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K R Bhaskar

Publications and source records attributed to K R Bhaskar.

35 records · Page 2Linked to original sources
PubMed1987

Pathophysiology of bronchial hypersecretion.

Density gradient ultracentrifugation has been used to analyse the lavage from the proximal intrapulmonary bronchi of normal human volunteers and of dogs. The secretion from explants of canine trachea and human lobar bronchi has been examined under basal conditions and after stimulation by secretagogues. Mucus from the hypersecretory airways has been obtained from tracheostomized patients and from a canine model of chronic bronchitis produced by exposure to SO2 gas. No macromolecule of density gradient typical of an epithelial glycoprotein was recovered from normal basal secretions: the glycoconjugate recovered was of higher density and has sugars typical of a proteoglycan. The explant produced typical epithelial glycoprotein if stimulated. As volume of secretion increases, macromolecular yield does also, both of epithelial glycoprotein and of a number of lipids that are synthesised by the airway and which are different from alveolar lipids.

Animals↗
PubMed1987

Lipids in airway secretions.

Lipids form a significant portion of airway mucus yet they have not received the same attention that epithelial glycoproteins have. We have analyzed, by thin layer chromatography, lipids present in airway mucus under "normal" and hypersecretory (pathological) conditions. The 'normals' included (1) bronchial lavage obtained from healthy human volunteers and from dogs and (2) secretions produced "in vitro" by human (bronchial) and canine (tracheal) explants. Hypersecretory mucus samples included (1) lavage from dogs made bronchitic by exposure to SO2, (2) bronchial aspirates from acute and chronic tracheostomy patients, (3) sputum from patients with cystic fibrosis and chronic bronchitis and (4) postmortem secretions from patients who died from sudden infant death syndrome (SIDS) or from status asthmaticus. Cholesterol was found to be the predominant lipid in 'normal' mucus with lesser amounts of phospholipids. No glycolipids were detected. In the hypersecretory mucus, in addition to neutral and phospholipids, glycolipids were present in appreciable amounts, often the predominant species, suggesting that these may be useful as markers of disease. Radioactive precursors 14C acetate and 14C palmitate were incorporated into lipids secreted "in vitro" by canine tracheal explants indicating that they are synthesised by the airway.

Animals↗
PubMed1986

Purification and chemical composition of gpL115, the human lymphocyte surface sialoglycoprotein that is defective in Wiskott-Aldrich syndrome.

gpL115, the surface sialoglycoprotein that is defective in lymphocytes of Wiskott-Aldrich syndrome patients has been purified from large scale cultures of the lymphoblastoid line CEM. The purification entails cell lysis and solubilization of gpL115 with the detergent Nonidet P-40, sequential affinity chromatography on lentil lectin-Sepharose, wheat germ lectin-Sepharose, and, after treatment with sialidase, on peanut lectin-Sepharose. Sepharose CL-6B gel filtration removes residual protein contaminants and transfers asialo-gpL115 from Nonidet P-40-containing to sodium dodecyl sulfate-containing buffer. The yield, 1300 micrograms of homogeneous protein/10(11) cells, represents greater than 60% recovery. The amino acid composition of gpL115 has several atypical features including low lysine content, high proline content, and very high content of hydroxyamino acids (12.5 residues of serine and 12.5 residues of threonine/100 amino acids). Total carbohydrate content of gpL115 is very high, i.e. 52% for the asialo-molecule. The major carbohydrate residues of asialo-gpL115 are galactose and N-acetylgalactosamine in approximately equimolar amounts (25 and 22 residues/100 amino acids, respectively) plus severalfold lower amounts of N-acetylglucosamine, fucose, and mannose.

Amino Acids↗
PubMed1986

Density gradient analysis of secretions produced in vitro by human and canine airway mucosa: identification of lipids and proteoglycans in such secretions.

Human and canine airway mucosal explants synthesize and secrete high molecular weight glycoconjugates, incorporating 14C-glucosamine, a radioactive precursor to epithelial glycoprotein. Our examination of secretions produced by several individual specimens, however, did not reveal epithelial glycoprotein of typical buoyant density (1.5 g/ml in CsBr); only a high-density component with features of glycoprotein and proteoglycan. To provide sufficient material for characterization, secretions from several specimens of human and canine explants were separately pooled and subjected to DGU in CsBr. After removal of lipids and proteins, the glycoconjugates were recovered into five fractions of different density. 14C-glucosamine had been incorporated in all five fractions. Fractions 1-4 together accounted for 88% of the radiolabel but gas chromatography indicated that none of these contained epithelial glycoprotein. Their amino acid compositions were similar to those of proteoglycans and electrophoresis confirmed the presence of chondroitin sulfates A, B, C, heparan sulfate and hyaluronic acid. Sugars typical of epithelial glycoprotein were identified only in the glycoconjugate subfraction 5 of lowest density (and also lowest in yield) in which glycosaminoglycans were also identified. By addition of radioactive precursors, 14C acetate, 14C palmitate and 14C mevalonic acid to the culture medium and autoradiography of the secreted lipids we have shown that the tracheal explants actively synthesize lipids. Lipids accounted for a high proportion, almost half by weight, of the explant secretion. While neutral and phospholipids predominate, glycolipids were also identified.

Amino Acids↗
PubMed1985

Density gradient study of bronchial mucus aspirates from healthy volunteers (smokers and nonsmokers) and from patients with tracheostomy.

Because it is difficult to obtain, little is known of bronchial mucus from the normal human airway; it has been mainly studied as sputum expectorated in chronic bronchitis with particular attention to epithelial glycoprotein. We have now applied density gradient methods to study this and other macromolecules and lipids in normal airway mucus. After lavage at bronchoscopy, mucus was aspirated from six normal volunteers, that include one light and two heavy smokers. This normal mucus has been compared with that obtained from four patients with tracheostomy because of respiratory muscle paralysis due to neurological disease. The normal aspirates contained small threads of mucus, the tracheostomy aspirates viscous blobs of jelly, a difference in physical appearance reflected in macromolecular yields, 0.3-1 mg/ml and 6-24 mg/ml respectively. On analytical ultracentrifugation normal mucus showed no discernible material in the buoyant density region typical of epithelial glycoprotein (1.5 g/ml): Virtually all the material migrated to the miniscus and was predominantly lipids and proteins. A trace amount of material recovered from a higher density region (greater than or equal to 1.6 g/ml) was found to contain both glycoprotein and proteoglycan. Aspirates from the heavy smokers contained appreciable amounts of material with typical buoyant density (approximately 1.5 g/ml) but still with features of proteoglycan. In contrast in tracheostomy aspirates epithelial glycoprotein of typical buoyant density and chemical composition accounted for up to 25% of nondialyzable material. We conclude that under normal conditions typical epithelial glycoprotein is virtually absent from airway mucus and that the glycoconjugate present has features of glycoprotein and proteoglycan.

Adult↗
PubMed1984

Airway mucus: composition and regulation of its secretion by neuropeptides in vitro.

Human and canine airway mucosa in vitro synthesizes and secretes mucus glycoprotein, proteoglycans and lipids which can be separated by density gradient ultracentrifugation in caesium bromide. In secretions from unstimulated explants, the small amount of mucus glycoprotein present is found in association with proteoglycans. 'Free' mucus glycoprotein of typical buoyant density is present only after stimulation of submucosal gland secretion by methacholine. Lipids are synthesized, at least in part, by the airway mucosa and occur in explant secretions as a viscoelastic gel, suggesting that they significantly influence the rheological properties of airway mucus. In addition to cholinergic and adrenergic secretomotor neurons, the airway mucosa is innervated by peptidergic fibres containing immunoreactivity to vasoactive intestinal peptide (VIP) and substance P (SP). In explants of non-bronchitic human airway, VIP inhibits baseline glycoprotein and lysozyme secretion; in canine airway mucosa, by contrast, VIP is a weak partial secretory agonist. SP is the most potent agonist of canine airway glycoprotein release described to date and appears to evoke secretion by a direct action on a stereospecific SP receptor rather than by inducing release of other endogenous secretagogues. VIP and SP have little effect on glycoprotein discharge by mucous and serous cells of the submucosal gland; SP appears to induce secretion by causing contraction of submucosal gland ducts. This may represent the most rapid way for delivering mucus into the airway in response to injury or irritation of airway epithelium.

Animals↗
PubMed1984

Extracellular calcium ion depletion induces release of glycoproteins by canine trachea.

The effect of depletion of extracellular Ca2+ on the release by canine trachea in vitro of [14C]glucosamine-labeled trichloroacetic-phosphotungstic acid-precipitable glycoproteins was evaluated. Incubation in Ca2+,Mg2+-free medium containing ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA, 10(-3) to 10(-5) M) induced a time-dependent increase in base-line release of high molecular weight, radiolabeled glycoprotein that was not blocked by 10(-5) M atropine, propranolol, or phentolamine. Maximal increase was proportional to EGTA concentration and was augmented by preincubation with methacholine chloride. The secretory response was reversed by reincubation of explants in medium containing Ca2+, Sr2+, or Ba2+ but not Mg2+. Assessment of explants denuded of epithelium or cartilage indicated that the secretory response to depletion of Ca2+ originated in the tracheal submucosa, probably the submucosal glands. Quantitative autoradiographic analyses showed, however, that incubation in Ca2+,Mg2+-free medium had no effect on radiolabel release from mucous or serous cells in the tracheal submucosal glands. Increased radiolabeled glycoprotein release in Ca2+,Mg2+-free medium was accompanied by exfoliation of the surface epithelium, though the two effects were apparently unrelated. The secretory response was not due to cell lysis or increased release of radiolabeled glycosaminoglycans, and the finding that the specific activity (ratio of bound radiolabel to protein content) of the glycoprotein released was not changed in Ca2+,Mg2+-free medium showed that it was not due to a change in the rate of glycoprotein synthesis. A model is proposed in which depletion of Ca2+ increases the rate of flow of mucus from the duct lumens of the tracheal glands.

Animals↗
PubMed1977

The separation and characterization of bronchial glycoproteins by density-gradient methods.

1. Sputum samples from a total of 18 asthmatic and chronic bronchitic patients were examined by analytical density-gradient ultracentrifugation. CsBr was used as the dispersal agent and dense electrolyte. 2. The patterns show two main groups of components, banding at about 1.3g/ml and 1.5g/ml; in addition, a few samples showed a further zone at approx. 1.65g/ml. These components were identified as protein, secretory glycoprotein and DNA respectively. The glycoprotein zone was frequently hypersharp, and usually contained two or more partially resolved bands; it was always well resolved from the protein. 3. The glycoprotein components were isolated from nine representative sputum samples by density-gradient ultracentrifugation on a preparative scale. Analytical density-gradient ultracentrifugation was used to monitor the efficiency of the separations. 4. Some sputum samples separated cleanly under these conditions, the glycoprotein being essentially devoid of free protein; in others, separation was apparently incomplete, although computer simulation indicated that the conditions were adequate to ensure separation. Further density-gradient separations in CsCl were necessary with several samples before satisfactory products were obtained; mixtures of CsCl with guanidinium chloride were no more effective than CsCl alone. The reluctance to separate indicates a very strong, but non-covalent, interaction between protein and glycoprotein, probably associated with the gelatinous character of the secretion. 5. The purified glycoprotein components were characterized analytically and physicochemically. They contained N-acetylgalactosamine, N-acetylglucosamine, galactose, fucose and N-acetylneuraminic acid, and had an amino acid composition in which serine, threonine and proline predominated; however, aspartic acid, glutamic acid and cystine were also appreciable. The glycoproteins were of very high molecular weight, and usually showed more than one component in sedimentation velocity; their distribution in a density gradient indicated a substantial, but largely monotonic, density heterogeneity. 6. Thiol reduction decreased the molecular weight very substantially, but the products were relatively more homogeneous than the native materials. The amino acid composition was changed significantly and a small and variable proportion of protein or peptide was liberated. It is concluded that the native materials are disulphide-linked aggregates, probably through a cross-linking peptide, in confirmation of earlier studies.

Amino Acids↗
PubMed1974

The macromolecular properties of blood-group-specific glycoproteins. Characterization of a series of fractions obtained by solvent fractionation.

1. The glycoprotein components of a human ovarian-cyst fluid were isolated by a solvent [95% (w/w) phenol]-extraction procedure; the phenol-insoluble water-soluble glycoprotein was further fractionated by (NH(4))(2)SO(4) and by ethanol to yield eight fractions. 2. The fractions were analysed in terms of amino acids, fucose, galactose, N-acetylglucosamine, N-acetylgalactosamine and sialic acid. Variations occurred, particularly in the proportion of peptide; these were partly correlated with varying extent of serological activity. 3. The fractions were characterized physicochemically in terms of buoyant density and degree of spreading in a density gradient, sedimentation velocity and molecular weight; their partial specific volumes and specific refraction increments were also determined. 4. The fractions showed wide variations in their sedimentation-velocity and density-gradient patterns, and gave evidence of pauci-dispersity in density. The fraction regarded as the most typical blood-group-specific glycoprotein sedimented as a single rapidly spreading peak and was of high molecular weight. 5. Significant correlations were observed between the physical properties of the glycoprotein fractions and the amount of their peptide component. The buoyant densities and sedimentation coefficients varied in a manner that suggested the existence of two families of glycoproteins. 6. It is suggested that variability in the extent of glycosylation, or in the degree of cross-linking, might account for the two families of glycoproteins, and that the extent of cross-linkage might also be a factor determining the solubility of these glycoproteins in hot saturated (NH(4))(2)SO(4).

Amino Acids↗
PubMed1974

The macromolecular properties of blood-group-specific glycoproteins. Characterization of a series of fractions obtained by density-gradient ultracentrifugation.

1. Equilibrium density-gradient ultracentrifugation in caesium salts was used in two stages in the isolation and subfractionation of the glycoprotein component from a human ovarian-cyst fluid. The eight main subfractions thus obtained were the subject of detailed physicochemical characterization. 2. The fractions were unimodal in buoyant-density distribution, but had discrete rho(0) values ranging from 1.31 to 1.35. 3. Weight-average molecular weights and sedimentation coefficients decreased regularly with decreasing density of the fraction, whereas the partial specific volumes and selective solvation parameters increased. The latter behaviour correlates well with the increasing peptide content of the lighter fractions. 4. The fractions exhibited a range of analytical composition, although all were within the limits previously observed for blood-group substances of Le(a) specificity. All fractions had approximately equal Le(a) activity. The peptide content varied systematically from 7% for the densest fraction to 15% for the lightest, but the relative distributions of the amino acids remained essentially constant throughout the series. In particular, serine plus threonine plus proline made up about 50% of the peptide content of all the fractions. Fucose, galactose and N-acetylglucosamine contents decreased with increasing peptide content of the fractions, but N-acetylgalactosamine and sialic acid exhibited the opposite trend. Molar ratios of N-acetylgalactosamine to the sum of serine and threonine remained essentially constant at 0.8-0.9, implying a high degree of glycosylation of all the molecules, but the ratio of N-acetylglucosamine to N-acetylgalactosamine decreased steadily with increasing peptide content, suggesting the presence of oligosaccharide side chains of various lengths. The results are discussed in terms of the accepted structure of glycoprotein molecules. 5. Experiments on the glycoproteins extracted with phenol from the same cyst fluid have confirmed that equilibrium centrifugation in caesium salts does not remove any non-covalently bound protein nor cause any changes in the tertiary structures of these glycoprotein molecules.

Acetylgalactosamine↗
PubMedDate not supplied

Lipids in airway mucus of acute quadriplegic patients.

Quadriplegic patients have difficulty in clearing lung mucus due to paralysis of muscles of respiration. In about 25% of these patients, excessive mucus in the airway necessitates tracheostomy, and in some patients it is fatal. In others there is spontaneous recovery. To determine if the excessive mucus results from secretion of abnormal mucus or from accumulation of normal mucus, we analyzed the lipids in mucus from eight quadriplegic patients. Lipids were separated from other constituents of the mucus by density gradient ultracentrifugation, extracted with chloroform-methanol (2:1), and examined by high-performance thin-layer chromatography (HPTLC). Cholesterol was the major neutral lipid; phosphatidylethanolamine, phosphatidylcholine, and sphingomyelin were the main phospholipids. Glycolipids were predominant, lactosylceramide (CDH) being the highest in amount. Two-dimensional HPTLC as well as high-performance lipid chromatography also revealed the presence of gangliosides: comparison with standards indicated the presence of GM1, GM2, GM3, and some unidentified gangliosides. In normal mucus, cholesterol is the predominant lipid; phospholipid is present in smaller amounts but glycolipids are not identified. Thus, results of our lipid analysis show that mucus from the quadriplegic patients is abnormal and similar to that in hypersecretory diseases such as chronic bronchitis and cystic fibrosis. Unlike these latter cases, hypersecretion in the quadriplegic has a rapid onset and, often, spontaneous recovery, suggesting that this is due to abnormal stimulation rather than an increase in the population of secretory cells.

Chromatography, High Pressure Liquid↗