scholarly journals Biosynthesis of respiratory-tract mucins. Incorporation of radioactive precursors into glycoproteins by canine tracheal explants in vitro

1973 ◽  
Vol 136 (4) ◽  
pp. 837-844 ◽  
Author(s):  
Daniel B. Ellis ◽  
Glenn H. Stahl
Keyword(s):  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Deae Cellulose ◽  
Sialic Acids ◽  
Equilibrium Density ◽  
Agarose Gels ◽  
Cscl Gradient ◽  
Tracheal Explants

1. Canine tracheal explants, cultured in medium 199, actively incorporated radioactive precursors into secreted macromolecules in vitro. 2. Puromycin, 6-diazo-5-oxo-l-norleucine and ouabain markedly inhibited the incorporation of these precursors. 3. Exogenous glucosamine at concentrations above 20mm caused a greater than 50% inhibition of the incorporation of l-[G-3H]fucose and l-[U-14C]serine. 4. Carbohydrate content of the purified secretions was approximately 50% and consisted principally of galactose, N-acetylglucosamine, N-acetylgalactosamine, fucose and sialic acids. 5. Chromatography on DEAE-cellulose and Bio-Gel A-150m and equilibrium density-gradient centrifugation in a CsCl gradient confirmed the presence of mucous glycoproteins. 6. Electrophoresis on 1% agarose gels gave profiles that were identical with canine respiratory mucus obtained in vivo. 7. These results support the utility of the explant system for studies of respiratory secretions.

scholarly journals Studies on gastric mucoproteins. The production of radioactive mucoproteins by pig gastric mucosal scrapings in vitro

1972 ◽  
Vol 127 (3) ◽  
pp. 577-587 ◽  
Author(s):  
D. Snary ◽  
A. Allen
Keyword(s):  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sedimentation Velocity ◽  
Water Soluble ◽  
Equilibrium Density ◽  
Optimum Conditions ◽  
Ph Values ◽  
Cscl Gradient ◽  
Sepharose 4B

1. Optimum conditions, including the effect of media of different pH values, were determined for the incorporation of radioactive precursors into mucoproteins by pig gastric mucosa in vitro. 2. Mucosal scrapings incorporated radioactivity from [U-14C]-glucose and from [G-3H]threonine or [G-3H]serine solely into the carbohydrate and protein portions respectively of the mucoprotein molecules. 3. Of the radioactive mucoprotein 22% was water-soluble and up to 80% of the remainder was soluble in other solvents. 4. Pronase was the most successful proteolytic enzyme tested for making the mucoprotein water-soluble, up to 94% dissolving after digestion. 5. The Pronase digestion products of the mucoproteins were separated from protein by equilibrium-density-gradient centrifugation in a CsCl gradient. 6. These Pronase-digested mucoproteins were further fractionated on Sepharose 4B and the isolated fractions analysed by chemical and sedimentation-velocity methods. 7. Pronase digestion and solvent extraction of mucosal scrapings labelled with 14C in the carbohydrate and 3H in the protein showed that one type of mucoprotein was the only non-diffusible biosynthetic product of the scrapings in vitro, and that this mucoprotein was the only mucoprotein constituent of the water-soluble and water-insoluble mucus.

scholarly journals Alteration of hepatic glucocorticoid receptor stability and nuclear binding in vitro by citrate

1983 ◽  
Vol 210 (1) ◽  
pp. 259-263 ◽  
Author(s):  
J Hubbard ◽  
M Kalimi
Keyword(s):  
Glucocorticoid Receptor ◽  
Glucocorticoid Receptors ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Deae Cellulose ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Nuclear Binding ◽  
Wide Range

Citrate greatly stabilized rat hepatic unbound glucocorticoid receptors in cell-free conditions at 4 degrees C with optimal effectiveness at 5-15 mM. Control receptors were inactivated at 4 degrees C with a half-life of less than 12 h. However, in the presence of 10 mM-citrate, unbound receptors were almost completely stabilized for 48 h at 4 degrees C. Citrate at a concentration of 1-2 mM yielded half-maximal stabilization. The stabilizing effect of citrate was rather specific, as succinate, alpha-oxoglutarate, oxaloacetate, malate and pyruvate had no apparent stabilizing action. Citrate stabilized receptors over a wide range of H+ concentrations, with complete protection between pH 6.5 and 8.5. In addition, citrate appeared to have a significant effect on glucocorticoid-receptor complex activation into a nuclear binding form. Thus 5-10 mM-citrate enhanced nuclear binding, with optimal activation achieved at 10 mM concentration. As analysed by sucrose-density-gradient centrifugation and DEAE-cellulose chromatography, no apparent change was observed in the physical characteristics of the glucocorticoid receptor in the presence of citrate.

scholarly journals LYSOSOMES IN RAT THORACIC DUCT LYMPHOCYTES FRACTIONATED BY ZONAL CENTRIFUGATION

1973 ◽  
Vol 59 (1) ◽  
pp. 177-184 ◽  
Author(s):  
William E. Bowers
Keyword(s):  
Thoracic Duct ◽  
Light Microscope ◽  
Cathepsin D ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Differential Centrifugation ◽  
Acid Hydrolases ◽  
Fractionation Method

A method of zonal centrifugation was developed which separates rat thoracic duct lymphocytes (TDL) mainly according to size. The validity of the fractionation method was supported by light microscope observations, Coulter Counter sizing, and in vivo and in vitro labeling of lymphocytes. The distributions of lysosomal acid hydrolases in TDL fractionated by zonal centrifugation are similar to the distribution obtained for the cells. This result indicates that the large lymphocyte is not the sole bearer of either lysosomes or the large amount of soluble cathepsin D found in homogenates of TDL. Both reside mainly in small lymphocytes. This point was clearly established by fractionating homogenates of purified small lymphocytes by means of differential centrifugation and isopycnic density gradient centrifugation.

scholarly journals Biosynthesis of proteoglycans in cartilage slices. Fractionation by gel chromatography and equilibrium density-gradient centrifugation

1972 ◽  
Vol 126 (4) ◽  
pp. 791-803 ◽  
Author(s):  
T. E. Hardingham ◽  
Helen Muir
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Chondroitin Sulphate ◽  
Gradient Centrifugation ◽  
Guanidine Hydrochloride ◽  
Specific Radioactivity ◽  
Equilibrium Density ◽  
Gel Chromatography ◽  
Sulphate Content

The kinetics of incorporation of [35S]sulphate into slices of pig laryngeal cartilage in vitro was linear with time up to 6h. The specific radioactivities of the extracted proteoglycans (containing about 80% of the uronic acid of the cartilage) and the glycosaminoglycans remaining in the tissue after extraction were measured after various times of continuous and ‘pulse–chase’ radioactivity incorporation. Radioactivity was present in the isolated chondroitin sulphate after 2 min, but there was a 35min delay in its appearance in the extractable proteoglycan fraction. Fractionation of the proteoglycans by gel chromatography showed that the smallest molecules had the highest specific radioactivity, but ‘pulse–chase’ experiments over 5h did not demonstrate any precursor–product relationships between fractions of different size. Equilibrium density-gradient centrifugation in 4m-guanidine hydrochloride showed that among the proteoglycan fractions the specific radioactivity increased as the chondroitin sulphate content decreased, but with preparations from ‘pulse–chase’ experiments there was again no evidence for precursor–product relationships between the different fractions. Differences in radioactive incorporation would seem to reflect metabolic heterogeneity within the proteoglycans extracted from cartilage. This may be due either to a partial separation of different types of proteoglycans or to differences in the rates of degradation of the molecules of different size and composition as a result of the nature and specificity of the normal degrading enzymes. The results suggest that molecules of all sizes were formed at the same time.

scholarly journals The effect of thrombin on the density distribution of blood platelets: detection of activated platelets in the circulation

Blood ◽  
1983 ◽  
Vol 62 (2) ◽  
pp. 433-438
Author(s):  
B van Oost ◽  
IH van Hien-Hagg ◽  
AP Timmermans ◽  
JJ Sixma
Keyword(s):  
Cardiopulmonary Bypass ◽  
Density Distribution ◽  
Density Gradient Centrifugation ◽  
Blood Platelets ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Human Platelets ◽  
Activated Platelets

The buoyant density of human platelets is decreased after they have been aggregated and induced to secrete their granule content by thrombin. This change in density was detected by discontinuous density gradient centrifugation using arabinogalactan (Stractan) solutions. The density decrease was dependent on the thrombin concentration and paralleled the extent of serotonin and beta-thromboglobulin secretion. The degranulated platelets maintained their integrity, and many of their functional properties. Mixtures of degranulated platelets and normal platelets could be resolved by Stractan gradient centrifugation and the number of degranulated platelets quantitated. Using this method, increased levels of less dense platelets were shown to occur after cardiopulmonary bypass. Assay of changes in platelet density by Stractan gradient centrifugation is a useful method for detection of activated platelets in vitro and in vivo.

scholarly journals The effect of thrombin on the density distribution of blood platelets: detection of activated platelets in the circulation

Blood ◽  
1983 ◽  
Vol 62 (2) ◽  
pp. 433-438 ◽  
Author(s):  
B van Oost ◽  
IH van Hien-Hagg ◽  
AP Timmermans ◽  
JJ Sixma
Keyword(s):  
Cardiopulmonary Bypass ◽  
Density Distribution ◽  
Density Gradient Centrifugation ◽  
Blood Platelets ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Human Platelets ◽  
Activated Platelets

Abstract The buoyant density of human platelets is decreased after they have been aggregated and induced to secrete their granule content by thrombin. This change in density was detected by discontinuous density gradient centrifugation using arabinogalactan (Stractan) solutions. The density decrease was dependent on the thrombin concentration and paralleled the extent of serotonin and beta-thromboglobulin secretion. The degranulated platelets maintained their integrity, and many of their functional properties. Mixtures of degranulated platelets and normal platelets could be resolved by Stractan gradient centrifugation and the number of degranulated platelets quantitated. Using this method, increased levels of less dense platelets were shown to occur after cardiopulmonary bypass. Assay of changes in platelet density by Stractan gradient centrifugation is a useful method for detection of activated platelets in vitro and in vivo.

The polypeptide components of scintillons, the bioluminescence organelles of the dinoflagellate Gonyaulax polyedra

1993 ◽  
Vol 71 (3-4) ◽  
pp. 176-182 ◽  
Author(s):  
Michel Desjardins ◽  
David Morse
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Binding Protein ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Close Packing ◽  
Gonyaulax Polyedra

Scintillons, the bioluminescence organelles of Gonyaulax polyedra, were purified by isopycnic density gradient centrifugation until only low levels of contaminating chloroplasts and mitochondria were detected by fluorescence and electron microscopy. Purified scintillons catalyzed the luminescent reaction with kinetics identical to those observed during the bioluminescence flash in vivo. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that the organelles appeared to contain only two proteins. These proteins were identified as luciferase (135 kilodaltons) and luciferin-binding protein (75 kilodaltons) based on their size and their known functions in the bioluminescence reaction in vitro. The staining of luciferin-binding protein by Coomassie blue was 2.4 ± 0.3 (n = 19) times greater than the luciferase, suggesting that there are four binding protein monomers for every luciferase monomer. A model is proposed for the close packing of the two proteins inside the scintillons.Key words: luciferase, luciferin-binding protein, density gradient centrifugation, dinoflagellate.

FATE OF NEUROHYPOPHYSIAL GRANULE MEMBRANES AFTER SECRETION IN THE RAT: GENERATION OF A NEW APPARENT ORGANELLE BY FREEZING AND THAWING SECRETORY GRANULES

1981 ◽  
Vol 88 (1) ◽  
pp. 115-123
Author(s):  
JEAN H. LACEY ◽  
B. T. PICKERING
Keyword(s):  
Density Gradient Centrifugation ◽  
Secretory Granules ◽  
Gradient Centrifugation ◽  
Hormone Release ◽  
Freezing And Thawing ◽  
Granule Fraction ◽  
Two Peaks ◽  
Stimulation Of

The contents and membranes of the secretory granules in the rat neurohypophysis were labelled in vivo with [35S]cysteine and [3H]choline respectively. Density-gradient centrifugation of the labelled granules showed the membrane label to be distributed largely between two peaks: one associated with intact granules and one with the characteristics of empty granule envelopes. Stimulation of hormone release in vitro led to the movement of membrane label from the intact granule fraction to the other one, consistent with the recapture of membrane as large vesicles. Freezing and thawing the crude granule fraction, ostensibly to aid osmotic fracture, produced a single membrane component with a density intermediate between the two original fractions.

Investigation of In Vivo Factors that May Influence Platelet Survival and Density

1979 ◽  
Author(s):  
M.A. Packham ◽  
J.F. Mustard ◽  
M.A. Guccione ◽  
P. D. Winocour ◽  
H.M. Groves ◽  
...  
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Platelet Membrane ◽  
Membrane Glycoproteins ◽  
Aortic Endothelium ◽  
Platelet Survival ◽  
Rabbit Platelets

Platelet survival CPS) is shortened in a number of conditions but the mechanisms responsible are unclear. In rabbits, removal of the aortic endothelium or injury of the neointima does not shorten PS. However, induction of thrombi in rabbit aortae with Indwelling cannulae (IDC) shortens FS (IDC 37.0 hr, control 79.6 hr), and Increases the proportion of platelets in the lightest fraction upon straetan density gradient centrifugation. Therefore we examined the effect of agents to which platelets may be exposed during thromboembolism (ADP, thrombin, plasmin) on PS and platelet density. ADP treatnent of washed rabbit platelets did not alter their survival but did increase the proportion in the lightest fraction. Treatment of platelets with thrombin did not shorten PS but increased the proportion in the lightest fraction. Treatment with plasmin in vitro shortened PS (plasmin, 57.6 ± 6.0 hr, control 80.2 ± 4,2 hr) and increased the proportion in the lightest fraction. Thus changes in platelet density are not necessarily associated with changes in PS. Of the factors investigated that are known to be involved in thromboembolism, only plasmin shortened PS. This may be due to its ability to alter major platelet membrane glycoproteins(principally glycoproteins I and II of rabbit platelets).

scholarly journals Glycosaminoglycan synthesis in mouse mastocytoma

1972 ◽  
Vol 126 (3) ◽  
pp. 587-592 ◽  
Author(s):  
T. Helting ◽  
S. Ögren ◽  
U Lindahl ◽  
H. Pertoft ◽  
T. Laurent
Keyword(s):  
Colloidal Silica ◽  
Density Gradient Centrifugation ◽  
Chondroitin Sulphate ◽  
Gradient Centrifugation ◽  
Barium Acetate ◽  
Chondroitinase Abc ◽  
Glycosaminoglycan Synthesis ◽  
Cell Fractions

The glycosaminoglycan synthesis in Furth solid mastocytoma tissue has been studied. Approx. 10% of the polysaccharide isolated after incubation in vitro with [14C]-glucosamine was digestible with chondroitinase ABC and the product of digestion was identified as 2-acetamido-2-deoxy-3-O-(β-d-gluco-4-enepyranosyluronic acid)-4-O-sulpho-d-galactose. Similarly, labelling of polysaccharide in vivo with35SO42-followed by isolation of mast-cell fractions by density-gradient centrifugation on colloidal silica revealed the presence of a polysaccharide which migrated as did chondroitin sulphate on electrophoresis in barium acetate. Chondroitinase ABC produced the same digestion product as before. Finally, the presence of the UDP-N-acetylgalactosamine–chondroitin 6-sulphate hexasaccharide N-acetylgalactosaminyltransferase previously implicated in chondroitin sulphate biosynthesis was demonstrated in microsomal particles from fractions of purified mast cells.

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