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 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 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 Reaction of dihydropyrrolizines with deoxyribonucleic acids in vitro

1972 ◽  
Vol 128 (2) ◽  
pp. 291-297 ◽  
Author(s):  
I. N. H. White ◽  
A. R. Mattocks
Keyword(s):  
Heat Treatment ◽  
Thermal Denaturation ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Equilibrium Density ◽  
Cross Linking ◽  
E Coli ◽  
Caesium Chloride ◽  
Monocrotaline Pyrrole

1. Thermal denaturation profiles of Escherichia coli DNA pretreated with monocrotaline pyrrole in vitro showed no difference from control DNA samples during heating. A substantial increase in the degree of renaturation during cooling of the pretreated DNA samples was observed. The degree of renaturation was dependent on the concentration of pyrrole used. 2. When rat liver DNA was pretreated with monocrotaline pyrrole there was a greater degree of renaturation after heat treatment than was found with E. coli DNA. 3. Equilibrium-density-gradient centrifugation in alkaline caesium chloride showed that DNA pretreated with monocrotaline pyrrole and heat denatured, renatured to a greater extent on quenching in ice than did untreated control DNA. The degree of renaturation was similar whether the initial treatment of the DNA with pyrrole was for 1 or for 15min. The reaction also appeared to be independent of pH between 5.5 and 9.0. 4. Retrorsine pyrrole was as effective as monocrotaline pyrrole in cross-linking the DNA, but monocrotaline pyrrole exposed to water for 5min before the addition of the DNA was ineffective. Pretreatment of E. coli DNA with synthetic bis-hydroxymethylpyrrole esters also caused renaturation after heat treatment. Monoesters were ineffective. 5. After treatment of rats with retrorsine, no cross-linking of the liver DNA could be demonstrated.

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.

Steroid sequestration within the rat adrenal zona glomerulosa

1988 ◽  
Vol 117 (2) ◽  
pp. 191-NP ◽  
Author(s):  
S. M. Laird ◽  
G. P. Vinson ◽  
B. J. Whitehouse
Keyword(s):  
Plasma Membrane ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Zona Glomerulosa ◽  
Membrane Preparation ◽  
The Media ◽  
Considerable Concentration ◽  
Highly Correlated ◽  
Plasma Membrane Preparation

ABSTRACT Accumulated data from in-vitro experiments have suggested that 18-hydroxysteroids may be stored within the intact rat adrenal zona glomerulosa. The phenomenon was further investigated by comparing the amount of steroid remaining in the zona glomerulosa tissue with that secreted into the media during incubation in vitro. The results showed that 18-hydroxydeoxycorticosterone (18-OH-DOC) and 18-hydroxycorticosterone (18-OH-B) were retained within the tissue against a considerable concentration gradient, with smaller amounts of aldosterone and corticosterone. Lysis of the intact zona glomerulosa, by preincubation in distilled water, yielded an enriched plasma membrane preparation. After subsequent incubation in Krebs–Ringer bicarbonate this preparation contained significantly more 18-OH-DOC than did the intact tissue, suggesting that tissuesequestered 18-OH-DOC is normally metabolized to other products. These may include 18-OH-B and aldosterone. Fractionation of homogenized intact zona glomerulosa and the enriched plasma membrane preparation by density gradient centrifugation showed that tissue 18-OH-DOC banded in fractions of density 1·063– 1·21 g/ml and that its distribution was highly correlated with protein. Corticosterone, 18-OH-B and aldosterone banded like added free [3H]18-OH-DOC in fractions of density < 1·006 g/ml. The results suggest that 18-OH-DOC is the major sequestered steroid within the rat adrenal zona glomerulosa and that this sequestration is attributable to the association of 18-OH-DOC with a high-density component of the plasma membrane. J. Endocr. (1988) 117, 191–196

scholarly journals Unstable association in vitro between donor and recipient DNA in Bacillus subtilis

1982 ◽  
Vol 152 (1) ◽  
pp. 275-283
Author(s):  
J Van Randen ◽  
K Wiersma ◽  
G Venema
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Gradient Centrifugation ◽  
Cross Linking ◽  
Dna Fragments ◽  
Dna Complexes ◽  
Cscl Gradient ◽  
Donor Moiety

In addition to stable donor-recipient DNA complexes, unstable complexes between donor and recipient DNA were formed in vitro with Bacillus subtilis. Whereas the stable complexes survived CsCl gradient centrifugation at pH 11.2 and phenol plus sodium p-aminosalicylate extraction with 0.17 M NaCl, the unstable complexes dissociated during these manipulations. The donor moiety from the unstable complexes remained associated with the recipient DNA during phenol plus sodium p-aminosalicylate treatment at 0.85 M NaCl. The unstable complexes could be stabilized artificially by cross-linking with 4,5',8-trimethylpsoralen. Dissociation of the complexes during CsCl gradient centrifugation could be prevented by centrifuging at pH 10. Heterologous DNA fragments derived from phage H1 DNA appeared to be unable to form complexes with the recipient B. subtilis DNA. Unstable complexes were also formed with Escherichia coli DNA, although under all conditions tested, more complex was detectable by using homologous B. subtilis DNA.

Dna Synthesis in Purified Populations of Avian Erythroid Cells

1972 ◽  
Vol 10 (1) ◽  
pp. 27-46
Author(s):  
A. F. WILLIAMS
Keyword(s):  
Biochemical Characterization ◽  
Bone Marrow Cells ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Experimental System ◽  
Equilibrium Density ◽  
Polychromatic Erythrocytes ◽  
Type Size ◽  
Haemoglobin Content

By the use of equilibrium density-gradient centrifugation erythroblasts and early polychromatic erythrocytes have been isolated from avian anaemic bone marrow. Cells from both the unfractionated and purified preparations have been characterized in terms of their histological type, size, haemoglobin content and ability to synthesize DNA. Erythroblasts were the only cells to synthesize DNA and it appeared that their progeny, the polychromatic erythrocyte, failed to enter a new S phase. The experimental system described allows biochemical characterization of earlier stages of avian erythropoiesis than has previously been possible.

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