Search PubMed⌕ Search

Biomedical subjects

A Wasteson

Publications and source records attributed to A Wasteson.

At least 55 records · Page 3Linked to original sources
PubMed1983

Storage and turnover of histamine, 5-hydroxytryptamine and heparin in rat peritoneal mast cells in vivo.

The biogenic amines and heparin of rat peritoneal mast cells were labelled in vivo by the injection of amine precursors (3H-histidine and 3H-5-hydroxytryptophan) and 35S-sodium sulfate. Uptake of label was rapid, probably reflecting the synthesis of new granule material, but the elimination was slow. Half-lives of radiolabelled histamine (23 days) and 5-hydroxytryptamine (5-HT; 25 days) did not differ statistically from that of heparin (35 days). The slow elimination rates suggest that mast cell secretion is of little biological significance under normal conditions but are well compatible with the idea that mast cell function is related to secretion evoked by appropriate immunological stimuli. It further permitted an analysis of the amine storage by repeated injections of unlabelled 5-HT. A 15-fold increase in 5-HT content was obtained while the total amine content remained constant. The uptake of 5-HT was balanced by a reduction of histamine in a molar 1:1 ratio. A displacement of histamine by 5-HT was further indicated by increased elimination rate of radiolabelled histamine in response to 5-HT injections. The results support previous binding studies in vitro and indicate that histamine and 5-HT are bound to identical storage sites in the mast cell granules.

Animals↗
PubMed1983

Cell surface components and growth regulation in cultivated arterial smooth muscle cells.

The surface of rat arterial smooth muscle cells was characterized with respect to some of its chemical and functional properties. The effects of selective enzymic degradations (hyaluronidase, chondroitinases, heparitinase or neuraminidase) on [35S]sulphate-prelabelled cells and on binding sites for cationized ferritin (CF) were examined to assess the presence and relative importance of individual species of macromolecules on the cell surface. The results indicate that about half of the strongly anionic sites on the cell surface (binding CF at pH 2.0) could be ascribed to sulphate groups of glycosaminoglycans and about half to carboxyl groups of sialic acid residues in glycoproteins and/or glycolipids. Weaker anionic sites (binding CF at pH 7.0) largely originated from carboxyl groups of glycosaminoglycans. Chondroitin sulphate and heparan sulphate were the main glycosaminoglycans. The surface of cells from young animals showed a higher glycosaminoglycan and a lower sialic acid content than that of cells from adult animals. Continuous treatment of the cultures with neuraminidase stimulated serum-induced initiation of DNA synthesis, while treatment with hyaluronidase or heparitinase inhibited it. Addition of hyaluronic acid, heparin or heparan sulphate to the culture medium inhibited initiation of DNA synthesis as well as cell proliferation. The effect was more marked in cultures of cells from young animals than from adults, although the latter cells were found to grow at a higher rate and to higher densities. These results suggest a role for cell-surface and pericellular glycoconjugates in growth regulation. A possible mechanism of action is that these molecules, due to their anionic charge or by steric exclusion, interfere with the binding of platelet-derived growth factor, a highly cationic polypeptide, to its cell-surface receptor.

Animals↗
PubMed1982

Enzymatic depolymerization of heparin-related polysaccharides. Substrate specificities of mouse mastocytoma and human platelet endo-beta-D-glucuronidases.

Two endo-beta-D-glucuronidases acting on heparin-related polysaccharides were investigated: a mouse mastocytoma enzyme previously implicated in the postbiosynthetic modification of the heparin proteoglycan and a human platelet-derived enzyme, capable of degrading heparan sulfate as well as heparin (with elimination of anticoagulant activity). The mastocytoma enzyme was found to depolymerize a heparin precursor polysaccharide containing both N- and O-sulfate groups to fragments somewhat smaller in molecular size than commercially available heparin. In contrast, another heparin precursor species, containing N- but no O-sulfate groups was resistant to degradation. Furthermore, incubation of the mastocytoma endoglucuronidase with a heparin octasaccharide having high affinity for antithrombin failed to cleave the beta-glucuronidic linkage in the antithrombin-binding region. Previous studies established that the platelet endoglucuronidase can degrade the exclusively N-sulfated as well as the N- and O-sulfated heparin precursor polysaccharides (Oldberg, A., Heldin, C.-H., Wasteson, A., Busch, C., and Höök, M. (1980) Biochemistry 19, 5755-5762). This enzyme has now been found to attack also the beta-glucuronidic linkage in the antithrombin-binding region of the heparin molecule. The loss of bio-affinity resulting from cleavage of this linkage in the antithrombin-binding heparin octasaccharide was utilized to construct a sensitive, specific, and simple assay method for the platelet endoglucuronidase. It is concluded that the platelet endoglucuronidase has a lower degree of substrate specificity than has the mastocytoma enzyme, in reflection of the surmised functional roles of the two enzymes.

Animals↗
PubMed1982

Interaction of platelet-derived growth factor with its fibroblast receptor. Demonstration of ligand degradation and receptor modulation.

Platelet-derived growth factor (PDGF) has previously been shown to bind to a specific high affinity receptor on human foreskin fibroblasts. The present study was carried out to characterize some of the cellular events resulting from the interaction of the ligand with its receptor. Radiolabeled PDGF was rapidly internalized and degraded after binding to the cells. The degradation was complete and was inhibited by low concentrations of the lysosomotropic agents, chloroquine, ammonium chloride, or methylamine, suggesting that the degradation occurs in the lysosomes. The cellular binding capacity for PDGF decreased after exposure of the cells to PDGF at 37 degrees C. This down regulation of the PDGF receptor was optimal after a 60-min incubation at 37 degrees C and half-maximal at 0.5 nM concentration of PDGF. The binding capacity was restored when the PDGF-containing medium was changed to medium without PDGF; the binding capacity increased from 40 to 80% od the initial value after a 4-h incubation at 37 degrees C. The reappearance on the cell surface of PDGF-binding sites was dependent on protein synthesis and totally blocked by cycloheximide (20 micrograms/ml). Thus, either the receptor has to be resynthesized after internalization or, alternatively, any step in the recycling of "used" receptors is dependent on protein synthesis. Exposure of the cells to PDGF also caused a dose-dependent decrease in the binding capacity for epidermal growth factor which has a distinct receptor on these cells. In contrast, epidermal growth factor did not modulate the PDGF binding capacity, lending no support to the idea that the receptors for epidermal growth factor and PDGF are processed in a common pathway.

Blood Platelets↗
PubMed1981

Somatomedin B: mitogenic activity derived from contaminant epidermal growth factor.

The mitogenic effect of somatomedin B on human cultured glial cells was neutralized by the addition of antibodies to mouse epidermal growth factor. Somatomedin B contained epidermal growth factor--like activity, competing for binding to the epidermal growth factor receptor. It is concluded that contaminating epidermal growth factor may explain the entire mitogenic activity of somatomedin B.

Cell Division↗
PubMed1981

Platelet-derived growth factor. Isolation by a large-scale procedure and analysis of subunit composition.

Platelet-derived growth factor was purified from fresh platelets by a large-scale procedure not involving the use of SDS (sodium dodecyl sulphate). The product, 0.5 mg of platelet-derived growth factor, obtained from about 3 x 10(13) platelets migrated as a single component in analytical gel electrophoresis in the presence of SDS and showed no inhomogeneity on sedimentation-equilibrium analysis in the ultracentrifuge. It had a high specific activity, 2 ng of platelet-derived growth factor/ml (70pM) being equivalent to 1% (v/v) human serum in an assay for multiplication-stimulating activity. Amino acid analysis revealed that platelet-derived growth factor contains all the common amino acids, except tryptophan, but no hexosamine. The molecular weight of platelet-derived growth factor, as determined by sedimentation-equilibrium analysis, was about 33 000. A similar value was obtained by gel electrophoresis in SDS under non-reducing conditions. In the presence of reducing agents the factor molecule was converted into two distinct components of lower molecular weight (17 000 and 14 000 respectively), as demonstrated by protein staining. The molecular model implicated by these findings is that platelet-derived growth factor consists of two different polypeptides chains, linked by disulphide bridges.

Amino Acids↗
PubMed1981

Specific receptors for platelet-derived growth factor on cells derived from connective tissue and glia.

A cellular receptor for platelet-derived growth factor (PDGF) was demonstrated by incubation of 125I-labeled PDGF with human foreskin fibroblast cultures followed by liberation of cell-bound radioactivity with Triton X-100. The cellular binding of labeled PDGF in the presence of increasing amounts of unlabeled PDGF showed saturation; Scatchard analysis of binding data indicated a single class of receptors having kd = 1 X 10(-9) M. The number of PDGF binding sites was approximately 3 X 10(5)/cell. Labeled PDGF binding reached an apparent equilibrium after 3 hr at 4 degrees C. At 37 degrees C, it passed a maximum after 30 min and then decreased with time due to degradation of the tracer. A large excess of unlabeled PDGF reduced labeled PDGF binding by more than 90% whereas similar doses of epidermal growth factor, fibroblast growth factor, or insulin had no effect. It was concluded that PDGF did not share receptors with these factors. PDGF receptors were found on skin fibroblasts, normal and malignant glial cells, smooth muscle cells, and 3T3 cells but not on epithelial-derived cells, neuroblastoma cells, endothelial cells, or peripheral lymphocytes.l As only the receptor-positive cells--i.e., the connective tissue- and glia-derived cells--are responsive to stimulation with PDGF, these findings imply a functional significance of the PDGF receptor.

Animals↗
PubMed1980

Characterization of a platelet endoglycosidase degrading heparin-like polysaccharides.

An endoglycosidase (heparitinase) acting on heparin and heparan sulfate was partially purified (approximately 300 times) from human platelets by affinity chromatography on heparan sulfate substituted Sepharose. Only heparin-like polysaccharides were degraded by the enzyme. The susceptibility of various biosynthetic heparin intermediates indicated that the platelet heparitinase had a requirement for sulfamino but not ester sulfate groups. No activity toward other uronic acid containing glycosaminoglycans could be demonstrated. Glucuronidic but not glucosaminidic linkages in heparin or heparan sulfate were attacked by the enzyme as shown by analysis of the reducing sugar moiety in oligosaccharide products. The anticoagulant activity of heparin, determined in an antithrombin III activation assay, was markedly reduced after treatment with the heparitinase. The enzyme was released from its storage site in platelets after induction of the platelet release reaction. The physiological function of platelet heparitinase is not known but may be to modify extracellular heparin-like polysaccharides in the vascular system.

Blood Platelets↗
PubMed1980

Chemical and biological properties of a growth factor from human-cultured osteosarcoma cells: resemblance with platelet-derived growth factor.

A human osteosarcoma cell line, U-2 OS, cultured under serum-free conditions, was shown to produce a growth factor (osteosarcoma-derived growth factor, ODGF) for human-cultured glial cells, fibroblasts, and other cells. ODGF, collected from the spent medium of 2 OS cultures, was purified by a sequence involving heparin-Sepharose chromatography, hydrophobic chromatography, gel chromatography, and preparative gel electrophresis in SDS. Purified ODGF, at a concentration of 3 ng/ml, elicited a mitogenic response in human glial cells equivalent to 50% of that afforded by human serum at a final concentration of 1%. The preparation was estimated to be > 50% pure. The biological activity of ODGF resided in a cationic, relatively heat-resistant, reduction-susceptible protein with a molecular weight of 30,000 (by gel chromatography and SDS-gel electrophoresis). The electrophoretic behaviour of radioiodinated ODGF suggested that the protein was composed of two different polypeptide chains (about 13,000-14,00 and 16,000-17,000 daltons, respectively) linked via disulphide bonds. The molecular makeup of ODGF was thus similar to that of platelet-derived growth factor. 125I-ODGF could be precipitated by an antibody to platelet-derived growth factor, indicating that the two factors were immunologically related. Resemblance with platelet-derived growth factor was also indicated by the finding that the latter (but not, e.g., fibroblast growth factor or epidermal growth factor) competed with 125I-ODGF for binding to human-cultured glial cells.

Blood Platelets↗
PubMed1980

Growth of normal human glial cells in a defined medium containing platelet-derived growth factor.

DNA synthesis and cell division were measured in serum-free cultures of human normal diploid glial cells maintained in MCDB 105 medium. In growth factor-free cultures the cells remained viable but the cell number was essentially constant. Supplementation with 10 ng of epidermal growth factor or platelet-derived growth factor per ml significantly stimulated DNA synthesis and cell multiplication. Growth occurred both when cells were allowed to settle in serum-containing medium and when cells were plated in serum-free medium. In the latter type of cultures, the cell yield was improved bu incubating the cells in collagen-coated dishes. The use of a miniclone technique allowed the analysis of cell multiplication induced by platelet-derived growth factor at the clonal level and demonstrated that the growth factor induced several cell cycle rounds in a large fraction of clones. The results show that normal cells grown in a recently developed synthetic medium (MCDB 105) supplemented with pure growth factors may multiply without the addition of plasma-derived factors ("progression factors"). It is suggested that the need fo progression factors may simply depend on the composition of the synthetic nutrient medium.

Blood↗
PubMed1979

Desensitisation of cultured glial cells to epidermal growth factor by receptor down-regulation.

Epidermal growth factor (EGF), which can be purified from the mouse submaxillary gland or from pregnant human urine, is a potent multiplication-stimulating factor for several types of cultured cells, including human fibroblasts and glial cells. The molecule binds with high affinity and saturation kinetics to a cell-surface receptor, is subsequently internalised and finally degraded. The binding event is accompanied by a reduction in the number of EGF receptors. This phenomenon--'receptor down-regulation'--has been demonstrated with several hormones and may be a general principle for the modulation of binding groups on the outer cell surface. Further, it has been proposed that receptor loss acts to regulate the cellular response to the binding ligand. The present study provides direct experimental support for this hypothesis. It demonstrates that down-regulation of EGF receptors on glial cells causes desensitisation of the mitogenic response of these cells to subsequent stimulation with EGF.

Cell Line↗
PubMed1979

Sequential degradation of a chondroitin sulphate trisaccharide by lysosomal enzymes from embryonic-chick epiphysial cartilage.

The disulphated trisaccharide D-N-acetylgalactosamine sulphate-beta-D-glucuronic acid-beta-D-N-acetylgalactosamine sulphate prepared from 35S- or 14C-labelled chondroitin sulphate was incubated with a preparation of lysosomal enzymes from embryonic-chick epiphysial cartilage. Degradation was demonstrated by analysis of the reaction products. By use of the appropriate intermediate products as substrates, in conjunction with specific enzyme inhibitors, it was shown that the degradation proceeded sequentially from the non-reducing end. It was initiated by sulphatase (preferentially hydrolysing sulphate ester groups at the 6-position), followed by beta-N-acetylgalactosaminidase and beta-glucuronidase, converting the substrate into monosaccharides and inorganic sulphate. The latter enzyme preferentially attacked disaccharides carrying their sulphate ester group at C-4 of the hexosamine residue. Generation of chondroitin sulphate oligosaccharides may occur by the action of an endoglycosidase, previously demonstrated in embryonic-chick cartilage. Endo- and exo-enzymes may thus form a functional unit in lysosomal degradation of chondroitin sulphate.

Animals↗