker for type II pneumocytes and as an indicator of secretory activity and maturation of type II pneumocytes.

Amino Acid Sequence↗

PubMed1988

Alkaline phosphatase: a marker of alveolar type II cell differentiation.

In an effort to identify type II cells by a method independent of staining phospholipid inclusions, we evaluated a histochemical technique for alkaline phosphatase activity in normal rat lung, in freshly isolated type II cells, and in primary culture of type II cells. In the adult rat alveolus, alkaline phosphatase staining selectively identified type II cells, although nonciliated bronchiolar (Clara) cells and loose perivascular connective tissue also stained for alkaline phosphatase activity. In cell suspensions of type II cells and other dissociated lung cells, alkaline phosphatase staining correlated closely with the modified Papanicolaou technique and was particularly useful in distinguishing type II cells from alveolar macrophages. To determine if alkaline phosphatase was related to the differentiated phenotype of type II cells, we studied conditions known to affect other type II cell functions. When type II cells were cultured on plastic substrata, the intensity of alkaline phosphatase staining decreased with increasing time in culture. To quantitate the apparent decrease in alkaline phosphatase activity, we used a biochemical assay to study the expression of alkaline phosphatase by type II cells. The specific activity of alkaline phosphatase in type II cells declined with increasing time in tissue culture on plastic substrata. Alkaline phosphatase activity was maintained, however, by culturing cells on Englebreth-Holm-Swarm (EHS) tumor matrix. Cells that had reduced levels of alkaline phosphatase activity following 48 h of culture on plastic substrata could be "rescued" by removing them from the plastic substratum and reculturing them for 48 h on EHS matrix. Alkaline phosphatase activity was also increased by culturing type II cells in the presence of cAMP or sodium butyrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗

PubMed1988

Pulmonary alveolar type II epithelial cells synthesize and secrete proteins of the classical and alternative complement pathways.

The serum complement system is a major mediator of inflammation reactions. Two of the complement proteins, the third (C3) and fifth (C5) components, are precursors of potent phlogistic molecules, C3a and C5a. C5a has potent chemotactic activity and plays an active role in pulmonary inflammation. We present evidence suggesting that several complement proteins, including C5, are synthesized locally in the lung in alveolar type II epithelial cells. Lung tissue from normal mice synthesized and secreted C5 protein similar to the C5 protein in mouse serum, whereas lung tissue from C5-deficient mice did not. Lung tissues from both normal and C5-deficient mice synthesized C3. Rat lung tissue synthesized and secreted C5, as well as C2, C4, C3, and factor B. Cultures of type II cells (95% type II cells, 5% macrophages) regularly synthesized all these proteins. In contrast, cultures of macrophages alone synthesized large amounts of C2 and factor B, and in some experiments C3 and C4, but never C5. The C5 synthesized by the rat cells was slightly larger than serum C5 (200 kD compared with 180 kD) and was not processed to the two-chain molecule seen in serum. Rat lung tissue and purified type II cells contained C5 mRNA with the same molecular mass as the C5 mRNA in rat liver and in mouse lung and liver. Human type II cells also synthesized C5, as well as C2, C4, C3, and factor B. Human pulmonary macrophages synthesized only C2, factor B, and, in some experiments, C3. Synthesis of complement proteins in cells that line the alveolar wall may provide a local source of these proteins for inflammatory responses in the lung. Local synthesis of complement proteins could be regulated independently of the synthesis in the liver.

Animals↗

PubMed1987

Functional differentiation of alveolar type II epithelial cells in vitro: effects of cell shape, cell-matrix interactions and cell-cell interactions.

Alveolar type II epithelial cells rapidly lose characteristics of differentiated function when cultured on plastic dishes. We have attempted to circumvent this problem by culturing type II cells under conditions that might better reproduce their environment in vivo. Cell-matrix interactions were studied by culturing isolated adult rat type II cells on Engelbreth-Holm-Swarm (EHS) tumor basement membrane. Aggregates of type II cells formed on the surface of the matrix during 4 days in culture. Microscopic examination of these aggregates revealed cuboidal cells that retained more characteristics of differentiated type II cells than did cells cultured on plastic. Type II cells cultured on EHS matrix incorporated a higher percentage of acetate into phosphatidylcholine (PC) than did cells on plastic, and a higher percentage of this PC was saturated. Phosphatidylglycerol (PG) synthesis by these cells was no different from that seen in cells on plastic. The effects of cell-cell interactions and cell shape were evaluated by culturing type II cells on feeder layers that in turn were grown on collagen gels. The feeder layer cells included fetal rat lung fibroblasts, adult rat lung fibroblasts, fetal rat skin fibroblasts, bovine aortic endothelial cells, and rat mammary tumor epithelial cells. One-half of the gels remained attached to the culture dish and one-half of the gels were detached after 24 h and allowed to float free in the medium. Type II cells grown in association with any of the attached feeder layers became flattened and lost their differentiated phenotype. These cells incorporated no greater percentage of acetate into PC than did cells on plastic. Saturated PC synthesis was modestly increased. PG synthesis declined in parallel with that seen in cells cultured on plastic. Type II cells cultured on feeder layers that were detached assumed their native cuboidal shape and also exhibited many morphological characteristics of differentiated function. These cells incorporated a significantly greater percentage of acetate into PC compared to cells on either plastic or attached feeder layers. Saturated PC synthesis also increased markedly. These cells, however, incorporated no greater percentage of acetate into PG than did cells on plastic or attached feeder layers. These data suggest an important role for cell shape and cell-matrix interactions and maintenance of type II cell differentiation. The effects of cell-cell interactions, while beneficial, appear to be non-specific.

Animals↗

PubMed1987

Tetradecanoylphorbol acetate and terbutaline stimulate surfactant secretion in alveolar type II cells without changing the membrane potential.

Alveolar type II cells were isolated from adult rat lungs after tissue dissociation with elastase. The effect of known secretagogues on transmembrane potential was examined in freshly isolated cells (day 0 cells) and in cells after one day of primary culture (day 1 cells). Freshly isolated type II cells were incubated with 3,3'-dipentyloxacarbocyanine (di-O-C5(3)) or 3,3'-dipropylthiadicarbocyanine (di-S-C3(5)), dyes whose intracellular fluorescence intensity is a direct function of the cellular transmembrane potential. Fluorescence was continuously recorded by fluorescence spectrophotometry. Type II cells rapidly incorporated the dyes, and the addition of gramicidin (1 microgram/ml) depolarized the cells as indicated by a change in fluorescence. Neither 12-O-tetradecanoylphorbol 13-acetate (TPA) nor terbutaline plus 3-isobutyl-1-methylxanthine (IBMX), which stimulate surfactant secretion from isolated alveolar type II cells, changed the transmembrane potential. The lipophilic cation triphenylmethylphosphonium (TPMP+) was used to quantitate the transmembrane potential of type II cells cultured for one day. Addition of TPA or terbutaline plus IBMX induced surfactant secretion but did not alter the transmembrane potential. To study further the relationship of secretion to the transmembrane potential, secretion was also determined in the presence of high extracellular potassium which depolarizes the cells and in the presence of choline in place of sodium. High potassium enhanced the basal secretion of phosphatidylcholine from 1.8% to 3.4% (P less than 0.01, n = 7). Substitution of sodium chloride by choline chloride had no effect on basal secretion but enhanced TPA-induced secretion (P less than 0.01). We conclude that high extracellular potassium induces membrane depolarization and stimulates surfactant secretion, but TPA or terbutaline plus IBMX stimulates secretion without detectable membrane depolarization and stimulation of secretion by TPA does not require extracellular sodium.

Animals↗

PubMed1987

Isolation and sequence of a cDNA clone for the rat pulmonary surfactant-associated protein (PSP-A).

Pulmonary surfactant is composed mainly of phospholipid and two groups of apoproteins. One of these apoproteins is a family of glycoproteins (pulmonary surfactant-associated protein A, PSP-A). We have isolated and sequenced a cDNA clone encoding for rat PSP-A and the full amino acid sequence has been deduced from the nucleotide sequence. The sequence of 56 amino acids at the N-terminus of PSP-A isolated from rats treated with silica was determined independently, and there is complete agreement with the sequence deduced from the cDNA. Isolated rat alveolar type II cells contain two species of mRNA for this protein.

Amino Acid Sequence↗

PubMed1987

Lasting changes in spontaneous multi-unit activity in the chick brain following passive avoidance training.

Day-old chicks were trained on a one-trial passive avoidance task by pecking at a small, shiny bead coated with either a bitter-tasting substance (methylanthranilate) or water. The undifferentiated spontaneous multi-unit activity recorded bilaterally from anaesthetized chicks 1-13 h after training on the above task exhibited a significant increase in the methylanthranilate-trained over water-control chicks within three structures of the right hemisphere: the hyperstriatum accessorium (47%, P less than 0.05), the medial hyperstriatum ventrale (49.1%, P less than 0.02) and the medial portion of the paleostriatum augmentatum (47.5%, P less than 0.02). Within the multi-unit record obtained from both groups of chicks there were periods of short duration (15-20 ms) containing high-frequency (400-450 Hz) large-amplitude (greater than or equal to 200 microV; 450 microV max peak-to-peak) spikes. As a consequence of training, there was a massive increase in the occurrence of these bursting epochs in the medial hyperstriatum ventrale of both the left (320%, P less than 0.001) and right (350%, P less than 0.001) hemispheres in methylanthranilate-trained compared to water-trained chicks. In addition, the mean number of spikes per burst at this site increased by 66% (P less than 0.001) with no change observed in any other structure sampled. The statistically significant increase in activity within the right hyperstriatum accessorium and medial paleostriatum augmentatum and the non-significant increase in these structures in the left hemisphere was produced almost entirely by tonic spiking. Enhanced spontaneous multi-unit activity recorded under anaesthesia following passive avoidance training in the chick is shown here to be a feature common to several medial forebrain structures. The magnitude of the elevation in bursting frequency and the degree of localization of this effect to the medial hyperstriatum ventrale of methylanthranilate trained chicks would appear to offer strong support to previous biochemical and morphological evidence implicating this structure in the process of memory consolidation for this task. The data reported here represent the first evidence of electrophysiological changes occurring as a consequence of passive avoidance training in the domestic chick.

Animals↗

PubMed1987

P1 plasmid replication: measurement of initiator protein concentration in vivo.

To study the functions of the mini-P1 replication initiation protein RepA quantitatively, we have developed a method to measure RepA concentration by using immunoblotting. In vivo, there are about 20 RepA dimers per unit-copy plasmid DNA. RepA was deduced to be a dimer from gel filtration of the purified protein. Since there are 14 binding sites of the protein per replicon, the physiological concentration of the protein appears to be sufficiently low to be a rate-limiting factor for replication. Autoregulation is apparently responsible for the low protein level; at the physiological concentration of the protein, the repA promoter retains only 0.1% of its full activity as determined by gene fusions to lacZ. When the concentration is further decreased by a factor of 3 or increased by a factor of 40, replication is no longer detectable.

Cloning, Molecular↗

PubMed1987

Effect of secretagogues on cytoplasmic free calcium in alveolar type II epithelial cells.

Pulmonary surfactant is synthesized and secreted by alveolar type II epithelial cells. Although intracellular calcium and other second messengers have been implicated in secretion by type II cells, this is the first report on measurement of cytoplasmic free calcium concentration ([Ca2+]i). Known secretagogues, 12-O-tetradecanoylphorbol-13-acetate (TPA) and terbutaline, were tested to see if they caused rapid increases in cytoplasmic calcium. Ionomycin, a calcium ionophore, was used to increase cytoplasmic free calcium concentration, to determine if a rapid increase in cytoplasmic free calcium would stimulate secretion, and to measure interactions with other secretagogues. Ionomycin increased both [Ca2+]i and pulmonary surfactant secretion from alveolar type II cells. A low concentration of ionomycin (100 nM) greatly enhanced secretion stimulated by terbutaline or by 8-bromo-cAMP but only had an additive effect on secretion stimulated by TPA. Terbutaline transiently increased [Ca2+]i by 24% over control basal condition, and the increase in [Ca2+]i produced by terbutaline occurred in the absence of extracellular calcium. TPA itself did not change [Ca2+]i. However, TPA completely inhibited the terbutaline-induced increase of [Ca2+]i but not the increase due to ionomycin. When alveolar type II cells were loaded with 2-(2-bis-[carboxymethyl]-amino-5-methyl-phenoxy)-methyl-6-methoxy-8-bis carboxymethylaminoquinoline (quin2) in calcium-free buffer, [Ca2+]i decreased from 143 +/- 10 to 31 +/- 8 nM. Lowering [Ca2+]i inhibited TPA- or terbutaline-induced secretion by 22 and 40%, respectively. Although the precise role of cytoplasmic free calcium on surfactant secretion cannot be established on the basis of current data, our results indicate that an increase in cytoplasmic free calcium produced by ionomycin stimulates secretion and that an increase in [Ca2+]i affects cAMP-induced secretion more than protein kinase C-mediated secretion in alveolar type II cells.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1987

Endogenous opioids modulate fetal rabbit lung maturation.

To test the hypothesis that endogenous opioids modulate fetal lung development, separate groups of pregnant rabbits received daily injections of saline, morphine (1 mg/kg body wt), or the opioid antagonist naloxone (0.4 and 5.0 mg) for 10 days during their last trimester of pregnancy. The corresponding groups of fetuses were then delivered prematurely on day 28 of gestation (term approximately 31 days) and evaluated with respect to differences in body weight, lung weight, and the ratios of wet to dry lung weight and lung dry weight to body weight, the static inflation and deflation air and saline pressure-volume (P-V) characteristics of the lungs, and lung morphology. Mean values for body weight, lung weight, and the ratios of lung wet to dry weight and lung dry weight to body weight were not significantly different among the saline control (C), morphine (M)-, and naloxone (NLX)-treated fetuses. On the other hand, the fetal air P-V curves varied significantly (P less than 0.001), wherein the M-treated group depicted increased lung distensibility and alveolar stability on lung deflation, whereas the opposite was obtained in the NLX-treated fetuses. Moreover, morphometric analyses demonstrated that the mean alveolar air space-to-tissue ratio in lungs from M-treated fetuses were significantly greater than that observed either in C or in NLX-treated fetuses (P less than 0.05); however, the air space-to-tissue ratio did not significantly vary between the C and NLX-treated animals. These observations provide new evidence that endogenous opioids enhance fetal lung maturation.

Animals↗

PubMed1987

Surfactant synthesis, secretion, and function in alveoli and small airways. Review of the physiologic basis for pharmacologic intervention.

Surface-active material is known to lower surface tension at the air/liquid interface and provide alveolar stability. Less well appreciated is the potential role of surface-active material in maintaining the patency of small airways and in transporting particles from the alveolar surface to the small airways. The interactions between the two dominant secretory cells in the terminal lung units, i.e., the alveolar type II cell and the Clara cell, are just starting to be discovered. The biochemical signals for secretion of surface-active material in vivo are still not known. The dominant physiologic stimulus is hyperventilation. Secretion in vitro is stimulated by activation of protein-kinase C (tetradecanoyl phorbol acetate), protein-kinase A (cyclic AMP, beta-adrenergic agonists, cholera toxin), and calcium ionophores (A23187 or ionomycin). The biochemical basis for secretion will be discussed.

Adrenal Cortex Hormones↗

PubMed1987

Insulin stimulates amino acid transport by alveolar type II epithelial cells in primary culture.

The present experiments were designed to investigate the effect of insulin on amino acid transport by alveolar type II epithelial cells isolated from adult rats. The initial rates of amino acid transport were determined by measuring the uptake of the nonmetabolizable amino acid, alpha-aminoisobutyric acid (AIB). Alveolar type II epithelial cells transported AIB by a sodium-dependent process that displayed saturable kinetics. Insulin stimulated transport of 0.1 mM AIB (82.3 +/- 15.0%, mean +/- SE, n = 4) with a half-maximally effective insulin concentration of 2.2 +/- 1.0 ng/ml (mean +/- SE), which is within the physiologic range of rat serum insulin concentration. Kinetic analysis of AIB transport showed that insulin increased the Vmax from 6.7 +/- 1.2 nmol/10(6) cells/min to 9.3 +/- 1.8 nmol/10(6) cells/min and had no effect on the Km value (5.2 +/- 0.8 mM). Alpha-(methylamino) isobutyric acid, a specific substrate for amino acid transport system A, inhibited both the sodium-dependent and insulin-modulated transport of AIB. Thus, alveolar type II epithelial cells transport amino acids in a sodium-dependent process and insulin stimulates this transport. The present study indicates that insulin may play a physiologic role in regulating amino acid transport in alveolar type II epithelial cells.

Amino Acids↗

PubMed1987

The effect of substratum and serum on the lipid synthesis and morphology of alveolar type II cells in vitro.

To determine the effect of various culture conditions on the maintenance of lipid synthesis and morphology in alveolar type II cells, we cultured isolated adult rat alveolar type II cells on either plastic or denuded human amnionic basement membrane (ABM) in medium supplemented with either fetal bovine, porcine, horse, rat, or human serum. Lipid synthesis was assessed by incubation with [1-14C]acetate and determination of the distribution of radiolabel into individual lipid classes. Cells cultured on ABM incorporated significantly higher percentages of acetate into either phosphatidylcholine (PC) or phosphatidylglycerol (PG), and retained lamellar inclusions and a more characteristic cuboidal shape for longer periods than did cells cultured on plastic. Compared to other sera, cells cultured in the presence of rat serum incorporated the highest percentages of acetate into PC and saturated PC, had the best preservation of lamellar-body ultrastructure, and also appeared to contain more multivesicular bodies. The percent composition of linoleic acid, an essential fatty acid, was found to vary widely among the different sera. Supplementing media with linoleic acid resulted in a marked increase in acetate incorporation into saturated PC and a decreased incorporation into PG. We conclude that for maintenance of differentiated function of adult rat alveolar type II cells in primary culture (1) ABM is preferable to plastic as a culture substratum, (2) rat serum is preferable to fetal bovine serum as a serum supplement, and (3) the regulation of lipid synthesis by linoleic acid causes disparate effects on PG and saturated PC synthesis.

Animals↗

PubMed1987

The coding sequence for the 32,000-dalton pulmonary surfactant-associated protein A is located on chromosome 10 and identifies two separate restriction-fragment-length polymorphisms.

The primary protein component of human pulmonary surfactant is a 32,000-dalton glycoprotein called surfactant-associated protein A. This protein is important for normal lung function, and its expression is developmentally regulated. Using a mapping panel of somatic-cell hybrids, we have localized the coding sequence for pulmonary surfactant-associated protein A to chromosome 10. Additionally, this sequence identifies two separate MspI restriction-fragment-length polymorphisms. Since there is a relative lack of polymorphic markers for chromosome 10, this sequence may be useful in linkage analysis.

Animals↗

PubMed1987

The effect of neutrophil migration and prolonged neutrophil contact on epithelial permeability.

The effect of neutrophil migration and prolonged neutrophil contact on epithelial permeability was examined. Although neutrophil migration was not associated with a change in epithelial permeability, prolonged neutrophil-epithelial contact following migration resulted in an increase in epithelial permeability. These results were not altered by catalase, a specific neutrophil elastase inhibitor, methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone or cyclohexamide. This suggests that neutrophil migration does not occur via an H2O2-induced reversible mechanism of junctional opening, which we describe herein.

Animals↗

PubMed1987

Surfactant in adult respiratory distress syndrome.

Adult Respiratory Distress Syndrome is a catastrophic disease which is characterized by tachypnoea, arterial hypoxemia, reduced pulmonary compliance, and diffuse alveolar infiltrates. The pathology is that of diffuse alveolar damage which includes injury to both endothelial cells and type I epithelial cells. The major physiological abnormality is reduced pulmonary compliance. The available data indicate that one of the causes for the reduced compliance is altered surface tension in the distal airspaces. Pulmonary surface material isolated from patients with Adult Respiratory Distress Syndrome has an altered phospholipid composition. Part of the current therapy is to overcome the reduced compliance by mechanical ventilation while allowing the lung time to heal. In the future, exogenous surfactant may also be used as part of the therapy in selected severely ill patients.

Detergents↗

PubMed1986

P1 plasmid replication. Role of initiator titration in copy number control.

The copy number control locus incA of unit copy plasmid P1 maps in a region containing nine 19 base-pair repeats. Previous results from studies in vivo and in vitro indicated that incA interacts with the plasmid-encoded RepA protein, which is essential for replication. It has been proposed that the repeat sequences negatively control copy number by sequestering the RepA protein, which is rate-limiting for replication. Our results lend further support to this hypothesis. Here we show that the repeats can be deleted completely from P1 miniplasmids and the deletion results in an approximately eightfold increase in plasmid copy number. So, incA sequences are totally dispensable for replication and have only a regulatory role. The copy number of incA-deleted plasmids can be reduced if incA sequences are present in trans or are reincorporated at two different positions in the plasmid. This reduction in copy number is not due to lowered expression of the repA gene in the presence of incA. We show that one repeat sequence is sufficient to bind RepA and can reduce the copy number of incA-deleted plasmids. When part of the repeat was deleted, it lost its ability to bind as well as influence copy number. These results show a strong correlation between the capacity of incA repeats to bind RepA protein both in vivo and in vitro, and the function of incA in the control of copy number.

Autoradiography↗