Biomedical subjects
Publications and source records attributed to R L Stevens.
Explore the source record for details and available documents.
125I-Insulin binding to rat chondrosarcoma chondrocytes increased, rather than decreased, when these transformed cells were maintained for 1-8 days in primary culture with incremental concentrations of insulin (3 to 1000 ng/ml). The increase in 125I-insulin binding was reversible and depended on the concentration of insulin in which the cells had been previously cultured. The chondrocytes did not change their phenotype in response to the incremental insulin concentrations in the culture medium. The variations in binding of radioligand were not due to different rates of internalization of the ligand-receptor complex, to protease degradation of the radioligand, to shedding of the insulin receptor, or to secretion of an inhibitory insulin binding component. Scatchard analyses of 125I-insulin binding data indicated the induction of a high affinity insulin receptor in the cells cultured in the presence of insulin. The insulin-induced increase in 125I-insulin binding was not associated with an increase in the binding of 125I-labeled multiplication-stimulating activity (125I-MSA), an insulin-like growth factor. Conversely, culturing the chondrocytes in 0.1-1.0 micrograms/ml of MSA did not alter the subsequent binding of either 125I-MSA or 125I-insulin. The insulin-induced increase in insulin binding to these transformed cells may be an abnormality in metabolic control that facilitates growth of the tumor in vivo.
Mouse bone marrow-derived mast cells differentiated in vitro and sensitized with monoclonal IgE respond to antigen-initiated activation with the release of histamine, beta-hexosaminidase, chondroitin sulfate E proteoglycan, and leukotriene C4 (LTC4). The chondroitin sulfate E nature of the glycosaminoglycan side chain was established by demonstrating that the chondroitinase ABC disaccharide digestion products were composed of equal quantities of 4-sulfated and 4,6-disulfated N-acetyl-galactosamine. The single immunoreactive sulfidopeptide leukotriene, released and quantitated with a class-specific antibody, was identified as LTC4 by its retention time on reverse-phase high-performance liquid chromatography and by its specific spasmogenic activity on the guinea pig ileum. The release of the preformed mediators, as well as of LTC4, was related in a dose-response fashion to the concentration of monoclonal IgE used during the sensitization step and to the concentration of specific antigen used to initiate the activation-secretion response. The optimal concentrations of IgE for sensitization and of antigen for challenge were the same for the release of preformed mediators and of LTC4. In addition, the time courses of their release were superimposable, with a plateau at 5 min after antigen challenge. The release of three preformed mediators and of LTC4 after fixation of IgE, washing of the sensitized cells, and antigen challenge unequivocally indicates a bone marrow-derived mast cell origin for these products. Linear regression analyses of the net percent release of beta-hexosaminidase to histamine and of 35S-chondroitin sulfate E to beta-hexosaminidase yielded straight lines that intersected at the origin, which indicates that the three preformed mediators are localized in the secretory granules of the bone marrow-derived mast cells. The concomitant generation of 23 ng of LTC4/10(6) sensitized bone marrow-derived mast cells represents the first example of IgE-dependent release of substantial amounts of LTC4, a component of slow reacting substance of anaphylaxis, from a mast cell population of greater than 95% purity. The IgE-dependent generation of LTC4, rather than prostaglandin D2, by the chondroitin sulfate E proteoglycan-containing bone marrow-derived mast cells contrasts with the predominant generation of prostaglandin D2 by heparin proteoglycan-containing mast cells. These differences together support the existence of two phenotypically different mast cell subclasses.
A differentiated population of cells with metachromatically staining granules and surface IgE receptors was obtained from mouse bone marrow cultured for 2 weeks in the presence of conditioned medium derived from concanavalin A-stimulated splenocytes. The cells were found to incorporate large amounts of [35S]sulfate into an intracellular 35S-labeled proteoglycan of Mr approximately 200,000 containing a maximum of seven glycosaminoglycan side chains (Mr = 25,000). After chondroitinase ABC treatment of density gradient-purified [3H] serine-labeled proteoglycan, the resulting core was Mr approximately 26,000 as assessed by gel filtration. Two-dimensional cellulose acetate electrophoresis of beta-eliminated 35S-labeled glycosaminoglycan revealed a single type of glycosaminoglycan that migrated at the position of oversulfated chondroitin sulfate E from squid cartilage. Chondroitinase ABC degradation of the 35S-labeled glycosaminoglycan yielded two cleavage products in approximately equal molar amounts which co-migrated in both descending paper chromatography and high voltage paper electrophoresis with a monosulfated disaccharide, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose, and a disulfated disaccharide, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-6-di-O-sulfo-D-galactose. The release of some free [35S]sulfate from the oversulfated disaccharide with either chondro-4-sulfatase or chondro-6-sulfatase and the complete desulfation by their combined action established that the oversulfated disaccharide contained N-acetylgalactosamine-4,6-disulfate. The 35S]labeled proteoglycan of these unique IgE receptor-bearing and histamine-containing cells, therefore, is composed of chondroitin sulfate E rather than heparin glycosaminoglycan, and thus is the first identification of such an intracellular localized proteoglycan in a mammalian cell.
Tunicamycin, an inhibitor of dolichol-diphospho-N-acetylglucosamine formation and hence an inhibitor of N-linked oligosaccharide biosynthesis, suppressed total proteoglycan synthesis by Swarm rat chondrosarcoma chondrocytes without affecting the size of the proteoglycan molecule, its secretion from the cell, or its ability to be retained in the extracellular matrix. In addition, tunicamycin did not substantially alter the ability of the chondrocytes to polymerize glycosaminoglycan onto an exogenous beta-D-xyloside acceptor. A secondary effect of tunicamycin suppression of proteoglycan synthesis was that a lesser amount of newly synthesized proteoglycan diffused from the extracellular matrix into the culture medium. The ability of exogenous hyaluronic acid and proteoglycan to increase the percentage of newly synthesized 35S-proteoglycan in the medium in tunicamycin-treated cultures indicates that matrix retention of 35S-proteoglycan is related to the total extracellular uronic acid content rather than to the presence or absence of mannose oligosaccharides bound to the proteoglycan molecule. These noncytotoxic concentrations of tunicamycin (33-333 ng/ml) decreased [3H]mannose incorporation to the same extent that they decreased total [35S]sulfate and [3H]serine incorporation, and caused the chondrocyte to synthesize and secrete a species of beta-hexosaminidase that was mannose-deficient as assessed by its failure to bind to concanavalin A. The additional finding of decreased insulin binding to tunicamycin-treated chondrosarcoma chondrocytes suggested that the inhibition of proteoglycan synthesis was due to diminution of receptors which respond to stimulatory hormones.
The insulin-like growth factor, multiplication stimulating activity (MSA), and insulin were recently shown to stimulate proteoglycan synthesis in monolayer cultures of chondrocytes derived from the Swarm rat chondrosarcoma. Insulin produced significant stimulation at a concentration of less than 1 ng/ml, suggesting that insulin was acting through the insulin receptor rather than through a somatomedin receptor. In this paper we have shown that the insulin-like growth factors IGF-I and IGF-II also are potent stimulators of [35S]sulfate incorporation into macromolecules recovered from the medium and cell layer matrix of the chondrosarcoma chondrocytes. Proinsulin was 3% as potent as insulin in stimulating [35S]sulfate incorporation. We identified receptors for insulin and the insulin-like growth factors, MSA, IGF-I, and IGF-II. Insulin, at concentrations 1000 times the concentration required to produce the biologic response, did not compete for binding of 125I-MSA-II-1 or of 125I-IGF-II and only partially competed for 125I-IGF-I binding. Anti-insulin receptor IgG stimulated proteoglycan synthesis and competed for 125I-insulin binding. Fab fragment prepared from anti-insulin receptor IgG completely blocked the stimulation of [35S]sulfate incorporation into macromolecules by insulin while only partially inhibiting the biologic response to insulin-like growth factors, MSA, IGF-I, and IGF-II. Similarly, the anti-insulin receptor IgG only partially inhibited the binding of 125I-IGF-I and 125I-IGF-II while completely blocking the binding of 125I-insulin. We conclude that insulin stimulates proteoglycan synthesis in the chondrosarcoma chondrocytes by acting through the insulin receptor whereas the insulin-like growth factors probably act through their own receptors.
Rat serosal mast cells cultured in the presence of heat-inactivated fetal calf serum incorporated (35S) sulfate into heparin proteoglycan of approximately Mr = 750,000 after a 3-h pulse and a 2-h chase. beta-D-Xyloside (0.1 mM) treatments of cultures of rat mast cells resulted in an insignificant increase in total (35S) sulfate incorporation and the appearance of free glycosaminoglycans without a change in proteoglycan size. At higher beta-D-xyloside concentrations, total (35S)sulfate incorporation was inhibited and an increase in the relative glycosaminoglycan content was observed concomitant with a reduction in proteoglycan amount and size. As assessed by susceptibility to digestion by chondroitinase ABC, hydrolysis by nitrous acid, [3H]hexosamine content, and electrophoretic mobility, only heparin chains were polymerized onto the proteoglycan core in all cultures. In contrast, individual glycosaminoglycans which appeared only after beta-D-xyloside treatment were predominantly chondroitin sulfate rather than heparin, indicating that the beta-D-xyloside acceptor supported polymerization of chondroitin sulfate but not of heparin glycosaminoglycan. Thus, the peptide core is an important determinant for the synthesis of heparin glycosaminoglycan by rat peritoneal mast cells.
Iduronate sulfatase was purified from human liver for an investigation of the degradative pathway of dermatan sulfate. An overall 80-fold purification was achieved and, more importantly, the preparation was free of alpha-L-iduronidase, beta-glucuronidase, N-acetylgalactosamine 4-sulfate sulfatase (arylsulfatase B) and highly enriched in beta-N-acetylhexosaminidase. The liver enzyme appeared to be composed of several molecular species. The enzyme activity was optimal at pH 4.0 and its Km was 10--20 microM with sulfoiduronyl sulfoanhydromannitol. Chloride was inhibitory at high concentration and among divalent metal ions, only copper was inhibitory. Nitrocatechol sulfate was not a substrate, but did show competitive inhibition. Its Ki for iduronate sulfatase was similar to its Km for arylsulfatase, suggesting a similarity in the substrate binding sites of iduronate sulfatase and arylsulfatases.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
The structures of proteoglycans synthesized and secreted by Swarm rat chondrosarcoma chondrocytes cultured in the presence of insulin, multiplication-stimulating activity (MSA), and fetal calf serum were investigated. Proteoglycans produced in all cultures were found as link protein stabilized aggregates. Monomeric proteoglycans from cultures treated with MSA or insulin were slightly larger in hydrodynamic size than those synthesized by chondrocytes maintained in unsupplemented medium. This increase reflected a 25 to 30% increase in the length of the covalently bound chondroitin sulfate side chains. No free glycosaminoglycans were detected in any cultures, although the presence of a lower molecular weight proteoglycan at a significant concentration was observed in the medium fraction of serum-maintained cultures. MSA and insulin treatments did not result in the synthesis of a proteoglycan containing other types of glycosaminoglycans nor did they alter the degree or position of sulfation of the chondroitin sulfate glycosaminoglycans. The small changes in proteoglycan structure do not account for the larger differences in the levels of incorporation of radioactivity indicating that the primary effect of MSA and insulin on proteoglycan synthesis in this in vitro system is an increased rate of synthesis and secretion of proteoglycan.
Two siblings of consanguineous parents had presented with a variety of findings indicative of juvenile metachromatic leukodystrophy (MLD). However, instead of the expected profound deficiency of arylsulfatase A (ARS A), their enzyme levels were about half-normal, and enzyme from fibroblasts had properties identical with the properties of enzyme from normal fibroblasts. Nevertheless, the hydrolysis of cerebroside sulfate by growing fibroblasts was markedly attenuated. Supplementation of the fibroblasts with cerebroside sulfatase activator normalized the response in the loading test. These results imply that the fibroblasts, and by extension the patients, are deficient in activator. Although the defective catabolism of cerebroside sulfate and the clinical manifestations in these patients mimic MLD, the molecular basis is distinct from the classical forms of the disorder.
Glycosaminoglycans in urine from patients representing the major different mucopolysaccharidoses were separated and measured by use of a procedure that requires only 2 mL of urine. The compounds were resolved by two-dimensional electrophoresis on cellulose acetate plates and made visible by staining with Alcian Blue. They were identified by co-migration with standard glycosaminoglycans, by digestion with specific glycosidases, and by specific degradation with HNO2. They were quantitated by comparing the absorbance of eluates of the stained spots to appropriate standard curves for each glycosaminoglycan. This study revealed additional findings. About half of the patients excreted small amounts of heparin. Further, the keratan sulfate in samples from Morquio's disease patients migrated differently from authentic keratan sulfate unless digested with chondroitinase ABC. Our results for these diseases are in harmony with earlier reports.
Explore the source record for details and available documents.
The cholate and taurodeoxycholate activations of cerebroside sulphate sulphohydrolase (cerebroside-3-sulphate 3-sulphohydrolase, EC 3.1.6.8) activity of arylsulphatase A (aryl-sulphate sulphohydrolase, EC 3.1.6.1) were compared. Taurodeoxycholate caused a sharp peak of response at a concentration of 1.25 mg/ml (type-I activation). Cholate also showed type-I activation but, in addition, it evoked a second, higher, response plateau at concentrations between 5 and 10 mg/ml (type-II activation). At the pH of the reaction, cholate is converted largely to the sparingly soluble free aicd, so at the high concentrations associated with type-II activation, copious precipitates were formed. It was found that the precipitated material was essential for the type-II activation. Type-I activation appears to involve bile salt interaction with substrate, while type-II activation appears to involve enzyme interaction with solid-phase cholic acid. the putative mutant arylsulphatase A in an unusual form of metachromatic leukodystrophy hydolysed cerebroside sulphate only in the presence of high levels of cholate. The type-II activation may thus be simulating a physiological desulphation reaction.
The structure of the proteoglycans from normal pig nucleus pulposus and relatively normal human annulus fibrosus and nucleus pulposus was investigated in detail and the results were compared with the current structural model of proteoglycans of hyaline cartilage. Like proteoglycans of cartilage, those of intervertebral disc contain keratan sulphate and chondroitin sulphate attached to a protein core; they are able to aggregate to hyaluronic acid; the protein core likewise has three regions, one lacking glycosaminoglycans, another rich in keratan sulphate and a third region rich in chondroitin sulphate. However, disc proteoglycans contain more keratan sulphate and protein and less chondroitin sulphate and are also considerably smaller than cartilage proteoglycans. In proteoglycans of human discs, these differences appeared to be due principally to a shorter region of the core protein bearing the chondroitin sulphate chains, whereas in proteoglycans of pig discs their smaller size and relatively low uronic acid content were due to shorter chondroitin sulphate chains. There were subtle differences between proteoglycans from the nucleus and annulus of human discs. In the latter a higher proportion of proteoglycans was capable of binding to hyaluronate.
Proteoglycans extracted with 4M-guanidinium chloride from pig intervetebral discs, and purified by equilibrium density-gradient centrifugation in CsCl, were of smaller hydrodynamic size than those extracted and purified in the same way from the laryngeal cartilage of the same animal. Whether this difference in size arose from degradation during the extraction and purification of the proteoglycans of the disc was investigated. Purified proteoglycans labelled either in the chondroitin sulphate chains or in the core protein were obtained from laryngeal cartilage by short-term organ culture. These labelled proteoglycans were added at the beginning of the extraction of the disc proteoglycans, and labelled cartilage and unlabelled disc proteoglycans were isolated and purified together. There was no appreciable loss of radioactivity after density-gradient centrifugation nor decrease in hydrodynamic size of the labelled cartilage proteoglycans on chromatography on Sepharose 2B, when these were present during the extraction of disc proteoglycans. It is concluded that disc proteoglycans are intrinsically of smaller size than cartilage proteoglycans and this difference in size does not arise from degradation during the extraction.
L-Tyrosine O-sulfate was hydrolyzed by pure human arylsulfatase A (arylsufate sulfohydrolase, EC 3.1.6.1). The rate of hydrolysis was 1/20 of the rate with nitrocatechol sulfate, but was comparable to the rate with cerebroside sulfate. The reaction was optimal at pH 5.3--5.5 and displayed zero order kinetics with time and enzyme concentration. The Km was about 35 mM. The enzyme showed no stereospecificity and hydrolyzed D-tyrosine O-sulfate with Km and V similar to those for the L-isomer. Arylsulfatase B was less than 5% as effective as arylsulfatase A in catalyzing the hydrolysis of the tyrosine sulfates. The daily urinary excretion of tyrosine sulfate by a patient with metachromatic leukodystrophy (arylsulfatase A deficiency) was comparable to the excretion by control subjects. The biological relevance of the tyrosine sulfatase activity of arylsulfatase A remains uncertain.