scholarly journals The biosynthesis in vitro of chondroitin sulphate in neonatal rat epiphysial cartilage

1972 ◽  
Vol 126 (2) ◽  
pp. 417-432 ◽  
Author(s):  
C. J. Handley ◽  
C. F. Phelps
Keyword(s):  
Hyaluronic Acid ◽  
Chondroitin Sulphate ◽  
Cetylpyridinium Chloride ◽  
Neonatal Rat ◽  
Turnover Rates ◽  
Glycosaminoglycan Synthesis ◽  
Epiphysial Cartilage ◽  
Sulphated Glycosaminoglycans ◽  
Tissue Contents

1. A system is described, which was used to incubate neonatal rat epiphysial cartilage in vitro with [U-14C]glucose and [35S]sulphate. 2. The acid glycosaminoglycans of neonatal rat epiphyses were extracted and fractionated on cetylpyridinium chloride–cellulose columns. The major components were chondroitin 4-sulphate (65%), chondroitin 6-sulphate (15%), hyaluronic acid (4%) and keratan sulphate (2%). 3. The acid-soluble nucleotides and intermediates of glycosaminoglycan synthesis were separated on a Dowex 1 (formate) system. The tissue contents and cellular concentrations of these metabolites were determined. 4. The rates of synthesis of UDP-glucuronic acid and UDP-N-acetyl-hexosamine from [U-14C]glucose were found to be 0.79±0.16 and 3.2±0.08nmol/min per g wet wt. respectively. 5. The incorporation of [U-14C]glucose into the uronic acid and hexosamine moieties of the polymers was also measured and the turnover rates of the glycosaminoglycans were calculated. It was found that chondroitin sulphate was turning over in about 70h and hyaluronic acid in about 120h. 6. The relative rates of synthesis of the sulphated glycosaminoglycans were calculated from [35S]sulphate incorporation and were found to be in good agreement with those obtained from [U-14C]glucose labelling.

In vitro synthesis of glycosaminoglycans in endocrine ophthalmopathy

1992 ◽  
Vol 127 (5) ◽  
pp. 397-402 ◽  
Author(s):  
G Kahaly ◽  
C Stover ◽  
J Beyer ◽  
E Otto
Keyword(s):  
Hyaluronic Acid ◽  
Acid Concentration ◽  
Cetylpyridinium Chloride ◽  
Acid Synthesis ◽  
Cell Mediated Immunity ◽  
Glycosaminoglycan Synthesis ◽  
In Vitro Synthesis ◽  
Significant Difference ◽  
Endocrine Ophthalmopathy

The effects of humoral and cell-mediated immunity on the glycosaminoglycan synthesis of retrobulbar fibroblasts was evaluated in patients with endocrine ophthalmopathy. After incubation with IgG and sera, secreted glycosaminoglycans, radiolabeled with D-6-3H-glucosamine and 35sulfate, were precipitated with cetylpyridinium chloride and ethanol. Hyaluronic acid synthesis of human retrobulbar fibroblasts after incubation with sera and IgG and after co-culture with lymphocytes was assessed by means of a radiometric test. Patients' IgG, compared to controls', accounted for a higher secretory stimulation of porcine retrobulbar fibroblasts (as measured by cetylpyridinium chloride precipitation) after 24 and 48 h. Contrasting with 24 h incubation time, glycosaminoglycan values after 48 h were increased two to threefold. Patients' and controls' sera caused earlier and stronger, yet indistinguishable glycosaminoglycan production. Non-sulfated hyaluronic acid was the preponderant glycosaminoglycan secreted into the media by retrobulbar fibroblasts. As assessed with the radiometric test, incubation with patients' and controls' sera and IgG did not reveal a significant difference in stimulating the hyaluronic synthesis of patients' and controls' retrobulbar fibroblasts. When measuring the hyaluronic acid synthesis of controls' and patients' retrobulbar fibroblasts after co-cultivation of lymphocytes, however, patients' lymphocytes had a marked ability to increase the hyaluronic acid concentration compared to controls' lymphocytes. The hyaluronic acid concentration after incubation of a patient's retrobulbar fibroblasts with autologous lymphocytes was markedly more elevated than the intrinsic hyaluronic acid production of retrobulbar fibroblasts. In conclusion, though a significant in vitro influence of patients' IgG and sera on the glycosaminoglycan release of both porcine and human (patients' as well as controls') retrobulbar fibroblasts could not be observed in this study, the indications of a marked stimulatory influence of lymphocytes on the hyaluronic acid secretion of retrobulbar fibroblasts demand further investigation.

scholarly journals Degradation of Sulphated Glycosaminoglycans at the Surface of Cultured Human Endothelial Cells by a Platelet Heparitinase

1977 ◽  
Author(s):  
C. Busch ◽  
B. Glimelius ◽  
Å Wastesson ◽  
B. Westermark
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Incubation Medium ◽  
Chondroitin Sulphate ◽  
Coagulation Factors ◽  
Platelet Lysate ◽  
Human Endothelial Cells ◽  
Endothelial Cell Surface ◽  
Sulphated Glycosaminoglycans

The non-thrombogenic property of the endothelial cell surface is a prerequisite for maintainance of blood circulation. The nature of this property is poorly understood. Recent advances in culturing techniques of endothelial cells in vitro may facilitate studies of the surface biochemistry. Human endothelial cells (EC) isolated from umbilical veins were shown to synthesize and secrete sulphated glycosaminoglycans (GAG). The recent finding of a platelet enzyme capable of degrading heparin sulphate (HS) raised the question:Can platelet lysate or a purified heparitinase detach and degrade endothelial HS? EC cultured in the presence of 35S-sulphate, produce 35S-labelled GAG which was isolated from the incubation medium from a cell associated trypsin labile pool and from a cellular pool not liberated by trypsin. After 48 hours of incorporation about 95% of 35S-GAG was found in the medium fraction, 5% in the trypsin fraction and negligible amounts in the cell fraction. In the trypsin pool (“surface fraction”) heparin sulphate comprised about 85%, while the remaining 15% consisted of chondroitin sulphate and/or dermatan sulphate. Incubation of 35S-labelled EC with platelet lysate or a partially purified preparation of the enzyme from the same source caused a marked release of cell-surface associated HS to the incubation medium as oligosaccharides. These effects could be ascribed to heparitinase activity and may alter the properties of the EC-surface and influence the interaction between these cells on one hand and blood cells or plasma components, e.g., coagulation factors on the other.

Identification of IHABP, a 95 kDa intracellular hyaluronate binding protein

1998 ◽  
Vol 111 (12) ◽  
pp. 1673-1684 ◽  
Author(s):  
M. Hofmann ◽  
C. Fieber ◽  
V. Assmann ◽  
M. Gottlicher ◽  
J. Sleeman ◽  
...  
Keyword(s):  
Hyaluronic Acid ◽  
Cell Surface ◽  
Tumour Cell ◽  
Chondroitin Sulphate ◽  
Binding Protein ◽  
Tumour Cell Line ◽  
Binding Motif ◽  
Variant Form ◽  
Hyaluronan Binding

The extracellular matrix component hyaluronan is believed to play important roles in various processes of organogenesis, cell migration and cancer. Recognition of and binding to hyaluronan is mediated by cell surface receptors. Three of them, CD44, ICAM-1 and RHAMM (receptor for hyaluronic acid mediated motility), have been identified. A cDNA clone designated RHAMM turned out to possess transforming capacity. Based on this published sequence, we isolated the complete cDNA of the murine gene. The cDNA comprises an open reading frame of 2.3 kb and encodes a 95 kDa protein. The protein carries a hyaluronan binding motif which binds to hyaluronan in vitro but not to heparin or chondroitin sulphate. It is ubiquitously expressed in normal cells and in all tumour cell lines irrespective of their metastatic properties. One tumour cell line, the metastatic Lewis lung carcinoma, expresses a larger 105 kDa variant form of the protein due to a genomic rearrangement. Antibodies raised against the 95 kDa protein were used for subcellular localization studies. The hyaluronan binding protein is not detectable at the cell surface but is rather localized exclusively intracellularly. Clearly, the sequence we have identified encodes a protein with properties substantially different to the RHAMM protein. We tentatively name the protein intracellular hyaluronic acid binding protein, IHABP.

scholarly journals The biosynthesis in vitro of keratan sulphate in bovine cornea

1972 ◽  
Vol 128 (2) ◽  
pp. 205-213 ◽  
Author(s):  
C. J. Handley ◽  
C. F. Phelps
Keyword(s):  
Uronic Acid ◽  
Cetylpyridinium Chloride ◽  
Corneal Stroma ◽  
Turnover Rates ◽  
Keratan Sulphate ◽  
Tissue Content ◽  
Dowex 1

1. Bovine corneas were incubated in vitro with [U-14C]glucose. 2. The glycosaminoglycans of corneal stroma were isolated and fractionated on cetylpyridinium chloride–cellulose columns. The major components were keratan sulphate (71%), chondroitin 4-sulphate (17%) and chondroitin 6-sulphate (4%). 3. The acid-soluble nucleotides and intermediates of glycosaminoglycan biosynthesis of corneal stroma were separated on Dowex 1 (formate form) and the tissue content and cellular concentrations were determined. 4. The rates of synthesis of the intermediates of glycosaminoglycan biosynthesis in corneal stroma were determined. 5. The incorporation of radioactivity from [U-14C]glucose into the uronic acid and hexosamine components of the glycosaminoglycans present in corneal stroma were measured and the turnover rates of these polymers were calculated. It was found that keratan sulphate was turning over in about 723h and chondroitin 6-sulphate in 251h.

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 Physicochemical characteristics of the glycosaminoglycan-lysosomal enzyme interaction in vitro. A model of control of leucocytic lysosomal activity

1976 ◽  
Vol 160 (2) ◽  
pp. 129-136 ◽  
Author(s):  
J L Avila ◽  
J Convit
Keyword(s):  
Phospholipase A2 ◽  
Intermolecular Interaction ◽  
Lysosomal Enzymes ◽  
Chondroitin Sulphate ◽  
Physicochemical Characteristic ◽  
Phagocytic Process ◽  
Ph Values ◽  
Ph Dependent ◽  
Sulphated Glycosaminoglycans

1. The activities of 30 different lysosomal enzymes were determined in vitro in the presence of the sulphated glycosaminoglycans, heparin and chondroitin sulphate, all the enzymes being measured on a density-gradient-purified lysosomal fraction. 2. Each enzyme was studied as a function of the pH of the incubation medium. In general the presence of sulphated glycosaminoglycans induced a strong pH-dependent inhibition of lysosomal enzymes at pH values lower than 5.0, with full activity at higher pH values. However, in the particular case of lysozyme and phospholipase A2 the heparin-induced inhibition was maintained in the pH range 4.0-7.0. 3. For certain enzymes, such as acid β-glycerophosphatase, α-galactosidase, acid lipase, lysozyme and phospholipase A2, the pH-dependent behaviour obtained in the presence of heparin was quite different to that obtained with chondroitin sulphate, suggesting the existence of physicochemical characteristic factors playing a role in the intermolecular interaction for each of the sulphated glycosaminoglycans studied. 4. Except in the particular case of peroxidase activity, in all other lysosomal enzymes measured the glycosaminoglycan-enzyme complex formation was a temperature-and time-independent phenomenon. 5. The effects of the ionic strength and pH on this intermolecular interaction reinforce the concept of an electrostatic reversible interaction between anionic groups of the glycosaminoglycans and cationic groups on the enzyme molecule. 6. As leucocytic primary lysosomes have a very acid intragranular pH and large amounts of chondroitin sulphate, we propose that this glycosaminoglycan might act as molecular regulator of leucocytic activity, by inhibiting lysosomal enzymes when the intragranular pH is below the pI of lysosomal enzymes. This fact, plus the intravacuolar pH changes described during the phagocytic process, might explain the unresponsiveness of lysosomal enzymes against each other existing in primary lysosomes as well as its full activation at pH values occurring in secondary lysosomes during the phagocytic process.

scholarly journals Degradation of heparin in mouse mastocytoma tissue

1971 ◽  
Vol 125 (4) ◽  
pp. 1119-1129 ◽  
Author(s):  
Sören Ögren ◽  
Ulf Lindahl
Keyword(s):  
Molecular Weight ◽  
Potassium Chloride ◽  
Chondroitin Sulphate ◽  
Cetylpyridinium Chloride ◽  
Supernatant Fraction ◽  
Gel Chromatography ◽  
Neutral Sugar ◽  
Pulse Labelling

1. Heparin was prepared from mouse mastocytoma tissue by mild procedures, including extraction of mast-cell granules with 2m-potassium chloride, precipitation of the extracted polysaccharide with cetylpyridinium chloride from 0.8m-potassium chloride and finally digestion of the isolated material with testicular hyaluronidase. The resulting product (fraction GEH) represented approx. 40% of the total heparin content of the tissue. 2. Fraction GEH was fractionated by gel chromatography on Sepharose 4B into three subfractions, with average molecular weights (¯Mw) of approx. 60000–70000 (highly polydisperse material), 26000 and 9000 respectively. Treatment of each of the subfractions with alkali or with papain did not affect their behaviour on gel chromatography. Amino acid and neutral sugar analyses indicated that the two low-molecular-weight fractions consisted largely of single polysaccharide chains lacking the carbohydrate–protein linkage region. It was suggested that these heparin molecules had been degraded by an endopolysaccharidase. 3. Pulse labelling in vivo of mastocytoma heparin with [35S]sulphate showed initial labelling of large molecules followed by a progressive shift of radioactivity toward fractions of lower molecular weight. Further, heparin-depolymerizing activity was demonstrated by incubating 35S-labelled heparin in vitro with a mastocytoma 10000g-supernatant fraction. Appreciable degradation of the polysaccharide occurred, as demonstrated by gel chromatography. In contrast, no depolymerization was observed on subjecting 14C-labelled chondroitin sulphate to the same procedure.

Cytochemical Evidence for Presynatic β-Adrenergic Regulation of Secretion in the Developing Rat Submandibular Gland

1977 ◽  
Vol 35 ◽  
pp. 430-431
Author(s):  
L.S. Cutler
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Submandibular Gland ◽  
Neonatal Rat ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Parotid Glands ◽  
Adrenergic Agonist ◽  
Rat Submandibular Gland

Many studies previously have shown that the B-adrenergic agonist isoproterenol and the a-adrenergic agonist norepinephrine will stimulate secretion by the adult rat submandibular (SMG) and parotid glands. Recent data from several laboratories indicates that adrenergic agonists bind to specific receptors on the secretory cell surface and stimulate membrane associated adenylate cyclase activity which generates cyclic AMP. The production of cyclic AMP apparently initiates a cascade of events which culminates in exocytosis. During recent studies in our laboratory it was observed that the adenylate cyclase activity in plasma membrane fractions derived from the prenatal and early neonatal rat submandibular gland was retractile to stimulation by isoproterenol but was stimulated by norepinephrine. In addition, in vitro secretion studies indicated that these prenatal and neonatal glands would not secrete peroxidase in response to isoproterenol but would secrete in response to norepinephrine. In contrast to these in vitro observations, it has been shown that the injection of isoproterenol into the living newborn rat results in secretion of peroxidase by the SMG (1).

Healing of Circular Defects in the Rabbit Medial Meniscus Can Occur Spontaneously and Is not Improved by Intra-Articular Hyaluronic Acid

1996 ◽  
Vol 09 (02) ◽  
pp. 60-5 ◽  
Author(s):  
N. Hope ◽  
P. Ghosh ◽  
S. Collier
Keyword(s):  
Hyaluronic Acid ◽  
Medial Meniscus ◽  
Chondroitin Sulphate ◽  
Rabbit Model ◽  
Type Ii Collagen ◽  
Type Ii ◽  
Keratan Sulphate ◽  
Meniscal Healing ◽  
Meniscus Healing ◽  
Made In

SummaryThe aim of this study was to determine the effects of intra-articular hyaluronic acid on meniscal healing. Circular defects, 1.0 mm in diameter, were made in the anterior third of the medial meniscus in rabbits. In one joint, 0.4 ml hyaluronic acid (Healon®) was instilled, and in the contralateral (control) joint, 0.4 ml Ringer’s saline. Four rabbits were killed after four, eight and 12 weeks and the menisci examined histologically. By eight weeks most of the lesions had healed by filling with hyaline-like cartilage. Healing was not improved by hyaluronic acid treatment. The repair tissue stained strongly with alcian blue, and the presence of type II collagen, keratan sulphate, and chondroitin sulphate was demonstrated by immunohistochemical localisation. In contrast to the circular defects, longitudinal incisions made in the medial menisci of a further six rabbits did not show any healing after 12 weeks, indicating that the shape of the lesion largely determined the potential for healing.The effect of hyaluronic acid on meniscal healing was tested in a rabbit model. With one millimeter circular lesions in the medial meniscus, healing by filling with hyalinelike cartilage was not significantly affected by the application of hyaluronic acid intra-articularly at the time of surgery, compared to saline controls, as assessed histologically four, eight and 12 weeks after the operation.

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