scholarly journals Metabolism of macromolecular heparin in mouse neoplastic mast cells

1976 ◽  
Vol 154 (3) ◽  
pp. 605-611 ◽  
Author(s):  
S Ögren ◽  
U Lindahl
Keyword(s):  
Mast Cells ◽  
Colloidal Silica ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Degradation Process ◽  
Subcellular Fractions ◽  
Gel Chromatography ◽  
Tissue Homogenates

1. Polysaccharide in a heparin-producing mouse mastocytoma was pulse-labelled in vivo with [35S] sulphate, and after various periods of time was isolated from subcellular fractions. Such fractions were recovered from tissue homogenates by consecutive centrifugations at 1000g for 10min, 20000g for 20min and 100000g for 1h. Initially the 35S-labelled polysaccharide formed occurred principally in the second centrifugal fraction (20000g precipitate), with small amounts in the first (granular) and third (microsomal) fractions. Analysis for glycosyltransferase activity confirmed that glycosaminoglycans were formed chiefly in particles sedimenting at 20000g. Molecules of this newly synthesized polysaccharide were considerably larger than those of commercially available heparin, as judged from gel chromatography. 2. Within the first hour after injection of [35S]sulphate, most of the labelled polysaccharide was redistributed from the second to the first centrifugal fraction. During, and possibly also after, this shift, the macromolecular polysaccharide was degraded, ultimately to the size of commercial heparin. The degradation process appeared complete 6h after injection of [35S]sulphate. 3. Particulate subcellular fractions were incubated with macromolecular [35S]heparin and the products were analysed by gel chromatography. Significant degradation of the substrate occurred only with the second centrifugal fraction. Further characterization of this fraction, by density-gradient centrifugation in iso-osmotic colloidal silica, revealed a single visible band of particles, at approximately the same density at lysosomes. This band contained all the β-glucuronidase, 35S-labelled endogenous polysacchride and heparin-degrading enzyme present in the second fraction.

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.

scholarly journals Characterization of an oxygen-stable nitrogenase complex isolated from Azotobacter chroococcum

1979 ◽  
Vol 181 (3) ◽  
pp. 569-575 ◽  
Author(s):  
R L Robson
Keyword(s):  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Azotobacter Chroococcum ◽  
Sucrose Density Gradient Centrifugation ◽  
Molybdenum Iron Protein ◽  
Iron Protein ◽  
Protective Proteins

In crude cell-free extracts of Azotobacter chroococcum, nitrogenase was much less sensitive to irreversible inactivation by O2 than was the purified enzyme. When nitrogenase was partially purified by anaerobic discontinuous sucrose-density-gradient centrifugation, O2-tolerance was retained. This preparation was considerably enriched in four polypeptides, three of which were derived from the Mo-Fe(molybdenum-iron) protein and Fe (iron) protein of nitrogenase. The fourth was purified to homogeneity and shown to be an iron-sulphur protein (mol.wt. 14000) probably containing a 2Fe–2S centre. When this protein was added to purified nitrogenase, the enzyme was rendered O2-tolerant, through stabilization was Mg2+-dependent. The isolated O2-tolerant nitrogenase was an equimolar stoicheiometric complex between the MO–Fe, Fe and protective proteins. It is likely that the formation of this complex in vivo is the mechanism of ‘conformational protection’ in this organism.

Characterization of particle-associated choriomammotrophin and progesterone in ovine placentomes

1986 ◽  
Vol 111 (2) ◽  
pp. 217-NP ◽  
Author(s):  
G. E. Rice ◽  
G. D. Thorburn
Keyword(s):  
Colloidal Silica ◽  
Specific Activity ◽  
Density Gradient Centrifugation ◽  
Secretory Granules ◽  
Gradient Centrifugation ◽  
Physicochemical Characteristics ◽  
Dense Granules ◽  
Endocrine Tissues

ABSTRACT The subcellular distribution and compartmentalization of choriomammotrophin (CM) and progesterone within ovine placentomes was investigated using differential and density gradient centrifugation techniques. Approximately 67% of placental CM and 45% of progesterone was associated with subcellular particles. The 10 000 g particulate fraction contained the highest specific activity of both CM and progesterone (19·1 ±3·8 (s.e.m.) μg/mg and 71·5 ± 9·2 pmol/mg protein respectively). This fraction was also shown to contain electron-dense granules with morphology similar to that of hormone-containing secretory granules isolated from other endocrine tissues. Particle-associated CM sedimented to a density of 1·051–1·054 g/ml in colloidal silica gradients and displayed physicochemical characteristics consistent with its storage in secretory granules. During in-vitro incubations, particle-associated CM was stable for up to 90 min, but dissociated when incubated in hypoosmotic medium. Particulate progesterone, which was also present in the CM-rich fraction and was stable for up to 90 min of incubation, was not affected by decreasing the osmolality of the incubation medium. These data suggest that ovine CM (but not progesterone) is stored within a population of secretory granules located within placentomes. J. Endocr. (1986) 111, 217–223

scholarly journals Oestradiol inhibits collagen breakdown in the involuting rat uterus

1972 ◽  
Vol 127 (4) ◽  
pp. 705-713 ◽  
Author(s):  
Janet N. Ryan ◽  
J. Frederick Woessner
Keyword(s):  
Cathepsin D ◽  
Density Gradient Centrifugation ◽  
Post Partum ◽  
Gradient Centrifugation ◽  
Specific Radioactivity ◽  
Rat Uterus ◽  
Oestradiol Treatment ◽  
Cathepsin D Activity ◽  
Stimulation Of

1. The earlier observation (Woessner, 1969) of oestradiol inhibition of collagen breakdown is confirmed and extended. Administration of 100μg of oestradiol-17β/day to parturient rats strongly inhibits the loss of collagen from the involuting uterus. Three experiments show that this effect is due to an inhibition of collagen degradation rather than to a stimulation of collagen synthesis. 2. Uterine collagen was labelled with hydroxy[14C]-proline by the administration of [14C]proline near the end of pregnancy. By 3 days post partum, control uteri lost 83% of their collagen and 90% of their hydroxy[14C]proline. Uteri from oestradiol-treated rats lost only 50% of both total and labelled hydroxyproline, with no decrease in the specific radioactivity of the hydroxyproline. 3. Incorporation of [14C]proline into uterine collagen hydroxyproline in vivo was not affected by oestradiol treatment. 4. Urinary excretion of hydroxyproline was increased in post-partum control rats and decreased in oestradiol-treated rats. 5. An enzyme capable of cleaving 4-phenylazobenzyloxycarbonyl-l-prolyl-l-leucylglycyl- l-prolyl-d-arginine (a substrate for clostridial collagenase) increased in activity in the post-partum uterus and was unaffected by oestradiol treatment. 6. Uterine homogenates digested uterine collagen extensively at pH3.2. This digestion was unaffected by the oestradiol treatment. 7. Lysosomal fractions prepared by density-gradient centrifugation of uterine homogenates contained coincident peaks of cathepsin D activity and peptide-bound hydroxyproline. The cathepsin D and hydroxyproline contents of this peak were unaffected by oestradiol treatment.

scholarly journals In vivo degradation of banana starch: Structural characterization of the degradation process

2010 ◽  
Vol 81 (2) ◽  
pp. 291-299 ◽  
Author(s):  
Fernanda H.G. Peroni-Okita ◽  
Renata A. Simão ◽  
Mateus B. Cardoso ◽  
Claudinéia A. Soares ◽  
Franco M. Lajolo ◽  
...  
Keyword(s):  
Structural Characterization ◽  
Degradation Process ◽  
In Vivo Degradation

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.

scholarly journals Characterization of Functional Vanilloid Receptors Expressed by Mast Cells

Blood ◽  
1998 ◽  
Vol 91 (4) ◽  
pp. 1332-1340 ◽  
Author(s):  
Tamás Bı́ró ◽  
Marcus Maurer ◽  
Shayan Modarres ◽  
Nancy E. Lewin ◽  
Chaya Brodie ◽  
...  
Keyword(s):  
Mast Cells ◽  
Ruthenium Red ◽  
Dorsal Root Ganglion Neurons ◽  
Interleukin 4 ◽  
Vanilloid Receptors ◽  
45Ca Uptake ◽  
Ganglion Neurons ◽  
Type Receptor

Abstract Capsaicin and its ultrapotent analog resiniferatoxin (RTX) act through specific vanilloid receptors on sensory neurons. The C-type receptor is coupled to 45Ca uptake, whereas the R-type is detectable by [3H]RTX binding. We describe here specific vanilloid responses in murine mast cells (MCs). In the MC lines and in bone marrow-derived mast cells, capsaicin and RTX induced45Ca uptake similarly to that observed for cultured rat dorsal root ganglion neurons (DRGs). This response was antagonized by the antagonists capsazepine and ruthenium red. As in DRGs, pretreatment of MCs with capsaicin or RTX induced desensitization to subsequent stimulation of 45Ca uptake. The potency for desensitization by RTX in the MCs corresponded to that for 45Ca uptake, whereas in DRGs it occurred at significantly lower concentrations corresponding to that for the high-affinity [3H]RTX binding site. Consistent with this difference, in MCs we were unable to detect [3H]RTX binding. Vanilloids were noncytotoxic to the MCs, in contrast to the DRGs. Although vanilloids did not cause degranulation in MCs, in the P815 clone capsaicin evoked selective interleukin-4 release. We conclude that certain MCs possess vanilloid receptors, but only the C-type that functions as a channel. Our finding that MCs can respond directly to capsaicin necessitates a reevaluation of the in vivo pathway of inflammation in response to vanilloids.

scholarly journals Separation of Degranulated Platelets from Normal Platelets

1977 ◽  
Author(s):  
P. Cieslar ◽  
J.P. Greenberg ◽  
M.A. Packham ◽  
R.L. Kinlough-Rathbone ◽  
J.F. Mustard
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Adenine Nucleotide ◽  
Thrombus Formation ◽  
Gradient Centrifugation ◽  
Rabbit Platelets ◽  
Fraction I

Platelets degranulated by thrombin (TDP) can be recovered, are effective in hemostasis and survive normally upon infusion into rabbits. Two approaches to determine whether platelets have been degranulated in vivo are: (1) measurement of circulating released materials; (2) detection of circulating degranulated platelets. We have used arabino-galactan (Stractan II) density gradient centrifugation to separate normal and degranulated platelets. The following distribution was obtained with washed rabbit platelets.The serotonin, PF4 and adenine nucleotide contents of the TDP were less than those of normal platelets and the TDP in fraction I had the lowest amounts. When TDP were labeled with 51cr and mixed with equal numbers of normal platelets, 85% of the platelets in fraction I were found to be TDP. 51Cr-TDP were injected into normal rabbits and reharvested after 18 hours. The greatest proportion of TDP was isolated in fraction I. Thus this method may make it possible to separate platelets that have lost their granule contents during participation in reversible thrombus formation in vivo.(* Visiting Fellow from the Faculty of Medicine, Charles University, Prague, Czechoslovakia.)

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